600-4-84-038
CHARACTERIZATION OF HAZARDOUS WASTE SITES
A METHODS MANUAL
VOLUME III. AVAILABLE LABORATORY ANALYTICAL METHODS
by
Russell "H. Plumb", Jr.
Lockheed Engineering Mamtgement Services Company
Las Vegas, Nevada 39114
Contract dumber 68-03-3050
Prsjtct Officer
Werner F. Beckert
Quality Assurance Division
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89114
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH *MD DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
LAS YE5AS. NEVADA "35114
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NOTICE
Although the research described in this manual has been funded
in part by the U.S. Environmental Protection Agency through Contract
69-03-3050 to the Lockheed Engineering and Management Services Comoa
Las Vegas, Nevada, it has not been subjected to Agency policy review
therefore does not necessarily reflect the views of the Agency. Men-
of trade names or commercial products does not constitute endorsemem
recommendation for »?e.
ii
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FOREWORD
This document is part of a multlvolume manual, entitled Characterization
of Hazardous Waste Sites--A Methods Manual, that is being prepared by the U.S.
Environmental Protection Agency.It is intended to serve a wide variety of
users as a source of Information on available methods for collection and
analysis of samples from hazardous waste disposal sites.
Volume I. Integrated Approach to Hazardous Waste Site Characterization
1 ncltides discussions on preliminary assessment, Initial data evaluation, ad-
minlstratlve procedures, offsite reconnaissance, site Inspection, chain of
custody, quality assurance, safety, and personnel training. In addition,
considerations to be included in the development of a sampling strategy and
the selection of analytical methods are presented.
Volume II. Available Sampling Methods 1s dedicated to sampling proce-
dures and sampling information.It is a description of methods and materials
available to field investigators ^or most sampling situations that arise
during routine hazardous waste disposal site and hazardous spill investiga-
tions.
This volume, Volume III. Available Laboratory Analytical Methods,
presents detailed methodology suitable for hazardous waste sample analysis.
The distinctions between this analytical compendium and existing contemporary
manuals are the scope of analytes to be potentially covered and the diversity
of sample matrices to be addressed. Thus, while existing manuals provide
detailed guidance for 50 to 100 specific analytes (and will be used as primary
sources for these procedures), Appendix VIII of the Resource Conservation and
Recovery Act lists more than 350 substances of concern. The purpose of this
compendium 1s to serve as a repository of available analytical techniques for
these substances. Also, existing manuals generally address a single sample
matrix whereas this compendium provides sample preparation guidance for as
many as five sample matrices. This 1s consistent with the common source intent
of the multivolume manual and the complex nature of hazardous wastes that
may require collection of samples of various sample matrices during a single
sampling event.
This volume 1s to be a dynamic document that will be updated and expanded
to reflect state-of-the-art methodology achieved by EPA, the regulated commu-
nity, and other members of ttre scientific community. Thus, as new compounds
are manufactured or classified as hazardous, the analytical part of the compen-
dium veil! have to be sxoanded- As analytical procedures are developed or
evaluated, the contents of the compendium wiil oe modified.
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The user is cautioned, however, that not all included procedures have
been used with all sample types. If problems are encountered with certain
sample types, or if there are any comments, corrections, suggested additions,
or questions concerning the material contained in, or omitted from this com-
pendium, the user is asked to direct comments to:
Hazardous Waste Metnods Evaluation Branch
Quality Assurance Division
Environmental Protection Agency
P.O. Box 15027
Las Vegas, Nevada 89114
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ACKNOWLEDGEMENT
The development of this analytical manual 1s the result of Initial
direction provided by the EPA-w1de Steering Committee. The contributions
of the committee members and additional reviewers Identified below are
appreciated.
Laboratory Committee
Individual
M. Birch
D. Friedman
F. Haeberer
E. Meier
T. Melggs
J. Poppitl
F. Richardson
D. Weltzman
Organization
Region IV
OSW
OERR
EMSL-LV
NEIC
OSW
OSW
DOHS
Additional Reviewers
W.
S.
w.
M.
A.
D.
D.
J.
W.
G.
Beckert
Bromberg
Budde
Dellarco
Galliart
Garnas
Gurka
Huang
Reynolds
Schweitzer
EMSL-LV
EMSL-RTP
EMSL-CIN
OMSQA
MCCC
NEIC
EMSL-LV
OMSQA
LEMSCO
EMSL-LY
Assistance provided by the following Individuals was essential to the
completion of this manual and is gratefully acknowledged: H. Kerfoot and
J. Sherma for drafting some of the analytical sections; H. Kerfoot and
J. Engels for proofing each of the analytical sections; M. Birch for
reviewing each of the manual jdrafts; and S. Brown, L. Steele, and D. Nidy
for word processing support.
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TABLE OF CONTENTS
Page
Foreword 111
Acknowledgement v
Chapter I. Introduction 1-1
A. Background 1-1
B. Objectives 1-1
C. Organization 1-4
Chapter II. Phase Separation and Screening Procedures for
Hazardous Waste Samples II-l
A. Introduction II-l
B. Storage and Handling of Hazardous Waste Samples II-l
C. Phase Separation of Hazardous Waste Samples 11-3
C.I Phase Separation II-3
C.2 Aliquot Preparation 11-5
D. Inorganic Screening Procedures 11-9
D.I Spot Test for Oxidants II-9
D.2 Spot Test for Cyanide ». , . II-12
D.3 Spot Test for Sulfide 11-16
D.4 Alternate Spot Test for Sulfide 11-18
D.5 Spot Test for pH 11-21
E. Organic Screening Procedures 11-22
E.I Extract Preparation 11-22
E.2 Extract Analysis 11-23
Chapter III. Procedures for Organic Compounds. ... III-l
Section 1. Volatile Organic Compounds III-2
J.I.I Analysis of Solid Hazardous Waste Samples for Volatile
Organic Compounds by Methanol Partitioning 111-21
J.I.2 Analysis of Solid Hazardous Waste Samples for Volatile
Organic Compounds by Polyethylene
Glycol Partitioning II1-25
J.2.1 Analysis of Water Samples for Volatile Organic
Compounds 111-31
0.3.1 Analysis of Soil/Sediment Samples 111-36
•J.4.1 Analysis of Volatile Organic Compounds in
Biological Tissue . . ,—.- , . , , !!!-AQ
vii
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TABLE OF CONTENTS (Continued)
0.4.2 Analysis of Biological Tissue Samples for Purgeable
Organic Compounds 111-44
J.5.1 Analysis of Air Samples for Volatile Organics 111-46
Section 2. Acid-Extractable Organic Compounds 111-56
J.I.I Analysis of Hazardous Waste Samples for
Acid-Extractable Organic Compounds 111-68
J.2.1 Analysis of Water Samples for
Acid-Extractable Organic Compounds 111-72
J.3.1 Analysis of Sediment Samples for Acid-Extractable
Organic Compounds by Hexane-Methanol Extraction . . . 111-77
J.3.2 Analysis of Sediment Samples for Acid-Extractable
Organic Compounds by Methylene Chloride Extraction. . 111-80
J.4.1 Analysis of Biological Tissue Samples for Acid-
Extractable Organic Compounds by Methylene
Chloride Extraction 111-85
Section 3. Base/Neutral-Extractable Compounds 111-93
J.I.I Analysis of Hazardous Wastes for Base/Neutral Compounds III-105
J.2.1 Analysis of Methylene Chloride Extracts of Aqueous
Samples for Base/Neutral Compounds III-112
J.3.1 Analysis of Sediment Samples for Base/Neutral Compounds 111-118
J.3.2 Analysis of Methylene-Chloride Sediment
Extracts for Base/Neutr?! Compounds .•...,.... !!I-!2
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TABLE OF CONTENTS (Continued)
H.I.I Determination of Organophosphorus Pesticides
1n Hazardous Wastes (Pesticide Formulations) III-231
H.2.1 Determination of Organophosphorus Pesticides
1n Water III-235
H.3.1 Determination of Organophosphorus Pesticides 1n Son. . III-243
H.4.1 Determination of Organophosphorus Pesticides
1n Fruits and Vegetables III-244
H.5.1 Determination of Organophosphorus Pesticides 1n A1r . . III-247
Section 6. Methods for the Determination of
Organonltrogen Pesticides III-255
1.1.1 Determination of Carbamates and Urea Pesticides
In Hazardous Waste Samples. Reserved III-267
1.2.1 Determination of Carbamate and Urea Pesticides
1n Industrial and Municipal Wastewater III-268
1.3.1 Determination of Carbamate Pesticides 1n Soil III-276
1.3.2 Determination of Urea Herbicides In Soil III-281
1.4.1 Determination of Carbamate Pesticides 1n
Fruits and Vegetables 111-284
1.5.1 Determination of Carbamate Pesticides In A1r III-2S3
Section 7. Methods for the Determination of Chlorinated
Phenoxy Add Herbicides III-303
F.I.I Analysis of Solid Waste Samples for Chlorinated
Herbicides by High Performance Liquid Chromatography. III-310
F.2.1 Analysis of Water Samples for Chlorinated Phenoxy
Add Herbicides by Chloroform Extraction III-312
F.2.2 Analysis of Water Samples for Chlorinated Phenoxy
Add Herbicides by Ethyl Ether Extraction 111-315
F.3.1 Analysis of Sediment Samples for Chlorinated Phenoxy
Add Herbicides by Acetone-Hexane Extraction III-318
F.3.2 Analysis of Soil Samples for Chlorinated Phenoxy
Add Herbicides by High Performance Liquid
Chromatography 111-322
Section 8. Methods for the Determination of D1ox1n (TCDD) . . . III-327
J.I.I Analysis of Hazardous Waste Samples for D1ox1n.
Reserved III-339
J.2.1 Analysis of Water Samples for D1ox1n III-340
J.2.2 Analysis of Water Samples for TCDD 111-348
J.3.1 Analysis of Methylene Chloride Extracts of
Sediment/Soil Samples for 2,3,7,8-TCDD III-351
J.2.2 Detsrwlnation of 2,3,7,8-TCDD 1n Methanol
Extracts of Soil and Sediment 111-25^
1x
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TABLE OF CONTENTS (Continued)
J.4.1 Analysis of Hexane Extracts of Biological
Tissue for TCDO 111-364
Section 9. Methods for the Determination of Polycyclic
Aromatic Hydrocarbons III-369
1.1.1 Analysis of Hazardous Waste for Polycyclic
Aromatic Hydrocarbons 111-381
1.2.1 Analysis for Polycyclic Aromatic Hydrocarbons
in Water Samples II1-386
1.3.1 Analysis of Sediments for Polycyclic
Aromatic Hydrocarbons 111-395
1.4.1 Analysis of Fish and Shellfish Tissue for PAH III-402
1.4.2 Analysis of Plant Tissue for PAH III-407
1.5.1 Analysis of Air for Polycyclic Aromatic Hydrocarbons. . III-414
Chapter IV. Procedures for Inorganic Substances IV-1
Section 10. Elemental Analysis by Atomic Absorption
Spectrometry IV-2
J.I.I Determination of Metals in LMB/LiF Fusion Pellets
of Waste Samples IV-13
J.2.1 Analysis of Water Samples for Metals IV-15
• J.3.1 Analysis of Soil/Sediment Samples for Heavy Metals. . . IV-33-
J.4.1 Analysis of Tissue Camples ror Heavy Metals 17-42
J.5.1 Analysis of Air Samples for Heavy Metals. ....... IV-44
Section 11. Methods for the Determination of Mercury IV-51
G.I.I Analysis of Hazardous Waste Samples for Mercury.
Reserved IV-57
G.2.1 Analysis of Water Samples for Mercury .... IV-58
G.3.1 Analysis of Sediment Samples for Mercury .... IV-60
G.4.1 Analysis of Fish Tissue Samples for Mercury IV-62
G.5.1 Analysis of Solid-Phase Collectors from Air
Sampling for Mercury IV-64
G.5.2 Sampling and Analysis of Air Samples Using
Liquid-Phase Collectors for Mercury IV-66
Section 12. Methods for the Determination of Methyl Mercury . . IV-70
G.I.I Analysis of Hazardous Waste Samples for
Methyl Mercury. Reserved IV-73
G.2.1 Analysis of Water Samples for Methyl Mercury IV-74
Jec-ion 13. Methods ror the Detern-;nation of Ar-jeni::. . . . . . TY-"T~>
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TABLE OF CONTENTS (Continued)
Page
F.I.I Analysis of Hazardous Waste Samples
for Arsenic. Reserved IV-80
F.2.1 Analysis of Aqueous Samples for Arsenic IV-81
F.2.2 Determination of Arsenic 1n Water Samples
with the Graphite Furnace Technique IV-83
F.3.1 Determination of Arsenic 1n Sediment Samples IV-85
Section 14. Methods for the Determination of Selenium IV-88
F.I.I Analysis of Hazardous Waste Samples
for Selenium. Reserved IV-91
F.2.2 Analysis of Aqueous Samples for Selenium Using
Hydride Generation IY-92
F.3.1 Analysis of Sediment Samples for Selenium
Using Graphite Furnace Techniques IV-94
F.3.2 Analysis of Sediment Samples for Selenium Using
Hydride Generation (AA) Techniques IV-96
Section 15. Methods for the Determination of Trace Metals
Using Inductively Coupled Plasma Atomic
Emission Spectroscopy IV-99
6.1.1 ICAP Determination of Metals in LMB/L1F
Fusion Pellets of Waste Samples IV-111
G.2,1 Determination of Metals in Aaueous Samples Using
Inductively Coupled Plasma Atomic Emission
Spectrometrlc Analysis IV-118
Section 16. Methods for the Determination of Cyanide IV-126
H.I.I Analysis of Hazardous Waste Samples for Cyanide.
Reserved IV-130
H.2.1 Analysis of Aqueous Samples for Cyanide IV-131
Section 17. Methods for the Determination of Sulflde IY-136
G.I.I Determination of Sulfide in Aqueous Phase
Hazardous Waste Disposal Site Samples IY-141
G.I.2 Determination of Sulflde in Solid Phase
Hazardous Waste Disposal Site Samples IV-143
G.2.1 Methylene Blue, Color1metr1c Determination
of Sulfide in Aqueous Samples IY-146
6.2.2 Determination of Sulflde in Aqueous Samples
by lodometric Titration IV-149
G.3.1 Determination of Sulfide in Sediment Samples
'Jsina *he Methylene Blue ColoHmetrfc Technique . . . IY-151
xi
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TABLE OF CONTENTS (Continued)
Page
Section 18. Methods for the Determination of Ammonia IV-156
G.I.I Analysis of Hazardous Waste Samples
for Ammonia. Reserved IV-164
G.2.1 Analysis for Ammonia in Aqueous Samples
Using an Automated Phenate Procedure IV-165
G.2.2 Automated o-Tolidine Colorimetric Analysis for
Ammonia in Aqueous Samples IV-169
G.2.3 Titrimetric or Colorimetric Determination of
Ammonia in Aqueous Samples IV-171
G.3.1 Determination of Ammonia in Sediments
Following Distillation IV-174
G.4.1 Determination of Ammonia in Biological Tissue.
Reserved IV-176
G.5.1 Determination of Ammonia in Air Samples IV-177
Chapter V. Screening and General Sample Characterization
Procedures V-l
Section 19. Methods for the Determination of Oxidants V-2
F.l.l Oxidant Capacity of Hazardous Waste Samples. Reserved. V-5
F.2.1 Oxidant Capacity of Aqueous Samples V-6
F.3.1 Determination of Oxidant Capacity in Soil Samples.
Reserved V-8
F.4.1 Determination of Oxidant Capacity in
Biological Tissue Samples. Reserved V-9
F.5.1 Determination of Oxidants in Air V-10
Section 20. Method for the Determination of
Reductant Capacity V-15
F.I Determination of Reductant Capacity Y-17
Section 21. Methods for the Determination of Acidity. ..... V-19
F.l.l Determination of Acidity in Hazardous Waste Samples.
Reserved V-21
F.2.1 Titrimetric Determination of Acidity in Aqueous
Samples V-22
Section 22. Methods for the Determination of Alkalinity .... V-24
F.l.l Determination of Alkalinity in Hazardous Waste
Samples. Reserved V-26
F.2.1 Titr^metric Determination of Alkalinity in
Aqueous Samples v-27
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TABLE OF CONTENTS (Continued)
Page
Section 23. Methods for the Determination of Percent
Moisture and Percent Solids V-29
E.I Percent Moisture Determination in Hazardous Waste
Samples V-31
E.2 Determination of Total Solids in Water V-32
E.3 Total Solids Determination for Sediment Samples .... V-34
Section 24. Methods for the Determination of Conductivity . . . V-36
6.1.1 Conductivity Measurements for Hazardous Wastes.
Reserved V-38
6.2.1 Measurement of Conductivity of Aqueous Samples V-39
xiii
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CHAPTER I
INTRODUCTION
A. BACKGROUND
Existing regulations require generators of waste material to determine
whether their waste products meet the established definition of hazardous
wastes [Section 3001 of the Resource Conservation and Recovery Act (RCRA), PL
94-580 (40 CFR Part 261)]* and are therefore subject to regulation. Since
no single characteristic can reliably establish the hazardous nature of all
wastes, the protocol developed for this purpose requires the measurement of
five behavioral characteristics of the waste and analysis of the waste for
the presence of specific constituents.2
The five behavioral characteristics are ignitability, corrosivity,
reactivity, EP toxicity (Teachability), and acute biological toxicity (Figure
1-1). Guidance for using these procedures has been developed by the EPA
Office of Solid Waste and Emergency Response.2 \ny waste that exceeds the
specified limits established for these procedures is considered hazardous.
A waste is also classified as hazardous if it contains any of the'spec'ffic
compounds listed in Subpart D or Appendix VIII of the RCRA regulations.* Since
chemical composition of a waste is an important part of the waste characteri-
zation protocol, the Environmental Protection Agency convened an Agency-wide
steering committee in August 1981 to assess the need for development of a
comprehensive manual to facilitate implementation of the analytical require-
ments of the hazardous waste regulations. The committee concluded that such a
hazardous waste manual was indeed necessary for the following reasons:
1. the number of specific compounds for which analysis may be required
exceeds the scope of existing analytical manuals (Appendix VIII of
RCRA lists 360 compounds),
2. hazardous waste analysis requires sampling and sample preparation
guidance for complex and diverse sample matrices whereas most existing
manuals only address a single matrix (usually water).
The purpose 1n preparing this hazardous waste manual is to provide a compen-
dium of methods 1n response to this regulatory need.
B. OBJECTIVES
This analytical compendium is Volume 3 of a broader compilation of informa-
tion on riazaroous *asta sampling and analysis that *s befng prepared by the
1-1
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Environmental Protection Agency, Environmental Monitoring Systems Laboratory
1n Las Vegas. The Individual volumes 1n this compilation are:
Volume 1. Integrated Approach to Hazardous Waste Site Characterization
This volume discusses the administrative and technical factors that
must be considered in planning and implementing a hazardous waste site
Investigation.
Volume II. Available Sampling Methods
This volume provides situational guidance on the use of sampling proce-
dures and equipment to obtain samples during hazardous waste site
investigations.
Volume III. Compendium of Procedures for the Analysis of Hazardous Wastes
This volume is a compilation of analytical procedures of known perform-
ance that are suitable for the analysis of hazardous waste samples.
The objectives established by the steering committee for this analytical
compendium include:
1. serving as a common source of analytical methods available to the
Hazardous Waste Program and other Individual EPA Program Offices
for use in their programs, as the need arises;
2. providing detailed guidance to the bench chemist 1n EPA and State
Regulatory laboratories on the use of these procedures, Including
sample handling and preparation;
3. serving as a planning document for EPA by identifying areas requiring
further analytical research and development work.
One additional requirement established by the steering committee is that
each procedure must be accompanied by a statement of performance (precision
and accuracy).
The compilation of analytical procedures 1n this volume obviously ad-
dresses the first two objectives. Hazardous waste analytical needs, objective
three, are identified by:
1. absence of analytical methods for specific compounds listed in
the regulations;
2. absence of sample preparation guidance for one or more sample
matrices;
3. absence of sufficient method performance data to Include a method or
to rate
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4. absence of necessary information on sample handling and sample
preservation.
Since the analytical needs are defined by the absence of essential information,
a standardized format was developed for presenting analytical procedures to
alert users to these .information gaps.
C. ORGANIZATION
This volume is divided into five Chapters. Chapter I identifies the
objectives and structure of the document. Chapter II presents an approach for
screening and pretreatment of hazardous waste samples. Chapter III and Chapter
IV present analytical protocols for organic compounds and inorganic substances,
respectively, listed in RCRA Appendix VIII. Chapter V presents analytical
procedures for general sample characterization.
The guidance presented in Chapter II is based on a protocol developed at
the National Enforcement Investigation Center, Denver, Colorado. It consists
of two aspects that must be considered when hazardous waste samples are to be
analyzed. The first part of Chapter II describes an approach to conduct a
safety screening of hazardous waste samples. The screening results alert the
analyst to potential hazards that may be encountered during the handling and
analysis of such samples due to the presence of strong oxidants, strong re-
ductants, the evolution of potential1y toxic gases such as hydrogen cyanide or
hydrogen sulfide, or extreme pH values. Appropriate precautions are recom-
mended that should be taken when such safety hazards and potential analytical
interferences are identified. In the second part of Chapter II, a protocol for
fractionating a complex hazardous waste sample into its component phases is
presented. This approach allows independent a.naly«is of each sample phase
according to the methods detailed in Chapters II! to V.
Available analytical procedures for the determination of a wide range of
sample constitutents and properties in as many as five sample matrices are
presented in Chapters III, IV, and V for organic compounds, Inorganic con-
stituents, and general sample characteristics, respectively. The sample
matrices that have been addressed are 1) hazardous wastes, 2) water, 3) soil/
sediment, 4) biological tissue, and 5) air. The distinction between the
hazardous waste category and the water or soil category may, at times, only
be a matter of concentration but the higher concentrations associated with
hazardous waste samples may require special safety precautions and special
sample handling (i.e. the use of glove boxes or regulated laboratories, smaller
aliquots, and/or special sample preparation methods).
The criteria for the selection of analytical procedures for inclusion in
this compendium are based on a combination of practical and analytical con-
siderations. Ideally, each procedure selected 1) should have been used in
numerous laboratories, 2) should have been evaluated for sensitivity, accuracy,
and precision, and 3) should have been evaluated for analytical interferences.
Also, effective sample preservation, maximum storage time, and sample prepara-
tion -^rocadurss *
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These considerations Included 1) an established regulatory need, 2) the
existence of only one procedure, 3) time and/or cost requirements, or
4) concern over safety in the use of a procedure. Another factor is that
many of the analytical techniques required for the analysis of hazardous
wastes are state-of-the-art developments, and sufficient data may not have
been generated to fully evaluate the performance of each procedure. Because
the analytical and practical considerations are not always compatible, a
ranking system is being used to identify the present status of each procedure
to the user.
Minimum requirements for selection of a procedure are: 1) an established
need exists for a specific procedure, 2) single-laboratory precision and
accuracy data exist for the procedure, and 3) the range of applicability of
the procedure has been defined. Analytical procedures-that meet these require-
ments are listed as "available". If multi-laboratory precision and accuracy
data exist for a procedure (suggesting more extensive evaluation and use),
the procedure is listed as "evaluated". The user is cautioned that the intent
is to assemble procedures of known performance and that these procedures may
not necessarily produce results with acceptable levels of precision and accu-
racy. Also, the ranking of each procedure is based on available data. It is
expected that, as more information becomes available, some of the included
procedures will be upgraded in status in future revisions of this compendium.
The analytical methods in each section are presented in a format that
follows the topical outline presented below:
A. Scope
B. Sample Handling and Storage
C. Interferences
0. Safety
E. Apparatus
F. Reagents
G. Quality Control
H. Calibration
I. Daily Performance Tests
J. Analytical Procedures
J.I. Analysis of Hazardous Wastes
J.2. Analysis of Water Samples
J.3. Analysis of Solid-Phase Samples
J.4. Analysis of Biological Tissue Samples
J.5. Analysis of Air Samples
K. Qualitative Identification
L. Calculations
M. References
Sample handling and storage information (Subsection B) for each analytical
section will be summarized in a flow diagram similar to that shown in Figure
1-2. When a particular method of samole ^and^'ng is inaDorooriate (i.e. air
drying of sediment samples scheduled for analysis of volatile organic com-
pounds), that portion of tne diagram is deleted. TMs approacfi visually
1-5
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Figure 1-2. Schematic diagram developed to summarize sample handling and storage information.
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reinforces the need to use certain types of samples, or that certain methods
of sample handling may be mandatory.
Diagrams of the sort presented in Figure 1-2 provided the analyst with
a general overview of the current state of knowledge regarding sample handling
and storage for various sample matrices. However, as discussed earlier, some
analytical procedures have been included based on practical need rather than
purely analytical considerations. For some of these procedures, information on
storage time and sample preservation is not available. The analyst is urged
to use professional judgement when this occurs and to minimize sample handling
and storage time prior to analysis. The absence of information in tables
similar to those indicated in Figure 1-2 identifies additional research and
development efforts needed to upgrade sample preparation techniques for each
analytical procedure.
Some of the topical headings in the procedure format do not apply to all
methods and have therefore been omitted where appropriate. For example, Daily
Performance Tests (I) and Qualitative Identification (K) usually refer to
GC/MS and GC procedures and are omitted from inorganic procedures sections.
However, within the Analytical Procedures Subsection (J), the matrices are
always presented in the order indicated. When information is lacking for one
or more sample matrix, that subsection has been reserved. This approach
was taken to allow inclusion of newly developed and reviewed methods as they
become available and to identify existing analytical needs.
Within the Analytical Procedures subsection, detailed guidance describing
the 'use of each method is being presented under the following headings.
J.I. Analysis of Hazardous Wastes .
J.I.I Reference
J.I.2 Method Summary
J.I.3 Applicability
J.I.4 Precision and Accuracy
J.I.5 Sample Preparation
J.I.6 Sample Analysis
Primary references are provided to allow users to confirm the appropriateness
or applicability of a procedure. Also, by maintaining visible integrity
between a method and the original author, a greater participation in future
revision of this document is anticipated.
The selected method of presenting analytical procedures resulted in a
certain amount of intentional redundancy. It is felt this approach will
provide continuity to the user while achieving the objective of providing
guidance for the analysis of samples of several matrix types.
1-7
-------
REFERENCES
1. Environmental Protection Agency. "Hazardous Waste Management System.
Identification and Listing of Hazardous Wastes." Federal Register Vol. 45
(92):33084-33133. May 19, 1980.
2. Environmental Protection Agency. "Test Methods for Evaluating Solid
Waste. Physical Chemical Methods." U.S. EPA, Office of Solid Waste and
Emergency Response, Washington, D.C. Report SW-846 (July 1982).
1-8
-------
CHAPTER II
PHASE SEPARATION AND SCREENING PROCEDURES FOR
HAZARDOUS WASTE SAMPLES
A. INTRODUCTION
The passage of time between sample collection and sample analysis is a
critical consideration in any analytical protocol. In addition to keeping
this time as short as possible, proper procedures must be instituted to
maintain sample integrity and minimize sample contamination. This is usually
accomplished by dividing the original sample into representative subsamples
or aliquots based on the number of specific analyses to be performed. Each
individual aliquot can then be treated with the appropriate preservative,
digestion solution or extraction solvent without fear of invalidating the use
of the sample for the determination of a second analyte or loss of the analyte
of concern.
Proper pretreatment of hazardous waste samples can serve an additional
function. Specifically, the use of screening techniques can identify poten-
'tial health or safety problems associated with the handling of samples. This
will permit.the necessary precautions to be implemented. For example, if
samoles known to contain cyanide are to be analyzed for total metals, they-
should be handled in a hood or pretreatea to remove cyanide to prevent prob-
lems associated with the evolution of hydrogen cyan-ide when the samples are
acidified. Furthermore, screening results can be used to judge the necessity
of performing more detailed and/or more expensive analyses on the same sample.
The purpose of this chapter is to provide general information for handling
and storing hazardous waste samples in the laboratory and to provide guidance
for implementing a standardized approach for pretreatment of hazardous waste
samples. The guidance addresses phase separation of complex, multi-phase
samples and screening of the resultant fractions for inorganic and organic
constituents.
B. STORAGE AND HANDLING OF HAZARDOUS WASTE SAMPLES
This section summarizes general safety procedures and good laboratory
practices that should be followed when handling hazardous waste samples in
the laboratory. Specific sample handling requirements are presented with
the appropriate analytical section in the following Chapters.
Samples of hazardous material should be stored at 4°C in a refrigerator
designed -for flammable materials or a specially designed cold room within a
regulated laboratory, if availaoie. Samples inouia ^e jtorea in the original
shipping container, Also, the refrigerator or cold room should only be for
II-l
-------
hazardous waste samples. This precaution is necessary to prevent cross-con-
tamination of samples or standards.
When transporting hazardous wastes within the laboratory, storage vessels
containing hazardous substances must be placed first in an unbreakable outer
container before being transported to laboratory work areas using good transfer
practices. Plastic-coated glass bottles with polypropylene caps, which can
satisfy a 4-foot drop test, are currently available which can serve as both
the storage vessel and the unbreakable outer container combined. Contami-
nated materials which are transferred from work areas to disposal areas must
first be placed in a closed plastic bag or other suitable impermeable and
sealed primary container. The primary container must be placed in a durable
outer container before being transported. The outer container must be labeled
with both the name of the hazardous substance and the warning: CAUTION -
HAZARDOUS SUBSTANCE.
Sample containers should only be opened in a glove box or operational
fume hood by personnel wearing protective clothing. The objective of this
procedure is to minimize exposure of the personnel to the samples.
No more than one sample container is to be opened at any one time. This
precaution will avoid cross-contamination of samples and potential synergistic
effects between components of different samples.
When samples are not being jssd for extended periods, they are to be
placed back into the original containers, resealed and returned to storage.
If stock quantities of hazardous reagents are used, they should be stored
in- a properly ventilated storage area that is secured at all times. The stor-
age area should be postad with ? s:gr bearing the ""egend: CAUTION - HAZARDOUS
MATERIALS - AUTHORIZED PERSONS ONLY. An inventory of stock quantities shall
be maintained by the laboratory Safety Officer. The inventory records shall
include the quantities of materials acquired and dates of acquisition and
disposition.
" No solvents, sample, or other contaminated materials, are to be poured
into any sink.- Generally, concentrated sample should only come in contact
with disposable glassware, unless the sample attacks glass, in which case
polypropylene labware should be used.
During sample preparation, contaminated glassware (glassware contacting
the sample) is separated from glassware for reagent handling. Disposable
glassware, pipets, sample vials, etc., are placed in waste containers for
disposal. Other glassware is rinsed thoroughly (at least 3 rinses) with an
appropirate solvent, the solvent placed in waste bottles and the glassware
washed with soap and water.
Any contaminated glassware or equipment that cannot be safely cleaned
is to be disposed of as hazardous material.
All -*tQrk rur^cas fw,«nch *.3os, htxxls, *1ocrs, «tc,^ shall be covered with
stainless steel, plastic sheets, dry adsorbent plastic-backed paper or other
II-2
-------
Impervious material. The protective surfaces shall be examined for possible
contamination immediately after each procedure involving the hazardous mate-
rial has been completed. Contaminated surfaces shall be decontaminated or
disposed of as is appropriate.
C. PHASE SEPARATION OF HAZARDOUS WASTE SAMPLES
1. Phase Separation
The separation of a sample into its component phases is accomplished by
centrifugation based on a procedure developed by the Environmental Pro-
tection Agency National Enforcement Investigation Center. This separation
procedure may yield as many as three distinct phases (Figure II-l). These
phases are 1) aqueous phase, 2) solid phase, and 3) non-aqueous phase(s).
The details of this separation method are presented below.
1.1 Reference
U.S. Environmental Protection Agency, "Hazardous Waste Site Samples -
Phase Separation." U.S. EPA, National Enforcement Investigation
Center, Denver, Colorado. 2 p. (February, 1980).
1.2 Summary
Samples are centrifuged in their original containers to achieve
phase separation. Individual phases are transferred to separate
containers for further processing.
- 1.3 Apparatus
Sample bottles, clear glass, screw-neck finish, wide-mouthed round
jars. (Kerr, A.C. No. 802; available from VWR Scientific, Catalog
No. 16194-063).
Teflon liners for sample bottles.
International Centrifuge, Model FXD with explosion-proof motor for
Class 1, group D location, or equivalent.
Automatic pipeting device, pipet-aid, or equivalent.
Disposable pi pets, 10 and 25 ml.
Polybags to fit sample bottles.
1.4 Procedure
Wipe the original sample container to remove any outside contamina-
tion. Label the bottle appropriately. Place the bottle in a poly
bag and seal with a rubber band or "twister".
II-3
-------
MuNf-phaM
Sampla
Cantrifuga
•t 2.0OO rpm
Saparata
_L
1.0ml
Attquot (aach)
Mbcibility
Mad
r w/
LJ
Miscibla
Only w/ H,O
Aquaoui
PhaM
1
Saa Figura 1
for Prap.
MiMiMa or
Not MitciMa
«v/ Both
1
Uta
Kari Fishar
Racult*
1
Mitcibla
Only w/ MaCI,
Nonaquaou*
Pttata
1
SM Figura 3
lor Prap.
It % H,O
U >60%
1
Aquaoui
PhaM
1
Saa Figura 1
for Prap.
If % H,O
te<60*
1
Nonaquaoui
Phaaa
1
Saa Figura 3
for Prap.
Figure II-l. Phase separation of hazardous waste samples.
-------
Heigh the sample bottle and prepare a counterbalance for the
centrifuge.
Place the sample and counterbalance in the centrifuge. Centrifuge
the sample for 30 minutes at 2000 RPM. Stop the centrifuge and
remove the sample.
Mark the interface levels between each phase on the sample bottle.
Relative volumes may be estimated by using a clean sample bottle
calibrated in 10-ml graduations.
Using an automatic pipeting device and disposable pipets, transfer
each distinct layer into a separate container. Label all phases
with the appropriate sample number. Classify each liquid phase as
either aqueous or nonaqueous based on sample miscibility with water.
(Procedure: Transfer 2 ml of each liquid phase Into separate test
tubes. Insert a 1-cm loosely packed plug of glass wool followed
by 1 cm of indicating silica gel. Cap the test tube and let stand
for 30 minutes. Disappearance of the blue indicator color indicates
that water is a major portion of the phase. Alternatively, use the
Karl Fischer titration procedure).
2. Aliquot Preparation
The centrifugation process may yield as many as three distinct phases.
These are the aqueous phase, the solid phase, and the non-aqueous phase(s).
The preparation of aliquots for screening and analysis of each phase is
summarized in the flow charts presented in Figures II-2, II-3, and II-4.
2.1 Aqueous Phase
Five aliquots should be set aside for screening of the aqueous phase
samples (Figure IJ-2). The first 1-ml aliquot is to be screened for
oxidant capacity, reductant capacity, cyanide, sulfide, and pH. A
second 2-ml aliquot is diluted to 200 ml with distilled water and
analyzed (using procedures in Chapter V) for acidity, alkalinity,
conductivity, oxidant capacity, and reductant capacity. Two separate
1-ml aliquots are set aside for cyanide and sulfide analyses. (These
two aliquots can be omitted if the spot tests for these analytes are
negative). If necessary, the sulfide aliquot may be preserved by the
addition of zinc acetate and the cyanide aliquot may be preserved by
the addition of sodium hydroxide and refrigeration. The final 1-ml
aliquot should be diluted to 100 ml with 2-percent nitric acid. This
solution is analyzed for mercury, other metals, and ammonia, if
desired.
2.2 Solid Phase
A schematic outline of the suggested approach to prepare solid-phase
nazaraous waste sample aVquots ror Anorganic screeninq or analysis
is presented in Figure II-3. The sulfide and cyanide procedures
II-5
-------
9-II
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-------
1
1
•
rSOml
Aliquot
Conductivity
determination
pHd
Acidify/
Afcaknity
2g
Aliquot
Extract w/
2OOml
Dist. H,O
lOOml
Aliquot
FHtar
0.450m
Strong Ik
Weak Add
Anion Analysla
BO ml
Aliquot
Oxidant
Spot Tart
Raductant
Spot Tart
Oxidant or
Raductant
Datarmination
Solid
Phasa
1
1
O 1 g
Aliquot
Digast w/ I
K,S,0. 4 1
1 KMnO, 1
Diluta to
1OO ml w/
Dist H,O
1
Marcury
Analysis
ig
Aliquot Al
Extr
Driad Ovar 1t
% Moistun) F
Weighing O 4
I Eh,
° ' • 1 & A
1 Alk;uot j Ar
J
tutu w/
LMti/LiF
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| UNO, 1 e=
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I 0.460m I
LgJMJBJg.K^BBjaj^BJ
_^^_^^__1. •
Elemental •
Analysis
I
'g
iquot
1=^
act w/
K> ml
HC1 1
T '
ig
Aliquot
Sumdeoi
Cyanide
Spot Tost
Hlar 1 g
60m Aliquot
ig
Aliquot
T '
•""^ Surflda
mmon— Datarminaaon
wrysis
Cyanide
Dataii mnalion
Task Always Parformad by Lab
« » I » Task Only Parformad by lab
Altar Positive Spot Test
1 Task Only Parformad by Lab
Whan Concuctivity is > 1.O000mhos
and pH is S 4 or > 9
••=> Task performed by Lab whan Subsequent
Analysis is Requested by Ragion
ajSBBSi Task Not Parformad by Lab
:«=• Task May Not be Performed by Lab
Figure II-3. Solid phase screening and sample preparation.
-------
I
00
1
0.1ml
Aliquot
I
Digest w/
K-.S-.0. &
KMnO,
i
Dilute to
1OOmlw/
Dist. H,0
I
Mercury
Analysis
Nonaqueous
Phase
1 ml
Aliquot
' I
Dilute to
1 00 ml w/
Xylene
1
Filter
0.45/jm
Teflon
' ' 1 J
Elemental •
Analysis
!
i
i
i
1 ml
Aliquot
I
Filter
0 45ijm
Teflon
1
Filtrate
!rnmn r^- __,
Lrnirni ryrr_ ^^
01 ml
Aliquot
1
Fuse w/
LMB/LiF
Flux
' 1 '
Dissolve Pellet in
1OO ml 4%
HNOj
i
Filter
045/jm
Elemental
Analysis
Residue
MHBHHB|===I
Discard
••••••iii
Figure II-4. Preparation of nonaqueous phase samples for inorganic analysis.
-------
simply require the setting aside of 1-g aliquots to be analyzed in
the event the spot test is positive. However, there are no known
effective preservatives for solid-phase samples. A fourth aliquot,
2 g, is extracted with 200 ml distilled water. The extract is
screened for the presence of oxidants and reductants, then analyzed
for oxidants and reductants, if necessary, and analyzed for conduc-
tivity, pH, acidity, alkalinity, and the anions of strong and weak
acids, if desired. A fifth aliquot is dried over ?2^S for percent
moisture detennination and subsequently fused with lithium metaborate
and lithium fluoride prior to metals analysis. A sixth aliquot is
digested with potassium persulfate and potassium permanganate and
analyzed for mercury. The seventh indicated aliquot is extracted
with 0.1 N HC1 and analyzed for ammonia.
2.3 Nonaqueous Phase
A schematic outline of the approach to prepare nonaqueous phase
hazardous waste samples for inorganic analysis is presented in
Figure 11-4. The two indicated fractions are for total mercury and
total metals. The aliquot preparation techniques are the same as
those provided for the preparation of solid phase aliquots for the
same analyses.
D. INORGANIC SCREENING PROCEDURES
Each sample phase separated from the original sample using procedures
specified in Subsection C should be qualitatively analyzed using procedures
specified in this section. Results of this testing will provide information
on the presence of strong oxidizing or reducing agents, cyanide, sulfide, or
extreme pH conditions. This information can be used to classify the waste
sample being analyzed as hazardous and to determine the most appropriate method
of sample handling.
Samples should be subjected to qualitative spot testing in the order pre-
sented in the text. For example, oxidizing spot tests should be performed
prior to CN spot tests which should be performed prior to pH tests. This
approach is suggested based on the relative order of importance of the informa-
tion to be obtained and partially out of convenience. By testing for the
presence of oxidizing agents first, the tests for cyanide and sulfide can
be omitted if oxidizing reagents are present. Similarly, if oxidizing agents
are absent, it would be beneficial to confirm the abence of cyanide or sulfide
prior to adjusting the pH of the sample.
Qualitative Screening Procedures for Hazardous Waste Samples
1. Spot Test for Oxidants in Aqueous-Phase Samples and Aqueous Extracts of
Solid-Phase Samples from Hazardous Waste Disposal Sites
^
1.1 Puroose
The objective of this test is to detect the presence of strong
oxidizing agents such as chlorine ana cnicnne uioxiae. Results
U-9
-------
are expressed as an equivalent amount of chlorine and are used to
determine the necessity of performing spot tests for cyanide and/or
sulfide.
This text can be performed on undiluted HWDS aqueous-phase and
water extracts of HWDS solid phase samples.
1.2 Procedure Summary
A drop of sample is placed on potassium iodide-starch test paper
which has been previously moistened with concentrated acetic acid;
a blue color indicates the presence of oxidants.
1.3 Safety Considerations
This test should be performed in a hood.
1.4 Appartus
1) Small white disposable weighing dishes.
2) 100-yl Eppendorf pipet and tips.
1.5 Reagents
1) Kl-starch test paper.
2) Glacial acetic acid, cone.
3) 1.0% w/v chloramine-T.
1.6 Detailed Procedure
1) Place a strip of Kl-starch paper in the weighing dish.
2) Moisten the paper with a drop of acetic acid.
3) Transfer 100 yl (2 drops) of the sample from the pH aliquot or the
aqueous extract from the solid-phase sample onto the test paper.
4) Wait approximately 5 minutes for color development.
5) A blue color will appear if an oxidant is present. Compare to a
blank prepared from distilled water.
1.7 Test Results
1) Record whether a positive or negative test was obtained.
2) If a positive test is obtained, determine the oxidant strength by
applying tne ox'aant quantIT^cation procedure.
11-10
-------
3) If a positive test result is obtained with an aqueous-phase sam-
ple, it is very unlikely that sulfide or cyanide will exist in
the sample. Therefore, these spot tests (sulfide and cyanide)
do not have to be performed.
4) If oxidant test results were negative, proceed with the test for
cyanide.
1.8 Quality Control Requirements
1) Check the spot test daily by testing 50 yl of the 1.0-percent
chloramine-T solution. A positive test should be obtained if
the test is proceeding properly. A negative test indicates
something is wrong and steps are to be taken to find the problem.
2) Do not perform the test on any samples until a positive test is
obtained for the chloramine-T solution.
3) Record the positive standard check on the bench sheet.
11-11
-------
2. Spot Test for Cyanide in Aqueous-Phase and Solid-Phase Hazardous Waste
Disposal Site Samples
2.1 Purpose
The object of the test is to quickly determine whether cyanide is
present in the sample. If present, precautions must be taken (such
as cyanide removal or working in a hood/glove box) to ensure that
acidification of the sample does not create hazardous conditions for
laboratory personnel.
2.2 Procedure Summary
Cyanide is reacted with chloramine-T in a buffered solution to pro-
duce cyanogen chloride. The cyanide is quantified based on the
intensity of the red-blue color that develops when cyanogen chloride
is mixed with pyridine-barbituric acid.
This procedure permits a rapid screening for the presence of cya-
nide above 60 ppb in aqueous samples (3-drop sample size) and 10
ug/g in solid samples (1-g sample size). The method will detect
cyanide in many of the common metallic cyanide complexes. However,
platinum, gold, and cobalt cyanide are not detected as cyanide.
2.3 Safety Considerations
Because of the volatility and hazards associated with cyanide, all
sample handling'should be done in a well-vented hood or glove box.
If cyanides are found to be oresent, all subsequent processing of
the sample should also be completed in a hood or glove box.
2.4 Interferences
1) Thiocyanate produces the same reaction in the spot test as
cyanide. These two substances can be distinguished by removing
the cyanide by reaction with formaldelhyde and quantifying the
remaining thiocyanate with the chloramine-T procedure.
2) A highly reduced sample interferes by consuming chloramine-T.
Additional chloramine-T should be added as necessary.
3) Aldehydes in excess of 0.5 mg/1 interfere by converting cyanide
to cyanohydrin.
2.5 Appartus
1) Plastic disposable 2-ml conical beakers.
2) Disposable capillary pipets and rubber bulb.
3) Rudoer, 2-noie stoppers r'or che test tuoes.
11-12
-------
4) Teflon connecting tube, 1 mm I.D.
5) Disposable, long-stem eye droppers.
6) Tygon rubber tubing, 1/4" I.D.
7) Compressed nitrogen and regulator.
8) Heating block capable of maintaining 75° ± 5°C with hole openings
for the test tubes.
2.6 Reagents
(All reagents to be made from ACS grade chemicals.)
1) Pyridine-barbituric acid reagent: place 3 g barbituric acid in
a 100-ml volumetric flask. Rinse the sides of the flask with a
minimum amount of distilled water. Add 15 ml pyridine and mix.
Add 3 ml cone. HC1 and mix. Add additional distilled water and
stir until the barbituric acid dissolves. Dilute to volume with
distilled water.
2) Chloramine-T solution: dissolve 1.0 g of white, water-soluble
chloramine-T in 100 ml distilled watar and refrigerate until
ready to use. Prepare fresh weekly.
3) Phosphate buffer: dissolve 138 g NaH2P04*H20 in distilled water
and dilute to 1 liter. Refrigerate solution after preparation.
4) Stock cyanide solution, 1000 mg/1 CN: dissolve 2.51 g KCN and .
2 g KOH in 1000 ml distilled water,
5) Cyanide spiking solution: add 20 ml 1.25-N NaOH solution to a
100-ml volumetric flask. Pipette 5.0 ml stock cyanide solution
to the flask and dilute to volume with distilled water. Store
solution in the dark. The cyanide concentration is 50 mg/1 CN.
6) Magnesium chloride solution: weigh 510 g MgCl2*6^0 into 1000 ml
volumetric flask, dissolve and dilute to volume with distilled
water.
7) Hydrochloric acid: dilute 50 ml concentrated HC1 with 50 ml
distilled water; and 0.1 g aluminum metal.
8) Sodium hydroxide solution: add 500 ml distilled water to 40 g
NaOH in a beaker, mix and cool. Transfer the NaOH solution to
a 1-liter volumetric flask and dilute to volume.
9) Methyl violet "indicator: dissolve 1 g methyl violet in 100 ml
ethyl alcohol.
11-13
-------
2.7 Detailed Procedure
Liquid Samples
1) Place 0.25 ml (approximately 5 drops) of sample in a disposable
beaker.
2) Add one drop phosphate buffer and mix. Check the resultant pH
with a pH indicator strip. If the pH is above 8, continue
adding buffer until a pH of approximately 8 is obtained.
3) Add 4 drops chloramine-T reagent and mix.
4) Add 4 drops pyridine-barbituric acid reagent and mix again.
Allow 8 minutes for full color development.
5) The appearance of a pink to red color indicates the presence of
1.0 mg/1 or more of cyanide. In comparison, a faint yellow
color should develop in a cyanide-free blank.
Solid Samples
1) One gram of sample is weighed into a disposable test tube to
which 1 ml MgCl2-solution is added; a small glass rod is used to
disperse the sample. The apparatus is shown in Figure II-5.
, 2) One drop methyl violet indicator is added to the sample tube.
The apparatus is assembled as shown in Figure II-5 and.l ml NaOH
is added to tube No. 2.
3) Nitrogen is bubbled through the tubes at the rate of 2 bubbles
per second for 25 minutes. Temperature of the heating block
is 75° ± 5°C.
4) Four drops (200 ul) 6-N aluminum-treated HC1 are added to the top
of the dropper with the apparatus at an angle such, that tubing
from the nitrogen supply can be attached to the dropper before
the acid hits the sample. The solution should turn yellow or the
violet coloration should be absent. If not, more acid must be
added.
5) One ml MgCl2-solution should be analyzed as a blank solution.
6) Transfer 250 ul of clear solution from tube No. 2 to a 2-ml
conical beaker. Add 100 ul phosphate buffer solution.
7) Add 4 drops chloramine-T reagent and mix.
8) Add 4 drops of pyridine-barbituric acid reagent and wait 8
minutes. A faint oink to violet color indicates the presence
of cyanide. —
11-14
-------
Ph Test Strip
Teflon Connecting Tube
Sai
s,
u
•nple
Tube#1
*N— • •*
Heating Block
Tube #2
Figure II-5. Appar:tj£ rcr cyanide and sulfide spot tests.
2.8 Test Results
Record the test results. If a positive cyanide test is obtained,
quantify the cyanide concentration using the procedure in Chapter
IV, Section 16.
2.9 Quality Control Requirements
1) Check the performance of the spot test daily by-analyzing the
cyanide spiking solution. If a positive test is not obtained,
measures must be taken to identify and correct the problem.
2) Perform sample spot test only after a positive test is obtained
for Step 1 above.
3) Record the positive standard check.
11-15
-------
3. Spot Test for Sulfide'in Aqueous-Phase Hazardous Waste Disposal Site
Samples
3.1 Purpose
The objective of the test is to quickly determine whether sulfide,
that may interfere with other analyses or create a hazard during
sample processing, is present in the sample.
3.2 Procedure Summary
One drop of the sample is placed on a lead acetate test paper
previously moistened with an acetic acid solution. The presence of
sulfide is indicated by a darkening of the paper. The method detec-
tion limit is approximately 4 mg/1.
If the aqueous sample is highly colored or turbid or the sample is
solid, then the Alternate Spot Test Procedure for Sulfide (paragraph
4) should be employed to check for sulfide.
3.3 Safety Considerations
This procedure should be carried out in a hood or glove box.
3.4 Interferences
None.
3.5 Apparatus
1) Disposable capillary.pipets and bulbs.
2) Small white weighing dish.
3.6 Reagents
1) Lead-acetate test paper [Fisher Scientific (Catalogue No.
14-862)].
2) Acetate buffer: dissolve 410 g sodium acetate trihydrate
(NaCpHTO?, 3H20) in 500 ml water. Add glacial acetic acid
to pH 4.5.
3.7 Detailed Procedure
1) Place a strip of the lead-acetate test paper in a white weighing
dish.
2) Wet the paper with 2 or 3 drops of the acetate buffer.
3) Add ore drop :arDla, A darkeninq of *;he *sst 'lane1"
11-16
-------
the presence of sulfide. Not less than 4 mg/1 sulfide can be
detected.
3.8 Test Results
Record the results. If a positive result is obtained, sulfide can
be quantified using the procedures given in Chapter IV, Section 17.
3.9 Quality Control Requirements
1) Check the performance of this spot test daily by performing the
test on a 10 mg/1 sulfide standard.
2) A positive test must be obtained before analyzing samples.
3) Record the positive standard check with the sample results.
11-17
-------
4. Alternate Spot Test Procedure for Sulfide in Highly Colored or Turbid
Aqueous Samples or Solid-Phase Samples from Hazardous Waste Disposal
Sites
4.1 Purpose
This method is for qualitatively determining the presence of sulfide
when the principal spot test cannot be used due to matrix inter-
ference.
The objective of the test is to quickly determine whether sulfide,
that may interfere with other analyses or create a hazard during
sample processing, is present in the sample.
4.2 Procedure Summary
This procedure allows for a quick screening of turbid aqueous samples
or semi-solid samples such as soil or sediment for the presence of
10 yg/g or greater of sulfide. Nitrogen is bubbled through a heated
sample mixed with MgCl2 and HC1. H2$ is evolved into a collection
media containing Cd(N(h)2« The presence of sulfide is indicated
by the discoloration or a lead acetate test strip suspended above
the sample. Larger amounts of sulfide are indicated by the formation
of a yellow precipitate in the collection medium.
4.3 Interferences
The treatments of acidification and gas-stripping release HCN and
H2$ in a reasonably pure state. However, cyanide and sulfide may
react in the collection medium to produce thiocyanate that can inter-
fere in the test.
The method detects all common metal sulfides except those of copper.
4.4 Apparatus
1) Disposable 1 x 7 cm test tubes.
2) Rubber, 2-hole stoppers for the test tube.
3) Teflon connecting tube, 1 mm 1.0.
4) Lead-acetate test strips.
5) Disposable, long-stem eye droppers.
6) Tygon rubber tubing, 1/4" I.D.
7) Compressed nitrogen and regulator.
3} .-Seating blocK :apable :f ,7iaint a living 75° - c°C *ith hole
openings for the test tubes.
11-18
-------
4.5 Reagents
(All reagents are to be prepared with ACS grade chemicals.)
1) Magnesium chloride solution: weigh 510 g of MgCl 2*6^0 into a
1000-ml volumetric flask, dissolve and dilute to volume with
distilled water.
2) Hydrochloric acid: dilute 50 ml concentrated HC1 with 50 ml
distilled water; add 0.1 g aluminum metal.
3) Cadmium nitrate solution: dissolve 30.9 g of Cd(N03)*4H20
in distilled water and dilute to 100 ml.
4) Sodium hydroxide solution: add 500 ml distilled water to 40 g
NaOH in a beaker, mix and cool. Transfer the NaOH solution to
a 1-liter volumetric flask and dilute to volume.
5) Methyl violet indicator: dissolve 1 g methyl violet in 100 ml
ethyl alcohol.
6) Acetate buffer: dissolve 410 g sodium acetate trihydrate in
500 ml water. Add glacial acetic acid to pH 4.5.
4.6 Detailed Procedure
Aqueous. Samp! es
1) One ml of the aqueous sample is oioetted into a disposable test
tube to which 1 ml MgCl? solution is added. A small glass rod is
used to disperse the sample. The necessary apparatus is shown in
Figure II-5.
2) One drop methyl violet indicator is added to tne sample tube and
a lead acetate test strip moistened with acetate buffer is sus-
pended above the sample between the stopper edge and the lip of
the tube.
3) The apparatus is assembled as in Figure I1-5 with 1 ml Cd (1^3)2
solution and 1 ml NaOH in tube No. 2.
4) Nitrogen is bubbled through the tubes at the rate of 2 bubbles
per second for 25 minutes. Temperature of the heating block
is 75° ± 5°C.
5) Four drops (200 yl) 6 N aluminum-treated HC1 is added to the top
of the dropper with the apparatus at an angle such that tubing
from the nitrogen supply can be attached to the dropper before
the acid hits the sample. The solution should turn yellow or the
violet coloration should be absent. If not. more acid must be
added. One ml MgCl£ solution should be analyzed as a blank
solution.
11-19
-------
Solid Samples
1) One gram of sample is weighed into a disposable test tube to
which 1 ml MgCl 2 solution is added; a small glass rod is used
to disperse the sample. The apparatus is shown in Figure II-5.
\
2) One drop of methyl violet indicator is added to the sample tube
and the lead-acetate test strip moistened with acetate buffer is
suspended above the sample between the stopper edge and the lip
of the tube.
3) The apparatus is assembled as in Figure II-5 with 1 ml
solution in tube No.' 2.
4) Nitrogen is bubbled through the tubes at the rate of 2 bubbles
per second for 25 minutes. Temperature of the heating block is
75° ± 5°C.
5) Four drops (200 yl) 6 N aluminum treated HC1 are added to the
top of the dropper with the apparatus at an angle such that
tubing from the nitrogen supply can be attached to the dropper
before the acid hits the sample. The solution should turn
yellow or the violet coloration should be absent. If not, more
acid must be added. One ml MgCl2 solution should be analyzed as
a blank solution.
*.7 Test Results
•»
- Any darkening of the lead-acetate test strip within 2 to 3 minutes
indicates the presence of suicide- Yellow coloration in tube. No. 2
indicates higher sulfide concentrations.
If a positive sulfide result was obtained, sulfide can be quantified
using the procedures in Chapter IV, Section 16.
4.8 Quality Control Requirements
Check the performance of this spot test daily by performing the test
on a standard sulfide solution (approximately 100 ppm). A positive
test must be obtained before analyzing samples. Record positive
standard test with the sample results.
11-20
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5. Spot Test for pH in Aqueous Samples and Aqueous Extracts of Solid Phase
Samples from Hazardous Waste Disposal Sites
5.1 Purpose
The objective of this test is to determine whether the pH of any
samples is extreme (<1 or >12) and would require special care in
handling and preparing the samples for further analysis.
5.2 Procedure Summary
The pH of the sample is approximated using pH indicator paper.
5.3 Sample Handling
Samples should be analyzed as soon as possible. This test should
be performed in the hood.
5.4 Apparatus
1) Disposable cups and caps.
2) pH test paper 0-14.
5.5 Procedure
Dip the pH test paper into the aqueous phase sample. Compare the
resultant color of the test paper with the chart on the package.
5.6 Test Results
1) Record the observed pH of the sample.
2) If the pH of the sample is less than 8, acidity of the sample
can be determined using procedures in Chapter V, Section 21.
3) If the pH of the sample is greater than 5, alkalinity of the
sample can be determined using procedures in Chapter V,
Section 22.
11-21
-------
E. ORGANIC SCREENING PROCEDURES
The following procedure is presented as a screening technique for hazard-
ous waste disposal site samples suspected of being highly contaminated with
organic compounds. Each of the sample phases prepared in Subsection C.I. can
be screened using GC/FID because of the wide linear range of the flame ioni-
zation detector and its relative insensitivity to water vapor. However, the
user is cautioned that the presence of a peak in the sample chromatogram is
only presumptive evidence for a specific component since one or more components
may co-elute at identical retention times under the stated analytical condi-
tions. The intent of the procedure is not to quantify specific compounds but
to identify those samples and/or sample phases that may require more time-
consuming and detailed analysis.
1. Extract Preparation
Separate the original sample into component phases by centrifugation
as described in Subsection C.I. Prepare an extract of each phase using
the appropriate guidance given below.
1.1 Direct Aqueous Screening
Analyze 1- to 5-ul aliquots of each liquid phase. Dilute samples
as necessary to obtain appropriately scaled responses.
Screen the samples and compare, by GC retention time, unknown sample
peaks to known standards. Prepare a iist of probable components
for each sample.
1.2 Aqueous-Phase Extraction
Transfer a 10-ml aliquot of the aqueous phase to a 17-ml vial con-
taining 2 g NaCl. Add 2 ml hexane. Cap with a Teflon-lined screw
cap and shake for 1 minute. Allow the layers to separate and trans-
fer 1 ml of the hexane layer into a 2-ml crimp-seal vial for analysis.
1.3 Nonaqueous Liquid Extraction
Dilute 1.0 ml of nonaqueous phase sample to 10 ml with hexane. Add
1 g anhydrous sodium sulfate and mix to dry the solvent. If the
sample phase is soluble in hexane, transfer 1.0 ml of the diluted
sample to a clean vial and add 9.0 mi hexane. Transfer 1.0 ml of
the second dilution to a 2-ml crimp-seal vial for analysis. Save
the dilutions until the analyses are completed.
If the sample phase is not soluble in hexane, repeat the procedure
using acetone as the solvent.
1.4 Solid and Sediment Extraction
Cheat che iolubi'i-'-y -f -.he :o1;d by placing ' i of samc'e 4n i
17-ml vial and adding 10 ml acetone. If more than 50 percent of
11-22
-------
the solid dissolves, proceed with (a). Otherwise, extract the sample
as described in (b).
a) Acetone-Soluble Samples
Dilute 1 ml of the acetone-solid suspension (if more than 50
percent of the solid has dissolved) to 10 ml with acetone and
analyze.
b) Acetone-Insoluble Samples
Mix 5 g sample with 10 g granular anhydrous sodium sulfate.
Place the mixture into a prerinsed cellulose thimble and place
the thimble into a micro-Soxhlet extractor (Kontes K292000, or
equivalent). Extract the sample for 3 hours (at least 15 cycles)
with acetone.
Transfer the extract to a vial and dilute to 25 ml with acetone.
Transfer 1.0 ml to a 2-ml crimp-seal vial for analysis. Save the
extract in a glass vial with a Teflon-lined cap until the analysis
is complete.
Extract Analysis
Sample extracts are screened by GC/FID. Screening of hexane and acetone
extracts is performed by injecting 1 to 2 yl of extract onto a 300-cm x
2-mm-I.D. glass column packed with 6 percent OV-101 on 60/80 mesh GC-Q
or equivalent. The temperature should be programmed to increase from
3Q°C 10 230°C at a rate of 6 to 8°/minute. A maximum run time of 75
minutes should be sufficient to observe most compounds of significance.
Dilute and/or concentrate the extracts, as necessary, to obtain appro-
priately scaled responses.
Volatile organic compounds at concentrations above 1 mg/1 can be deter-
mined by direct aqueous injection gas chromatography. Lower concentra-
tions, based on the ratio of sample volume to extraction solvent volume,
should be detectable in the other sample phases.
11-23
-------
REFERENCES
1. U.S. Environmental Protection Agency. "Hazardous Waste Site Samples -
Phase Separation." National Enforcement Investigation Center, U.S. EPA,
Denver, Colorado. 9 p. (February, 1980).
2. U.S. Environmental Protection Agency. "Laboratory Preparation Procedures
for Analytical Screening of Hazardous Waste Site Samples." National
Enforcement Investigations Center, U.S. EPAj Denver, Colorado. 9 p.
(No date).
3. American Society for Testing and Materials. "Measuring Volatile Organic
Matter in Water by Aqueous Injection Gas Chromatography." Method D-2908.
1983 Annual Book of ASTM Standards. Section 11. Philadelphia,
.Pennsylvania. (1983)
11-24
-------
III. PROCEDURES FOR ORGANIC COMPOUNDS
-------
SECTION 1
VOLATILE ORGANIC COMPOUNDS
A. SCOPE
Volatile organic compounds are characterized by high vapor pressure. This
common property is used to isolate the compounds from the sample matrix prior
to GC/MS identification and measurement. Detailed methods are presented in
Subsection J for the determination of volatile organic compounds in hazardous
wastes, municipal and industrial discharges, ground and surface water, sedi-
ments, biological tissue, and air. Method detection limits for these compounds
using multi-component, screening procedures will vary with sample matrix and
sample size.
B. SAMPLE HANDLING AND STORAGE
The same physical property that is used to isolate volatile compounds
from the sample matrix can create potential problems when trying to maintain
sample integrity prior to analysis. Because of the volatility of the analytes,
samples should be collected in as quiescent a manner as possible, and the
sample bottles should be comoletely Billed (no head spaca). These steps are
intended to minimize the loss of volatile constituents through degassing.
Also, the sample bottle should remain hermetically sealed until the time of
analysis.
All samples must be iced or refrigerated from the time of collection
until the time of analysis (Figure 1). This step should also counter the
tendency for these compounds to vaporize. If the sample source is known to
contain residual chlorine, sodium thiosulfate should be added as a preser-
vative to the sample bottles just prior to sample collection. Addition of 10
mg/40 ml is sufficient to neutralize 5 ppm chlorine. If necessary, a separate
sample aliquot can be analyzed for total chlorine content using a field kit.
A stoichiometric quantity of sodium thiosulfate plus a 10-percent excess can
then be added to neutralize the chlorine.! The completely filled (no head
space) and sealed sample bottle should be shaken vigorously for 1 minute to
ensure uniform distribution of the preservative. There are no known chemical
preservatives for solid-phase samples due to the uncertainty of attaining homo-
geneous mixing.
Evidence suggests that certain aromatic compounds, such as benzene,
toluene, and ethyl benzene, are susceptible to rapid biological degradation.2
Refrigeration alone w-5"P not orovide ^deauate *amole observation *cr these
compounds in all types of waters. Therefore, when these compounds are of
interest, a separata sample should be collected for analyses and preserved
III-2
-------
SAMPll
NATRII
UATEM ON
LEACHATE
SAMPLE
PROCESSING
PURPOSE TOTAL CONttNTRATIOR TOTAL CONCENTRATION
IN HASTE IN MATER
SAMPLE
CONTAINER
SAMPLE
PRESERVATIVE
STORAGE
TIME
SAMPLE
SIZE
14 d
25
SLUDGE. SOIL
OR SCUINCNT
MET
SI OH AGE
PURGE
ANALYZE
DDT
STORAGE
1
FROZEN
STORAGE
T;)TAl
LONCEMRATION IN
SID INC NT
0
BIOLOGICAL
TISSUE
FROZEN
STORAGE
I>URGE
ANALYZE
TOTAL
TISSUE
ONUNTRAT10N
CO
Air
TENAX
CARTRIDGE
PURGE
ANALYZE
TOTAL
NCENTRATIOI
IN AIR
14
1-10 g
CARTRIDGE
-tt'C
4 UCEKS
VARIABLE
Figure 1. Handling and sample storage Information for volatile organic analysis.
-------
with hydrochloric acid. It is suggested that a 500-ml sample be collected in a
clean glass container (no head space). The pH of this sample should be adjusted
to about 2 with 1:1 HC1. (This sample should be stirred to ensure adequate mix-
ing of the preservative, but in such a manner as to prevent or minimize degassing
of sample constituents.) The preserved sample should be quiescently transferred
to a clean 25-ml sample bottle and hermetically sealed to exclude all trapped
air bubbles. If residual chlorine is known or suspected to be present, sodium
thiosulfate should also be added to the sample.
Soil, sediment, or sludge samples should be stored in a field-wet condition
with refrigeration. Drying or freezing are not recommended as storage techniques
since volatile constituents may be lost during the drying or thawing cycles.
Organic vapors collected from ambient air on Tenax GC cartridges are
stable and can be quantitatively recovered from the cartridge sampler up to 4
weeks after sampling when the cartridges are tightly enclosed in cartridge
holders and placed in a second container that can be sealed, protected from
light, and stored at -20°C.3»4,5
All samples must be analyzed within 6 to 28 days of collection, 1»6
depending on the sample matrix (Fiaure 1).
C. INTERFERENCES
The majority of contamination problems encountered in volatile organic
analyses occur due to either impurities in the purge gas or organic compounds
outgassing from the plumbing ahead of the trap. The analytical system must be
demonstrated to be free from contamination under the conditions of the analysis •
by running laboratory reagent blanks, me use of non-TFE plastic tubing,
non-TFE thread sealants, or flow controllers with rubber components in the
purging device should be avoided.
Samples.can be contaminated by diffusion of volatile organics (partic-
ularly fluorocarbons and methylene chloride) through the septum seal into the
sample container during shipment and storage. A field reagent blank con-
sisting of reagent water and treated by the sampling and sample processing
protocol used can serve as a check on such contamination.
Contamination by carry-over can occur whenever high-level and low-level
samples are sequentially analyzed. In order to reduce carry-over, the purging
device and sample syringe must be rinsed with reagent water between sample
analyses. Whenever a sample is encountered that shows an unexpectedly high
concentration of volatile organics, it should be followed by an analysis of
reagent water to check for possible cross-contamination. For samples containing
large amounts of water-soluble materials, suspended solids, high-boiling com-
pounds or high levels of purgeable compounds, 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 tne entire jyscem ;nay oe require'.;.
III-4
-------
Purging may cause some samples to foam excessively. Any foam that passes
through the gas transfer lines and enters the sorbent trap can have a negative
effect on resultant sample analyses due to deactivation of the trap or the
introduction of nonvolatile artifacts.7 These problems can be countered by
the use of surfactants such as silicone antifoaming agents, the application of
heat to dissipate the foam where possible, or cleaning of the analytical
system when necessary.
D. SAFETY
The following chemicals covered by this method have been tentatively
classified as known or suspected carcinogens to humans or other mammals:
benzene, carbon tetrachloride, chloroform, 1,4-dichlorobenzene, and vinyl
chloride. Primary standards of these hazardous compounds should be prepared
in a functional fume hood. A NIOSH/MESA-approved toxic gas respirator should
be worn when the analyst handles samples containing high concentrations of
these compounds.
• 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 chem-
icals must be reduced to the lowest possible level by whatever means avail-
able. Each 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.8.3,19
E. APPARATUS
1. Sampling equipment, for discrete sampling.
1.1 Vial - 25 ml capacity or larger, equipped with a screw cap with hole
in center (Pierce No. 13075, or equivalent). Wash with detergent,
rinse with tap water and distilled water, and dry at 105°C before use.
1.2 Septum - Teflon-faced silicone (Pierce No. 12722 or equivalent).
Wash with detergent, rinse with tap water and distilled water, and
dry at 105°C for 1 hour before use.
2. Purge-and-Trap device - The purge-and-trap device consists of three
separate pieces: the sample purger, the trap, and the desorber.
2.1 The sample purger must be designed to accept 5 ml-samples with a
water column at least 3 cm deep. The head space between the water
column and the trap must have a total volume of less than 15 ml.
The ourae qas must oass through the water column as finely divided
bubbles with buobie diameters of less :nan j .Tin jt the origin. The
purge nas must be introduced no more than 5 mm from the base of the
water column. The sample purger illustrated in Figure i meecs cnese
design criteria.
III-5
-------
Optional
Foam
Trap
xit 1/4 in.
O. D.
•—14 mm.
O. D.
Inlet 1/4 in.
O. D.
1 /4 in.
O. O. Exit
Sample Inlet
2 Way Syringe Valve
17 cm. 20 Gauge Syringe Needle
'6 mm. O. D. Rubber Septum
10 mm. O.D.
10.mm. Glass Frit
Medium Porosity
Inlet
1/4 in. O. D.
^1/16 in. O.D.
Stainless Steel
13X Molecular
Sieve Purge
Gas Filter
Purge Gas.
Flow Control
Figure 2. Purging device.
2.2 The trap must be at least 25 cm long and have an inside diameter of
at least 0.25 cm. The trap must be packed to contain the following
minimum lengths of adsorbents: 1.0 cm of methyl silicone-coated
packing (Subsection F.3.2), 15 cm of 2,6-diphenylene oxide polymer
(Subsection F.3.1), and 8 cm of silica gel (Subsection F.3.3), The
minimum specifications for the trap are illustrated in Figure 3.
III-6
-------
Packing Procedure
Contraction
Smm
8cm
15 cm
1cm
Smm
Glass
Wool
Grade IS
Silica Gel
Tenax
Trap Inlet
3% OV-1
Glass
Wool
Compression
Fitting Nut
and Ferrules
14ft. 7 n /Foot
Resistance Wire
Wrapped Solid
Thermocouple/
Controller
Sensor
Tubing 25 cm.
0.105 in. I. D.
0 125 in. O.D.
Stainiess Steel
Figure 3. Trap packings and construction to include desorb capability.
2.3 "ITie desorber must be capable of rapidly heating the trap tc 130°C.
The polymer section of the trap should not be heated higher thai
180°C and the temperature of the remaining sections should not
exceed 220°C. The desorber design illustrated in Figure 3 meets
these criteria.
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 4 and 5.
3. Vacuum extraction equipment for the recovery of volatile organic com-
pounds from sediments and biological tissue. A diagram of the apparatus
for vacuum extraction and cryogenic concentration of volatile organic
compounds from sediments and fish tissue is shown in Figure 6. The
vacuum extractor can be assembled from materials normally available in
the laboratory. The low pressure necessary for extraction is supplied by
a vacuum pump capable of producing a 10~3-torr vacuum and a flow rate
of 25 1/min. The concentrator traps (25-ml Tekmar purging tubes, or
equivalent) are used for condensing the volatile vapors and transferring
the extract to the purge-and-trap device. The concentrator trap is
connected to the transfer lines of the vacuum extractor with 1/8-inch
compression Sittings and graphite farruies. The cransfar iir.es ure mace
of glass-lined 1/4 inch O.D, stainless steel tubinq. Gas-tiqht valves
vVl-V'4), Hupro 8-48KT, are connected with compression fittings and
graphite ferrules. The 125-irl septum vial containing the sample aliquot
III-7
-------
13X Motacuiai
Swira Filter
bqu*4
lnr*ctton
Port»
Option*! 4-Port Column
Salocnon Varw
Column Own
(I I~I P — » Confirmatory Column
-L4J-L i >T.D~«»
^»» Analytical Column
- Trip ( Ott ] Haat»»
22 "C V J Control
Purging
O*viet
Not*.
All lin*t b«nw««n
trap and GC
«nouM IM rw«t«d
toBO C
Figure 4. Schematic of purge-and-trap device - purge mode.
Prvtiur*
A«?ulator
Purge
G*
Purg* I——
s*«F.o»* •[
Control ^^\ t
13X Mot*>cul*f -
S»*v« Ftlt*tr
Column Qv*n
^.» Confirmatory Column
To O«t«ctof
«•.> Anatyticjl Coi
-------
Velva 1
Sampla
Valve 4
Vacuum
Pump
Concentrator
Trap
Figure 6. Vacuum extractor.
is connected to the system with a one-hole rubber stopper pierced with
the 1/4-inch O.D. tubing. A liquid nitrogen cold trap is placed between
the vacuum pump and concentrator trap in order to prevent condensation of
pump oil vapors in the concentrator trap. The helium line and pressure
gauge are connected at a junction in the transfer line between sample and
V2 and are used primarily to test ihe apparatus for 1-aaks. The helium
used is 99.999 percent pure, and normally, because of the small quantities
used, it is not necessary to purify che gas further.. If it is desired,
however, a-gas filter trap can be added to the helium line to ensure
purity. An ultrasonic vibrator is used to agitate the sample during extrac-
tion.
4. Gas chromatograph/mass spectrometer system.
4.1 Gas chromatograph - An analytical system complete with a tempera-
ture-programmable gas chromatograph suitable for on-column injection
and all required accessories including syringes, analytical columns,
and gases.
4.2 Column - 1.8 m long x 0.25 cm I.D. stainless steel or glass, packed
with 1 percent SP-1000 on Carbopack B (60/80 mesh), or equivalent.
This column was used to develop the method performance statements in
Subsection J.2.1.4. Capillary columns can be used, as long as the
criteria of Subsection G are met.
4.3 Mass spectrometer - Capable of scanning from 20 to 260 amu every 7
seconds or less, utilizing 70 volts (nominal) electron energy in the
electron impact ionization mode and producing a mass spectrum which
meets all the criteria 1n Table 1 when 50 ng of 4-bromofluorobenzene
(BFB) is injected through the GC inlet.
4.4 oC/HS interface - .-my GC-oo-MS ;ntsr*ace that jives acceptable cal-
ibration ooints at 50 ng per injection for each of the parameters of
interest and achieves all acceptaoie performance criteria (see
III-9
-------
TABLE 1. BFB KEY ION ABUNDANCE CRITERIA*!
Mass Ion Abundance Criteria
50 15 to 40% of mass 95
75 50 to 70% of mass 95
95 Base peak, 100% relative abundance
96 5 to 9% of mass 95
173 <1% of mass 95
174 >50% of mass 95
175 5 to 9% of mass. 174
176 >50% of mass 95
177 5 to 9% of mass 176
======================3==========3============================.
Subsection I) may be used. GC-to-MS interfaces constructed of
all-glass or glass-lined materials are recommended. Glass can be
deactivated by silanizing with dichlorodimethylsilane.
4.5 Data system - 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 fons Of a
specific mass and plotting sucn ion aoundances 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 1imits.
5. Syringes - 5 ml glass hypodermic with Luerlok tip (2 each), compatible
with the purging device.
6. Micro syringes - 25 ul, 0.15 mm I.D. needle.
7. Syringe valve - 2-way, with Luer ends (3 each), compatible with the
purging device.
8. Syringe - 5 ml, gas-tight with shut-off valve.
9. Bottle - 15 ml, screw-cap, with Teflon cap liner.
10. Balance - analytical, capable of accurately weighing 0.0001 g.
TII-10
-------
11. Sample transfer implements - A series of implements are suggested for the
rapid transfer of hazardous waste aliquots from sample containers to
laboratory glassware. The intent is to provide a simple and quick
transfer process that avoids or minimizes the loss of volatile compon-
ents. Liquids of low to moderate viscosity may be transferred using
conventional laboratory pipets. Non-tacky solids may be transferred
using conventional laboratory spatulas. Spoon-shaped porcelain spatulas
(Coors No. 60478, or equivalent) are useful in that they have a measure-
able bowl-volume. Samples having a desired approximate volume can thus
be obtained. Transfer of tacky or non-tacky and semi-solids may be
simplified using the implements described below.
11.1 Implement for transfer of non-tacky semi-solids. A 3-ml glass
hypodermic syringe is modified. The plunger is removed and the
normally closed end of the barrel is cut away. To use this imple-
ment, the plunger is replaced flush with the cut-away, open end.
The device is pressed into a semi-solid sample, thereby forcing the
plunger out of the barrel. When the plunger has been displaced by a
volume equal to the approximate sample volume desired, the syringe
is withdrawn and the semi-solid plug is transferred to a tared
vessel by displacing the material with the plunger.
11.2 Implement for the transfer of tacky semi-solids and solids. This
approach will be useful for the transfer of some tacky or tarry
materials. Glass tubing of approximately 1 cm I.D. is cut into short
sections having a desired approximate volume (i.e., 1 ml = 1.0 cm I.D.
x 1.3 cm length). To obtain a desired volume of sample take a tared
tubing section of that volume, and using a Teflon-coated laboratory
spatula, press a portion of tarry sample into the tubing section.
The sample-filled tubing section is then placed directly into a
centrifuge tube containing PEG (see Subsection J.I.2); the centrifuge
tube and PEG are weighed before the sample is added.
11.3 Implement for the transfer of viscous liquids. This device is fash-
ioned by cutting the constricted end from a 5-ml graduated pipet.
The large-bore pipet thereby obtained is used in conjunction with a
conventional laboratory pipetting aid, preferably of the syringe
type. This implement allows convenient transfer and approximate
volumetric measurement of some viscous liquids.
F. REAGENTS
1. Reagent water - Reagent water is defined as a water in which an inter-
ferent is not observed at the method detection limit of the parameters of
Interest.
1.1 Reagent water may be generated by passing tap water through a carbon
filter bed containing approximately 1 Ib. of activated carbon (Calgon
Corp., Filtrasorb-300, or equivalent).
III-ll
-------
1.2 A water purification system (Millipore Super-Q, or equivalent) may be
used to generate reagent water.
1.3 Reagent water may also be prepared by boiling water for 15 minutes.
Subsequently, while maintaining the temperature at 90°C, bubble a
contaminant-free inert gas through the water for 1 hour. While
still hot, transfer the water to a narrow-mouth screw cap bottle and
seal with a Teflon-lined septum and cap.
2. Sodium thiosulfate - (ACS) Granular.
3. Trap materials
3.1 2,6-Diphenylene oxide polymer - Tenax (60/80 mesh), chromatographic
grade, or equivalent.
3.2 Methyl silicone packing - 3% OV-1 on Chromosorb-W (60/80 mesh), or
equivalent.
3.3 Silica gel, (35/60 mesh) Davison Chemical grade-15, or equivalent.
4. Methanol - Pesticide quality, or equivalent.
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 gasses 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 potentially hazardous amounts of these materials.
5.1 Place 9.8 ml of methanol into a 10-ml ground glass stoppered vol-
umetric 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.
5.2 Add the assayed reference material as described below:
5.2.1 Liquids - Using a 100-ul syringe, immediately add 2 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.
5.2.2 Gases - To prepare standards for any of the four halocarbons
that boil below 30°C (bromomethane, chloroethane, chloro-
methane, 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 "•eference standard above the surface of the
liquid, fhe neavy gas rapiaiy dissolves in cne methanol.
T T T ,1 O
-------
5.3 Reweigh, dilute to volume, stopper, then mix by inverting the flask
several times. Calculate the concentration in micrograms per micro-
liter from the net gain in weight. When compound purity is assayed
to be 96 percent or greater, the weight may be used without correc-
tion to calculate the concentration of the stock standard. Com-
mercially prepared stock standards may be used at any concentration
if they are certified by the manufacturer or by an independent source.
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.
5.5 Prepare fresh standards weekly for the four gases and 2-chloro-
ethylvinyl ether. All other standards must be replaced after 1
month, or sooner if comparison with check standards indicates a
problem.
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 stan-
dards should be prepared at concentrations such that the aqueous cali-
bration standards prepared in Subsections H.3 or H.4 will bracket the
working range of the analytical system.12 Secondary dilution standards
should be stored with minimal neadspace and should be checked frequently
for signs of degradation or evaporation, especially just prior to pre-
paring calibration standards from them. Standards should be discarded if
evaporation losses exceed 5 percent of the initial volume. Quality
control check standards that can he-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.
Surrogate standard spiking solution - Select a minimum of three surrogate
compounds from Table 2. Prepare stock standard solutions for each sur-
rogate standard in methanol as described in Subsection F 5. Prepare a
surrogate standard spiking solution from these stock standards at a
concentration of 150 pg/10 ml in water. Store the spiking solution at
4°C in Teflon-sealed glass containers with a minimum of headspace. The
solutions should be checked frequently for stability. They should be
replaced after 6 months. The addition of 10 yl of this solution to 5 ml '
of sample or standard is equivalent to a concentration of 30 yg/1 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.
111-13
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TABLE 2. SUGGESTED SURROGATE AND INTERNAL- STANDARDS3
Retention Time Primary Secondary
Compound (Min.) Ion Ions
Surrogate Standards
Benzene-ds 17.0 84
4-Bromofluorobenzene 28.3 95 174, 176
l,2-Dich1oroethane-d4 12.1
1,4-Difluorobenzene 19.6 114 63, 83
Ethylbenzene-d5 26.4
Ethylbenzene-d^o 26.4 98
FTuorobenzene 18.4 96 70
Pentafluorobenzene 23.5
Internal Standards
Bromochloromethane 9.3 128 49, 130, 51
2-Bromo-l-chloropropane nd 77 79, 156
1,4-Dichlorobutane nd 55 90, 92
aFor chromatographic conditions, see Tabie 3.
G. QUALITY CONTROL
1. A formal quality control program should be an integral component of the
analytical methods presented in this section. The minimum requirements
for this program consist of an initial demonstration of laboratory capa-
bility, the analysis of spiked samples as a continuing check on method
performance, and the analysis of blanks to demonstrate that reagent
interferences are under control.
2. To demonstrate the ability to generate data with acceptable accuracy and
precision, the analyst must perform the following operations:
2.1 For each parameter to be measured, select a spike concentration
representative of the expected levels 1n the samples. Using stock
standards, prepare a quality control check sample concentrate in
methanol 500 times more concentrated than the selected concentra-
tions. 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.
111-14
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2.2 Using a syringe, add 10 pi of the check sample concentrate and 10 yl
of the surrogate standard dosing solution (Subsection F.7) to each
of a minimum of four 5-ml aliquots of reagent water. A representa-
tive 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 Subsection J.2.1.
2.3 Calculate the average percent recovery (R), and the standard devi-
ation of the percent recovery (s), for all parameters and surrogate
standards. Wastewater background corrections must be made before R
and s calculations are performed.
3. The analyst must calculate method performance criteria for each of the
surrogate standards.
3.1 Calculate upper and lower control limits for method performance for
each surrogate standard, using the values for R and s calculated in
Subsection G.2.3:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) * » - 3 s
The UCL and LCL can be used to construct control charts^ that are
useful in observing trends in performance.
2.2 For each surrogate standard, the ""aboratory -nust deve^oo 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 anal-
ysis of four aliquots of wastewater as described in Subsection
G.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 Subsection G.4. The
accuracy statements should be updated regularly.i3
4. The laboratory is required to spike all samples with the surrogate stan-
dard spiking solution to monitor spike recoveries. If the recovery for
any surrogate standard does not fall within the method performance con-
trol limits, the results should be labeled as suspect. The laboratory
should monitor the frequency of qualified data to insure that it remains
at or below 5 percent of total output.
5. Each day, the analyst must demonstrate, through the analysis of reagent
water, that interferences from the analytical system are under control.
6. It is recommended that the laboratory adopt additional quality assurance
practices *or use with this method. The soec-if'ic oracticss that, are -nost
productive depend upon the needs of the laboratory and the nature of the
samples, field duplicates may oe analyzed to monitor the prec'slon of
the sampling technique. Whenever possible, the laboratory should perform
iil-i5
-------
analysis of standard reference materials and participate in relevant per-
formance evaluation studies. Also, any modifications of the procedure in
response to changes in the state-of-the-art should be evaluated in terms
of method performance.
H. CALIBRATION
1. Assemble a purge-and-trap device that meets the specifications in Sub-
section E.2. Condition the trap overnight at 180°C by backflushing with
an inert gas flow of at least 20 ml/min. Prior to use, condition traps
daily by backflushing for an additional 10 minutes at 180°C.
2. Connect the purge-and-trap device to a gas chromatograph. The gas chro-
matograph must be operated using temperature and flow rate parameters
equivalent to those in Table 3. Calibrate the purge-and-trap GC/MS
system using either the internal standard technique (Subsection H.3) or
the external standard technique (Subsection H.4).
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 comoounds 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 limita-
tions, no internal standard can be suggested tnat is applicable to all
samples. Due to their generally unique retention times, bromochloro-
methane, 2-bromo-l-chloropropane, and 1,4-dichlorobutane have been used
successfully as internal standards. Additional compounds that may be
used are listed in Table 2.
3.1 Prepare calibration standards dt a minimum of three concentration
levels for each parameter of interest.
3.2 Prepare a spiking solution containing each of the internal standards
using the procedures described in Subsections F.5 and F.6. It is
recommended that the secondary dilution standard be prepared at a
concentration of 15 yg/ml for each internal standard compound. The
addition of 10 yl of this standard to 5.0 ml of sample or calibra-
tion standard would be equivalent to 30 yg/1.
3.3 Analyze each calibration standard, according to Subsection J, adding
10 yl of internal standard spiking solution directly to the syringe
(Subsection J.2.1.5). Tabulate the area response of the character-
istic ions (Table 4) against concentration for each compound and
internal standard and calculate response factors (RF) for each
compound using Equation 1.
RF « (AsC1s)/(A1sCs) Eq. 1
TIJ-16
-------
TABLE 3. CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION LIMITS
=========================================================
Parameter
Retention Time
(Min.)
Column 1
Method
Detection Limit
(ug/D
Chloromethane
Bromomethane
Vinyl chloride
Chloroethane
Methyl ene chloride
Tri chl orof 1 uoromethane
1,1-Dichloroethene
1,1-Dichloroethane
Trans-1, 2-dichl oroethene
Chloroform
1,2-Di chloroethane
1 , 1 , 1-Tri chl oroethane
Carbon tetrachloride
Bromodi Chloromethane
1 ,2-Di chl oromethane
Trans-1 ,3-dichloropropene
Trichl oroethene
Benzene
Di bromochl oromethane
1,1 ,2-Tri chl oroethane
Ci s-1 ,3-dichl oropropene
2-Chloroethyl vinyl ether
Bromoform
1,1,2,2-Tetrachloroethane
Tetrachl oroethene
Toluene
Chlorobenzene
Ethyl benzene
1,3-Di chlorobenzene
1 ,2-Di chl orobenzene
1 ,4-Di chl orobenzene
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
Ib.o
17.0
17.1
17.2
17,2
18.6
19.8
22.1
22.2
23.5
24.6
26.4
nd
nd
nd
nd
nd
nd
nd
2.8
nd
2.8
4.7
1.6
1.6
2.8
3.3
2.8
2.2
6.0
5.0
1.9
4.4
3.1
5.0
nd
nd
4.7
6.9
4.1
6.0
6.0
7.2
nd
nd
nd
x==============================================================================
nd = not determined
Column Conditions:. Carbopak B (60/80 mesh) coated with 1 percent SP-1000
packed in 1.8 m x 2-mm i.D. giass column with hen urn jsrr-ier gas at 2 -"! ow
rate of 30 ml/min. Column temoerature is isothermal at 45°C for 3 min, then
programmed at 8°C per minute to 220° and neid for 15 mm.
111-17
-------
TABLE 4. CHARACTERISTIC IONS FOR PURGEABLE ORGANICS
Parameter
Primary
Ion
Secondary Ions
Chioromethane
Bromomethane
Vinyl chloride
Chloroethane
Methylene chloride
Trichlorof1uoromethane
1,1-Dichloroethene
1,1-Dichloroethane
Trans-l,2-dichloroethene
Chloroform
1,2-Dichloroethane
1,1,1-Trichloroethane
Carbon tetrachloride
Bromodi chloromethane
1,2-Dichloropropane
Trans-l,3-dichloropropene
Trichloroethene
Benzene
Di bromochloromethane
1,1,2-Trichloroethane
Ci s-1,3-dichloropropene
2-Chloroethylvinyl ether
Bromoform
1,1,2,2-Tetrachloroethane
Tetrachloroethene
Toluene
Chlorobenzene
Ethyl benzene
1,3-Di chlorobenzene
1,2-Dichlorobenzene
1,4-Dichlorobenzene
50
94
62
64
84
101
96
63
96
83
98
97
117
127
112
75
130
78
127
97
75
106
173
168
164
92
112
106
146
146
146
52
96
64
66
49, 51, 86
103
61, 98
65, 83, 85, 98, 100
61, 98
85
62, 64, 100
99, 117, 119
119, 121
83, 35, 129
63, .65, 114
77
95, 97, 132
129, 208, 206
83, 85, 99, 132, 134
77
63, 65
171, 175, 250, 252, 254, 256
83, 85, 131, 133, 166
129, 131, 166
91
114
91
148, 113
148, 113
148, 113
111-18
-------
where:
AS = Area of response of the characteristic ion for the
analyte being measured
Ais = Area response of the characteristic ion for the internal
standard.
C.jS * Concentration of the internal standard.
Cs * Concentration of the analyte being measured.
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-JS, vs RF.
3.4 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 ±10%, the test must be repeated using a fresh calibration
standard. Alternatively, a new calibration curve must be prepared
for that compound.
4. External standard calibration procedure.
4.1 Prepare calibration standards at a minimum of three concentration
levels for each parameter by carefully adding 20.0 yl of one or more
secondary dilution standards to 50, 250, or 500 ml of reagent water.
A 25 yl syringe with a 0.15 mm I.D. needle should be used for this
operation. On» of the external standards should be at a concentra-
tion near, but above, the method detection limit (see Table 3) 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. If not so
stored, they must be discarded after 1 hour.
4.2 Analyze each calibration standard according to Subsection J 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. Alterna-
tively, if the ratio of response-to-concentration (calibration
factor) is a constant over the working range (<10% relative stan-
dard 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.
4.3 The working calibration curve or calibration factor must be veri-
fied on each working day by the measurement of one or more calibra-
tion standards. If the response for any parameter varies from the
predicted -espouse by more than ilO%, the test must be repeated
using a fresh calibration standard. Alternatively, a new calibra-
tion curve or calibration factor tiust be prepared *c- that
parameter.
111-19
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I. DAILY GC/MS PERFORMANCE TESTS
1. At the beginning of each day that analyses are to be performed, the GC/MS
system must be checked to see if acceptable performance criteria are
achieved for BFB.H 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 day.14
2. These performance tests require the followtng 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.
3. At the beginning of each day, inject 2 pi of BFB solution directly on
column. Alternately., add 2 ul of BFB solution to 5.0 ml of reagent water
or standard solution and analyze according to Subsection J.2. Obtain a
background-corrected mass spectrum of BFB and verify that all the key ion
criteria in Table 1 are achieved. If all the criteria are not achieved,
the mass spectrometer must be retuned and the test repeated until all
criteria are met.
111-20
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J. ANALYTICAL PROCEDURES
1.1 Analysis of Solid Hazardous Waste Samples for Volatile Organic
Compounds by Methanol Partitioning
Analytical Procedure: available
Sample Preparation: available
1.1.1 Reference/Title
U.S. Environmental Protection Agency, "Method for Preparation
of Medium Concentration Hazardous Waste Samples." U.S. EPA
Region IV, Athens, Georgia, p. 7. 1981.
1.1.2 Method Summary
One gram aliquots of soil, solid, aqueous liquid, or non-
aqueous liquid are transferred to vials inside a chemical
carcinogen glovebox. The samples are then removed from the
glovebox and diluted with methanol. After mixing, an aliquot
of the methanol solution is added to reagent water and the
resultant solution is analyzed by the purge-and-trap GC/MS
technique.
1.1.3 Applicability
This procedure is designed for the safe handling and prep-
aration of potentially hazardous samples from hazardous waste
sites to be analyzed for organic chemicals including priority
pollutants. The method is directed to contaminated soil
samples and waste samples that may be solid, aqueous liquid,
or non-aqueous liquid and suspected to contain less than 10
percent of any one organic chemical component. Method detec-
tion limits and retention times for compounds quantitated by
this procedure are summarized in Table 3.
1.1.4 Precision and Accuracy
This extraction and preparation procedure was developed for
rapid and safe handling of hazardous samples. The design of
the method thus did not stress efficient recoveries of all
components. Rather, the procedure was designed for a mod-
erate recovery of a broad spectrum of organic chemicals.
Thus, analytical results may sometimes only reflect the mini-
mum amount of analyte in the sample.
1.1.5 Sample Preparation
Place the oriainal samcle container into the glovebox.
Additional items tnat snouia oe in cne gioveoox inciuae 1}
:3libr.itad 2nd *ared ?0-fi via1-; with cans, ?> ;» soatula, 3)
a balance, 4) a capped vial containing 10 ml of interference-
free methanol, 5) a capped vial containing reagent water and
111-21
-------
6) a medicine dropper. The vial of methanol is to be
used as a method blank. (One method blank should be run
for each batch of 20 samples or less.) Open the sample
transportation can and remove the sample vial. Note and
record the physical state and appearance of the sample. If
the sample bottle is broken, immediately repackage the
sample and terminate the analysis.
It is easy to contaminate a sample with purgeable organic
materials. Therefore, the following precautions must be
followed once sample processing has been initiated.
a. There should be no solvent fumes or solvents, other
than the methanol blank, inside the glovebox during the
time the samples are being weighed out.
b. Tightly recap each sample container after weighing.
Allow fumes from the first sample to be exhausted
before opening the next sample.
Open the sample vial and mix the sample. If the sample is
a liquid, transfer a drop to the vial containing water to
determine if the sample is aqueous or non-aqueous. Record
the result. Transfer approximately 1 gram (or 1 ml) of
sample to the tared calibrated vial. Wipe the mouth of the
vial with tissue to remove any sample material and cap tne
vial tigntly. Record the exact weight of sample taken.
Reseal the original sample and replace it in the original
packing.
Remove the vials frcm the glovebox and place them in an
efficient hood. If the sample is liquid, dilute the sanple
as indicated in paragraph a. If the sample is solid,
dilute the sample as indicated in paragraph b.
a. Dilute the VOA sample to the 10 ml-mark with
interference-free methanol. Recap the sample vial and
shake.
b. If the sample is soil or solid waste, add 10 ml of
interference-free methanol. Recap the sample vial and
shake.
Methylene chloride or other solvents should not be used in
the hood when samples are being diluted. Also, the samples
should be stored in a solvent-free atmosphere at 4°C prior
to purge-and-trap analysis. Sample analyses should be
completed within 14 days^ (Figure 1).
111-22
-------
1.1.6 Sample Analyses
Transfer a 25-pl aliquot of the methanol extract to 5 ml of
reagent water in a purge device. Process as a liquid
sample (Subsection J.2.).
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.
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 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. The second subsample can:
1. be used to prepare dilutions ^f ^analysis is required,
2. serve as a backup in the event the original subsample
is lost, or
o. serve as a Jupllcate.
Add 10.0 ill of the surrogate spiking solution (Subsection
F.7) and, if applicable, 10.0 yl of the internal standard
spiking solution (Subsection H.4.2) through the valve bore,
then close the valve. The surrogate and internal standards
may be mixed and added as single spiking solution. 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.
Close both valves and purge the sample for 11.0 ± 0.1
minutes at ambient temperature.
After purging the sample, attach the trap to the chromato-
graph, adjust the device to the desorb mode, and begin the
GC temperature program. Concurrently, introduce the
trapped materials to the GC column by rapidly heating the
trap to 180°C while backflushing the trap with an inert gas
between 20 and 60 ml/min for 4 minutes. If this rapid
heating requirement cannot be met, the gas chromatographic
column must De usea as a secondary trap by cool Inn *t to
30°C (or subambient, if problems persist) Instead of the
recommended initial temperature of 45"C.
111-23
-------
While the trapped material 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.
After desorbing the sample for 4 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. Maintain
the trap temperature at 180°C. Do not allow the trap tem-
perature to exceed 180°C, since the sorption/ desorption is
adversely affected by heating the trap to higher temperatures,
After approximately 7 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.
If the response for any ion exceeds the working range of
the system, dilute the sample aliquot in the second syringe
(paragraph 1.1.6) with reagent water and re-analyze.
If nothing is detected in the methanol extract of the solid
phase sample, the process can be repeated using a larger
sample aliquot.
Calculate and report results as indicated in Subsections K
and L.
111-24
-------
1.2 Analysis of Solid Hazardous Waste Samples for Volatile
Organic Compounds by Polyethylene Glycol Partitioning
Analytical Procedure: available
Sample Preparation: available
1.2.1 Reference/Title
Battelle Laboratories, "Manual of Collaborators on Evaluation
of Methods for Analysis of Hazardous Wastes." Prepared under
EPA Contract 68-03-3098. Battelle Columbus Laboratories,
Columbus, Ohio (1981).I6
1.2.2 Method Summary
A portion of solid waste is dispersed in polyethylene qlycol
(PEG) to dissolve the purgeable organic constituents.1' A
portion of the PEG solution is combined with water in a spec-
ially designed purging chamber. An inert gas is then bubbled
through the solution at ambient temperature and the purgeable
components are efficiently transferred from the aqueous phase
to the vapor phase. The vapor is swept through a sorbent
column where the purgeable components are trapped. After
purging is completed, the sorbent column is heated and back-
flushed with inert gas to desorb the purgeable components
onto a gas chromatographic column. The gas chromatographic
column is heated to elute the purgeable components which are
detected with a mass spectrometer.!»6
1.2.3 Applicability
This method covers the determination of purgeable organic
compounds in a variety of solid waste materials. It is
applicable to nearly all types of samples, regardless of
water content, including aqueous sludges, caustic liquors,
acid liquors, waste solvents, oily wastes, tars, fibrous
wastes, polymeric emulsions, filter cakes, spent carbon,
spent catalysts, soils, and sediments. The method is based
upon a purge and trap/gas chromatographic/mass spectrometric
procedure.18
The method detection limits for the procedure can be expected
to vary with the sample matrix. A detailed round-robin
testing program is presently (1981-82) underway to define
these limits.
This method shoul.d be restricted to use by or under the super-
vision of analysts experienced in the use of purge-and-trap
systems and gas chromatograph/mass spectrometers and skilled
-------
1.2.4 Precision and Accuracy
These variables are presently being evaluated. Analytical
precision and accuracy should be similar to the limits pre-
sented in Subsection J.2.1.
1.2.5 Estimation of Total Volatiles
In order to avoid overloading the detector when analyzing
samples for volatile organic compounds, an approximate
determination of total volatiles is made by extracting or
dissolving a portion of sample with n-hexadecane. An aliquot
of the n-hexadecane solution is then analyzed by gas chro-
matography. The estimate of total volatile content is based
on the total area response for all components eluting prior
to n-dodecane. The estimated total volatile content is
calculated by applying a response factor obtained for n-
nonane to the total area response defined above.
The response factor and retention time for n-dodecane are
determined by analyzing a solution containing 0.20 mg/ml
n-nonane and 0.20 mg/ml n-dodecane in n-hexadecane. Inject a
2-yl aliquot of this solution into a gas chromatographic
system operated under the following conditions:
Column - 30 m x 0.25 mm I.D. DB-5 fused silica capillary
column.
Column Temperature - maintain the initial column tempera-
ture at 20°C for 4 minutes and then increase to 300°C at
8°C per minute. The final column temperature is maintained
for 10 minutes.
Detector - Flame ionization detector.
Determine the area response for n-nonane, and divide by 0.2
to obtain the area response factor for n-nonane. Record the
retention time of n-dodecane.
Add 1.0 gm of sample to 20 ml of n-hexadecane and 2 ml of 0.5
M_ N32HP04 contained in a 50-ml glass centrifuge tube. Cap
securely with a Teflon-lined screw cap. Shake the mixture
vigorously for 1 minute. If the sample does not disperse
during the shaking process, sonify the mixture in an ultra-
sonic bath for 30 minutes. Allow the mixture to stand until i
clear supernatant is obtained. Centrifuge if necessary to
facilitate phase separation.
Analyze a 2-ul aliquot of the n-hexane supernatant using the
thromatograohic
-------
corresponding area of an n-hexadecane blank. Using the area
response factor determined for n-nonane in paragraph 1.2.5,
estimate the total volatile content of the sample as mg of
volatile components per gm of sample as follows:
TVC = TARsample - TAR blank x 20
n-Nonane Area Response factor
where:
TVC - total volatile content of sample in mg/g
TARsamp-]e = total area response obtained for the sample
T"W*blank = total area response obtained for a blank.
When using the purge-and-trap GC/MS analytical procedure, the
total quantity of volatile components injected should not
exceed approximately 10 yg. The volume of PEG solution con-
taining sample that is analyzed is selected based on the GC
screen of the n-hexadecane solution.
a) If the total volatile content (TVC) of the sample, as
determined in paragraph 1.2.5, is 1.0-mg/g or less, use a
200-yl aliquot of the-PEG extract prepared according to
paragraph 1.2.6.
b) If the TVC is greater than 1.0 mg/g, use an aliquot of
PEG extract that contains approximately 10 yg. The volume
(in yl) of the aliquot to be taken is calculated by dividing
200 by the TVC.
c) If the TVC is greater than 20 mg/g, dilute a 200-yl aliquot
of PEG extract to 10 ml with reagent PEG. For this case,
calculate the volume of the diluted extract to be taken for
analysis by dividing 10,000 by the TVC.
1.2.6 Preparation of Polyethylene Glycol (PEG) Solution
To a 50-ml glass centrifuge tube with Teflon-lined cap, add
15 ml of reagent PEG using a graduated pi pet. Weigh the cap-
ped centrifuge tube and PEG on an analytical balance to 1 mg.
Using an appropriate implement (see Subsection E.10),
transfer 1 gm (±10%) of sample to the PEG in the centrifuge
tube. Take care not to touch the sample-transfer implement
to che PEG.
111-27
-------
The transfer should be carried out in such a fashion that the
sample is dissolved in or submerged in the PEG as expedi-
tiously as possible. This technique is intended to prevent
loss of volatiles from the sample to a degree consistent with
the requirements of this method.17 After the sample has
been transferred, recap the centrifuge tube and weigh on an
analytical balance to 1 mg. After the sample weight has been
obtained, add reagent PEG to the 20-ml mark on the centrifuge
tube and securely recap the tube.
Disperse the sample by vigorous agitation for 1 minute. The
mixture may be agitated manually or with the aid of a vortex-
mixer. If the sample does not disperse during this process,
sonify the mixture in an ultrasonic bath for 30 minutes.
Allow the mixture to stand until a clear supernatant is
obtained. Centrifuge if necessary to facilitate phase separa-
tion.
The supernatant solution may be stored for future analytical
needs. If this is desired, transfer the solution to a 10-ml
screw cap vial with Teflon cap liner. Store at -10 to -20°C,
and protect from light.
1.2.7 GC/MS Analysis of PEG Solution
To the purging device, add 5.3 ml of reagent water. The add-
ition is made using a 5-ml -glass syringe equipped with a
15-cm 20-22-gauge needle. The needle is inserted through the
sample inlet shown in Figur? 2. The intsrnal diameter of the
14-gauge needle that forms the sample ^nlet will permit
insertion of a 20-gauge needle.
Using a 25-vl microsyringe equipped with a long needle,
enrich the reagent water in the purging device with the ap-
propriate internal standards, surrogate standards, and BFB.
Add the aliquot directly to the reagent water in the purging
device by inserting the needle through the sample inlet (see
Figure 2). When discharging the contents of the micro-
syringe, be sure that the needle is well beneath the surface
of the water.
Based on the calculations in paragraph 1.2.5., take an
appropriate aliquot of the PEG solution described in para-
graph 1.2.6. Use a microsyringe equipped with a long needle
to measure and dispense the aliquot. In order to load the
PEG solution into the microsyringe it will be necessary to
draw the solution into a disposable glass pipet and dis-
charge the viscous solution directly into the barrel of the
microsyringe. Completely fill the barrel with PEG solution,
and than ''nsert the plunger ind ""owe*- to ^he .Jesired -nark.
Dispense the aliquot directly into the aqueous solution in
the purging device by inserting che neeale througn the
111-28
-------
sample Inlet. When discharging the contents of the micro-
syringe, be sure that the end of the needle is well below the
surface of the water.
Close the 2-way syringe valve at the sample'in-let.
Adjust the 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.
Close both valves and purge the sample for 11.0 ± 0.1 minutes
at ambient temperature.
At the conclusion of the purge time, attach the trap to the
chromatograph, adjust the device to the desorb mode, and
begin the GC temperature program.
Temperature - Isothermal at 45°C for 3 minutes, then in-
creased at 8°C per minute to 220°C, and maintained at 220°C
for 15 minutes.
Concurrently, introduce the trapped materials to the GC col-
umn by rapidly heating the trap to 180°C while backflushing
the trap with an inert gas between 20 and 60 ml/min for 4
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 suDamoient, if proolems per- •
sist) instead of the recommended initial temperature of 45°C.
While the trapped material is being desorbed into the gas
chromatograph, empty the purging chamber using a glass
syringe equipped with a long needle. Rinse the chamber with
two 5-inl flushes of reagent water.
(Between each use, wash the purging device with a detergent
solution, rinse it with distilled water, and then dry it in
an oven at 105°C. It is convenient to have several purging
devices available so that a clean unit 1s available as
required.)
After desorbing the sample for 4 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. Maintain
the trap temperature at 180°C. Do not allow the trap tem-
perature to exceed 180eC, since the sorption/desorption is
adversely affected by heatinq the trap to higher tempera-
tures. After approximately 7 minutes, turn off the crap
heater ind ooen the syringe valve to stoo the gas flow
through the trap. When cool, the trap is ready for the next
sample.
111-29
-------
If the response for any ion exceeds the working range of the
system, repeat the analysis using a correspondingly smaller
aliquot of the PEG solution described in paragraph 1.2.6.
Calculate results as indicated in Subsections K and'L.
111-30
-------
2.1 Analysis of Water Samples for Volatile Organic Compounds
Analytical Procedure: evaluated
Sample Preparation: available
2.1.1 Reference/Title
U.S. Environmental Protection Agency, "Purgeables - Method 624."
Federal Register, Vol. 44, No. 233:69532-69539. December 3,
1979.18
2.1.2 Method Summary
An inert gas is bubbled through a 5-ml water sample contained
in a specially designed purging chamber at ambient temper-
ature. 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 back-
flushed with the inert gas to desorb the purgeables onto a
gas chromatographic column. The gas chromatograph is temper-
ature programmed to separate the purgeables which are then
identified and quantified with a mass spectrometer.^*^
2.1.3 Applicability
This method is appropriate for the determination of the
listed volatile organic compounds in municipal and industrial
discharges, ground water, and surface water samples.
2.1.4 Precision and Accuracy
The method was tested in 2 to 4 laboratories.*9 Reported
method detection limits ^minimum concentration that can be
measured and reported with 99 percent confidence that the
value is greater than zero),20 average recoveries, and
average standard deviations for the recovery data are pre-
sented in Table 3 and Table 5.
2.1.5 Sample Purging
Table 3 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 separations achieved by Column
1 is shown in Figure 7. Other packed columns or chromato-
graphic conditions may be used if the requirements of Sub-
section G.2 are met.
^fter ?chiev?nn the key •''on abundance criteria in Subsection
H, calibrate the system dally as aescnbea in ^uosection I.
-------
TABLE 5. ACCURACY AND PRECISION FOR PURGEABLE ORGANICS
Parameter
Benzene
Bromodi chl oromethane
Bromoform
Bromomethane
Carbon tetrachloride
Chlorobenzene
Chl oroethane
Chloroform
Chl oromethane
Di bromochl oromethane
1,1-Dichloroethane
1,2-Dichl oroethane
1,1-Dichloroethene
Trans-1, 2-dichloroethene
1 ,2-Dichl oroorooane
Cis-l,3-dichlorooropene
Trans-1 ,3-di chl oropropene
Ethyl benzene
Methyl ene chloride
1 , 1 ,2 ,2-Tetrachl oroethane
Tetrachl oroethene
Toluene
1,1, 1-Trichl oroethane
1,1,2-Tri chl oroethane
Tri chl oroethane
Trichlorofluoromethane
Vinyl chloride
Reagent Water
Average
Percent
Recovery
89
97
94
90
91
94
67
90
91
«5
83
102
74
90
94
95
91
109
82
81
97
96
92
102
106
59
103
Standard
Deviation
(%)
12
11
14
16
23
23
22
18
22
12
10
12
24
25
26
15
13
19
46
31
13
22
21
14
14
23
30
Wastewater
Average
Percent
Recovery
93
103
88
78
91
103
60
91
64
99
87
103
80
85
99
98
93
106
66
78 '
99
97
94
103
110
67
79
Standard
Deviation
(%)
24
31
12
15
33
24
23
26
28
17
21
. 27
32
35
30
' 20
16
28
66
31
26
25
36
19
22
48
22
Samples were spiked between 10 and 1000 yg/1.
Column Conditions: Carbopak B (60/80 mesh) coated with 1 percent SP-1000
packed in a 1.8 m x 2-mm I.D. glass column with helium carrier gas at a flow
rate of 30 ml/min. Column temperature is isothermal at 458C for 3 min.,
then programmed at 8°C per min. to 220° and held for 15 min.
111-32
-------
Column 1% grMOOO on *up«leacort
Program 48'C. 3 rnin . »> p*> nwi to 220°C
O*Mctor. M«M SpocrranwMr
I I I
24*
I I I I I I I I I I I
• 10 12 14 II 11 20 . 22 24 2« 21
Mmutn
Figure 7. Gas chromatogram of volatile organics by purge and trap.
111-33
-------
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.
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. The second subsample can:
1. be used to prepare dilutions if reanalysis is required,
2. serve as a backup in the event the original subsample is
lost, or
3. serve as a duplicate.
Add 10.0 pi of che surrogate spiking solution (Subsection F.7)
and, if applicable, 10.0 yl of the internal standard spiking
solution (Subsection ri.4.2) through the valve bore, then close
the valve.' The surrogate and internal standards may be mixed
and added as single spiking solution.
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.
Close both valves and purge the sample for 11.0 ± 0.1 minutes
at ambient temperature.
2.1.6 Sample Analysis
When sample purging is complete, attach the trap to the
chromatograph, adjust the device to the desorb mode, and
begin the GC temperature program. Concurrently, introduce
the trapped materials to the GC'column by rapidly heating the
trap to 180°C while backflushing the trap with an inert gas
between 20 and 60 ml/min for 4 minutes. If this rapid heat-
ing 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.
'•/hile the Crapped •na^'-'al ;s beinn desorbed *ntc the gas
chromatograph, empty the purging chamber using the sample
111-34
-------
Introduction syringe. Wash the chamber with two 5-ml flushes
of reagent water.
After desorbing the sample for 4 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. Maintain t^e trap
temperature at 180°C. Do not allow the trap temperature to
exceed 180°C, since the sorption/desorption is adversely
affected by heating the trap to higher temperatures. After
approximately 7 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.
If the response for any ion exceeds the working range of the
system, dilute the sample aliquot in the second syringe with
reagent water and reanalyze.
Calculate results as indicated in Subsections K and L.
111-35
-------
3.1 Analysis of Soil/Sediment Samples
Analytical Procedure: evaluated
Sample Preparation: available
3.1.1 Reference/Title
Hiatt, M., "Analyses of Fish and Sediment for Volatile
Priority Pollutants." Anal. Chem. £3:1541-1543 (1981).21
3.1.2 Method Summary
This method is a modification of that used for Purgeable
Organics in Water.2 However, instead of stripping the
volatile organic compounds of interest from the sample matrix
with an inert gas, the compounds are drawn off in a vacuum
and condensed in a super-cooled trap. The condensed vola-
tile compounds are then transferred to a conventional purge-
and-trap device and the sample is processed as a water
sample. The sample is vaporized and swept through a sorbent
column where the purgeables are trapped. After purging is
completed, the sorbent column is heated to desorb the purge-
ables and the sample is backflushed onto a gas chromato-
graphic column with an inert gas. The gas chromatograph is
temperature-programmed to separata the purgeables which are
then identified and quantified with a mass spectrometer.
3.1.3 Applicability
This method is appropriate for the determination of the
listed volatile organic compounds in sediments. Limited
testing has shown this method to produce better compound
recovery than conventional purge-and-trap or sample dilution
techniques.21
3.1.4 Precision and Accuracy
Data for the recovery of known spikes from a sediment matrix
are presented in Table 6. The average compound recovery
using the vacuum technique was 96 ± 7 percent compared to 82
t 18 percent for direct purge and trap with a diluted sample
and 83 ± 10 percent for direct purge and trap with thermal
desorption.2l
3.1.5 Sample Preparation
The vacuum extractor (Figure 6) must be airtight: and free of
moisture before an extraction can be started. A clean 125-ml
septum vial is connected, the vacuum pump started, and V2 to
V4 are opened to evacuate the apparatus. The elimination of
''ine condensation is iccomolished by warming the transfer
lines while evacuating the system. Heating tape is effective
in creating even cransfer 1;ne temperatures and can be used
111-36
-------
TABLE 6. SPIKE RECOVERY DATA FOR VOLATILE ORGANICS FROM SEDIMENT
MATRIX USING THE VACUUM EXTRACTION TECHNIQUE*
====z======r=========================r================a=================
Compound Percent Recovery Standard Deviation
Chl oromethane
Bromomethane
Vinyl chloride
Chl oroethane
Methyl ene chloride
Tri chl orof 1 uoromethane
1,1 -Di chl oroethene
1 ,1-Di chl oroethane
Trans-1 ,2-dichl oroethene
Chloroform
1,2-Di chl oroethane
1,1,1-Tri chl oroethane
Carbon tetrachloride
Acrylonitrile
Bromodi chl oromethane
1 ,2-Di chl oromethane
Trans-1, 3-dichl oropropene
Trichl oroethene
Benzene
Di bromochl oromethane
1 , 1 , 2-Tri chl oroethane
Ci s-1 ,3-di chl oropropene
Bromoform
Tetrachl oroethene
1 , 1 ,2,2-Tetrachl oroethane
Toluene
Chlorobenzene
Ethyl benzene
98
86
108
106
Sample
80
82
101
92
102
96
106
100
89
96
96
31
98
94
98
98
32
90
104
98
102
101
97
22
24
35
27
Contaminated
8
9
7
10
11
17
11
13
8
8
4
6
6
4
10.
5
•7
9
13
8
4
5
5
:a=a=a=====================:===a=a===============s=z===:============== ====== = = = = =
*After Hyatt21
continuously during extraction. The vacuum extractor is
pressurized with helium by closing V3 and opening VI
(Figure 6). The apparatus is then leak-tested by applying
soapy water on all connections and making the appropriate
adjustments when leaks are located. When the apparatus is
airtight, close VI and open V3. Heat the transfer lines and
concentrator trap" for 5 minutes to eliminate any contamina-
tion from previous extracts. The system is now ready for
sample extraction.
Weigh out 10 g of sediment sample and transfer to a 125-ml
septum vial.
111-37
-------
To begin the extraction process, close V2 (V3 and V4 remain
open), cool the concentrator trap with a liquid nitrogen
bath, and replace the empty 125-ml vial with the sample
vial. Disconnect the vacuum source by closing V3. Open V2
to permit vapors from the sample vial to reach the concen-
trator trap. The sample vial, containing 10 g of sediment
sample, is then immersed in the ultrasonic water bath. The
equilibrium temperature generated in the ultrasonic bath is
50°C. Therefore, the bath is initia-lly filled with water at
50°C and that temperature is maintained by continuous ultra-
sonic operation.
After 5 minutes of ultrasonic agitation, the vacuum source is
connected by opening V3 (Figure 6). The lower pressure
hastens the transfer of volatile compounds from the sample to
the super-cooled concentrator trap. After 15 minutes of
vacuum, close V3 and open VI to fill the system with helium
until atmospheric pressure is obtained. Close VI and V2 to
isolate the concentrate. The sample extraction is now com-
plete and the concentrate is ready for transfer to a purge-
and-trap device. The concentrate can be held in the liquid
nitrogen bath for up to an hour prior to analysis.
3.1.6 Sample Analysis
Disconnect the sample concentrator trap from the vacuum
extractor and connect it to the purge-and-trap device. Some
outgassing.may be observed when the sample extract is melted;
therefore, the axtract should be kept frozen until the
concentrator trap is attached to the purge-and-trap device.
Warm the concentrator trap walls to loosen the extract and
allow the ring of ice formed during condensation to drop to
the bottom of the trap. To this partially melted extract,
add 5 ml of distilled, deionized water containing the in-
ternal standard. Continue processing the sample using a
modified purge-and-trap procedure. Place the concentrator
trap in an ice water bath and purge for 5 minutes. Then
immerse the concentrator trap in a 55°C water bath and purge
for an additional 7 minutes. This modification is intended
to standardize sample handling procedures in order to produce
reproducible purging efficiencies.
At the conclusion of the purge time, attach the trap to the
chromatograph, adjust the device to the desorb mode, and
begin the GC temperature program. Concurrently, introduce
the trapped materials to the GC column by rapidly heating the
trap to 180°C while backflushing the trap with an inert gas
between 20 and 60 ml/min for 4 minutes. If this rapid heat-
ting requirement cannot be met, the gas chromatographic
column nust ba used as 3 secondary two by -oolinq *t to 30°C
(or subambient, if problems persist) instead of the recom-
mended initial temperature of 453C.
111-38
-------
While the trapped material 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.
After desorbing the sample for 4 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. Maintain the trap
temperature at 180°C. Do not allow the trap temperature to
exceed 180°C, since the sorption/desorption is adversely
affected by heating the trap to higher temperatures. After
approximately 7 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.
If the response for any ion exceeds the working range of the
system, dilute the sample aliquot in the second syringe with
reagent water and reanalyze.
Calculate results as indicated in Subsections K and L.
-------
4.1 Analysis of Volatile Organic Compounds in Biological Tissue
Analytical Procedure: evaluated
Sample Preparation: available
4.1.1 Reference/Title
Hiatt, M., "Analysis of Fish and Sediment for Volatile
Priority Pollutants." Anal. Chem. £3:1541-1543 (1981).21
4.1.2 Method Summary
This method is a modification of that used for Purgeable
Organics in Water.2 However, instead of stripping the
volatile organic compounds of interest from the sample matrix
with an inert gas, the compounds are volatilized in a vacuum
system and condensed in a super-cooled trap. The condensed
volatile compounds are then transferred to a conventional
purge-and-trap device and the sample is processed as a water
sample. The sample is vaporized and swept through a sorbent
column where the purgeables are trapped. After purging is
completed, the sorbent column is heated to desorb the purge-
ables and the sample is backflushed onto a gas chromato-
graphic column with an Inert gas. The gas chromatograph is
temperature-programmed to separate the purgeables which are
then identified and quantified with a mass spectrometer.
4.1.3 Applicability
This method is appropriate for the determination of the
listed volatile organic compounds in fish tissue. Limited
testing has shown this method to produce better compound
recovery than conventional purge-and-trao or sample dilution
techniques.21
4.1.4 Precision and Accuracy
Data for the recovery of known spikes from a fish tissue
matrix are presented in Table 7. The average compound
recovery using the vacuum technique was 76 ± 20 percent
compared to 44 ± 16 percent for direct purge and trap with a
diluted sample and 64 ± 12 percent for direct purge and trap
with thermal desorption.21
4.1.5 Sample Preparation
The vacuum extractor (Figure 6) must be airtight and free of
moisture before an extraction can be started. A clean 125-ml
septum vial is connected, the vacuum pump started, and V2 and
VA are ooened to evacuate the ^ooaratus, The elimination of
line condensation is accomplished by warming the transfer
lines while evacuating the system. Heating tape J;s af'ecflve
in creating even transfer line temperatures and can be used
-------
TABLE 7. SPIKE RECOVERY DATA FOR VOLATILE ORGANICS FROM FISH TISSUE
USING THE VACUUM EXTRACTION TECHNIQUE*
;==========================================================================
Compound
Percent Recovery
Standard Deviation
Chi oromethane
Bromomethane
Vinyl chloride
Chi oroethane
Methyl ene chloride
Trichlorofluoromethane
1,1-Dichloroethene
1,1-Di chl oroethane
Trans-l,2-dichloroethene
Chloroform
1 ,2-Dichl oroethane
1,1,1-Tri chl oroethane
Carbon tetrachloride
Bromodi chl oromethane
1,2-Dichloropropane
1,2-Dichloropropane
Trans-l,3-dichloropropene
Tricnl oroetnene
Benzene
Di bromochl oromethane
1 ,1 ,2-Tri chl oroethane
Ci s-1 ,3-dichl oropropene
Bromoform
Tetrachl oroethene
1 , 1 ,2 ,2-Tetrachl oroethane
Toluene
Chlorobenzene
Ethyl benzene
85
' 126
64
69
Sample
99
74
90
86
107
92
92
91
64
54
54
52
65
57
56
56
54
ND
NO
61
ND
64
ND
22
75
11
22
Contaminated
2
8
6
9
31
5
8
9
11
7
7
9
11
10
9
7
9
-
-
10
-
15
-
*After Hyatt21
continuously during extraction. The vacuum extractor is
pressurized with helium by closing V3 and opening VI
(Figure 6). The apparatus is then leak-tested by applying
soapy water on all connections and making the appropriate
adjustments when leaks are located. When the apparatus is
airtight, close V-l and open V3. Heat the transfer lines and
concentrator trap for 5 minutes to eliminate any contamina-
tion from previous extracts. The system is now ready for
sample extraction.
Weign out 10 g of homogenized fish t'ssue and transfer to a
125-ml septum vial.
•
111-41
-------
. To begin the extraction process, close V2 (V3 and V4 remain
open), cool the concentrator trap with a liquid nitrogen
bath, and replace the empty 125-ml vial with the sample
vial. Disconnect the vacuum source by closing V3» Open V2
to permit vapors from the sample vial to reach the concen-
trator trap. The sample vial, containing the fish tissue, is
then immersed in the ultrasonic water bath. The equilibrium
temperature generated in the ultrasonic bath is 50°C. There-
fore, the bath is initially filled with water at 50°C and
that temperature is maintained by continuous ultrasonic
operation.
After 5 minutes of ultrasonic agitation, the vacuum source is
connected by opening V3. The lower pressure hastens the
transfer of volatile compounds from the sample to the super-
cooled concentrator trap. After applying a vacuum for 15
minutes, close V3 and open VI to fill the system with helium
until atmospheric pressure is obtained. Close VI and V2 to
isolate the concentrate. The sample extraction is now com-
plete and the concentrate is ready for transfer to a purge-
and-trap device. The concentrate can be held in the liquid
nitrogen bath for up to an hour prior to analysis.
4.1.6 Sample Analysis
Disconnect the sample concentrator trap from the vacuum ex-
tractor and connect it to the purge-and-trap device. Some
outgassing may be observed when the sample extract is melted;
therefore, the extract should be keot frozen until the con-
centrator trap is attached to the purge-and-trap device.
Warm the concentrator trap walls to loosen the extract .and
allow the ring of ice formed during condensation to drop to
the bottom of the trap. To this partially melted extract,
add 5 ml of distilled, deionized water containing the in-
ternal standard. Continue processing the sample using a
modified purge-and-trap procedure. Place the concentrator
trap in an ice water bath and purge for 5 minutes. Then •
immerse the concentrator trap in a 55°C water bath and purge
for an additional 7 minutes. This modification is intended
to standardize sample handling procedures in order to produce
reproducible purging efficiencies.
At the conclusion of the purge time, attach the trap to the
chromatograph, adjust the device to the desorb mode, and
begin the GC temperature program. Concurrently, introduce
the trapped materials to the GC column by rapidly heating the
trap to 180° while backflushing the trap with an inert gas
between 20 and 60 ml/min for 4 minutes. If this rapid heat-
ing requirement cannot be met, the gas chromatographic column
must be used as a secondary trao by cooling it to 30°C (or
suoambient, if prodiems persist; instead of the recommenGea
initial temperature of 45°C,
111-42
-------
While the trapped material 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.
After desorbing the sample for 4 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. Maintain the trap
temperature at 180°C. Do not allow the trap temperature to
exceed 180°C, since the sorption/desorption is adversely
affected by heating the trap to higher temperatures. After
approximately 7 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.
If the response for any ion exceeds the working range of the
system, dilute the sample aliquot in the second syringe with
reagent water and reanalyze.
Calculate results as indicated in Subsections K and L.
111-43
-------
4.2 Analysis of Biological Tissue Samples for Purgeable Organic
Compounds
Analytical Procedure: available
Sample Preparation: available
4.2.1 Reference/Title
U.S. Environmental Protection Agency, "Extraction and Analysis
of Priority Pollutants in Biological Tissue." Method PPB 10/80.
U.S. EPA, S&A Division, Region IV. Laboratory Services Branch,
Athens, Georgia, p. 7. (1980).22
4.2.2 Method Summary
A 1-gram aliquot of homogenized fish tissue is diluted with
organic-free water. The volatile compounds are stripped from
the sample at 55°C and collected in a purge-and-trap appa-
ratus. The compounds are backflushed from the trap, identi-
fied and quantified using computerized GC/MS methodology.
4.2.3 Applicability
The limit of detection for chis methoa is usually dependent
upon the level of interferences rather than instrumental
limitations. Where interferences are not a problem, the
limit of detection for most compounds analyzed by GC/MS is 2
mg/kg (wet weight basis).
This method is recommended for use only by experienced
residue analysts or under the close supervision of such
qualified persons.
4.2.4 Precision and Accuracy
This information is not presently available.
4.2.5 Sample Preparation
The fish must be ground in an area free of volatile organic
compounds. The preferred procedure is to usa a blender and
blend equal amounts of dry ice with the fish.
If the sample is very large, a food processor or meat grinder
is used.
Immediately after homogenizing the fish sample, weigh 1 g
into a screw cap tube lined with aluminum foil. Store in a
freezer until analyzed on GC/MS for volatile organic
compounds.
Add 5 mi of organic-free water already spiked with the sur-
rogate spike to the sample. Replace the cap and shake the
111-44
-------
contents until the solids are dispersed throughout the
water.
Immediately place the tube on the purge-and-trap apparatus
and heat at 55°C for 12 minutes while purging.
4.2.6 Sample Analysis
The volatiles are trapped on a 24" Tenax trap and backflushed
onto the GC column at 180°C for 4 minutes while the column is
held at room temperature (50°C). The GC is then programmed
to 210°C at 8°C/min and held for 11 minutes.
The volatile compounds are identified and quantified by the
MS computer system.
Calculate results as indicated in Subsections K and L.
111-45
-------
5.1 Analysis of Air Samples for Volatile Organics
Analytical Procedure: available
Sample Preparation: available
5.1.1 Reference/Title
Erickson, M. D., C. M. Sparacino, M. K. Alsup, M. T. Giguere,
R. W. Handy, and E. D. Pellizzari, "Preliminary Study on
Toxic Chemicals in Environmental and'Human Samples: Part II.
Protocols for Environmental and Human Sampling and Analysis."
EPA Contract No. 68-01-3849. Research Triangle Institute,
Research Triangle Park, North Carolina. Prepared for U.S.
EPA, Office of Research and Development, Washington, D.-C.
p. 304, (1981).
5.1.2 Method Summary
Recovery of volatile organics from Tenax GC is accomplished
by thermal desorption and purging witn helium into a liquid
nitrogen cooled nickel capillary trap4,5,23 ancj ^nen intro-
ducing the vapors into a high resolution gas chromatographic
glass column where the constitutents are separated from
each other.5>24 Characterization and quantification of the
constituents in the sample are accomplished by mass spec-
trometry either by measuring the intensity of the total :on
current signal or by mass fragmentography,5
5.1.3 Applicability
The linear range for the analysis of volatile organic com-
pounds is a function of the breakthrough volume of each
specific compound trapped on the Tenax GC sampling cartridge
and of the inherent limits of detection of the mass spec-
trometer for each organic compound.5,25 The breakthrough
volume is defined as that point at which 50 percent of a
discrete sample introduced into a sampling cartridge is lost.
Although the identity of a compound is not known during
ambient air sampling (therefore its breakthrough volume is
also unknown), the compound can still be quantified after
GC/MS/COMP identification once the breakthrough volume has
subsequently been established. The breakthrough volumes for
some volatile organics, and verified by a previously de-
scribed technique,3.5 are shown in Table 8.5t23,26 The
linear range for quantisation using glass capillary columns
on a gas chromatograph/mass spectrometer/computer (GC/MS/COMP)
is generally three orders of magnitude.3 Table 9 lists the
overall detection limits for some examples of volatile
organics.25
111-45
-------
TABLE 8. TENAX GC BREAKTHROUGH VOLUMES FOR TARGET COMPOUNDS (LITERS)
Temperature (°C)
Compound BP 50 60 70 80 90 100
Chloroform
Carbon tetrachloride
1,2-Dichloroethane
1,1,1-Trichloroethane
Tetrachl oroethyl ene
Trichl oroethyl ene
Chlorobenzene
61
77
83
75
121
87
132
56
45
71
31
481
120
1989
41
36
55
24
356
89
871
32
28
41
20
261
67
631
24
21
31
16
. 192
51
459
17
17
24
12
141
37
332
13
13
19
9
104
28
241
BP = Boiling Point in °C.
TABLE 9. OVERALL THEORETICAL SENSITIVITY OF HIGH-RESOLUTION GAS
CHROMATOGRAPHY/MASS SPECTROMETRY/COMPUTER ANALYSIS
FOR VOLATILE ORGANIC POLLUTANTS
Estimated Detection Limit3
Compound ng/m^ ppt
Chloroform
Carbon tetrachloride
1,2-Dichloroethane
1,1,1-Trichloroethane
Tet rachl oroethyl ene
Tri chl oroethyl ene
Chlorobenzene
Benzene
200
250
32
66
2.5
10
2.10
100
420
400
8.15
12.45
0.38
1.92
0.47
210
aLimits are calculated, on the basis of the breakthrough
volume for 2.2 g of Tenax GC (at 70°F) capillary column
performance and sensitivity of the mass spectrometer to that
comoound in the mass fraamentoaraohv mode of most intense ion.
-------
5.1.4 Precision and Accuracy
The reproducibility of this method has been determined to
range from ±10 to ±30 percent of the relative standard devia-
tion for different substances when replicate sampling car-
tridges are examined.24,25,26,27 jne inherent analytical
errors are a function of several factors: 1) the ability to
accurately determine the breakthrough volume and its relation
to field sampling conditions for each of the organic compounds
identified; 2) the accurate measurement of the ambient air
volume sampled; 3) the percent recovery of the organic from
the sampling cartridge after a period of storage; 4) the
reproducibility of thermal desorption for a compound from the
sampling cartridge and its introduction into the analytical
system; 5) the accuracy of determining the relative molar
response ratios between the identified substance and the
external standard used for calibrating the analytical system;
6) the reproducibility of transmitting the sample through the
high resolution gas chromatographic column; and 7) the
day-to-day reliability of the MS/COMP system.3-5,23-28
The accuracy of analysis is generally ±30 percent but depends
on the chemical and physical nature of the compound.3>5
5.1.5 Sampling Equipment
a) Personnel Monitor Pump
A personnel monitor pump (MSA Co. - Model C-2QO} is ifsed
for sample collection. Flow rates are adjusted to 0.05
1/min for an 8-hour collection period. Flows are ad-
justed such that a total volume of 0.024 m3 air is
sampled for a given collection period.
b) Sampling Cartridges
The sampling cartridges are prepared by packing a 10 cm x
1.5 cm I.D. glass tube containing 8 cm of 35/60 mesh Tenax
GC with glass wool in the ends to provide support.5*28
Sampling cartridges with longer Tenax flow paths can be
used to achieve larger breakthrough volumes.25 Virgin
Tenax (or material to be recycled) is extracted in a
Soxhlet apparatus for a minimum of 18 hours each with
methanol and n-pentane prior to preparation of sampling
cartridges.5*" After purification of the Tenax GC
sorbent and drying in a vacuum oven at 120°C for 3 to 5
hours at 28 inches of water, the Tenax is sieved to
provide a 35/60 particle size range. The sieving and all
further sampling cartridge preparation steps should be
conauctea .r. j ''^earr' ~oom, Camping cartridges are
then oreoared and conditioned at 270°C with helium flow
111-48
-------
at 30 ml/min for 30 minutes. The conditioned cartridges
are transferred to Kimax (2.5 cm x 150 cm) culture
tubes, immediately sealed using Teflon-lined caps, and
cooled. This procedure is performed in order to avoid
recontamination of the sorbent bed.5
5.1.6 Sample Analysis
The instrumental conditions for the analysis of volatile
organics on the sorbent Tenax GC sampling cartridge are shown
in Table 10. The thermal desorption chamber and the six port
Valco valve are maintained at 270° and 240°, respectively.
The jet separator is maintained at 240°. The mass spectrom-
eter is set to scan the mass range from approximately 20 to
350. The helium purge gas through the desorption chamber is
adjusted to 15-20 ml/min. The nickel capillary trap on the
inlet manifold is cooled with liquid nitrogen. In a typical
thermal desorption cycle, a sampling cartridge is placed in
the preheated desorption chamber and the helium gas is chan-
neled through the cartridge to purge the vapors into the
liquid nitrogen capillary trap (the inert activity efficiency
of the trap has been shown in a previous study).24,27 After the
desorption has been completed, the six-port valve is rotated
and the temperature at the capillary loop is rapidly raised
(greater than 10°/min); the carrier gas cnen sweeps the vapors
onto the high resolution GC column. The glass capillary
column is temperature programmed from ambient to 240°C at
4°C/min and held at che upper limit r'or a minimum of 10 min-
utes. After all the components have eluted, the column is
cooled to ambient temperature and the next sample is pro-
cessed.5
The standard can be added as an internal standard during
sampling. Since, however, the volume of air taken to produce
a given sample is accurately known, it is also possible and
more practical to use an external standard where the standard
is introduced into the cartridge prior to its analysis. Two
standards, hexafluorobenzene and octafluorotoluene, are used
for the purpose of calculating RMR's (Relative Molar Re-
sponses). It has been determined that the retention times
for these two compounds are such that they elute from the
glass capillary column (SE-30) at a temperature and retention
time which does not interfere with the analysis of unknown
compounds in ambient air samples.
Since the volume .of air taken to produce a given sample is
accurately known and an external standard is added to the
sample, then the weight per cartridge and hence the concen-
tration of the jnxnown can je Jeterminea. The approach for
quantitating ambient air pollutants requires that the RMR be
determined for each constituent of interest during the
analysis of field samples. Every sixth cartridge is a
111-49
-------
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1.1 The characteristic ions for each compound of interest must have
their maxima in the same or within one scan of each other.
1.2 The retention time must fall within ±30 seconds of the retention
time of the authentic compound.
1.3 The relative peak heights of the three characteristic ions in the
EICP's must fall within ±20 percent 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.
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 percent of the sum of the two peak
heights. Otherwise, structural isomers are identified as isomeric pairs.
L. CALCULATIONS
1. When a compound has been identified, the quantitation of that parameter
should be based on the Intsgratsd abundance from the E!C? 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.
2. 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
Subsection H.3.2. In order to eliminate the need to obtain complete
calibration curves for each compound for whicn quantitative information
is desired, use the method of relative molar response (RMR) fac-
tors. 26t27,28 Successful use of this method requires information on
the exact amount of standard added and the relationship of RMR (unknown)
to the RMR (standards). The method of calculations is as follows:
(1)
Astd/Molesstd
A » system response, height or area determined by
integration or triangulation.
3. The value of RMR is determined from at least six inaepenaent ana-
lyses. -^ Linearity over the dynamic range and an •'ntercept of z
has been previously described.^
-------
, %
(2) RMRunk/std =
A = system response, as above
g = number of grams present
GMW * gram molecular weight .
, % Aunk'GMWunk-9 std
(3) Sunk '
Astd.6MWstd.RMRunk/std
4. If the internal standard calibration procedure is used, calculate the
concentration in the sample using the response factor (RF) determined in
Subsection H.4.3 and Equation 2.
Concentration yg/l = (^Cis)/(Ais)(RF) Eq. 2
where:
As = Area of the characteristic ion for the parameter or
surrogate standard to be measured
ATS = Area °^ the characteristic ion for the internal standard
C-jS * Concentration of the internal standard.
5. If a sample is analyzed, and the ratio of the internal standard to sur-
rogate standard is more than 20 percent higher than that observed for the
calibration solution, these results indicate that the internal standard
in question was present as a sample component prior to spiking. When
this condition is observed, then an alternate calculation is employed in
which the response factors (RF) are determined based on the surrogate
standards rather than the internal standards normally used. Calculations
are carried out using Eq. 2 with the exception that the concentration and
area response of the surrogate standard are substituted for the internal
standard.
6. Report results in micrograms per liter. The results for cis- and
trans-l,3-dichloropropene should be reported as total 1,3-dichloropropene
(Storet No. 34561, CAS No. 542-75-6). When duplicate and spiked samples
are analyzed, report all data obtained with sample results.
7. If any of the surrogate standard recoveries fall outside the control
limits which were established as directed -'n Subsection G.3, data for all
parameters in that sample must be labeled as suspect.'
-------
REFERENCES
1. Analytical Sciences Division. "Master Scheme for the Analysis of Organic
Compounds in Water. Interim Protocols." Chemistry and Life Sciences
Group, Research Triangle Institute. Research Triangle Park, North
Carolina. Prepared for Environmental Research Laboratory, U.S.
Environmental Protection Agency, Athens, Georgia. 1980.
2. 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, Pennsylvania. Special Technical Publication 686. 1978.
3. Erickson, M. D, C. M. Sparacino, M. K. Alsup, M. T. Giguere, R. W. Handy
and E. D. Pellizzari. "Preliminary Study on Toxic Chemicals in Environ-
mental and Human Samples. Part II. Protocols for Environmental and
Human Sampling and Analysis." EPA Contract No. 68-01-3849. Research
Triangle Institute, Research Triangle Park, North Carolina. Prepared for
U.S. Environmental Protection Agency, Office of Research and Development,
Washington, D. C. p. 304, 1981.
4. Pellizzari, E. D. Development of Method for Carcinogenic Vapor Analysis
in Ambient Atmospheres. Publication No. EPA-650/2-74-121, Contract No.
68-02-1228, p. 148, July, 1974.
5. Pellizzari, E. 0. Development of Analytical Techniques for Measuring
Ambient Atmospheric Carcinogenic Vapors. Puoiication No. EPA-600/
2-75-075, Contract No. 68-02-1228, p. 187, November, 1975.
6. "Preservation and Maximum Holding Time for the Priority Pollutants."
U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Cincinnati, Ohio 45268. In preparation.
7. Rose, M. E. and B. N. Colby. "Reduction in Sample Foaming and Purge and
Trap Gas Chromatography/Mass Spectrometry Analysis." Anal. Chem. 51:
2176-2180. 1979.
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 22-6. (Revised,
January, 1976).
10. "Safetv in Academic Chemistry Laboratories." American Chemical Society
Publication, Committee on Chemicai .Safety, 3rd edition. 197S.
-------
11. Budde, W. L., and J. W. Eichelberger. "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,
Ohio. 45268. p. 16. April 1980.
12. Bellar, T. A., and J. J. Lichtenberg. "Determining Volatile Organics at
Micrograms per Liter Levels by Gas Chromatography." Journal American Water
Works Association. £6_ pp. 739-744. 1974.
13. U.S. Environmental Protection Agency. "Handbook of Analytical Quality
Control in Water and Wastewater Laboratories." EPA 600/4-79-019,
U.S. Environmental Monitoring and Support Laboratory, Cincinnati, Ohio.
45268. March 1979.
14. Eichelberger, J. W., L. E. Harris, and W. L. Budde. "Reference Compound
to Calibrate Ion Abundance Measurement in Gas Chromatography - Mass
Spectrometry Systems." Analytical Chemistry, £7, 995-1000. 1979.
15. U.S. Environmental Protection Agency. "Method for Preparation of Medium
Concentration Hazardous Waste Samples." U.S. Environmental Protection
Agency, Region IV, Athens, Georgia, p. 7. May, 1981.
16. Battelle Laboratories. "Manual of Collaborators on Evaluation of Methods
for Analysis of Hazardous Wastes." Prepared under EPA Contract
68-03-3098. Battelle Columbus Laboratories, Columbus, Ohio. 1981.
17. Ligon, Jr., W. V., ana ri. Grace. '?oiy\ethylene glycol) as a Diluent
for Preparation of Standards for Volatile-Drganics in Water." Analytical
Chemistry, 53:920-921. 1981.
18. U.S. Environmental Protection Agency. "Purgeables - Method 624."
Federal Register. 44 No. 233:69532-69539. December 3,,, 1979.
19. Kleopfer, R. D. "Priority Pollutant Methodology Quality Assurance
Review." U.S. Environmental Protection Agency, Region VII, Kansas City,
Kansas. Seminar for Analytical Methods for Priority Pollutants, Norfolk,
Virginia. January 17-18, 1980. U.S. Environmental Protection Agency,
Office of Water Programs, Effluent Guidelines Division, Washington, D. C.
20460.
20. U.S. Environmental Protection Agency. "Analytically Determined Method
Detection Limits for Priority Pollutant Methodology as Method Performance
Criteria." U.S. Environmental Protection Agency, Environmental Monitoring
and Support Laboratory, Cincinnati, Ohio. In preparation.
21. Hiatt, M. "Analysis of Fish and Sediment for Volatile Priority
Pollutants." Anal. Chem. 53:1541-1543. 1981.
111-54
-------
22. U.S. Environmental Protection Agency. "Extraction and Analysis of
Priority Pollutants in Biological Tissue." Method PPB 10/80. U.S.
Environmental Protection Agency, SAA Division, Region IV, Laboratory
Services Branch, Athens, Georgia, p. 7.
23. Pellizzari, E. D., J. E. Bunch, B. H. Carpenter and E. Sawicki.
"Collection and Analysis of Trace Organic Vapor Pollutants in Ambient
Atmospheres. Technique for Evaluating Concentration of Vapors by Sorgent
Media." Environ. Sci. Technol. 9: pp. 552-555. 1975.
24. Pellizzari, E. D. The Measurement of Carcinogenic Vapors in Ambient
Atmospheres. Publication No. EPA-600-7-77-055, Contract No. 68-02-1228.
p. 288. June, 1977.
25. Pellizzari, E. D. "Analysis of Organic Air Pollutants by Gas
Chromatography and Mass Spectroscopy." EPA-600/2-77-100. p. 114.
26. Pellizzari, E. D. "Analysis of Organic Air Pollutants by Gas
Chromatography and Mass Spectroscopy." EPA-600/2-79-057. p. 243.
27. Pellizzari, E. D. "Ambient Air Carcinogenic Vapors Improved Sampling and
Analytical Techniques and Field Studies." EPA-600/2-79-0081. p. 340.
May, 1979.
28. Pellizzari, E. D., J. E. Bunch, R. E. Berkley and J. McRae.
"Determination of Trace Hazardous Organic Vapor Pollutants in Ambient
Atmospheres by Gas Chromatography/Mass Spectrometry/Computer." Anal.
Chem. 48:803-807. 1976.
-------
SECTION 2
ACID-EXTRACTABLE ORGANIC COMPOUNDS
A. SCOPE
Acid-extractable organic compounds can be measured using the analytical
procedures presented in Subsection J. These compounds are extracted from the
initial sample matrix under acidic conditions and identified/quantified using
GC/MS procedures.
B. SAMPLE HANDLING AND STORAGE
Conventional water sampling practices should be followed except that the
bottle should not be prerinsed with sample oefore cai'ec'ion. Grsb samoles
should be collected in glass containers and refrigerated immediately. Com-
posite samples should preferably be collected in glass containers and, if
possible, refrigerated during the period of compositing. ATI automatic
sampling equipment must be free of Tygon and other potential sources of
organic contamination. When necessary, *he equipment :hould be prerinsed with
hexane prior to use.
Water samples should be iced or refrigerated from the,time of collection
until extraction.! Chemical preservatives should not be used in the field
unless more than 24 hours will elapse before sample delivery to the lab-
oratory. If the samples cannot be extracted within 48 hours of collection,
the recommended method of sample preservation is as follows:
1. If the sample contains residual chlorine, add 35 mg of sodium thiosulfate
per 1 ppm of free chlorine per liter of sample.1
2. Adjust the pH of the water sample to pH of 7 to 10 using sodium hydroxide
or sulfuric add. Record the volume of acid or base used.1
All water samples must be extracted within 7 days and completely analyzed
within 30 days of collection (Figure 1).
Sediment and soil samples may be stored field-moist (refrigerated)
frozen, or dried. The effective storage period is not known.
111-56
-------
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Hastes
1 Analyze 1 I Analyze
P. rpwe Total Total "Dissolved" Total Cone. Total Cone. Total Cone. Total Cone. Total
Cone. Cone. Cone. In Nobility Mobility In Soil. In Soil. In Soil. In Biological Cone. I
In Art In Mater Hater at pH S at p»l S Sediment Sediment Sediment Tissue Waste
Container 666 C
S«*ple
Preser-
vative 4*C 4»C 4"C 4"C 4'c
Storage '
Tlae 7d 7d 7d
Staple
Si'e 11 11 11 Ju 9 - 50 9 |0 g la
Mgure 1. Sample handling and storage Information for samples scheduled for acid-extractable analysis.
-------
C. INTERFERENCES
Solvents, reagents, glassware, and other sample processing hardware may
yield discrete artifacts and/or elevated baselines causing misinterpretation
of chromatograms. All of these materials must be demonstrated to be free from
interferences under the conditions of the analysis by running method blanks.
Specific selection of reagents and purification of solvents by distillation in
all-glass systems may be required.
Analytical interferences associated with samples will vary considerably
from source to source, depending on the diversity of the industrial complex,
municipality, or system being sampled. Matrix interferences may be caused by
components that are coextracted from the sample but are not normally of inter-
est. The most common such components are petroleum-derived naphthenes, high-
molecular-weight polymeric components, and.long-chain components such as waxes
and triglycerides. The extent of such matrix interferences will vary consid-
erably from source to source. A cleanup procedure using gel permeation
chromatography has been incorporated into the method for certain cases to
remove long-chain and high-molecular-weight material. No cleanup procedure is
available for the removal of naphthenes. When such matrix interferences are
present, the sample extract is diluted arid the method detection limit is
increased proportionately. Many of the matrix interferences are solvent-
sxtractabla nonvolatile components whicn necessitate the -ncre frequent clean-
ing of the GC injection port and the more frequent removal of the injection
end of the GC capillary column.
Interferences due to fish oil in tissue samples can be eliminated by
acetonitrile partitioning.2 The ultrasonic probe used to assist in the
partitioning process must be scrupulously cleaned between samples. The pro-
cedure consists of rinsing the probe with solvent (acetonitrile) into the
sample, removing residue on the probe with a wet tissue, rinsing the probe
with methylene chloride, and sonicating in hexane for 3 to 4 minutes on 50
percent pulse.
D. 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 chem-
icals must be minimized 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. All operations Involving
the use of methylene chloride, including the extraction of the waste sample,
filtration of the extract, and concentration of the extract, must be performed
in a fume hood. Care should be taken to avoid skin contact with methylene
chloride.
i 1 4- ,/
-------
E. APPARATUS
1. Oven, drying.
2. Desiccator.
3. Crucibles, porcelain, squat form, Size 2 or equivalent.
4. Sonicator Cell Disruptor - Heat Systems - Ultrasonics, Inc., with a 3/4 in.
high gain probe, 375 watt or equivalent.
5. Beakers, 400-ml.
6. Separatory funnels - 250 ml, 500 ml, and 2 1 with Teflon stopcocks.
7. Drying tubes - 180 mm x 25 mm.
8. Glass wool, Pyrex.
9. Furnace, muffle.
10. Buchner funnels.
11. Filtrator - Fisher-9-789 or equivalent.
12. Drying column - Twenty (20) mm I.D. pyrex chromatographic column equipped
with coarse glass frit or glass wool plug.
13. Kuderna-Danish (K-D) apparatus.
13.1 Concentrator tube - Ten (10) ml, graduated (Kontes K-570050-1025, or
equivalent). Calibration must be checked. Ground glass stopper
(size 19/22 joint) is used to prevent evaporation of extracts.
13.2 Evaporative flask - Five hundred (500) ml (Kontes K-57001-0500, or
equivalent). Attach to concentrator tube with springs (Kontes
K-662750-0012).
13.3 Snyder column - Three-ball macro (Kontes K-503000-0232, or
equivalent).
13.4 Snyder column - Two-ball micro (Kontes K-569002-0219, or
equivalent).
13.5 Boiling chips - Extracted, approximately 10/40 mesh.
14. Water bath - Heated, with concentric ring cover, capable of temperature
control (±2°n.
15. Pins, ipproximately 35 cm x ?5 cm-x 6 cm.
111-59
-------
16. Gas chromatograph - Analytical system complete with chromatographic
column suitable for on-column injection and all required accessories
including flame ionization detector and electron capture detector, column
supplies, recorders, gases, and syringes.
17. Acid column and analytical conditions - Supelcoport (100/120) coated with
1% SP-1240 DA, packed in a 10 cm x 2 mm I.D. pyrex glass column. Use ultra
pure nitrogen carrier gas at a flow rate of-30 ml/min. Column tempera-
ture held at 80°C for 2 minutes programmed to 290°C at 8°C/min., and held
at 290°C for 16 minutes.
18. Gas chromatograph/mass spectrometer, Finnigan 4000 and ..INCOS 2300 Com-
puter Data System - Capable of scanning from 35 to 450 a.m.u. every 7
seconds or less at 70 V (nominal) and producing a recognizable mass
spectrum at unit resolution from 50 ng of DFTPP when the sample is intro-
duced through the GC inlet. The mass spectrometer must be interfaced
with a gas chromatograph equipped with an injector system designed for
splitless. injection and glass capillary columns or an injector system
designed for on-column injection with all-glass packed columns. All
sections of the transfer lines must be glass or glass-^ined and must be
deactivated (use Sylon-CT, Supelco, Inc., or equivalent, to deactivate).
NOTE: Systems utilizing a jet separator for the GC effluent are recom-
mended since membrane separator: may lose 'ansitivity *or light molecules
and glass frit separators may inhibit the. elution of polynuclear aro-
matics. Any of these separators may be used provided that it gives
recognizable mass spectra and scceotable calibration ooints at the limit
of detection specified for each individual compound.
A computer system must be interfaced to the mass spectrometer to allow
acquisition of continuous mass scans for the duration of the chromato-
graphic program. The computer system should also be equipped with mass
storage devices for saving all data from GC/MS runs. There must be
computer software available to allow searching any GC/MS run for specific
ions and plotting the intensity of the ions with respect to time or scan
number. The ability to integrate the area under any specific ion plot
peak is essential for quantification.
19. Extracting equipment.
19.1 ADT tissumizer (Tekmar SDT 182EN or equivalent).
19.2 Centrifuge (IEC CU-5000 or equivalent).
19.3 Screw-capped centrifuge bottles, 200 ml (Scientific Products C4144)
with TFE-lined screw caps.
19.* Peakers 'or beaked - 200 nil or eouivalent.
19.5 Glass jyringe - 53 il squipped with i ISO Tit x "5 mm T.D. TFE tube.
111-60
-------
20. Gel permeation chromatography cleanup.
20.1 Chromatography column - 500 mm x 19 mm I.D. (Scientific Products
C-4670-106 or equivalent).
20.2 Bio-Beads S-X3, 200/400 mesh (Bio-Rad Laboratories 152-2750).
20.3 Glass wool.
20.4 Graduated cylinders - 100 ml.
20.5 GPC Autoprep or equivalent (Analytical Biochemistry Labs, Inc. 1002
or equivalent with 25-mrn I.D. column containing 50 to 60 g of Bio-
Beads S-X3) (Optional).
21. Continuous Liquid-Liquid Extractors - Teflon- or glass-connecting joints
and stopcocks, no lubrication. (Hershberg-Wolf Extractor - Ace Glass
Co., Vineland, New Jersey. P/N 6841-10, or equivalent).
F. REAGENTS
1. Sodium sulfate, granular anhydrous and reagent grade. Rinse with
methyiene cnioriae (20 mi/g) dna condition at 500°C for a minimum of 2
hours. Cool in a desiccator and store in a glass bottle.
2. Hexane - Pesticide quality, distilled in glass.
o. rtcetone - Pesticide quality, distilled in glass. .
4. Methyiene chloride - Pesticide quality, distilled in glass.
5. Petroleum ether - Pesticide quality, distilled .in glass.
6. Diethyl ether - Preserved with 2% ethanol, pesticide quality and dis-
tilled in glass. The ether must be free of peroxides as indicated by EM
Quant Test Strips (EM Laboratories, Inc., 500 Executive Blvd., Elmsford,
New York. 10523).
7. Florisil - PR grade (60/100 mesh).
8. Methanol - Pesticide quality, distilled in glass.
9. Sodium hydroxide solution, 50%, extracted 3 times with methyiene
chloride.
10. Sodium hydroxide (ACS),-ION in distilled water.
11. Sodium hydroxide (ACS), 6N in distilled water.
12. Sulfuric acid (ACS), 6N in distilled water.
13. Hydrochloric acid (ACS), concentrated, 12N.
111-61
-------
14. Stock Standards - Prepare stock standards from EPA Priority "Pollutants
Kits containing the preanalyzed compounds or from alternative commercial
sources. Dissolve 0.100 g of assayed reference material in pesticide-
quality isooctane or other appropriate solvent and dilute to volume in a
100-ml ground glass stoppered volumetric flask. The concentration of
this solution is 1.00 yg/ul. Transfer the stock solution to a 15-ml
Teflon-lined screw cap vial, store in a refrigerator, and check fre-
quently for signs of degradation or evaporation, especially just prior to
preparing working standards.
15. Calibration Standards - Prepare calibration standards that contain the
compounds of interest, either singly or mixed together. The standards
should be prepared at concentrations that will completely bracket the
working range of the chromatographic system (two or more orders of magni-
tude are suggested). For example, if the limit of detection can be cal-
culated as 20 ng injected, prepare standards at 10 ug/ml, 100 yg/ml,
1,000 wg/ml, etc. so that injections of 1 to 5 yl of the calibration
standards will define the linearity of the detector in the working range.
16. Surrogate Standards Addition - Surrogate standards should be stable
isotope derivatives of compounds that will aid in the monitoring of the
analysis procedures. "Tie number snd composition of surrogate standards
will, to some extent, be dependent on compound avaiiaoility. Where
possible the stable isotope-labeled compounds (which are mass resolved
from the unlabeled compound) will be utilized to simplify calculations in
the measurement of recovery. The surrogate standards should be added to
the chilled ?olid sample via injection of an appropriate volume of the
surrogate standard mix. [The sample sh'ould be laced with a quantity of
surrogate standard which is equivalent to 5 ppm (e.g., 250 yg/50 g
sample)]. The surrogate spike should be added to the solid samp^ in
aliquots of the total volume to be added (e.g., five 20-ul injections
from a 100-yl syringe) while manually stirring the material with a clean
glass stirring rod to facilitate dispersion of surrogate compounds. It
is important that the sample and sample container are chilled during the
surrogate standard addition. Surrogate standard addition should be
accomplished as quickly as possible to minimize the loss of the most
volatile analytes covered by this method. Upon surrogate standard
addition, the isolation/cleanup procedure can be initiated.
It is recognized that uniform distribution of surrogate standards to a
solid phase sample will not be obtained by this procedure. Also, it must
be recognized that the surrogates will not mirror the properties of organic
compounds which have been associated with soil or sediment samples for
decades. Nevertheless, data generated from surrogate standards will
serve to exemplify a "best" case recovery for the sample in question, and
will provide QA/QC data for every sample.
G. QUALITY CONTROL
Before processing any samples, demonstrate through the analysis of a
method blank that all glassware and reagents are interference-free. Eacn time
a set of samples is extracted or there is a change in reagents, a method blank
111-62.
-------
should be processed as a safeguard against chronic laboratory contamination.
Field replicates should be collected and analyzed to determine the precision
of the sampling technique. Laboratory replicates should be analyzed to deter-
mine the precision of the analysis. Fortified samples should be analyzed to
determine the accuracy (recovery) of the analysis. Field blanks should be
analyzed to check for contamination introduced during sampling and transpor-
tation.
H. CALIBRATION
Prepare calibration standards that contain the compounds of interest,
either singly or mixed together. The standards should be prepared at concen-
trations that will bracket the working range of the chromatographic system
(two or more orders of magnitude are suggested). Assemble the necessary gas
chromatographic apparatus and establish operating parameters equivalent to
those required. By injecting calibration standards, establish the linear
range of the analytical system and demonstrate that the analytical system
meets the detection limits requirements specified in Table 1. Characteristic
ions for compounds in the acid-extractable fraction are summarized in Table 2.
If the sample gives peak areas above the working range, dilute and reanalyze.
Internal standard method - The internal standard approach is acceptable
for all of the semivolatile organics. The uti^'zatfon of the internal
standard method requires the periodic determination of response factors (RF)
that are defined in Equation H-l.
RF = (AsCis)/(AisCs) Eq. H-l
where:
As = the integrated area or peak height of the characteristic
ion for the analyte standard
ATS = the integrated-area or peak height of the characteristic
ion for the internal standard
Cis * the amount (pg) of the internal standard
Cs = the amount (pg) of the analyte standard.
The relative response ratio for the analytes should be known for at least
two concentration values - 20 ng injected to approximate 10 pg/1 and 200 ng
injected to approximate the 100-pg/l level (assuming 1 ml final volume and a
2-pl injection). Those compounds that do not respond at either of these
levels may be run at concentrations appropriate to their response.
The response factor (RF} should be determined over all concentration
ranges for the standards (Cs) that are being determined. [Generally, the
amount of •'nternal standard idded *o <»ach extric*. *s the *ame '20 -jg)
-------
TABLE 1. GAS CHROMATOGRAPHY OF ACID-EXTRACTABLE COMPOUNDS
Storet Retention time(a)
Compound No. (minutes) Limit of detection^)
2-Chlorophenol
2-Nitrophenol
Phenol
2, 4-Dimethyl phenol
2,4-Dichlorophenol
2,4,6-Trichlorophenol
4-Chloro-3-methyl-
phenol
2,4-Oinitrophenol
2-Methyl-4,6-dinitro-
phenol
Pentachlorophenol
4-Nitrophenol
34586
34591
34694
34606
34601
34621
34452
34616
34657
29094
34646
==================
5.9
6.4
8.0
9.4
9.8
11.8
13.2
1-.5
-.5.:
17-5
20.3
=====
50
50
50
50
50
50
50
500
500 •
C~C
50
==================
25
25
25
25
25
25
25
250
250
25
25
(a)l.8 m glass column (6.4 mm O.D. x 2 mm I.D.) packed with 1 percent SP-1240
DA coated on 100/120 mesh Supelcoport. Carrier gas: helium at 30 ml per
min. Temperature program: 2 min isothermal at 70°, then 8° per min to
200°C. If desired, capillary or SCOT columns may be used.
(b)This is a minimum level at which the entire analytical system must give
mass spectral confirmation. (Nanograms injected is based on a 2-yl
injection of a 1-liter sample that has been extracted and concentrated
to a volume of 1.0 ml.)
Once this calibration curve has been determined, it should be verified daily
by injecting at least one standard solution containing internal standard. If
significant drift has occurred, a new calibration curve must be constructed.
To quantify, add the internal standard *:o *>e ~oncantratad samole extract, no
more than a few minutes before injecting into the GC/MS system to minimize the
possibility of losses due to evaporation, adsorption, or chemical reaction.
Calculate the concentration by using the previous equation using the
II1-64
-------
TABLE 2. CHARACTERISTIC IONS OF ACID-EXTRACTABLE COMPOUNDS
Characteristic ions
Compound
Electron Impact
Chemical lonization
(methane)
2-Chl orophenol
2-Nitrophenol
Phenol
2, 4-Dimethyl phenol
2, 4-Di chl orophenol
2,4 ,6-Tri chl orophenol
4-Chloro-3-methyl phenol
2,4-Dinitrophenol
2-Methyl-4,6-dinitrophenol
Pentachl orophenol
4-Nitrophenol
Phenanthrene (d-l°)(a)
========= ==================
128
139
94
122
162
196
142
184
198
266
65
188
========
64
65
65
107
164
198
107
63
182
264
139
94
130
109
66
121
98
200
144
154
77
268
109
80
129
140
95
123
163
197
143
185
199
267
140
189
:==========
131
168
123
151
165
199
171
213
227
265
168
217
157
122
135
163
167
201
183
225
239
269
122
==============
(a) Suggested internal standard.
appropriate response factor taken from the calibration curve. Either deute-
rated or fluorinated compounds can be used as internal standards and surrogate
standards. Phenol-de, pentafluorophenol, 2-perfluoromethyl phenol, and
2-fluorophenol have been suggested as appropriate internal standards or
surrogates for the acid compounds. The compounds used as internal standards
must be different from the surrogate standards.
I. DAILY INSTRUMENT CALIBRATION
At the beginning of each day, the mass calibration of the GC/MS system
must be checked and adjusted,.if necessary, to meet specifications, using
decafluorotriphenylphosphine (DFTPP). Each day acid-extractable compounds are
measured, the column performance specification with pentachlorophenol must be
met. DFTPP can be mixed in solution with the pentachlorophenol to complete two
specifications with one injection, if desired.
To perform the mass calibration test of the GC/MS system, the following
Instrumental parameters are required:
Electron energy - 70 V (nominal)
Mass range - 35 to 450 amu
Scan time - 7 seconds or less
Source temperature - ^3G-OOQ0C
II!-65
-------
GC/MS system calibration - Evaluate the system performance each day that it is
to be used for the analysis of samples or blanks by examining the mass spec-
trum of DFTPP. Inject a solution containing 50 ng of DFTPP and check to
ensure that performance criteria listed in Table 3 are met. If the system
performance criteria are not met, the analyst must retune the spectrometer and
repeat the performance check. The performance criteria must be met before any
samples or standards may be analyzed.
Column performance is evaluated by injecting 50 ng of pentachlorophenol
into the instrument. The tailing factor for the resultant peak, as calculated
in Figure 2, must be less than five for the performance to be considered
acceptable.
TABLE 3. DFTPP KEY IONS AND ION ABUNDANCE CRITERIA
==============================================================
Mass Ion Abundance Criteria
51 30 to 60 percent of mass 198
68 Less than 2 percent of mass 69
70 Less than 2 percent of mass 69
127 40 to 60 percent of mass 198
197 Less than 1 percent of mass 198
198 Base peak, 100 percent relative abundance
199 5 to 9 percent of mass 198
275 10 to 30 percent of mass 198
365 Greater than 1 percent of mass 198
441 Present but less than mass 443
442 Greater than 40 percent of mass 198
443 17 to 23 percent of mass 442
======3======================================================================
111-66
-------
Tailing Factor =
BC
AB
Example Calculation: Peak Height = OE = 100 mm
10% Peak Height = BD = 10 mm
Peak Width at 10% Peak Height = AC = 23 mm
AB =11 mm
BC - 12 mm
Therefore: Tailing Factor = — = 1.1
Figure 2. Tailing factor calculation.
111-67
-------
J. ANALYTICAL PROCEDURES
1.1 Analysis of Hazardous Waste Samples for Acid-Extractable Organic
Compounds.
Analytical Procedure: available
Sample Preparation: available
1.1.1 Reference
U.S. Environmental Protection Agency, "Method for Preparation
of Medium Concentration Hazardous Waste .Samples." U.S. EPA,
Region IV, Athens, Georgia, May, 1981.
1.1.2 Method Summary
Approximately one-gram aliquots of soil, solid, aqueous
liquid, or non-aqueous liquid are transferred to vials inside
a chemical carcinogen glove box. The acidified samples are
then extracted with methylene chloride. The methylene chlor-
ide extract is screened by GC/FIQ and, based on the screening
results, the sample extracts are appropriately concentrated
and analyzed with a GC/MS system.
1.1.3 Applicability
This procedure is designed for the safe handling and prepar-
ation of potentially hazardous samples from hazardous waste
sites for analysis for organic acid-extractable compounds.
The method is directed to contaminated soil samples and waste
samples that may be solid, aqueous liquid, or non-aqueous
liquid and suspected to contain less than 10% of any single
organic chemical component. The method is not designed for
use with samples expected to contain less than 10 ppm of
specific acid-extractable compounds. This type of sample,
such as sediment samples taken from leachate streams, should
be analyzed using a method for sediment/soil samples (Sub-
section J.3.1 or J.3.2).
1.1.4 Precision and Accuracy
These extraction and preparation procedures were developed
for rapid and safe handling of hazardous waste samples. The
design of the method thus did not stress efficient recoveries
of all components. Rather, the procedure was designed for
moderate recovery of a broad spectrum of organic acids. The
results of the analyses thus may sometimes reflect only the
minimum amount of the constituent present in the sample.
The procedure is designed to allow detection limits as low as
10 pom for orgamc acld-extractaoie compounds. Some samples,
however, iiay contain high concentrations of chemicals that
interfere with the analysis of other components at low levels.
TII-68
-------
The detection limits in those cases may be significantly
higher. Percent recovery and standard deviation information
on the use of this method in a single laboratory is presented
in Table 4.
TABLE 4. RECOVERY DATA FROM SOIL BY REGION IV MEDIUM CONCENTRATION
HAZARDOUS WASTE METHOD3
r================:
Acid Fraction Cone, yg/gm Avg. % Rec. Std. Dev.*
2-Chlorophenol
2-Nitrophenol
Phenol
2 ,4-Dimethyl phenol
2,4-Dichlorophenol
2,4,6-Trichlorophenol
4-Chloro-m-cresol
2,4-Dinitrophenol
4,6-Dinitro-2-cresol
Pentachlorophenol
4-Nitropnenol
20
20
20
20
20
20
20
160
40
40
130 .
89
87
85
89
92
89
89
52
87
88
31
3.4
4.1
5.0
4.1
4.9
5.2
5.0
3.3
3.4
2.8
5.0
:=========================
* - ± one standard deviation based on three trials.
1.1.5 Sample Preparation
Place the sample container into the glovebox. Additional
items that should be in the glovebox include (1) calibrated
and tared 20-ml vials with caps, (2) a spatula, (3) a
balance, (4) a capped vial containing 10 ml of interference-
free methanol, (5) a vial of water, and (6) a medicine
dropper. The vial of methanol is to be used as a method
blank (One method blank should be run for each batch of 20
samples or less). Open the sample transportation can and
remove the sample vial. Note and record the physical state
and appearance of the sample. If the sample bottle is
broken, immediately repackage the sample and terminate the
analysis.
Open the sample vial and mix the sample. If the sample is i
liquid, transfer one drop to a vial containing water to
determine whether the sample is aqueous or non-aqueous.
Record the results based on the degree of mixing of the
sample with the *ater.
111-69
-------
Transfer approximately 1 gram (or 1 ml) of the sample to a
calibrated and tared 20-ml vial. Wipe the mouth of the vial
with tissue to remove any excess sample material. Cap the
vial. Record the exact weight of sample taken.. Reseal the
original sample and replace it in the original packaging.
Proceed with a methylene chloride extraction of the organic
acid compounds in the sample based on the miscibility of the
original sample with water. Follow paragraph (a) for an
aqueous sample, paragraph (b) for a non-aqueous sample, and
paragraph (c) for a solid phase sample.
a. If the sample has been classified as aqueous, dilute the
sample with 10 ml of methylene chloride. Cap the vial and
shake the sample for 2 minutes. Add 2 grams of anhydrous
sodium sulfate to the vial to absorb the water. Shake the
sample.
b. If the sample has been classified as non-aqueous, dilute
the sample to a final volume of 10 ml with methylene
chloride. Cap the vial and mix for 2 -inutes. Add 1
gram of anhydrous sodium sulfate to absorb any water that
may be present. Shake the sample.
c. If the sample has been classified as a solid, add 10 ml of
methylene chloride. Cap the sample and shake for 1 hour
on a wrist action shaker. Add 1 gram of anhydrous sodium
sulfate to the sample and thoroughly mix.
1.1.6 The extract should be screened by GC/FID using the column
indicated in Subsection E. Prior to use, standardize the
GC/FID detector for full scale response to 40 ng/yl of
dig-phenanthrene.
If the response of any sample component is greater than 25%
of the dio-phenanthrene response, analyze the 10-ml ex-
tracts prepared in paragraph 1.1.5 by GC/MS (paragraph 1.1.7).
If the sample extract does not produce a reponse that is
greater than 25% of the dlO-phenanthrene response, concen-
trate the extract under a gentle stream of nitrogen to a
final volume of 1 ml and analyze by GC/MS (paragraph 1.1.7).
GC/MS Analysis of the Acid Fraction.
1.1.7 At the beginning of each day that analyses are to be per-
formed, inject 50 ng of pentachlorophenol either separately or
as part of a standard mixture that may also contain 50 ng of
OFTPP, *nto the ^nstrument. """he tailing *actor for penta-
chlorophenol, calculated as indicated in Figure 2, should be
less than 5.
f ^ —
-/U
-------
Establish instrument operating conditions equivalent to those
provided below:
Mass Spectrometer
Mass range m/e 41-475
Scan time 7 seconds or less
Electron energy 70 eV
Source temperature 280-300°C
Start acquisition 0.1 min. after stopping flow
Column Conditions
1.8 m glass column (6.4 mm 0.0., 2 mm I.D.) packed with 3%
SP-2250 coated on 100/120 mesh Supelcoport; carrier gas:
helium at 30 ml/min. Temperature program: isothermal for
4 minutes at 50°C, then increasing at 8°/min to 270°C, and
hold at 270°C for 30 minutes. If desired, capillary or
SCOT columns may be used in place of the packed column.
Program the GC/MS to operate in the Extracted Ion Current
Profile (EICP) mode, and colleci EICP's for the three char-
acteristic ions listed in Table 2 for each compound being
quantitated. Operating in this mode, calibrate the system
response for each compound using either the internal or
external standard procedure.
If the internal standard approach is being used, the standard
is not to be added to the sample extract until immediately
before injection into the instrument. Mix the extract thor-
oughly before withdrawing an aliquot for analysis. Inject 2
to 5 yl of the sample extract using the solvent flush tech-
nique.
If external calibration is employed, record the volume of
extract and standard solution injected to the nearest 0.05
pi. If the response for any ion exceeds the linear range of
the system, dilute the extract and reanalyze.
When the extracts are not being used for analysis, they
should be stored in vials capped with unpierced septa, in the
dark, and at 4°C.
Proceed to Subsection K for qualitative identification
criteria and calculation of the results.
111-71
-------
2.1 Analysis of Water Samples for Acid-Extractale Organic Compounds
Analytical Procedure: evaluated
Sample Preparation: available
2.1.1 Reference
U.S. Environmental Protection Agency, "Semi-Volatiles Determina-
tion." Method 625. Federal Register 44 No. 233: 69540-69551.
December 3, 1979.
2.1.2 Method Summary
The pH of a 1-liter water sample is adjusted to 2 or less and
the sample 1s extracted with methylene chloride. Following
concentration of the extract, it is analyzed on a calibrated
GC/MS. Qualitative Identification is performed using the
retention time and the relative abundance of three charac-
teristic ions. Quantitative analysis is performed using
internal standard techniques with a single characteristic ion.
2.1.3 Applicability
This method is applicable to the determination of those
organic acids listed in Table 5 when they occur in aqueous
samples such as municipal and industrial discharges. The
method is designed to be used to meet the monitoring re-
quirements of the National Pollutants Discharge Elimination
System (NPDES). Method detection limits and characteristic
ions for compounds measured by this procedure are summarized
in Tables 2 and 5.
The method should be restricted to use by, or under the
direct supervision of, analysts experienced in the operation
of gas chromatograph/mass spectrometers and skilled in the
interpretation of mass spectra.
2.1.4 Precision and Accuracy
Average recovery data and method detection limits based on
the analysis of spiked reagent water by a single laboratory
are presented in Table 5. Analytical recovery data for
wastewater and reagent water samples are presented in
Table 6.
2.1.5 Sample Extraction
Samples may be extracted using separatory funnel techniques
or with a continuous extractor. Where emulsions prevent the
111-72
-------
TABLE 5. METHOD DETECTION LIMITS FOR ACID-EXTRACTABLE ORGANIC COMPOUNDS
IN REAGENT WATER ANALYZED BY METHOD 6254.5
====================================
Spike
Compounds ug
4-Chl oro-3-methyl phenol a
2, 4-Dichl orophenol3
2, 4-Dimethyl phenol3
2,4-Dinitrophenola
2-Methyl -4 ,6-di ni trophenol a
4-Nitrophenola
Pentachl orophenol3
Phenol3
2, 4, 6-Trichl orophenol3
BHC
BHC
4, 4 '-DDE
Endosulfan sul^ate
======
Level
/I
10
10
10
40
40
10
10
10
10
6
6
10
7
Average %
Recovery
71
60
57
94
77
52
87
28
64
69
56
69
79
MDL
ug/i
3.0
2.7
2.7
42
24
2.4
3.6
1.5
2.7
4.2
3.1
5.6
5.6
aMDL based on 8 aliquots of reagent water
TABLE 6. PRECISION AND ACCURACY DATA FOR THE DETERMINATION
OF ACID-EXTRACTABLE COMPOUNDS6
==========================================================
Parameter
Reagent Water
AverageStandard
Percent Deviation
Recovery* %
Wastewater
Average Sta naa rd
Percent Deviation
Recovery* %
4-Chl oro-3-methyl phenol
2-Chlorophenol
2 , 4-Dichl orophenol
2, 4-Dimethyl phenol
2,4-Dinitrophenol
2-Methyl -4 ,6-di ni trophenol
4-Ni trophenol
2-Ni trophenol
Pentachl orophenol
Phenol
96
80
86
71
89
87
65
95
87
61
16
22
24
19
22
34
33
22
24
11
2,4,6-Trichlorophenol 91 22
*c= ==============================================
99
71
84
72
92
102
59
87
84
54
19
23
23
16
40
23
46
22
22
24
80 24
==============================
*Soikes ranged from 20 to 2,500 ug/liter.
111-73
-------
use of the separatory funnel technique, the continuous
extractor technique is recommended (paragraph 2.1.6).
Mark the water meniscus on the side of a 1-liter sample bottle
for later determination of the volume extracted. Pour the
entire sample into a 2-liter separatory funnel. Adjust the pH
of the sample with hydrochloric acid to a pH of 2 or less.
Thoroughly mix the sample and measure the pH to ensure that
it is 2 or less.
Add 60 ml methylene chloride to the original sample bottle.
Cap the bottle and shake for 30 seconds to rinse the con-
tainer. Transfer the solvent into the separatory funnel and
extract the sample by shaking for 2 minutes with periodic
venting to release excess vapor pressure.
Allow the solvent layer to separate from the water phase for
a minimum of 10 minutes. If the emulsion interface between
layers is more than one-thinj the size of the solvent layer,
mechanical techniques such as stirring, filtration of the
emulsion through glass wool, or cantrifugation should be
attempted. Collect the methylene chloride extract in a
250 ml ^r1 enmeyer -''ask. If the emulsion cannot be broken
or the amount of solvent recovered is less than 80 percent
(after correcting for water soluDility) of that initially
added, the sample, solvent, and emulsion should be trans-
ferred into a continuous extractor to complete the extraction
process I paragraph 2.1.6).
Add a second 60-ml portion of methylene chloride to the
original sample container. Rinse the container and transfer
the solvent to the separatory funnel. Extract the sample for
an additional 2 minutes. Add the solvent layer to the first
extract in the Erlenmeyer flask.
Repeat the extraction process a third time with a final 60-ml
portion of methylene chloride. Combine the extracts in the
Erlenmeyer flask. (The sample can be discarded or retained
for extraction of base/neutral compounds following pH adjust-
ment).
Pour the combined extracts through a drying column containing
7 to 10 cm of anhydrous sodium sulfate, and collect it in a
500-ml K-D flask equipped with a 10 ml concentrator tube.
Rinse the Erlenmeyer flask with 20 to 40 ml of methylene
chloride. Pour the rinse through the drying column and
combine with the sample extract. Proceed to paragraph 2.1.7
2.1.5 Continuous Samole Extraction.
Place 100 to 150 ml of mettry^ene chloride in the extractor
and 200 to 500 ml of methylene chloride in the distilling
-------
flask. Add the aqueous sample (pH 2 or less) to the extrac-
tor. Add distilled water as necessary to operate the
apparatus and extract for 24 hours. Remove the distilling
flask and pour the contents through a drying column contain-
ing 7 to 10 cm of anhydrous sodium sulfate. Collect the
extract in a 500-ml K-D evaporator flask equipped with a
graduated 10-ml concentrator tube.
2.1.7 Sample Extract Concentration
Add 1 or 2 clean boiling chips to the 500-ml K-D flask and
attach a three-ball macro-Snyder column. Prewet the Snyder
column by adding about 1 ml of methylene chloride through the
top of the column. Place the K-D apparatus on a warm water
bath (60 to 65°C) so that the concentrator tube is partially
immersed in the water and the entire lower rounded surface of
the flask is bathed with water vapor. Adjust the vertical
position of the apparatus and the water temperature as required
in order to complete the concentration process in 15 to 20
minutes. At the proper rate of distillation, the balls of the
column actively chatter but the chambers do not flood. When
the liquid has reached an apparent volume of 1 ml, remove the
K-D apparatus and allow the solvent co drain for ^t 1-,aast 10
minutes while cooling. Remove the Snyder column and Hnse
the flask and its lower joint into the-concentrator tube with
1 to 2 ml of methylene chloride. A 5-ml syringe is recom-
mended for this operation.
Add a clean boiling chip and attach a two-ball micro-Snyder
column to the concentrator tube. Prewet the column by adding
about 0.5 ml methylene chloride through the top of the col-
umn. Place the K-D apparatus on a.warm water bath (60 to
65°C) so that the concentrator tube is partially immersed in
the water. Adjust the vertical position of the apparatus and
the water temperature as necessary to complete the concen-
tration process in 5 to 10 minutes. At the proper rate of
distillation, the balls of the column actively chatter but
the chambers do not flood. When the liquid reaches an
apparent volume of approximately 0.5 ml, remove the K-D
apparatus from the water bath and allow the solvent to drain
and cool for at least 10 minutes. Remove the micro-Snyder
column and rinse its lower joint into the concentrator tube
with approximately 0.2 ml of methylene chloride. Adjust the
final volume to 1.0 ml, seal, and label as the acid fraction.
Determine the original sample volume by refilling the sample
container to the meniscus mark and transferring the liquid to
a 1,000-ml graduated cylinder. Record the sample volume to
the nearest 5 ml -
ill-75
-------
2.1.8 GC/MS Analysis of the Acid Fraction
Establish instrument operating conditions equivalent to those
provided below:
Mass Spectrometer
Mass -range
Scan time
Electron energy
Source temperature
Start acquisition
Column Conditions
me/41-475
1 second or less
70 eV
280-300°C
0.1 min after stopping flow
1.8 m glass column (6.4 mm O.D., 2 mm I.D.) packed with 1%
SP-1240 DA coated on 100/120 mesh Supelcoport; carrier gas:
helium at 30 ml /min. Temperature program: isothermal for
4 min at 50°C, then increasing at 8°C/min to 270°C, and
hold at 270°C for 30 min. If desired, capillary or SCOT
columns may be used.
At the beginning of eacn aay tnat acia rraction analyses are
to be performed, inject 50 ng of pentachlorophenol , either
separately or as part of a standard mixture tnat may also
contain 50 ng of DFTPP, into the instrument. "Die tailing
factor for pentachlorophenol, calculated as indicated in
Figure 2, inouid oe less than 5.
Program the GC/MS to operate in the Extracted Ion Current
Profile (EICP) mode, and collect EICP's for the three char-
acteristic ions listed in Table 2 for each compound being
quantitated. Operating in this mode, calibrate the system
response for each compound using either the internal or
external standard procedure.
When the internal standard procedure is being used, the
standards should not be added to the sample extracts until
immediately before injection Into the instrument. Mix the
extract thoroughly before withdrawing an aliquot for anal-
ysis. Inject 2 to 5 pi of the sample extract. The preferred
method is the solvent-flush technique.
When the extracts are not being used for analysis, they
should be stored in vials with unpierced septa in the dark at
4°C.
Calculate the concentration of sample constituents as
in uasecfions
anc ..
111-76
-------
3.1 Analysis of Sediment Samples for Acid-Extractable Organic Compounds
by Hexane-Methanol Extraction
Analytical Procedure: available
Sample Preparation: available
3.1.1 Reference/Title
U.S. Environmental Protection Agency, "Extraction and Analysis of
Priority Pollutants in Sediment." U.S. EPA, Region IV, SiA
Division, Athens, Georgia. Method PPS-9/80, p. 7, (1980).
3.1.2 Method Summary
A 30-gram sample of field-moist sediment is mixed with anhy-
drous sodium sulfate and extracted with 40 percent hexane in
methanol using an ultrasonic probe. Extraneous compounds are
removed by extraction under basic conditions. The extract
containing acid-extractable compounds is then dried and con-
centrated. The extracts are screened on GC/FID and analyzed
on GC/MS if peaks are noted on the FID chromatogram.
3.1.3 Applicability
This method covers the determination of oriority pollutants
in soils and sediment.
The limit of detection for this method is usually dependent
upon the level of interferences rather than instrumental
limitations. Where interferences are not a problem, the
limit of detection for most compounds analyzed by GC/MS is
1,000 ug/kg.
This method 1s recommended for use only by experienced
residue analysts or under the close supervision of such
qualified persons.
3.1.4 Precision and Accuracy
Information is not available at this time.
3.1.5 Sample Preparation
Decant and discard the water layer over the sediment. Mix
samples thoroughly, especially composited samples. Discard
any foreign objects such as sticks, leaves, and rocks.
Weigh 30 grams of sample into a 400-ml beaker and add 30
grams of anhydrous sodium sulfate. Mix well and allow to dry
to a sandy texture.
Immediately after weighing the sample for extraction, weigh
separate 5- to 10-grams sliqucts of the partially-dried
111-77'
-------
sediment into a tared crucible. Determine the percent solids
by drying overnight at 103°. Allow to cool in a desiccator
for half an hour before weighing. If percent volatile solids
is to be determined, place the oven-dried sample into a
muffle furnace and heat to 550°C for 60 minutes. Allow to
cool in a desiccator before weighing. Discard when finished.
Begin the extraction process by adding 100 ml of 40-percent
hexane 1n methanol to the sample. Adjust the pH of the
mixture to 2. Place an ultrasonic probe approximately 1/2"
below the surface of the solvent but above the sediment layer
and sonicate the sample for 3 minutes at full power with
pulse set at 50 percent. Decant the solvent into a Buchner
funnel.
Repeat the extraction two more times using 100 ml of 40-
percent hexane in methanol for each extraction. Prior to the
sonicatlon step, check the pH of the sample suspension and
adjust to pH 2, as necessary. Collect the extracts in the
Buchner funnel and concentrate to a volume of 100 ml.
3.1.6 Acid Extract Cleanup
Transfer the extract to a 500-ml separatory funnel containing
250 ml of distilled water and 25 ml of saturated sodium
sulfate solution. Adjust the pH to about 11. Shake the
separatory funnel for 2 minutes.
Drain the water layer into a beaker and discard the hexane
layer. Return the aqueous phase to the separatory funnel and
add 25 ml of hexane. Check the pH and adjust to 11 if
necessary. Shake for 2 minutes. Discard the hexane layer.
Adjust the pH of the aqueous layer to 2. Extract with three
separate 25-ml portions of methylene chloride and combine the
extracts in a clean 250-ml separatory funnel. Wash the
extract with two 100-ml portions of acidified water (pH <2).
Pass the methylene chloride extract through a drying column
packed with 7 to 10 cm of anhydrous sodium sulfate and 2 to
3 cm of acid-washed glass wool. The glass wool should also
be solvent-rinsed with methanol, acetone, and methylene
chloride prior to use.
Concentrate the extract to a volume of approximately 6 ml
using a K-D apparatus. Rinse the Snyder column and the
concentrator tube with a small volume of methylene chloride
and reduce the extract to a final volume of 1 ml under a
stream of dry nitrogen. Transfer the extract to a GC vial
and label. If the sample will not be analyzed Immediately,
store at 4°C.
i i
1-78
-------
3.1.7 Sample Analysis by Gas Chromatography/Flame lonization
Screening.
Determine the FID response of the analytical system to 100 ng
pentachlorophenol.
Screen the acid extract on GC/FID to determine whether GO/MS
analysis is necessary. If any of the peaks in the sample
extract produce a response that is greater than 100 ng pent-
achlorophenol , calculate the concentration of the largest
peak.
(a) If the concentration is greater than 1,000 yg/kg (dry
weight basis), analyze the extract using GC/MS.
(b) If the concentration is less than 1,000 yg/kg, report
the sample concentration as <1,000 yg/kg.
(c) If all peaks in the sample extract are less than that
produced by 100 ng pentachlorophenol, record the minimum
detection limit for the sample.
(d) Analyze ail blanks and spikes, xecora ^ne precision and
accuracy data.
111-79
-------
3.2 Analysis of Sediment Samples for Acid-Extractable Organic Compounds
by Methylene Chloride Extraction
Analytical Procedure: available
Sample Preparation: available
3.2.1 Reference
Jacobs Engineering Group, "Manual of Methods for the Analysis
of Hazardous Wastes." Contract Report 68-03-2569 Task 8008
prepared for EPA Environmental Monitoring Systems Laboratory -
Las Vegas, Nevada. Jacobs Engineering Group, Pasadena,
California (1981).
Method Summary
A 50-g sample of soil or sediment is extracted with methylene
chloride using wet residual waste/solvent techniques. Aided
by a high speed homogenizer, samples are extracted at pH 2 to
Isolate the organic acid compounds. The extract is cleaned
up using gel permeation chromatography prior to analysis.
3.2.3 Applicability
This method is suitaoie for che determination of acid-
extractable compounds in solid-phase samples such as soil and
sediment. Method detection limits will vary with sample size
and co-extracted interferences.
3.2.4 Precision and Accuracy
No explicit information is available at this time. However,
analytical performance should be similar to that reported for
acld-extractable compounds in water (Subsection J.2.1).
3.2.5 Sample Preparation
Thoroughly mix the sample by homogenizing 1t in the original
sample bottle. Weigh into a 200-ml centrifuge bottle a 50-g
aliquot or an appropriate weight based on screening analysis.
Add surrogate standards and mix the aliquot to be analyzed.
Adjust the pH of the sample with hydrochloric acid to a pH of
2 or less. The add should be added slowly and with constant
mixing to minimize foaming of the sample. Mix briefly with a
homogenizer to ensure uniform sample pH.
Add 60 ml of methylene chloride to the sample bottle and
homogenize briefly. Rinse the homogenizer with a minimum of
water and then with 5 to 10 ml of methylene chloride. Addi-
tional liiethyiene ;h1or"'de ?ay be jdded 'intil *he total "Mould
1n the centrifuge bottle is near the top.
TII-80
-------
Centrifuge the samples for 15 minutes. The mixture will
separate into an aqueous layer over the methylene chloride
extract. A solid cake or emulsion may form at the water-
methyl ene chloride interface. If the emulsion interface
between layers is more than one-half the size of the solvent
layer, a smaller sample size should be used to complete the
phase separation.
Withdraw the organic extract from the centrifuge bottle with
a 50-ml glass syringe that has been equipped with a 150-mrn x
5-mm I.D. TFE tube. Discharge the extract into a 300-ml beaker.
Repeat the sample extraction procedure a second time with a
60-ml portion of methylene chloride. Combine the extracts.
Perform a third extraction with a final 60-ml portion of
methylene chloride and combine the extracts.
3.2.6 Sample Extract Drying
Pour the combined extract resulting from the extraction
procedure through a drying column containing 7 to 10 cm of
organics-free anhydrous sodium sulfate. Collect the dried
extract in a 500-ml K-D flasK equipped with a 10-mi concen-
trator tube.
Wash the flask that original-ly contained the extract and the
drying tube three times with 30-ml portions of methylene
chloride. Add these washes to uhe sample extract in the K-D
flask.
3.2.7 Sample Extract Concentration
Add one or two clean boiling chips to the flask and attach a
three-ball macro-Snyder column. Prewet the column by adding
approximately 1 ml of the extracting solvent (methylene
chloride) through the top of the column. Place the apparatus
in a 60 to 65°C water bath so the concentrator tube is par-
tially immersed in the water and the lower rounded surface of
the flask is bathed with water vapor. Adjust the apparatus
as necessary to complete concentration to approximately 10 ml
in 15 minutes. (At the proper rate of distillation, the
balls of the column will chatter but the chambers will not
flood.)
Remove the Snyder column, and rinse the flask and its lower
joint into the concentrator tube with 1 to 2 ml of methylene
chloride.
HI-81
-------
Fit the concentrator tube with a modified macro-Snyder
column. Organic-free nitrogen is employed to reduce the
volume of the extract to approximately 5 ml. Wash the
concentration tube with two 0.2-ml volumes of methylene
chloride.
Adjust the final extract volume to 5 ml for subsequent internal
standard addition and GC/MS analysis. If the extract obtained
above is "clean", then a final extract volume of 1 ml is
required.
3.2.8 Gel Permeation Cleanup
Determine the residue weight of the concentrated sample
extract by placing a 1-ml aliquot on a tared aluminum foil
pan, allowing the solvent to evaporate, and reweighing the
pan. These results are used to determine the volume of
extract to be applied to the column for cleanup. The volume
of extract applied to the column should not exceed the capa-
city of the column, approximately 200 mg. If the residue
weight is on the order of 1 to 5 mg, cleanup by gel permea-
tion can, in many cases, be avoided.
Transfer 5 ml of the GPC calibration solution to the Bio-
Beads S-X3 column. Drain the column into a 100»ml graduated
centrifuge tube until the liquid is just above the surface of
the GPC packing. Wash the calibration solution on the column
with several 1-ml aliquots of methylene chloride. Elute the
columns with 200-ml aliquots of methylene chloride and col-
lect 10-ml fractions.
Analyze the fractions for bis-[2-ethylhexyl]phthalate and
pentachlorophenol by GC/FID on a 1% SP-1240 DA column.
Determine the corn oil elution pattern by evaporation of each
fraction to dryness followed by gravimetric determination of
the residue. Plot the concentration of each component in
each fraction versus the total eluent volume.
The first fractions of the elution volume that represent an
approximate 85% removal of the corn oil and 85% recovery of
the bis-(2-ethylhexyl)phthalate can be discarded. Collect
the fractions that elute up to a retention volume represented
by 50 ml after the elution of pentachlorophenol. (Typical
procedures are to discard the first 60 ml, to collect the
next 110 ml, and to wash the column with 250 ml of methylene
chloride between 'samples.)
Select 3 'olume if 'amole extract 'based on the -esidue
weight determination) that will not overload the column.
Apply an aliquot (i to 4 ml; of the extract to the column and
drain the column until the sample is just above the surface
111-82
-------
of the GPC packing. Wash the extract onto the column with
several 1-ml portions of methylene chloride. Elute the
column with 200-ml portions of methylene chloride.
Collect the first 60 ml of eluent in a 100-ml graduate
cylinder, pass the next 110 ml of eluent through a drying
column containing 6 cm of anhydrous sodium sulfate and
collect it in a 500-ml K-D flask equipped with a 10-ml concen-
trator tube. Rinse the drying column with three 25-ml por-
tions of methylene chloride.
Add one or two clean boiling chips to the flask and attach a
three-ball macro-Snyder column. Prewet the column by adding
approximately 1 ml of methylene chloride through the top of
the column. Place the apparatus in a 60 to 65°C water bath
so that the concentrator tube is partially immersed in the
water, and the lower rounded surface of the flask is bathed
with water vapor. Adjust the apparatus to complete concen-
tration to approximately 10 ml in 15 minutes. (At the proper
rate of distillation, the balls of the column will chatter
but the chambers will not flood.)
Semove the Snyder column, and rinse the -"ask 2nd 't.s * awer
joint into the concentrator tube with 1 to 2 ml of methylene
chloride.
Fit the concentrator tube with a modified macro-Snyder col-
umn. Organic-free nitrogen is employed to reduce the volume
of the extract to approximately 5 ml or 1 ml (but not below
0.5 ml). Wash the concentrator tube with two 0.2-ml volumes
of methylene chloride.
Adjust the final extract volume to 5 ml or 1 ml for subse-
quent internal standard addition and GC/MS analysis. If the
extract obtained above is "clean", then a final extract
volume of 1 ml is required.
If the extract is to be stored before GC/MS analysis, trans-
fer the extract to an appropriately-sized serum vial equipped
with a Teflon-lined rubber septum and crimp cap. The extract
volume should be scored on this vial, and appropriate sample
identification must be affixed. Store the extracts in the
dark at 4°C.
It is possible that samples which contain high concentrations
of extractable organic compounds will not be amenable for
concentration to 5 ml. For extracts of this type the final
volume after concentration should be adjusted to a minimal
volume *hat permits extract samoling with a Tncro-syHnqe.
Obvious remedies will likely include either starting with
Iii-83
-------
smaller sample size or concentration to a volume greater than
5 ml.
Transfer the cleaned, concentrated extract to a 6-ml serum
TFE capped bottle and store at 4°C for 6C/MS analysis.
Qualitatively identify specific compounds in the sample
extract and calculate their concentrations as indicated in
Subsection K.
111-84
-------
4.1 Analysis of Biological Tissue Samples for Acid-Extractable Organic
Compounds by Methylene Chloride Extraction
Analytical Procedure: available
•Sample Preparation: available
4.1.1 Reference
U.S. Environmental Protection Agency, "Extraction and Analyses
of Priority Pollutants in Biological Tissue." U.S. EPA S4A
'Division, Region IV, Laboratory Services Branch, Athens, Georgia.
Method PPB 10/80. p. 7, (1980).2
4.1.2 Method Summary
A 10-g sample of homogenized fish tissue is mixed with 40 g
of sodium sulfate, and extracted which methylene chloride
using an ultrasonic probe. The samples are filtered, con-
centrated to 10 ml or less, cleaned up with acetonitrile
partitioning, and concentrated to 1 ml. The extract is
screened using gas chromatography and quantified using mass
spectrometry.
4.1.3 Applicability
The limit of detection for this method 1s usually dependent
upon the level of interferences rather than instrumental
limitations. Where interferences are not a problem, the
limit of detection for most compounds analyzed by GC/MS is
2 mg/kg (wet.weight basis). .
The method is recommended for use only by experienced residue
analysts or under the close supervision of such qualified
persons.
4.1.4 Estimates of Precision and Accuracy
No information is presently available. However, analytical
performance should be similar to that reported for Subsection
J.2.1.
4.1.5 Sample Extraction
Blend equal amounts of fish tissue and dry ice. If a large
sample is being processed, a food processor or meat grinder
may be convenient.
Weigh 10 g of homogenous sample into a 400-ml beaker and mix
with 40 g of sodium sulfate. Ensure that the sample is
thoroughly dry.
111-85
-------
Add 100 ml methylene chloride to the tissue mixture. Place
an ultrasonic probe in the mixture and sonicate at 50 percent
pulse for 3 minutes. Transfer the methylene chloride phase to
a 500-ml K-D flask.
Repeat the methylene chloride sonication/extraction of the
tissue sample with a second 100-ml portion of methylene
chloride. Combine the extracts in a K-D flask. Extract the
sample residue a third time with 100 ml methylene chloride.
Combine the extracts.
NOTE: The probe should be carefully cleaned between samples
as indicated in Subsection C (Interferences).
Add a clean boiling chip to the K-D flask and attach a three-
ball macro-Snyder column. Place the K-D apparatus on a water
bath and concentrate the extract to 10 ml.
Quantitatively transfer the concentrated extract to a 125-ml
separatory funnel. Add enough hexane to bring the final
volume to approximately 15 ml. Extract the sample four times
by shaking vigorously with 30-ml oortions of hexane-saturated
acetonitrile for one minute.
Combine and transfer the acetonitrile phases to a 1-liter
separatory funnel and add 650 ml of distilled water and 40 ml
of saturated sodium chloride solution. ~MTx "thoroughly for 30
to 45 seconds. Adjust tne pri of tne aqueous pnase to 2 and
extract with two 100-ml portions of methylene chloride.
Shake the sample vigorously for 15 to 30 seconds during each
extraction.
Combine the methylene chloride extracts in a 1-liter separ-
atory funnel and wash with two 100-ml portions of distilled
water. Discard the water layer and pour the methylene
chloride layer through a drying column containing 7 to 10 cm
of anhydrous sodium sulfate and 2 to 3 cm of glass wool.
Collect the extract in a 500 ml K-D flask equipped with a 100-
ml ampul. Rinse the separatory funnel and drying column with
three 10-ml portions of methylene chloride. Add the rinsings
to the K-D flask.
Attach a three-ball macro-Snyder column and place the K-D
apparatus in a hot water bath (60-65°C). Concentrate the
extract to 6 to 10 ml. Use a stream of dry nitrogen to
concentrate the extract to 1 ml.
Transfer the extract to a GC vial and label as the acid-
extractaoie fraction of che semi-volatile compounds. Tins
extract is now readv for analysis.
111-86
-------
4.1.6 Analysis by Gas Chromatopraphy
The acid-extractable compounds are screened with GC/FID using
the column Indicated in Subsection E to determine whether
GC/MS analyses are necessary. Relative retention times and
limits of'detection are summarized in Tables 1 and 5. A
representative chromatogram is presented in Figure 3.
Determine the FID response of 100 ng pentachlorophenol. If
any peaks in the sample extract produce a greater instrument
response than pentachlorophenol, calculate the concentration
of the largest peak:
(a) If the calculated concentration of the sample component
is greater than 2 mg/kg (wet weight basis), analyze the
extract by GC/MS.
(b) If the calculated concentration of the sample component
is less than 2 mg/kg, report the concentration as less
than 2 mg/kg.
(c) If all sample peaks in the chromatograms are less than
the pentachlorophenol peak, "ecord the minimum detection
limit for the sample.
Analyze all blanks and spikes. Record the pertinent pre-
cision and accuracy data with the sample information.
4.1.7 Gas Chromatography/Mass Spectroscopy Analysis for cne Acia-
Extractable Compounds
At the beginning of each day that acid extractable compound
analyses are to be performed, inject 50 no pentachlorophenol,
either separately or as part of a standard mixture that may
also contain 50 no DFTPP. The tailing factor for pentachloro-
phenol, calculated as shown in Figure 2, should be less
than 5.
Establish chromatographic conditions equivalent to those
listed in Table 1. Included in this table is information on
estimated retention times and sensitivities that can be
achieved by this method. An example of the pre-separation
achieved by. the column is shown in Figure 3.
Program the GC/MS to operate in the Extracted Ion Current
Profile (EICP) mode, and collect EICP spectra for the three
characteristic ions listed in Table 2, page 111-65 for each
compound being quantified. Operating in this node, calibrate
the system response for each compound by using the internal
standard procedure.
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If the internal standard approach is used, the standards
should not be added to the sample extracts until immediately
before injection into the instrument. Mix the spiked extract
thoroughly. Inject 2 to 5 pi of the extract using the
solvent-flush technique.
If the instrument response for any ion exceeds the linear
range of the system, dilute the extract as necessary and
reanalyze.
Qualitative identification and quantitative calculations are
completed as indicated in Subsection K. When the extracts
are not being used for analysis, store them in vials capped
with unpierced septa in the dark at 4°C.
K. QUALITATIVE IDENTIFICATION
To qualitatively identify a compound, obtain an Extracted Ion Current
Profile (EICP) for the primary ion and the two other ions listed in Table 2.
The criteria below must be met for a qualitative identification.
1. The characteristic ions for each compound must have their maxima in the
same or within one scan of each other.
2. The retention time for the experimental mass spectrum must be within ±30
seconds of the retention time.of the authentic compound.
3. The ratios of the three EICP oeak heiahts must agree within ±20% with the
ratios of the relative intensities for these ions in a reference mass
spectrum. The reference mass spectrum can be obtained from either a
standard analyzed through the GC/MS system or from a reference library.
4. Structural isomers that have very similar mass spectra can be explicitly
identified only if the resolution between the isomers in a standard mix
is acceptable. Acceptable resolution is achieved if the valley height
between isomers is less than 25% of the sum of the two peak heights.
Otherwise, structural isomers are identified as isomeric pairs.
For samples that contain an inordinate number of interferences, the
chemical ionization (CI) mass spectrum may make identification easier.
Characteristic CI ions for most of the compounds are given in Table 2.
The use of chemical ionization MS to support El MS is encouraged but not
required.
L. CALCULATIONS
1. Acid-Extractable Compounds in Water.
When a comoound has been identified, the ouantification of that compound
will be based on the integrated area from tne specific ion plot of the
first listed characteristic ion in Table 2.
111-39
-------
If the sample produces an Interference for the first listed ion, use a
secondary ion to quantify. Quantification can be accomplished by the
internal standard method.
Internal standard - By adding a constant known amount of internal
standard (C^s in yg) to every sample extract, the concentration o
(C0) in yg/1 in the sample is calculated using Equation L-l.
(As)(Cis)
C0 = Eq. L-l
(Ais) (RF) (Y0)
where:
V0 = the volume of the original sample in liters; the other
terms are defined in text above (Eq. H-l).
Report all results to two significant figures. Report results in v9/l
without correction for recovery data. When duplicate and spiked samples
are analyzed, all data obtained should be reported.
In order to minimize unnecessary GC/MS analysis of method blanks and
field blanks, the f;eld blank Tiay be screened on a FTD/GC equipped with
an SP-1240 DA column.
2. Acid-Extractable. Compounds in Solid-Phase Samples (Soil/Sediment).
2.1 Percent Dry Solids
gm of dried sample
x 100 = % dry solids
gm of wet sample
2.2 Percent Volatile Solids
gm of dried sample - gm ignited sample
x 100 = % volatile solid
gm of wet sample
2.3 Concentration of acid-extractable compounds
C • 0
Cone. Acid Cmods (wet weight) =
F
111-90
-------
C • D
Cone. Acid Cmpds (dry weight) =
F-S
where:
C = concentration of acid compound in sample extract, pg/1
D = final volume of sample extract, 1
F = wet weight of sample initially extracted, g
S = percent solids of initial sample.
111-91
-------
REFERENCES
1. U.S. Environmental Protection Agency. "Base/Neutrals, Acids, and
Pesticides - Method 625." Federal Register Vol. 44: No. 233:
69540-69552. December 3, 1979.
2. U.S. Environmental Protection Agency. "Extraction and Analysis of
Priority Pollutants in Biological Tissue." Method PPB 10/80. U.S. EPA,
Region IV, S&A Division, Laboratory Services Branch, Athens, Georgia.
p. 7, (1980.).
3. U.S. Environmental Protection Agency. "Method for Preparation of Medium
Concentration Hazardous Waste Samples." U.S. EPA, Region IV, Athens,
Georgia, p. 8, May 1981.
4. Glaser, J. A., D. L. Foerst, G. D. McKee, 'S. Quave and W. L. Budde.
"Theory and Application of Method Detection Limit. A new Performance
Criterion for Chemical Analysis." U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio.
September 1981.
5. Glaser, J. A., 0. L. Foerst, G. 0. McKse, S. Ouave and W. L. Budde.
"Trace Analyses for Wastewaters." ES&T .!£: 1426-1435 (1981).
6. U.S. Environmental Protection Agency. "IFB WA-81-H017." U.S. EPA,
Washington, D.C. July 6, 1981.
•
7. U.S. Environmental Protection Agency. "Extraction and Analysis of
Priority Pollutants in Sediment." U.S. EPA, Region IV, S&A Division,
Athens, Georgia. Method PPS 9/80, p. 7, (1980).
8. Jacobs Engineering Group. "Manual of Methods for the Analysis of
Hazardous Wastes." Contract Report 68-03-2569 Task 8008 prepared for EPA
Environmental Monitoring Systems Laboratory-Las Vegas, Nevada. Jacobs
Engineering Group, Pasadena, California (1981).
9. U.S. Environmental Protection Agency. "Extraction and Analysis of
Priority Pollutants in Biological Tissue." U.S. EPA, Region IV, S&A
Division, Laboratory Services Branch, Athens, Georgia. Method PPB 10/80
p. 7, (1980).
-------
SECTION 3
BASE/NEUTRAL-EXTRACTABLE COMPOUNDS
A. SCOPE
Base/neutral compounds in the semi-volatile fraction of the priority
pollutants (Table 1) can be quantified using the analytical procedures
presented in Subsection J. These compounds are solvent-extractable under
alkaline conditions and identified/quantified using GC/MS procedures.
TABLE 1. BASE-NEUTRAL EXTRACTABLES
ComDOund
Storet No.
Comnound
Storet Mo.
Acenaphthene 34205
Acenaphthylene 34200
Anthracene 34220
Benzo[a]anthracene 34526
Benzo[b]fluoranthene 34230
Benzo[k]fluoranthene 34242
Benzo[a]pyrene 34247
Benzo[ghi]pery1ene 34521
Benzidine 39120
Bis[2-chloroethyl]ether 34273
Bis[2-chloroethoxy]methane 34278
Bis[2-ethylhexyl]phthalate 39100
Bis[2-chloroisopropyl]ether 34283
4-Bromophenyl phenyl ether 34636
Butyl benzyl phthalate 34292
2-Chloronaphthalene 34581
4-Chlorophenyl phenyl ether 34641
Chrysene 34320
Dibenzo[ah]anthracene 34556
Di-n-butyl phthalate 39110
1,3-Dichlorobenzene 34566
1,4-Dichlorobenzene " 34571
1,2-Dichlorobenzene 34536
34631
Diethyl phthalate ' 34336
Dimethyl phthalate 34341
2,d-0initrotoluene 34611
2,6-Dinitrotoluene 346?6
Di-n-octyl phthalate 34596
1,2-Diphenylhydrazine 34346
Fluoranthene 34376
Fluorene 34381
Hexachlorobenzene 39700
Hexachlorobutadiene 34391
Hexachloroethane 34396
Hexachlorocyclopentadiene 34386
Indeno[l,2,3-cd]pyrene 34403
Isophorone 34408
Naphthalene . 39250
Nitrobenzene 34447
N-Nitrosodimethylamine 34438
N-Ni trosodi-n-propylami ne 34428
N-Nitrosodiphenylamine 34433
Phenanthrene 34461
Pyrene 34469
2,3,7,8-Tetrachloro- 34675
dibenzo-p-dioxin
1.2,4-Trichlorobenzene 34551
===============
111-93
-------
B. SAMPLE HANDLING AND STORAGE
Conventional water sampling practices should be followed except that the
bottle should not be prerinsed with sample before collection. Grab samples
should be collected in glass containers and refrigerated immediately. Com-
posite samples should preferably be collected in glass containers" and, if
possible, refrigerated during the period of compositing. All automatic
sampling equipment must be free of Tygon and other potential sources of
organic contamination. When necessary, the equipment should be prerinsed with
hexane prior to use.
Water samples should be iced or refrigerated from the time of collection
until extraction.1 Chemical preservatives should not be used in the field
unless more than 24 hours will elapse before sample delivery to the labora-
tory. If the samples cannot be extracted within 48 hours of collection, the
recommended method of sample preservation is as follows:
1. If the sample contains residual chlorine, add 35 mg of sodium thiosulfate
per 1 ppm of free chlorine per liter of sample.1
2. Adjust the pH of the water sample to a pH of 7 to 10 using sodium
hydroxide or sulfuric acid. Record the volume of acid or base used.1
VP water samples must be extracted within 7 days and completely analyzed
within 30 days of collection (Figure 1).
Sediment and soil samples may be stored field-moist (refrigerated),
frozen, or dried. The effective storage period is not known (Figure 1).
C. INTERFERENCES
Solvents, reagents, glassware, and other sample processing hardware may
yield discrete artifacts and/or elevated baselines causing misinterpretation
of chromatograms. All of these materials must be demonstrated to be free from
interferences under the conditions of the analysis by running method blanks.
Specific selection of reagents and purification of solvents by distillation in
all-glass systems may be required.
Analytical interferences associated with samples will vary considerably
from source to source, depending on the diversity of the Industrial complex,
municipality, or system being sampled. Matrix Interferences may be caused by
components that are coextracted from the sample but are not normally of
Interest. The most common such components are petroleum-derived naphthenes,
high-molecular-weight polymeric components, and long-chain components such as
waxes and triglycerides. The extent of such matrix interferences will vary
considerably from source to source. A cleanup procedure using gel permeation
chromatography has been incorporated into the method for certain cases to
remove long-chain and high-molecular-weight material. No cleanup procedure 1s
available for the removal of naphthenes. When such matrix Interferences are
present, the samole extract is diluted and the method detection limit is
Increased proportionately. Many of the matrix interferences are ^olvent-
extractable nonvolatile components which necessitate the more frequent
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cleaning of the GC injection port and the more frequent removal of the
injection end of the GC capillary column.
Interference due to fish oil in tissue samples can be eliminated by
acetonitrile partitioning,2 but the ultrasonic probe used to assist in the
partitioning process must be scrupulously cleaned between samples. The
procedure consists of rinsing the probe with solvent (acetonitrile) into the
sample, removing residue on the probe with a wet Kimwipe, rinsing the probe
with methylene chloride, and sonicating in hexane for 3-4 minutes on 50%
pulse.
The recommended analytical procedure may not have sufficient resolution
to differentiate between certain isomeric pairs. These are anthracene and
phenanthrene, chrysene and benzo(a)anthracene, and benzo(b)fluoranthene and
benzo(k)fluoranthene. The GC retention time and mass spectral data are not
sufficiently unique to make an unambiguous distinction between these com-
pounds. Alternative techniques should be used to identify and quantify these
specific compounds.
D. SAFETY
The toxicity or carcinogenicity of each reagent used in this method has
not been precisely defined; however, -?ach x;hemical compound should be treated
as a potential health hazard. From this viewpoint, exposure to these chem-
icals must be minimized by whatever means available. The laboratory manager
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 .-natsrlal data handling sheets should also be made available
to all personnel involved *n the chemical analysis. All operations involving
the use of methylene chloride, including the extraction of the waste sample,
filtration of the extract, and concentration of the extract, must be performed
in a fume hood. Care should be taken to avoid skin contact with methylene
chloride.
E. APPARATUS
1. Class I Biological safety cabinet (Glovebox) suitable for handling
chemical carcinogens, as described on page 123 of "Laboratory Safety
Monograph, A Supplement to the NIH Guidelines for Recombinant DNA
Research," U.S. Department of Health, Education and Welfare, PHS, NIH,
January 1979. The cabinet should have an interchange panel for intro-
ducing materials, a retaining tray to catch spills, and a static pressure
gauge.*
2. Spatula. Stainless stee.l or Teflon. Fisher Scientific catalog No.
14-375-10 or equivalent.
*Kewaunee, Inc. model SH-3704-MS-X, or equivalent is acceptable.
111-96
-------
3. Balance capable of weighing 100 grams to the^nearest 0.01 gram.
4. Vials, specimen, screw cap, approximately 20 ml. Use Teflon liner.
Calibrate at 10 ml by pipetting 10 ml of solvent into the tube and
marking the bottom of the meniscus.
5. Vials and caps, 2 ml for GC auto-samplers.
6. Disposable pipets, Pasteur.
7. Test tube rack.
8. Oven, drying.
9. Desiccator.
10. Crucibles, porcelain, squat form, Size 2 or equivalent.
11. Sonlcator Cell Disrupter - Heat Systems - Ultrasonics, Inc., with a
3/4"-high gain probe, 375 watt or equivalent.
12. Beakers, 400 ml.
13. Separatory funnels - 120 ml, 250 ml, 500 ml, and 2 1 with Teflon
stopcocks.
14. Pyrex glass wool.
15. Furnace, muffle. -
16. Biichner funnels.
17. Kuderna-Danish (K-D) apparatus.
17.1 Concentrator tube - 10 ml, graduated (Kontes K-570040-1025 or
equivalent).
17.2 Evaporative flask - 500 ml (Kontes K-570001-0500 or equivalent).
17.3 Snyder column - three-ball macro (Kontes K-50300-0121 or
equivalent).
17.4 Snyder column - Two-ball micro (Kontes K-569002-0219, or
equivalent).
18. Boiling chips - Beryl saddles (Fisher, 91915) crushed.
19. Water bath - Heated, with concentric ring cover, capable of temperature
control (±2'C).
111-97
-------
20. Pans, approximately 35 cm x 25 cm x 6 cm.
21. Filter paper - Whatman 41, ashless.
22. Vacuum filtration apparatus (Fisher 9-788) or 500-ml suction filtration
flasks.
23. Vacuum pump.
24. Drying columns - 25 mm x 200 mm packed with 4 cm of glass wool.
25. Florisil columns - Pyrex, 400 mm x 25 mm O.D. with Teflon stopcock, but
without glass frit.
26. Food Processor - (Hobart food processor - 8181D or equivalent).
27. Gas chromatograph - analytical system complete with gas chromatograph
suitable for on-column injection and all required accessories including
flame ionization detector and electron capture detector, column supplies,
recorders, gases, and syringes.
27.1 Column 1 - for base/neutral and pesticides, a 6-ft (1.83 m) glass
column (6 mm 0.0. x 2 mm I.D.) packed with 3% SP-2250 coated on
100/120 mesh Supelcoport, or equivalent. This column was used to
generate tne precision ana accuracy cata summarized in Table 5.
27.2 Column 2 and analytical conditions - Chromosorb W (100/120 mesh),
coated with 3% OV-17 packed fn a 1.83 m x 2 mm I.D. Pyrex glass
- column. Use ultra pure nitrogen at a flow rate of 30 ml/min.
Column temperature is held at 30"C for 2 min., programmed-to 290°
at 8e/min., and held at 290"C for 16 min.
28. Mass spectrometer - capable of scanning from 35 to 450 a.m.u. every 7
seconds or less at 70 V (nominal) and producing an acceptable mass
spectrum at unit resolution from 50 ng of DFTPP when the sample is
introduced through the GC inlet. The mass spectrometer must be inter-
faced with a gas chromatograph equipped with an injector system designed
for splitless injection and glass capillary columns or an Injector system
designed for on-column injection with all-glass packed columns. All
sections of the transfer lines must be glass or glass-lined and must be
deactivated (use Sylon-CT, Supelco, Inc., or equivalent, to deactivate).
NOTE: Systems utilizing a jet separator for the GC effluent are recom-
mended since membrane separators may lose sensitivity for light molecules
and glass frit separators may inhibit the elution of polynuclear aromat-
tics. Any of these separators may be used provided that it gives
recognizable mass spectra and acceptable calibration points at the limit
of detection specified for each individual compound.
A computer system must be interfaced to the mass spectrometer to allow
continuous acquisition of -nass :cafis *or *.he luraf'on of the chromato-
graphic program. There must be computer software available to allow
III-9R
-------
searching any GC/MS run for specific Ions and plotting the Intensity of
the ions with respect to time or scan number. The ability to integrate
the area under any specific ion plot peak is essential for quantification.
29. Extracting equipment
29.1 ADT tissumizer (Tekmar SDT 182EN or equivalent).
29.2 Centrifuge (IEC CU-5000 or equivalent).
2^.3 Screw-capped centrifuge bottles, 200 ml (Scientific Products C4144)
with TFE-lined screw caps.
29.4 Fleakers (or beakers) - 300 ml or equivalent.
2<>.5 Glass syringe - 50 ml equipped with a 150 mm x 5 mm I.D. TFE tube.
30. Continuous liquid-liquid extractors - Teflon or glass connecting joints
and stopcocks, no lubrication. (Hershberg-Wolf Extractor - Ace Glass
Co., Vineland, New Jersey, P/N 6841-1D, or equivalent).
31. Gel permeation chromatography cleanup equipment.
31.1 Chromatography column - 500 mm x 19 rnm T.D. 'Scientific Products
C-4670-106 or equivalent).
31.2 Bio-Beads S-X3, 200/400 mesh (Bio-Rad Laboratories 152-2750).
31.3 Glass vool.
31.4 Graduated cylinders - 100 ml.
31.5 GCP Autoprep or equivalent (Analytical Biochemistry Labs, Inc. 1002
or equivalent with 25 mm I.D. column containing 50 to 60 g of Bio-
Beads S-X3). (Optional)
F. REAGENTS
1. Sodium sulfate, anhydrous, reagent grade - heated 2 hours at 500°C,
cooled in a desiccator for 4 hours, and stored in a glass bottle.
2. Methylene chloride - pesticide residue analysis grade or equivalent.
3. Hexane - pesticide residue analysis grade or equivalent.
4. Reagent water. Water purified by passage through activated charcoal or
equivalent. When aliquots of this water are analyzed using the pro-
cedure, the Impurities measured shall be below the detection limits based
on 3 1-gram samole aliquot.
ni-99
-------
5. Methanol, pesticide residue analysis grade, free of purgeable organics.
(Open a fresh bottle and check It by adding 100 pi to 5 ml of organic-
free water. Analyze on the GC/MS system using the purge-and-trap
technique.)
6. Acetone - pesticide quality and distilled in glass.
7. Petroleum ether - pesticide quality and distilled in glass.
8. Diethyl ether - preserved with 2% ethanol, pesticide quality and dis-
tilled 1n glass. NOTE: Ether must be free of peroxides as indicated by
EM Quant Test Strips (test strips are available from EM Laboratories,
Inc., 500 Executive Blvd., Elmsford, New York 10523).
9. FTorisIl - PR grade (60/100 mesh).
10. Sodium hydroxide (ACS), 6 N in distilled water.
11. Sodium hydroxide (ACS), 10 N 1n distilled water.
12. Sodium hydroxide (ACS), 50% in distilled water. Extracted 3 times with
methylene chloride.
13. Hydrochloric acid (ACS), concentrated ;12 ;j).
14. Sulfuric add (ACS), 6 N in distilled water.
15. Stock standards - Prepare stock standard solutions at a concentration of
1.00 ug/ul. For example, dissolve 0.100 3 of assayed reference material
in pesticide-quality i-^ooctane or other acpropr^ate solvent and dilute to
volume in a 100-ml ground glass stoppered volumetric flask. The stock
solution is transferred to 15-ml Teflon-lined screw cap vials, stored in
a refrigerator, and checked frequently for signs of degradation or
evaporation, especially just prior to preparing working standards from
them. Protect PNA standards from light.
16. Calibration standards - Prepare calibration standards that contain the
compounds of Interest, either singly or mixed together. The standards
should be prepared at concentrations that will completely bracket the
working range of the chromatographic system (two or more orders of mag-
nitude are suggested). For example, if the limit of detection can be
calculated as 20 ng Injected, prepare standards at 10 ug/ml, 100 pg/ml,
1000 ug/ml, etc. so that Injections of 1-5 ul of the calibration stan-
dards will define the linearity of the detector in the working range.
17. Surrogate standards - Surrogate standards should be stable isotope
derivatives of compounds -which will aid 1n the monitoring of the
analysis procedures. The number and composition of surrogate standards
will to some extent be dependent on compound availability. Where pos-
sible the stable-"!sotope-labeled compounds (which are TWSS resolved from
III-100
-------
the unlabeled compound) will be utilized ^to simplify calculations in the
measurement of recovery. The surrogate standards should be added to
the chilled solid sample via injection of an appropriate volume of the
surrogate standard mix. [The sample should be spiked with a quantity of
surrogate standard which is equivalent to 5 ppm (e.g., 250 ug/50 g
sample).] The surrogate spike should be added to the solid sample in
aliquots of the total volume to be added (e.g., five 20-ul injections
from a 100 jil syringe) while manually stirring the material with a clean
glass stirring rod to facilitate dispersion of surrogate compounds. It
is important that the sample and sample container are chilled during the
surrogate standard addition. Surrogate standard addition should be
accomplished as quickly as possible to minimize the loss of the most
volatile analytes quantitated with this method. Upon surrogate standard
addition, the isolation/cleanup procedure can be initiated.
It is recognized that uniform distribution of surrogate standards in the
sample will not be obtained via this procedure. Also, it is recognized
that the surrogates will not mirror the properties of organic compounds
which have been associated with soil or sediment samples for decades.
Nevertheless, data generated from surrogate standards will serve to
exemplify a "best" case recovery for the sample in question, and will
provide QA/QC data for every sample.
G. QUALITY CONTROL
. Before processing any samples, demonstrate through the analysis of a
method blank that all glassware and reagents are interference-free. Each time
a set of samples is extracted or there is a change in reagents, a method blank
should ba procsssed as a safeguard against chronic laboratory contamination.
Standard quality assurance practices should be used to document the
performance of this method. Field replicates should be collected and analyzed
to determine the precision of the sampling technique. Laboratory replicates
should be analyzed to determine the precision of the analysis. Fortified
samples should be analyzed to determine the accuracy (recovery) of the
analysis. Field blanks should be analyzed to check for contamination intro-
duced during sampling and transportation.
Five percent of the samples (1 of 20), or one sample each time a set of
samples is prepared, whichever frequency is higher, should be blanks of the
reagents, water and solvents. Contaminants shall be below the detection limits
based on a 1-gram (1-ml) sample aliquot.
Five percent of the samples (1 of 20), or one sample each time a set of
samples is prepared, whichever frequency is higher, should be spiked, prepared,
and analyzed in duplicate.
H. CALIBRATION
Prepare calibration standards that contain the compounds of interest,
either singly or mixed together. The standards should be prepared at concen-
trations tnat will bracket tne working range of the -ftromatograpfrfc .system
III-101
-------
(two or more orders of magnitude are suggested). If the limit of detection
can be calculated as 20 ng injected, for example, prepare standards at 1
ug/ml, 10 ug/ml, 100 wg/ml, etc. so that injections of 1-5 ul of the calibra-
tion standards will define the linearity of the detector in the working
range.
Assemble the necessary gas chromatographic apparatus and establish
operating parameters equivalent to those indicated. By injecting calibration
standards, establish the linear range of the analytical system and demonstrate
that the analytical system meets the detection requirements. If the sample
gives peak areas above the working range, dilute and reanalyze.
Internal standard method - The internal standard approach is acceptable
for all of the semivolatile organics. The utilization of the internal stan-
dard method requires the periodic determination of response factors (RF) that
are defined in Equation 1.
RF = (AsC1s)/(AisCs) Eq. 1
where:
As = the integrated area or peak height of the characteristic ion
for the contaminant being measured
A^s » the integrated area or peak height of the characteristic ion
•• for the internal standard
Cfs » the amount (jig) of the internal standard
Cs * the amount (ug) of the contaminant.
The relative response ratio for the analytes should be known for at least
two concentration values - 20 ng injected to approximate 10 ug/1 and 200 ng
injected to approximate the 100-ug/1 level (assuming 1 ml final volume and a
2-ul injection). Those compounds that do not respond at either of these
levels may be run at concentrations appropriate to their response.
The response factor (RF) should be determined over all concentration
ranges for the standards (Cs) that are being determined. [Generally, the
amount of internal standard added to each extract is the same (20 ug) so that
C-is remains constant.] This should be done by preparing a calibration curve
where the response factor (RF) is plotted against the standard concentration
(Cs), using a minimum of three concentrations over the range of interest.
Once this calibration curve has been determined, 1t should be verified daily
by injecting at least one standard solution containing internal standard. If
significant drift has occurred, a new calibration curve must be constructed.
To quantify, add the internal standard to the concentrated sample extract no
more than a few iirinutes before Injecting Into the GC/MS system to minimize the
possibility of losses due to evaporation, adsorption, or chemical reaction.
III-102
-------
Calculate the concentration by using the previous equations with the approp-
riate response factor taken from the calibration curve. Either deuterated or
fluorinated compounds can be used as Internal standards and surrogate
standards. Napththalene-dg, anthracene-dig, pyridine-ds, aniline-ds,
nitrobenzene-dc, l-fluoronaphthalene, 2-ftuoronaphthalene, 2-fluorobiphenyl,
2,2'-difluorobiphenyl, and 1,2,3,4,5-pentafluorobiphenyl have been used or
suggested as appropriate Internal standards/surrogates for the base/neutral
compounds. Compounds used as Internal standards are not to be used as surrogate
standards. The Internal standard must be different from the surrogate standards.
External standard method - The external standard method can also be used
at the discretion of the analyst. Prepare a master calibration curve using a
minimum of three standard solutions of each of the compounds that are to be
measured. Plot concentrations versus integrated areas or peak heights (selected
characteristic ion for GC/MS). One point on each curve should approach the
limit of detection. After the master set of Instrument calibration curves has
been established, the curves should be verified daily by Injecting at least one
standard solution. If significant drift has occurred, a new calibration curve
must be constructed.
I. DAILY GC/MS PERFORMANCE TESTS
At the beginning of each day, the mass calibration of the GC/MS system
must be cnecxed ind Adjusted, if necessary, to meet DFTPP specifications.
Additionally, each day base/neutral compounds are to be analyzed, the column
performance specifications with benzidine must be met. DFTPP can be -nixed -'n
solution with benzidine to complete the two specifications with one Injection,
if desired. The performance criteria must be met before any samples or
standards are analyzed.
Evaluate the analytical system performance each day that It 1s to be used
for the analysis of samples or blanks by examining the mass spectrum of DFTPP.
The following instrumental conditions are required to perform the mass cali-
bration test of a GC/MS system:
Electron Energy - 70 volts (nominal)
Mass Range - 35-450 amu
Scan Time - 7 seconds or less
Inject a solution containing 50 ng DFTPP and check to ensure that established
performance criteria listed in Table 2 have been satisfied. If the system
performance criteria are not met, retune the spectrometer and repeat the
performance check.
Column performance 1s evaluated by injecting 100 ng of benzidine into the
Instrument. The tailing factor for the resultant peak, as calculated in
Figure 2, must be less than 3-for the performance to be considered acceptable.
The user is cautioned that some problems may be encountered due to the oxida-
tion of benzidine. However, benzidine has been specified for this purpose.1
Also, tailing -factor criteria have not been established *or any other ral-J-
bration material to be used with this fraction (base/neutral compounds).
III-103
-------
TABLE 2. OFTPP KEY IONS AND ION ABUNDANCE CRITERIA
Mass
Ion Abundance Criteria
51
68
70
127
197
198
199
275
365
441
442
30 to 60 percent of mass 198
Less than 2 percent of mass 69
Less than 2 percent of mass 69
40 to 60 percent of mass 198
Less than 1 percent of mass 198
Base peak, 100 percent relative abundance
5 to 9 percent of mass 198
10 to 30 percent of mass 198
Greater than 1 percent of mass 198
Present but less than mass 443
Greater than 40 percent of mass 198
Tailing Factor = —-
AB
Example Calculation: Peak Height = DE = 100 mm
10% Peak Height - BO = 10 mm
Peak Width at 10% Peak Height = AC = 23 mm
AB = 11 mm
BC = 12 mm
12
Therefore: Tailing Factor = —- - ". 1
Figure 2. Tailing factor calculation.
!!1-104
-------
J. ANALYTICAL PROCEDURES
1.1 Analysis of Hazardous Wastes for Base/Neutral Compounds
Analytical Procedure: available
Sample Preparation: available
1.1.1 Reference
U.S. Environmental Protection Agency, "Method for Preparation
of Medium Concentration Hazardous Waste Samples." U.S. EPA,
Region IV, Athens, Georgia. May 1981.3
1.1.2 Method Summary
Approximately one-gram allquots of soil, solid, aqueous
liquid or non-aqueous liquid are transferred to vials inside
a chemical carcinogen glove box. The samples are then
extracted with methylene chloride. The methylene chloride
extract is screened by GC/FID using the appropriate base/
neutral column. Based on initial screening "esults, the
sample extracts are appropriately concentrated and analyzed
with a GC/MS system.
1.1.3 Applicability
This procedure is designed for the safe handling jnd prepar-
ation of potentially hazardous samples from hazardous waste
sites for analysis of organic chemicals Including priority
pollutants. The method is directed to contaminated soil
samples and waste samples that may be solid, aqueous liquid,
or non-aqueous liquid and suspected to contain less than 10%
of any one organic chemical component. The method is not
designed for samples expected to contain less than 10 ppm of
base/neutrals and acids, such as many sediment samples taken
from leachate streams. This type sample should be analyzed
using a method for sediment/soil samples (Subsection J.3.1
or J.3.2.
1.1.4 Precision and Accuracy
These extraction and preparation procedures were developed
for rapid and safe handling of hazardous samples. The design
of the methods thus did not stress efficient recoveries of
all components. Rather, the procedures were designed for
moderate recovery of a broad spectrum of organic chemicals.
The results of the analyses thus may sometimes reflect only
a minimum of. the amount present In the sample.
The procedure is designed to allow detection limits as low as
10 ppm for base/neutrals. Some samples, however, may contain
high concentrations of chemicals that interfere with the
III-105
-------
analysis of other components at low levels. The detection
limits 1n those cases may be significantly higher. Percent
recovery and standard deviation Information on the use of
this procedure 1n a single laboratory is presented in
Table 3.
TABLE 3. RECOVERY DATA FROM SOIL BY REGION IV MEDIUM
CONCENTRATION HAZARDOUS WASTE METHOD3
Base/Neutral
Fraction
Cone.
ug/gm
Avg.
% Rec.
Std.
Dev.*
1,3-Dichl orobenzene 20 92 8.2
Bis[2-chloro1sopropyl] ether 20 83 2.8
1, 2, 4-Tr1chl orobenzene 20 94 2.8
Naphthalene 20 90 2.9
2-Chloronaphthalene 20 78 10
Dimethyl phthalate 20 79 2.9
4-Chlorophenyl phenyl ether 20 93 1.4
Di ethyl phthalate 20 83 2.1
4-Bromophenyl phenyl sther 45 ?1 - 0.47
Dibutyl phthalate 20 99 8.6
Butyl benzyl phthalate 20 "5 9.3
Chrysene 20 90 13
Di-n-octyl phthalate ' 20 53 11
Benzo[a] pyrene 40 92 25
BenzoCghi] perylene 20 89 30
Acenaphthylene 20 83 4.1
1,2-Di phenyl hydrazlne 20 85 9.8
Phenanthrene 20 85 3.4
Fluoranthene 20 85 4.1
Benzldine 20 47 4.1
Benzanthracene 20 86 4.3
Ideno[l,2,3-cd] pyrene 30 83 4.1
* ± one standard deviation based on three trials.
1.1.5 Sample Preparation
Place the sample container Into the glovebox. Additional
Items that should be 1n the glovebox Include (1) calibrated,
tared 20-ml vials with caps, (2) a spatula, (3) a balance,
(4) a capped vial containing 10 ml of Interference-free
methanol, (5) a vial of water, and (6) an eye dropper. The
vial of methanol 1s to be used as a method blank. (One
method blank should be run for each batch of 20 samples or
less.) Open the sample transoortatlon can and remove the
sample container. Note and record the physical state and
appearance of *he -amole, !f the samole bottle 1s broken,
Immediately repackage the sample and terminate the analysis.
-------
Open the sample bottle and mix the sample. If the sample is
a liquid, transfer one drop to a vial containing water to
determine whether the sample is aqueous or non-aqueous.
Record the result. Transfer approximately 1 gram (or 1 ml)
of the sample to a calibrated and tared 20-ml vial. Wipe the
mouth of the vial with tissue to remove any excess sample
material. Cap the vial. Record the exact weight of sample
taken. Reseal the original sample and replace it in the
original packaging.
Proceed with a methylene chloride extraction of the base/
neutral compounds in the sample based on the miscibility of
the original sample with water. Follow paragraph (a) for an
aqueous sample, paragraph (b) for a non-aqueous sample, and
paragraph (c) for a solid sample.
a. If the sample has been determined to be aqueous, dilute
the sample with 10 ml of methylene chloride. Cap the
vial and shake the sample for two minutes. Add 2 grams
of anhydrous sodium sulfate to the vial to absorb all
water. Shake the sample.
b. If the sample was determined to be non-aqueous, dilute
the sample to 5 final volume of 10 ml with methylene
chloride. Cap the vial and mix for two minutes. Add one
gram of anhydrous sodium sulfate to absorb all water.
Shake the sample.
c. If the sample is a solid matrix, add 10 ml methylene
chloride. Cap the sample and shake for one hour on a
wrist-action shaker. Add one gram of anhydrous sodium
sulfate to the sample and thoroughly mix.
Withdraw a sample of the organic extract with a syringe for
analysis as described in paragraph 1.1.6.
1.1.6 Screening of Base/Neutral Extracts
The base/neutral extracts should be screened by GC/FID using
appropriate columns. If packed columns are used, the
extracts must be screened on both base/neutral and acid GC
columns specified in Method 625.*
Using the GC analytical conditions given in Reference 1,
analyze the base/neutral extracts by GC/FID. Standardize the
GC/FIO for full-scale response with 40 ng/ul of djQ-phen-
anthrene for .the base/neutrals.
If the response of any component is greater than 25% of the
resoonse of the di^-phenanthrene, analyze the extract (para-
graph 1.1.5) by GCVMS.— In some cases 1t may be necessary to
dilute the extract prior to analysis. If no "ssponse exceeds
III-107
-------
25% of the response of the d^o-phenanthrene, concentrate the
extract under a gentle stream of nitrogen to 1 ml and perform
the GC/MS analysis.
1.1.7 GC/MS Analysis of the Base/Neutral Fraction
Establish Instrument operating conditions equivalent to those
provided below:
Mass Spectrometer
Mass Range m/e 41-475
Scan Time 7 seconds or less
Electron Energy 70 eV
Source Temperature 280-300eC
Start Acquisition 0.1 min after stopping
flow.
Column Conditions
Column: 1.8 m glass (6.44 mm O.D. x 2 mm I.D.), packed with 3%
SP-2250 coated on 100/120 mesh Supelcoport. carrier gas:
helium at 30 ml/min. Temperature program: isothermal for 4
•nin it SO'C, then increasing at 8*/m1n to 270°C, and hold at
270°C for 30 min. If desired, capillary or SCOT columns may
be used.
Program the GC/MS to operate in the Extracted Ion Current
Profile (EICP) mode, and collect EICP's for the three char-
acteristic ions listed in Table 4 for eacn compound oeing
quantltated. Operating in this mode, calibrate cne system
response for each compound using either the internal or
external standard procedure.
If the internal standard approach 1s being used, the analyst
should not add the standard to the sample extracts until
immediately before Injection into the instrument. Mix the
extract thoroughly before withdrawing an aliquot for
analysis. Inject 2 to 5 ul of the sample extract. The
solvent-flush technique 1s preferred.
If external calibration is employed, record the volume of
extract and standard solution injected to the nearest
0.05 ill. If the response for any ion exceeds the linear
range of the system, dilute the extract and reanalyze.
When the extracts are not being used for analyses, they should
be stored in vials with unpierced septa in the dark at 4°C.
Proceed to Subsection K for qualitative Identification
criteria and calculation of the results.
III-108
-------
TABLE 4. CHARACTERISTIC IONS FOR BASE/NEUTRAL EXTRACTABLES
Characteristic Ions
Compound
1,3-Dichl orobenzene
1 ,4-Di chl orobenzene
Hexachi oroethane
Bi s[2-chl oroethyl ]ether
1 ,2-Dichl orobenzene
Bis[2-chloroisopropyl]-
ether
N-Ni trosoai -n-propy i ami ne
Isophorone
Nitrobenzene
Hexachi orobutadi ene
1 ,2,4-Trichlorobenzene
Naphthalene
B1 s[2-chl oroethoxy]-
me thane
Hexachi orocycl opentadi ene
2-Chloronaphthalene
Acenaphthylene
Acenaphthene
Dimethyl phthalate
2,6-Dinitrotoluene
Fluorene
Electron Impact
146
146
117
63
146
45
130
32
77
225
180
126
83
237
162
152
154
63
. 165
166
148
148
201
83
148
77
42
95
123
223
182
129
95
235
164
151
153
194
63
165
113
113
199
95
113
79
101
138
65
227
145
127
123
272
127
153
152
164
121
167
Chemical lonization
(methane)
146
146
199
63
146
77
--
129
124
223
181
129
65
235
163
152
154
151
83
166
148
148
201
107
148
135
—
157
152
225
182
157
107
237
191
153
155
163
211
167
150
150
203
109
150
137
—
175
164
227
209
169
137
239
203
181
183
164
223
195
(continued)
III-109
-------
TABLE 4. (Continued)
Characteristic Ions
Compound
4-Chl orophenyl phenyl ether
2,4-Dinitrotoluene
1,2-Di phenyl hydrazine*
Di ethyl phthalate
N-Ni trosodi phenyl ami ne**
Hexachl orobenzene
4-Bromophenyl phenyl ether
Phenanthrene
Antnracene
Oibutyl pnthalate
Fluoranthene
Pyrene
Benzldine
Butyl benzyl phthalate
B1s[2-ethylhexyl]-
phthalate
Chrysene
Benzo[a]anthracene
3,3-Dichlorobenzidine
D1-n-octyl phthalate
Benzo[b]fluoranthene
Benzo[k]fluoranthene
Electron Impact
204
165
77
149
169
284
248
178
173
149
202
202
184
149
149
228
228
252
149
252
252
206
89
93
177
168
142
250
179
179
150
101
101
92
91
167
226
229
254
—
253
253
141
163
105
150
167
249
141
176
175
104
100
100
185
—
279
229
226
126
--
125
125
Chemical lonization
(methane)
--
183
185
177
169
284
249
i78
173
149
203
203
185
149
149
228
228
--
—
252
252
—
211
213
223
178
286
251
179
173
205
231
221
213
299
--
229
229
—
--
253
253
--
223
225
251
198
288
277
207
207
279
243
243
225
327
--
257
257
--
--
281
281
(continued)
III-110
-------
ssaasaasaaasaasaaaaasaaaaaasa
TABLE 4. (Continued)
SSSBSSBSBaBBas-=SS = SSSBaS8a =
Characteristic Ions
Compound
Benzo[a]pyrene
Indeno[l ,2 ,3-cd]pyrene
Dibenzo[ah]anthracene
Benzo[ghi]perylene
N-Ni trosodimethyl ami ne
Bis[ch1oromethy1]ether
2,3,7,8-Tetrachlorodibenzo-
Electron Impact
252
276
278
276
42
45
__
253
136
139
138
74
49
322
125
277
279
277
44
51
320
Chemical lonization
(methane)
252 253
276 277
278 279
276 277
...
—
59
281
305
307
305
--
--
— .
p-dioxin
168
94
80
Oeuterated anthracene
[d-10]***
=====================================================
*Detected as azooenzene
**Detected as diphenylanrine
***Suggested internal standard
189 217
==========================
III-lll
-------
2.1 Analysis of Methylene Chloride Extracts of Aqueous Samples for
Base/Neutral Compounds
Analytical Procedure: available
Sample Preparation: available
2.1.1 Reference
U.S. Environmental Protection Agency, "Semi-Yolatiles
Determination." Method 625. Federal Register 44
No. 233:69540-69551. December 3, 1979.1 ~
2.1.2 Method Summary
The pH of a one-liter water sample is adjusted to a pH of 11
or greater and the sample is extracted with methylene chlor-
ide. Following extract concentration, the sample is analyzed
on a calibrated GC/MS.
2.1.3 Applicability
This method is applicable to the determination of those
compounds listed 1n Table 1 when they occur in aqueous
samples such as -nunicipal and industrial discnarges. The
method is designed to be used to meet the monitoring require-
ments of the National Pollutant Discharge Elimination System
(NPDES).
TM3 method should be restricted to use by, or under the
supervision of, analysts experienced 1n the operation of gas
chromatograph/mass spectrometers and skilled in the inter-
pretation of mass spectra.
2.1.4 Precision and Accuracy
Precision and accuracy performance data for this procedure,
based on the analysis of reagent water and wastewater in a
single laboratory, are summarized in Table 5.
2.1.5 Separatory Funnel Sample Extraction
Samples may be extracted by separatory funnel techniques or
with a continuous extractor. Where emulsions prevent accept-
able solvent recovery with the separatory funnel technique,
the continuous extraction technique is recommended.
Mark the water meniscus on the side of a one-liter sample
bottle for later determination of the volume extracted. Pour
the entire sample Into a 2-1 separatory funnel. Adjust the
pH if khe samel? with 6 N MaOH to 11 or greater, Thorouahlv
mix the sample and measure the pH to ensure that it 1s 11 or
greater.
m-112
-------
TABLE 5. ACCURACY AND PRECISION FOR BASE/NEUTRAL EXTRACTABLES
r====z================s:=s==z=======================«============
Reagent Water Hastewater
Parameter
Acenaphthene
Acenaphthylene
Aldrin
Anthracene
Benzo[a]anthracene
Benzo[b]f 1 uoranthene
Benzo[k]f 1 uoranthene
Benzo[ghi]perylene
Benzo[a]pyrene
Benzidine
Butyl benzyl phthalate
Beta-BHC
Delta-BHC
Bi s[2-chl oroethoxylmethane
Bi s[2-chl oroethyl ]ether
3ix[2-chloroisopropy1 ]ether
Bi s[2-ethyl hexyl ]phthal ate
-------
TABLE 5. (Continued)
===============================================================================
Reagent Water Wastewater
AverageStandardAverageStandard
Parameter % Recovery Deviation % % Recovery Deviation %
Indeno[l ,2 ,3-cd]pyrene
Isophorone
Naphthalene
Nitrobenzene
N-Nitrosodi-n-propyl ami
N-Nitrosodiphenylamine
PCB-1221
PCB-1254
Phenanthrene
Pyrene
1 ,2 ,4-Trichl orobenzene
65
75
6
72
ne 68
84
77
80
84
86
64
37
33
32
31
39
24
11
13
14
15
16
===================
81
77
75
82
76
86
__,
_..
76
80
69
43
42
35
54
45
31
--
__
22
23
26
Enrichment concentrations ranged from 5 to 2400 ug/1
Add 60 ml methylene chloride to the original sample bottle.
Cap the bottle and shake for 30 seconds to rinse the container.
Transfer the solvent into the separatory funnel and extract
tne sample by shaking for two minutes with periodic venting to
release excess vapor 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 size of the solvent layer,
mechanical techniques such as stirring, filtration of the
emulsion through glass wool, or centrifugation should be
attempted. Collect the methylene chloride extract in * 250-ml
Erlenmeyer flask. If the emulsion can not be broken and/or
the amount of solvent recovered is less than 80% (after cor-
recting for water solubility) of that Initially added, the
sample, solvent, and emulsion should be transferred into a
continuous extractor to complete the extraction process
(paragraph 2.1.6).
Add a second 60-ml portion of methylene chloride to the
original sample container. Rinse the container and transfer
the solvent to the sample in the separatory funnel. Extract
for an additional two minutes and combine extracts 1n an
Erlenmeyer flask.
Repeat the extraction process a third time with a final 60 ml
portion of methylene chloride. Combine the extracts 1n the
Erlenmeyer flask. (The sample can be discarded or retained
for extraction of the acidic organic compounds following ?K
adjustment.)
TTT.IId
-------
Pour the combined extracts through a drying column containing
7 to 10 cm of anhydrous sodium sulfate, and collect it in a
500-ml K-D flask equipped with a 10-ml concentrator tube.
Rinse the Erlenmeyer flask with 20 to 40 ml of methylene
chloride. Pour the rinse through the drying column and combine
with the sample extract. Proceed to paragraph 2.1.7.
2.1.6 Continuous Sample Extraction
Place 100 to 150 ml of methylene chloride in the extractor
and 200 to 500 ml of methylene chloride in the distilling
flask. Add the aqueous sample (pH 11 or greater) to the
extractor. Add distilled water as necessary to operate the
extractor and extract for 24 hours. Remove the distilling
flask and pour the contents through a drying column contain-
ing 7 to 10 cm of anhydrous sodium sulfate. Collect the
extract in a 500-ml K-D evaporator flask and label as the
base/neutral fraction.
2.1.7 Sample Extract Concentration
Equip the K-D flask with a 10-ml concentrator tube. Add 1 *o
2 clean boiling chips to the flask and attach a three-ball
macro-Snyder column. Pr-swet the Snyder column by adding
about 1 ml of methylene chloride through the top. Place the
K-D apparatus on a warn water bath (50 to 65*C) so that the
concentrator tube is partially immersed in the water and the
entire lower rounded surface of the flask is bathed with
water*vapor. Adjust the vertical position of the aoparatus
and the water temperature as required in order to complete
the concentration process in 15 to 20 minutes. At the proper
rate of distillation, the balls of the column actively
chatter but the.chambers do not flood. When the liquid has
reached an apparent volume of 1 ml, remove the K-D apparatus
and allow the solvent to drain for at least 10 minutes while
cooling. 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.
Add a clean boiling chip and attach a.two-ball micro-Snyder
column to the concentrator tube. Prewet the column by adding
about 0.5 ml methylene chloride through the top. Place the
K-D apparatus on a warm water bath (60 to 65*C) so that the
concentrator tube is partially Immersed in the water. Adjust
the vertical position of the apparatus and the water temper-
ature as necessary to complete the concentration process in 5
to 10 minutes. At the proper rate of distillation, the balls
of the column actively chatter but the chambers do not flood.
When the liquid caches an aooarent volume of aooroximately
0.5 ml, remove the K-D apparatus from the water bath and
aV.ow the solvent to drain and cool for at least 10 minutes.
III-115
-------
Remove the micro-Snyder column and rinse its lower joint into
the concentrator tube with approximately 0.2 ml of methylene
chloride. Adjust the final volume to 1.0 ml, seal, and label
as the base/neutral fraction.
Determine the original sample volume by refilling the sample
container to the meniscus mark and transferring the liquid to
a 1,000-ml graduated cyclinder. Record the sample volume to
the nearest 5 ml.
2.1.8 GC/MS Analysis of the Base/Neutral Fraction
At the beginning of each day that base/neutral analyses are
to be performed, inject 100 ng of benzidine, either separ-
ately or as part of a standard mixture that may also contain
50- ng of DFTPP, into the instrument. The tailing factor for
benzidine, calculated as indicated in Figure 2, should be
less than 3.
Establish instrument operating conditions equivalent to those
provided below:
Mass Spectrometer
Mass Range m/e 41-475
Scan Time 7 seconds or less
Electron Energy 70 eV
Jource Temperature 230-200*0
Start Acquisition 0.1 min after stooping flow.
Column Conditions
Column: 1.8 m glass column (6.4 mm O.D. x 2 mm I.D.), packed with
3% SP-2250 coated on 100/120 mesh SupeTcoport. Carrier gas:
helium at 30 ml/min. Temperature program: isothermal for 4
min at 50'C, then increasing at 8°C/min to ?70°C, and hold
at 270eC for 30 minutes. If desired, capillary or SCOT
columns may be used.
Program the GC/MS to operate in the Extracted Ion Current
Profile (EICP) mode, and collect EICP's for the three char-
acteristic Ions listed in Table 4 for each compound being
quantitated. Operating in this mode, calibrate the system
response for each compound using either the internal or
external standard procedure.
If the internal standard approach is being used,, the analyst
should not add the standard to the sample extracts until
immediately before Injection
-------
If external calibration is employed, record the volume of
extract and standard solution injected to the nearest
0.05 ul. If the response for any ion exceeds the linear
range of the system, dilute the extract and reanalyze.
When the extracts are not being used for analyses, tfiey
should be stored in vials with unpierced septa in the dark at
4'C.
Proceed to Subsection K for qualitative identification
criteria and calculation of the results.
III-117
-------
3.1 Analysis of Sediment Samples for Base/Neutral Compounds
Analytical Procedure: available
Sample Preparation: available
3.1.1 Reference/Title
U.S. Environmental Protection Agency, "Extraction and Analysis
of Priority Pollutants in Sediments." PPS-9/80, U.S. EPA,
Region IV, S&A Division, Athens, Georgia. 7 pp. (1980).4
3.1.2 Method Summary
A 30-gram sample is mixed with anhydrous sodium sulfate and
extracted with 1:1 acetone:hexane using an ultrasonic probe.
The base/neutral extract 1s washed with water to remove the
acetone, dried, and concentrated. The extracts are screened
on GC/FID and analyzed on GC/MS if peaks are noted on the FID
chromatogram.
3.1.3 Applicability
This method covers the determination of priority pollutants
in soils and sediment. The limit of detection for this
method is usually dependent upon the level of interferences
rather than instrumental limitations. Where interferences
are not a problem, the limit of detection fo^ most compounds
analyzed by GC/MS is 1,000 ug/kg.
This method is recommended for use only by experienced
residue analysts or under the close supervision of such
qualified persons.
3.1.4 Precision and Accuracy
This information is not presently available.
3.1.5 Sample Preparation
Decant and discard the water layer over the sediment. Mix
samples thoroughly, especially composited samples. Discard
any foreign objects such as sticks, leaves and rocks.
Weigh 30 g of sample Into a 400-ml beaker and add 30 g of
anhydrous sodium sulfate. Mix well and allow to dry to a
sandy texture.
Immediately after.weighing the sample for extraction, weigh
5 to 10 g of the partially-dried sediment into a tared cruci-
ble. Determine the percent solids by drying overnight at
103'C to 105°C. Allow ;o cool In a desiccator for half an
hour before weighing. If percent volatile solids are to be
TIT-118
-------
determined, place the oven-dried sample into a muffle furnace
and ignite at 550DC for 60-minutes. Allow to cool in a
desiccator before weighing.
Add 100 ml of 1:1 acetone:hexane to the sampTe-sodium sulfate
mixture. Place a sonification probe about 1 cm below the
surface of the solvent but above the sediment layer. Sonicate
for 3 minutes at full power with pulse set at 50%. Decant the
solvent into a Biichner funnel. Add 100 ml of 1:1 acetone:
hexane. Sonicate for 3 minutes at full power with the pulse
set at 50%. Decant the solvent into the Buchner funnel. Add
a third 100-ml portion of 1:1 acetone:hexane to the residual
sample. Sonicate for 3 minutes at full power with a 50%
pulse.
Pour the entire sample into the Biichner funnel and rinse with
hexane.
Concentrate the B/N extract to about 100 ml.
3.1.6 Extract Cleanup
Transfer the extract to a 500-ml separatory funnel containing
250 ml of distilled water and 25 ml of saturated sodium sul-
fate solution. Chake the separatory funnel for 2 minutes.
Drain the water layer into a clean beaker and the hexane
layer into a clean 250-ml separatory funnel.
Transfer the ^atsr *ntc the 500-nil separatory funnel and
re-extract with 25 ml of methylene chloride by shaking the
separatory funnel for 2 minutes. Combine extracts.
Repeat the process by adding an additional 25 ml of methylene
chloride to the separatory funnel and extracting for an
additional 2 minutes. Transfer the solvent phase to the
250-ml separatory funnel containing the first two extracts.
Wash the extracts with 2 x 100 ml of distilled water. Pass
the solvent extract through a drying column packed with 7 to
10 cm of organic-free anhydrous sodium sulfate and an inch of
glass wool, solvent-rinsed with methyl alcohol, acetone, and
hexane.
Concentrate the extract to 10 ml with a K-D apparatus on a
steam bath. Remove the concentrator tube and concentrate the
extract to 1 ml with nitrogen.
Remove one-half of the extract and place in a GC vial.
Dilute to 1 ml with methylene chloride. This is the B/N
fraction of the sample, '.ibel the volume as ? ml.
III-119
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3.1.7 Gas Chromatography/Flame lonization Screening of the
Base/Neutral Extract
Calculate the FID response of the Instrument to a 50-ng
Injection of hexachlorobenzene (HCB). {The GC/MS requires
about 50 ng HCB to give a complete mass spectra.) Screen the
prepared base/neutral extract on GC/FID.
Calculate the concentration of the sample peak that produces
the greatest Instrument response above that observed for the
HCB standard.
If the concentration 1s less than 1,000 ug/kg, report the
sample concentration as <1,000 ug/kg.
If the concentration is greater than 1,000 ug/kg, analyze the
base/neutral extract by GC/MS (paragraph 3.1.8).
If the sample peaks 1n the chromatogram are less than the
responses produced by the HCB standard, record the sample
concentrations as less than the minimum detection limit.
Analyze all reolicate samples, blanks, and spiked samples in
a similar fashion. Record the variability, spike recovery,
and blank analysis information in a QC log book.
3.1.8 Gas Chromatography/Mass Spectroscopy Analyses of the
Base/Neutral Extracts
At the beginning of each day that base/neutral analyses are
to be performed, inject 100 nanograms of benzidine either
separately or as part of a standard mixture that may also
contain 50 ng of DFTPP. Calculate the tailing factor as
shown 1n Figure 2 and discussed elsewhere.5 The tailing
factor for benzidine should be less than 3.
Establish chromatographic conditions equivalent to those
presented 1n Table 6. Included 1n these tables are estimated
retention times and sensitivities that can be achieved by
this method. An example of pre-separation achieved by this
column is shown 1n Figure 3.
Establish the GC/MS operating conditions Indicated below:
Mass Range m/e 41-475
Scan Time 7 seconds or less
Electron Energy 70 eV
Source Temperature 280-300eC
Start Acquisition 0.1 minute after stopping flow.
Program the GC/MS to operate in the Extracted Ion Current
Profile uICP) mode, and collect ZIC? for the thj-ee ions
III-120
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TABLE 6. GAS CHROMATOGRAPHY OF BASE/NEUTRAL EXTRACTABLES
==============s==========s:=============================================
Retention Time (fl)
Compound (minute) Limit of Detection
1,3-Dichlorobenzene
1,4-Dichl orobenzene
Hexachloroethane
Bi s[2-chl oroethyl ]ether
1 ,2-Dichl orobenzene
81 s[2-chl oroi sopropyl ]ether
N-Ni trosodi-n-propyl ami ne
Nitrobenzene
Hexachlorobutadiene
1 ,2 ,4-Trichl orobenzene
Isophorone
Naphthalene
Bis[2-chloroethoxy]me thane
Hexachl orocycl opentadiene
2-Chloronaphthalene
Acenaphthylene
Acenaphthene
Dimethyl phthalate
2,6-Dinitrotoluene
Fluorene
4-Chlorophenyl phenyl ether
2,4-Dinitrotoluene
1 ,2-Di phenyt hydrsrrine'0'
Di ethyl phthalate
N-NitrosodiphenyTaminefd)
Hexachl orobenzene
4-Bromophenyl phenyl ether
Phenanthrene
Anthracene
Di-n-butyl phthalate
Fluoranthene
Pyrene
Benzidine
Butyl benzyl phthalate
Bi s[2-ethyl hexyl ]phthal ate
Chrysene
BenzoFalanthracene
3,3'-Dichlorobenz1dine
Di-n-octyl phthalate
Benzo[b]f 1 uoranthene
Benzo[k]fl uoranthene
Benzo[a]pyrene
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
13.3
18.7
19.3
19.5
19.8
20.1
20.1
20.5
21.0
21.2
22.8
22.8
24.7
26.5
27.3
28.8
29.9
30.6
31.5
31.5
32.2
32.5
34.9
34.9
36.4
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
(continued)
III-121
-------
TABLE 6. (Continued")
8=======================================================================
Retention Time (a)
Compound (minute) Limit of Detection vb)
Indeno[l,2,3-cd]pyrene
Di benzo[ah]anthracene
Benzo[ghi]perylene
N-Nitrosodimethyl amine
2 ,3 ,7 ,8-Tetrachl orodibenzo-p-
dioxin
42.7
43.2
45.1
50
50
50
25
25
25
(a)Co1umn: 1.8 m glass (6.4 mm O.D. x 2 mm I.D.), packed, with 3% SP-2250 coated
on 100/120 mesh Supelcoport. Carrier gas: helium at 30 ml per min.
Temperature program: isothermal for 4 minutes at 50"C, then 8°C per min to
270eC; hold at 270°C for 30 minutes. If desired, capillary or SCOT columns
may be used.
(b'This is a minimum level at which the entire analytical system must give
mass spectral confirmation. (Nanograms injected is based on a 2-ul
injection of a 1-1 sample that has been extracted and concentrated to a
.-olume of 1.0 ml.)
(c)[)etected as azobenzene.
.^Detected as diphenyl amine.
listed in Table 4 for each compound being measured. Opera-
ting in this mode, calibrate the system response for each
compound by using either the internal or external standard
procedure.
If the internal standard approach is used, the standards
should not be added to the sample extracts until immediately
before injection into the instrument. Mix the spiked extract
thoroughly. Inject 2 to 5 ul of the sample extract. The
solvent-flush technique is the preferred procedure.
If the external calibration approach is used, record the
volume of extract injected to the nearest 0.05 ul.
If the instrument response for any ion exceeds the linear
range of the system, dilute the extract as necessary and
reanalyze.
Qualitative identification criteria and calculations are
described in Subsection K. When the extracts are not being
used for analysis, store them in vials with unpierced septa
in the dark at 4*C.
HI-122
-------
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3.2 Analysis of Methylene Chloride Sediment Extracts for Base/Neutral
Compounds
Analytical Procedure: available
Sample Preparation: available
3.2.1 Reference
Jacobs Engineering Group, "Manual of Methods for the Analyses
of Hazardous Wastes." Contract Report 68-03-2569 prepared for
Environmental Protection Agency, Environmental Monitoring
Systems Laboratory, Las Vegas, Nevada. Jacobs Engineering
Group, Pasadena, California. 1981.6
3.2.2 Method Summary
A 50-g sample of soil/sediment is extracted with methylene
chloride using wet residual waste/solvent techniques. Aided
by a high-speed homogenizer, samples are extracted at pH 11
to isolate the base/neutral compounds. The extract is
cleaned up using gel permeation chromatography.
3.2.3 Applicability
The procedure is for use with solid-phase samples such as soil
and sediment. The detection limit of the procedure will be
influenced by sample size, co-extracted materials, and sample
cleanup.
3.2.4 Precision and Accuracy
This information is not presently available.
3.2.5 Sample Preparation
Thoroughly mix the sample by homogenizing it in the original
sample bottle. Weigh into a 200-ml centifuge bottle a 50-g
aliquot or an appropriate weight based on screening analysis.
Add surrogate standards and mix the aliquot to be analyzed.
Adjust the pH of the sample with 10 N sodium hydroxide to a
pH of 11 or greater. Mix briefly with the homogeni-zer to
ensure uniform sample pH.
Add 60 ml of methylene chloride to the sample bottle and
homogenize briefly. Rinse the homogenizer off with a minimum
of water and then with 5 to 10 ml of methylene chloride.
Additional methylene chloride may be added until the liquid
surface 1n the centrifuge bottle is close to the top.
Centrifuge the sample for 15 minutes. The mixture wiil
separate into an aqueous "layer over the methyl *>ne chloride
extract. A solid cake or emulsion may form at the water-
methyl ene chloride interface. If the emulsion interface
III-124
-------
between layers is more than one-half the size of the solvent
layer, a smaller sample size should be used to complete the
phase separation. Withdraw the organic extract from the
centrifuge bottle with a 50-ml glass syringe that has been
equipped with a 150-mm x 5-mm I.D. TFE tube. Discharge the
extract into a 300-ml beaker.
Repeat the sample extraction procedure a second time with a
60-ml portion of methylene chloride. Combine the extracts.
Perform a third extraction with a final 60-ml portion of
methylene chloride and combine the extracts.
3.2.6 Sample Extract Drying
Pour the combined extract resulting from the extraction proce-
dure through a drying column containing 7 to 10 cm of organic-
free anhydrous sodium sulfate.. Collect the dried extract in a
500-ml Kuderna-Danish flask equipped with a 10-ml concentrator
tube.
Wash the flask that originally contained the extract and the
drying tube three times with 30-ml aliquots of methylene chlo-
ride. Add these washes to the sample extract in the Kuderna-
Danish flask.
3.2.7 Sample Extract Concentration
Add one or two clean boiling chips to the flask anoV attach a
three-bal" siacro-Snyder column. P'-swet the column by adding
approximately 1 ml of the extracting solvent through the top
of the column. Place the apparatus in a 60 to 65"C water
bath so that the concentrator tube is partially immersed in
the water and the lower rounded surface of the flask is
bathed with water vapor. Adjust the apparatus as necessary
to complete concentration to approximately 10 ml in 15
minutes. (At the proper rate of distillation, the balls of
the column will chatter but the chambers will not flood.)
Remove the Snyder column, and rinse the flask and its lower
joint into the concentrator tube with 1 to 2 ml of methylene
chloride employed in the extraction.
Fit the concentrator tube with a modified macro-Snyder column.
Organic-free nitrogen is employed to reduce the volume of the
extract to approximately 5 ml or 1 ml (but not below 0.5 ml).
Wash the concentrator tube with two 0.2-ml volumes of methylene
chloride.
Adjust the final sxtract volume to S TI"! or 1 ml eor subse-
quent internal standard addition and GC/MS analysis. If the
111-125
-------
extract obtained above 1s "clean," then a final extract
volume of 1 ml 1s required.
3.2.8 Gel Permeation Cleanup
Determine the residue weight of the concentrated sample
extract by placing a 1-ml aliquot on a tared aluminum foil
pan, allowing the solvent to evaporate, and reweighing the
pan. These results are used to determine the volume of
extract to be applied to the column for cleanup. The volume
of extract applied to the column should not exceed the
capacity of the column, approximately 200 mg. If the residue
weight is on the order of 1 to 5 mg, cleanup by gel permea-
tion can, 1n many cases, be avoided.
Transfer 5 ml of the GPC calibration solution to the Bio-
Beads S-X3 column." Drain the column Into a 100-ml graduated
centrifuge tube until the liquid is just above the surface of
the GPC packing. Wash the calibration solution on the column
with several 1-ml aliquots of methylene chloride. Elute the
columns with 200-ml aliquots of methylene chloride and
collect 10-ml fractions.
'Analyze the fractions for bis[2-ethylhexyl]phthalate and
pentachlorcphenol by GC/FID on a 1 percent SP-1240 DA column.
Determine the corn oil elution pattern by evaporation of each
fraction to dryness followed by gravimetric determination of
*he residue. Plot the concentration of each component in
each fraction versus the total eluant volume.
The first fractions of the eluant that represent an approximate
85 percent removal of the corn oil and 85 percent recovery
of the bis[2-ethylhexyl]phthalate can be discarded. Collect
the fractions that elute up to a retention volume represented
by 50 ml after the elution of pentachlorophenol. (Typical
procedures are to discard the first 60 ml, to collect the
next 110 ml, and to wash the column with 250 ml of methylene
chloride between samples.)
Select a volume of sample extract (based on the residue
weight determination) that will not overload the column.
Apply an aliquot (1 to 4 ml) of the extract to the column and
drain the column until the sample 1s just above the surface
of the GPC packing. Wash the extract onto the column with
several 1-ml portions of methylene chloride. Elute the
column with 200-ml aliquots of methylene chloride.
Collect the first 60 ml of eluant 1n a 100-ml graduate
cylinder »,nd nass the next 110 ml of eluant through a drying
column containing 6 cm of anhydrous soaium suifate and
et In a 500-inl Kuderna-Oanish flask equipped with 10-ml
II I-i.26
-------
concentrator tube. Rinse the drying column with three 25-ml
portions of methylene chloride.
Add one or two clean boiling chips to the flask and attach a
three-ball macro-Snyder column. Prewet the column by adding
approximately 1 ml of the extracting sol vent, through the top
of the column. Place the apparatus in a 60 to 65°C water
bath so that the concentrator tube is partially immersed in
the water, and the lower rounded surface of the flask is
bathed with water vapor. Adjust the apparatus to complete
concentration to approximately 10 ml in 15 minutes. (At the
proper rate of distillation, the balls of the column will
chatter but the chambers will not flood.)
Remove the Snyder column, and rinse the flask and its lower
joint into the concentrator tube with 1 to 2 ml of the
solvent employed in the extraction.
Fit the concentrator tube with a modified macro-Snyder
column. Organic-free nitrogen is employed to reduce the
volume of the extract to approximately 5 ml or 1 ml (but not
below 0.5 ml). If the extract obtained above is "clean,"
then a final extract volume of 1 ml is required. After the
desired volume '">as been reached, wash the Snyder column joint
and the concentrator tube with two 0.2-ml volumes of the
extracting solvent. Adjust the final extract volume to 5 ml
or 1 ml for subsequent internal standard addition and GC/MS
analysis.
If the extract is to be stored before GC/MS analysis, trans-
fer the extract to an appropriately sized serum vial equipped
with a Teflon-lined rubber septum and crimp cap. The extract
volume should be scored on this vial, and appropriate sample
identification must be affixed to the vial. Store the
extract in the dark at 4°C.
It is possible that samples which contain high concentrations
of extractable organic compounds will not be amenable for
concentration to 5 ml. For extracts of this type, the final
volume after concentration should be adjusted to a minimal
volume which results in an extract viscosity that allows sam
pllng with a micro-syringe. Obvious remedies will likely include
either starting with smaller sample size or concentration to a
volume greater than 5 ml.
Qualitative Identification criteria and calculations are
described in Subsection K.
III-127
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4.1 Analysis of Base/Neutral Compounds in Biological Tissue
Analytical Procedure: available
Sample Preparation: available
4.1.1 Reference/Title
U.S. Environmental Protection Agency, "Extraction and Analyses
of Priority Pollutants in Biological Tissue." Method 10/80.
U.S. EPA, S&A Division, Region IV, Laboratory Services Branch,
Athens, Georgia, p. 7. (1<580).2
4.1.? Method Summary
A 10-g sample of homogenized fish tissue is mixed with 40 g
of sodium sulfate and extracted with methylene chloride using
an ultrasonic probe. The sample is filtered, concentrated
to 10 ml or less, cleaned up using acetonitrile partitioning,
and concentrated to 1 ml. The extract is screened using gas
chromatography and quantified using mass spectrometry.
4.1.3 Applicability
The limit of detection for this method is usually dependent
upon the level of interferences rather than instrumental
limitations. Where interferences are not a problem, the
limit of detection for most compounds analysed Dy GC/MS is
-2 mg/kg (wet weight basis).
The method is recommended for use only by experienced residue
analysts or under the close supervision of such qualified
persons.
4.1.4 Estimates of Precision and Accuracy
No information is presently available.
4.1.5 Sample Extraction
Blend equal amounts of fish tissue and dry ice. If a large
sample is being processed, a food processor or meat grinder
may be convenient.
Weigh 10 g of homogeneous sample into a 400-ml beaker and mix
with 40 g of sodium sulfate. Ensure that the sample is
thoroughly dry.
Add 100 ml of methylene chloride to the tissue mixture.
Place an ultrasonic probe in the mixture and sonicate at 50
percent pulse for 3 minutes. Transfer the methylene chloride
phase to a 500-ml K-D flask.
III-128
-------
Repeat the methylene chloride/sonication extraction of the
tissue sample with a second 100-ml portion of methylene
chloride. Combine the extracts in the K-D flask and extract
the residue a third time with 100 ml methylene chloride.
Combine the extracts.
NOTE: The probe should be carefully cleaned between each
sample as indicated in Subsection C (Interferences).
Add a clean boiling chip to the K-D flask and attach a
three-ball macro-Snyder column. Place the K-D apparatus on a
water bath and concentrate the extract to 10 ml.
Quantitatively transfer the concentrated extract to a 125-ml
separatory funnel. Add enough hexane to bring the final
volume to approximately 15 ml. Extract the sample four times
by shaking vigorously for 1 minute with 30-ml portions of
hexane-saturated acetonitrile.
Combine and transfer the acetom'trile phases to a one-liter sep-
aratory funnel and add 650 ml of distilled water and 40 ml of
saturated sodium chloride solution. Mix thoroughly for 30 to
45 seconds. Adjust the oH of the aqueous phase to 12 and ex-
tract with two iOO-inl portions of .methylene :n!oride. Shane tne
sample vigorously for 15 to 30 seconds during each extraction.
The residual aqueous sample phase may be retained for extrac-
tion of acidic compounds or discarded.
Combine the methylene chloride extracts in a one-liter separa-
tory funnel and wash with two 100-ml portions of distilled
water. Discard the water layer and pour the methylene chloride
layer through a drying column packed with 7 to 10 cm of organic-
free anhydrous sodium sulfate and one inch of glass wool. Col-
lect the extract in a 500-ml K-D flask equipped with a 100-ml
ampul. Rinse the separatory funnel and drying column with
three 10-ml portions of methylene chloride. Add the rinsings
to the K-D flask.
Attach a three-ball macro-Snyder column and place the K-D
apparatus in a hot water bath (60-65*C). Concentrate the ex-
tract to 6 to 10 ml. Use a stream of dry nitrogen to concen-
trate the extract to 1 ml.
Transfer the extract to a GC vial and label as the base/
neutral fraction of the semi-volatile compounds. This
extract is now ready for analysis.
4.1.6 Analysis by Gas Chromatography
The base/neutral extracts are screened on GC/FID using the
appropriate column to determine whether GC/MS analyses are
III-129
-------
necessary. Relative retention times and limits of detection
are summarized in Table 6; a representative chromatogram is
presented in Figure 3.
Calculate the FID response of 50 ng hexachlorobenzene (HCB)
for base/neutral compounds. (The GC/MS requires approxi-
mately 50 ng HCB to give a complete mass spectrum.)
If any sample peaks produce a greater response than hexa-
chlorobenzene, calculate the concentration of the largest
peak.
a. If the calculated concentration of the sample component
is greater than 2 mg/kg (wet weight basis), analyze the
extract by GC/MS.
b. If the calculated concentration of the sample component
is less than 2 mg/kg, report the concentration as less
than 2 mg/kg.
c. If all sample peaks in the chromatogram are less than the
hexachlorobenzene peak, record the minimum method
detection limit for the sample.
Analyze all blanks and spikes and record the pertinent
precision and accuracy data with the sample Information.
4.1.7 Gas Chromatography/Mass Spectroscopy Analysis of the Base/
Neutral Extracts
At the beginning of each day that base/neutral analyses are
to be performed, inject 100 ng of benzidine either separately
or as part of a standard mixture that may also contain 50 ng
DFTPP. The tailing factor for benzidlne, calculated as shown
in Figure 2, should be less than 3.
Establish chromatographic conditions equivalent to those
presented in Table 6. Included in this table is information
on estimated retention times and sensitivities that can be
achieved by this method. An example of the pre-separation
achieved by this column is shown in Figure 3.
Establish the GC/MS operating conditions Indicated below:
Mass Range m/e 41-475
Scan Time 7 seconds or less
Electron Energy 70 eV
Source Temperature 280-300"C
Start Acquisition 0.1 minute after stopping flow.
Program the GC/MS to ooerate in the Extracted Ion Current
Profile (EICP) mode, and collect EICP for the tnree
IH-130
-------
characteristic ions listed in Table 4 for each compound being
measured. Operating in this mode, calibrate the system
response for each compound by using either the internal or
external standard procedure.
If the internal standard approach is used, the standards
should not be added to the sample extracts until immediately
before injection into the instrument. Mix the spiked extract
thoroughly. Inject 2 to 5 yl of the sample extract. The
solvent-flush technique is the preferred procedure.
If the external calibration approach is used, record the
volume of extract injected to the nearest 0.05 yl.
If the instrument response for any ion exceeds the linear
range of the system, dilute the extract as necessary and
reanalyze.
Qualitative identification criteria and quantitative calcula-
tions are described in Subsection K. When the extracts
are not being used ^or analyses, store then in vials with
unpierced septa in the dark at 4°C.
K. QUALITATIVE AND QUANTITATIVE DETERMINATION
To qualitatively identify a compound', obtain an Extracted Ion Current
profile (EICP^ for the primary ion and the two other ions listed in Table 4.
The criteria below must be met for a qualitative identification.
1. The characteristic ions for each compound must have their maxima in the
same or within one scan of each other.
2. The retention time for the experimental mass spectrum must be within ±30
seconds of the retention time of the authentic compound.
3. The ratios of the three EICP peak heights must agree within ±20% with the
ratios of the relative intensities for these ions in a reference mass
spectrum. The reference mass spectrum can be obtained from either a
standard analyzed through the GC/MS system or from a reference library.
4. Structural isomers that have very similar mass spectra can be explicitly
identified only if the resolution between the isomers in a standard mix
is acceptable. Acceptable resolution is achieved if the valley height
between isomers is less than 25 percent of the sum of the two peak heights.
Otherwise, structural isomers are identified as isomeric pairs.
In samples that contain an inordinate number of interferences, the
chemical ionization (CD mass spectrum may make identification easier.
In Table 4, characteristic CI ions for most; of cne compounds are given.
The ':se o-f7 chemical ''onization MS to support EI/MS is encouraged but not
!! 1-1.31
-------
required. When a compound has been identified, the quantification of
that compound will be based on the integrated area from the specific ion
plot of the first listed characteristic ion in Table 4. If the sample
produces an interference for the first listed ion, use a secondary ion to
quantify. Quantification can be done by the external or internal
standard method.
Internal standard - By adding a constant known amount of internal stan-
dard (Cis in yg) to every sample extract, the concentration of contam-
inant (C0) in yg/1 in the sample is calculated using Equation 2.
(As)(C1s)
C0 = '— Eq. 2
(Ais) (RF) (V0)
where:
V0 = the volume (in liters) or mass (in grams) of the original
sample; the other terms are defined in text (Subsection H).
External standard - The concentration of the unknown is calculated from
the slope and intercept of the calibration curve. The unknown
concentration is determined using Equation 3.
(A)(Vt)
ug/1 = ng/ml = Eq. 3
(Vi)(Vs)
where:
' A = mass of compound from the calibration curve (ng)
V-j = volume of extract injected (ul)
Vt = volume of total extract (ul)
Vs = sample volume (ml) or sample mass (g) extracted.
Report all results in yg/1 to two significant figures without correction
for recovery data. When duplicate and spiked samples are analyzed, all
data obtained should be reported.
III-132
-------
REFERENCES
1. U.S. Environmental Protection Agency. "Base/Neutrals, Acids, and
Pesticides - Method 625." Federal Register Vol. 44: No. 233
69540-69552. December 3, 1979.
2. U.S. Environmental Protection Agency. "Extraction and Analyses of
Priority Pollutants in Biological Tissue." Method 10/80. U.S.
Environmental Protection Agency, S&A Division, Region IV, Laboratory
Services Branch, Athens, Georgia. 7 p. (1980.).
3. U.S. Environmental Protection Agency. "Method for Preparation of Medium
Concentration Hazardous Waste Samples." U.S. Environmental Protection
Agency, Region IV, Athens, Georgia. May 1981.
4. U.S. Environmental Protection Agency. "Extraction and Analysis of
Priority Pollutants in Sediments." PPS-9/80, U.S. Environmental
Protection Agency, Region IV, S&A Division, Athens, Georgia. 7 p.
(1980).
5. Eichelberger, J. W., L. E. Harris and W. L. Budde. "Reference Compound
to Calibrate Ion Abundance Measurement in Gas Chromatography - Mass
Soectrometry Systems." Anal. Chem. Vol. 47:995-1000 (1975).
6. Jacobs Engineering Group. "Manual of Methods for the Analysis of
Hazardous Wastes." Contract Report 68-03-2569 prepared for Environmental
Protection Agency, Environmental Monitoring Systems Laboratory, Las
Vegas, Nevada. Jacobs Engineering Group, Pasadena, California. (1981).
III-133
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SECTION 4
PESTICIDES AND POLYCHLORINATED BIPHENYLS
A. SCOPE
The analytical procedures provided 1n Subsection J of this Section cover
the determination of chlorinated pesticides and PCBs in hazardous waste, waste
oil, water, soil/sediment, biological tissue, and air samples. The compounds
of interest are extracted, the extracts are concentrated, and the compounds are
identified/quantified using GC or GC/MS procedures.
B. SAMPLE HANDLING AND STORAGE
Conventional water sampling practices should be followed except that the
bottle should not be prerinsed with sample prior to collection. Grab samples
should be collected in glass containers and refrigerated immediately. Com-
posite samples should preferably be collected in glass containers and, if
possible, refrigerated during the period of compositing. All automatic
sampling equipment must be free of Tygon and other potential sources of
organic contamination. Wh'en necessary, the equipment should be prerinsed with
hexane prior to use.
Water 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, they should be adjusted to a pH range of 5.0 to 9.0 with
sodium hydroxide or sulfuric acid, as appropriate. Record the volume of acid
or base used. If aldrin is to be determined, add sodium thiosulfate when
residual chlorine is present. EPA Methods 330.4 and 330.5 may be used to
measure chlorine residual.1 Field test kits are also available for this
purpose.
Plastic bottles must not be used since they are known to introduce
interferences and absorb pesticides. The sample size is dictated by the
sensitivity required for a particular purpose and the detection system to be
employed. Sufficient sample should be collected to permit the analysis of
duplicate and spiked analyses. Breakage of glass sample bottles can be
minimized by shipping them in expanded polystyrene containers molded to fit
the bottles.
Ml samples must be extracted within 7 days of collection and the
extracts completely analyzed within 40 days or extraction^ (Figure i).
HT-134
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Satole 1 Air I Hater or
Matrix I 1 teachate
nut
Sample Paper
Pro- or
cess Ing Tenax
Cart-
ridge
| Slwdoe. Soil or Sedlwnt
|
\ \ \
r | Preserve Centri-
1
, * ,,
1 Slortl Preserve
Extract]! Extract I 1 Store
*-« I Analyze 11 Analyie I Extract/
7* 1 II 1 Ol«e*t
»-«
in
1 Analyte 1
Ha f Uet Storage 1 Or
Treat- 1 1 Stor
•ent
di
I ^
*
IP f IP [Extract]
lox 1 loxlctty | |
lelt* 1 .
Anal
1 t Hr.ct 1 | Analyie |
Extract
I Analyie j
L— -- - — *
Analyie 1
•lolog- Haiardousl
leal Haste
Tissue |
I i
y frwen Frtutn
age. Storage Storage Store
I
act Extract Extract | Extract 1
jr/e Analyte Analyte 1 | Analyte
Purpose Total Cone. Total "Dissolved* Total Cone. Total Cone. Total Cone. Total Total
In Air Cone. Cone. In Nobility Mobility In Sedlwnt. In Seotccnt. In Sedlxnt. Cone. In Cone. In
In Hater Hater at pM S *t pH S Sludge. Soil. Sludge. Soil. Sludge. Soil. Tissue HasU
S*«plt
Container foil. 6 6 G 6 G G 6 6
Sample
Preser-
vative 4t 4*C 4*C
Storage
T1o» 74 74 74
S*"p1t *
Site la1 it 11 II 10 g 10 g 10 g 10 g 1 •! I fl
Figure 1. Handling and sample storage Information for pesticide and PCB samples.
-------
Sediment/soil samples should also be stored 1n glass containers. To
prevent sample contamination from sample bottle caps or cap liners, the
containers should be sealed with either Teflon or acetone/hexane-washed
heavy-duty aluminum foil. Sediment samples may be stored in a field-moist,
air-dried, or frozen condition if a total sediment concentration is to be
determined. When operational procedures such as the EP Toxicity Test are to
be performed, the samples should be stored in a field-moist condition.
Air can be sampled for PCB analysis using polyurethane cartridges as the
collection medium.3 Place a P-4000 pump and sampling cartridge on a tripod
or other support so that the intake is at least 1 meter above the ground. A
shelter (such as an umbrella) should be used to protect the sampler in the
event of precipitation. Operate the sampler for a minimum of 4 hours. A
nominal flow of 3.8 1/min. will provide a total sampled volume of approxi-
mately 1 m^ in 4 hours. This volume should not exceed the breakthrough
volumes of the cartridges for the compounds to be analyzed. Package and
remove the collected samples from the sampling area.
All polyurethane plugs should be kept in hexane-rinsed aluminum foil and
individual glass containers before and after sampling to minimize possible
contamination.
Oil samples snould oe collected in glass containers with a volume of at
least 20 ml and equipped with Teflon-lined screw caps.4 Prior to sample
collection, the containers should be cleaned in the following manner:
a) - Wash all sample bottles and seals in detergent solution. Rinse with tap
*ater ind l^en distilled water. Allow the bottles and seals to dry in a
contaminant-free area. Rinse the seals with pestle:de-grade hexane ind
allow to air dry.
b) Heat the sample bottles to 400*C for 15 to 20 minutes or rinse with
pesticide grade acetone or hexane and allow to air dry.
c) Store the clean bottles inverted or sealed until used.
Oil samples should be stored in a cool, dry, dark area until analyzed.
Storage times in excess of 4 weeks are not recommended for unknown or
undefined sample matrices.*
C. INTERFERENCES
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.
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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. After the glassware
has dried, heat in a muffle furnace at 400°C for 15 to 30 minutes.
Some thermally stable materials, such as PCBs, may not be elimi-
nated 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 inter-
ference. 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.
1.2 The use of high-purity reagents and solvents helps to minimize
interference problems. When necessary, purification of solvents by
distillation in all-glass systems may be required.
2. Interferences by phthalate esters can pose a major problem in pesticide
analysis when using the electron capture detector. These compounds gener-
ally appear in the chromatogram as large late eluting peaks, especially in
the 15- and 50-percent fractions from Florisil column cleanup. Common
flexible plastics contain varying amounts of phthalates that are easily
extracted or leached during laboratory operations. Cross-contamination of
clean glassware, especially solvent-wetted surfaces, routinely occurs
when plastics are handled during extraction steps. 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.°»? The interferences
from phthalate esters can be avoided by using a microcoulometric or
electrolytic conductivity detector.
3. Matrix interferences may be caused by contaminants that are coextracted
from the sample. The extent of matrix interferences will vary consider-
ably from source to source, depending upon the nature and diversity of
the industrial complex or municipality being sampled. The cleanup
procedures in Subsection J can be used to overcome many of these inter-
ferences, but unique samples may require additional cleanup approaches to
achieve the MDL listed in Table 1.9
4. Interference from fish oil can be eliminated by acetonitrile parti-
tioning. 8 However, the ultrasonic probe must be scrupulously cleaned
between samples. The procedure is:
1) Rinse the probe with solvent into the sample.
2) Remove residue on the probe with a wet tissue.
3) Rinse the probe with methylene chloride.
4) Sonicate with hexane for 3 to 4 minutes on 50 percent pulse.
5. Special attention is called to industrial plasticizers and hydraulic
fluids such as the chlorinated biphenyls which are potential sources of
interference in pesticide analysis.10 Chlorinated biphenyls containing
111-137
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TABLE 1. METHOD DETECTION LIMITS FOR ORGANOCHLORINE
PESTICIDES AND PCBs9 BY ECD GAS CHROMATOGRAPHY
Method
Parameter Detection Limit, ug/1
Aldrin
Alpha-BHC
Beta-BHC
Delta-BHC
Gamma- BHC
Chlordane
4,4'-DDD
4,4'-DDE
4, 4 '-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan Sulfate
Endrin
Endrln aldehyde
Heptachlor
Heptachlor Epoxide
Toxaphene
OC3-1016
PC8-1221
PCB-1232
PCB-1242
PC8-1248
PCB-1254
0.004
• 0.003
0.006
0.009
0.004
0.014
0.011
0.004
0.012
0.002
0.014
0.004
0.066
0.006
0.023
0.003
0.083
0.024
nd
nd
nd
0.065
nd
nd
PCB-1260 nd
================================ ====================
nd * not determined
4 to 8 chlorine atoms per molecule have been reported in extracts of
birds, fish, mussels, and water. Possible Interferences from these
compounds are Indicated by unresolved peaks (shoulders and non-gaussian
peaks) slight discrepancies in retention times, and peaks that elute
later than p,p'-DDT. A number of chlorinated biphenyl Isomers may
Interfere with the determination of DDE, ODD, and DDT Isomers. The
purity of compounds associated with specific peaks 1n the DDT chromato-
gram should be periodically examined by repeating the chromatographic
analysis after conversion of DDT to DDE with KOH in ethanol. Particu-
lar1;-' savere *nter*erencss will require a more detailed examination of
the sample extract.
111-138
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6. The electron capture detector responds to a wide variety of organic
compounds. It is likely that some compounds will be encountered as
Interferences during GC-EC analysis. Periodic mass spectrometric
analyses on composited and/or selected samples will provide for positive
identification of specific compounds and interferents.
D. SAFETY
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 1s 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 sampling and analysis of samples containing these compounds. Addi-
tional references on laboratory safety have been identifiedll-13 for
the information of the analyst.
2. The following parameters covered by this method have been tentatively
classified as known, or suspected, human or mammalian carcinogens:
4,4'-DDT, 4,4i-DDD, and the PC8s. Primary standards of these toxic
compounds should be prepared 1n a glovebox and/or containment laboratory.
3. Diethyl ether should be monitored regularly to determine the peroxide
content. Under no circumstances should diethyl ether be used with a
peroxide content in excess of 50 ppm as an explosion eculd result.
Peroxide test strips manufactured by EM Laboratories (available from
Scientific Products Co., Cat. No. P1126-8, and other suppliers) are
recommended for this test. Procedures for removal of peroxides from
diethyl ether are included in the instructions supplied with the per-
oxide test kit.
E. APPARATUS
1. Sample Transfer Implements - Implements are required to transfer portions
of solid, semi-solid, and liquid wastes from sample containers to lab-
oratory glassware. The transfer must be accomplished rapidly to avoid
loss of volatile components during the transfer step. Liquids of low to
moderate viscosity may be transferred using conventional laboratory
pipets. Non-tacky solids may be transferred using conventional labora-
tory spatulas. Spoon-shaped porcelain spatulas (Coors No. 60478, or
equivalent) are useful in that they have a measureable bowl-volume.
Samples having a desired approximate volume can thus be obtained. Trans-
fer of tacky or non-tacky solids and semi-solids may be simplified using
the imolements described below. A modified pipet suitable for transfer
of some viscous liquids is also described.
III-139
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1.1 Implement for transfer of non-tacky semi-solids - A 3-ml glass
hypodermic syringe is modified. The plunger is removed and the
normally closed end of the barrel is cut away. To use this
implement, the plunger is replaced flush with the cut-away, open
end. The device is pressed into a semi-solid sample, thereby
forcing the plunger out of the barrel. When the plunger has been
displaced by a volume equal to the approximate sample volume
desired, the syringe is withdrawn and the semi-solid plug is
transferred to a tared vessel by displacing the material with the
plunger.
1.2 Implement for the transfer of tacky semi-solids and solids - This
approach will be useful for the transfer of some tacky or tarry
materials. Glass tubing of approximately 1 cm diameter is cut into
short sections having a desired approximate volume (i.e., 1 ml = 1.0
cm I.D. x 1.3 cm length). To obtain a desired volume of sample, take
a weighed tubing section of that volume, and, using a Teflon-coated
laboratory spatula, press a portion of tarry sample into the tubing
section. The sample-filled tubing section is then placed directly
into a centrifuge tube containing polyethylene glycol (PEG); the
centrifuge tube and PEG are weighed before the sample is added.
1.3 Implement for the transfer of viscous liquids - This device is
fashioned oy cutting the constricted end from a 5-ml graduatsd
pi pet. The large-bore pi pet thereby obtained is used in conjunction
with a conventional laboratory pipetting aid, preferably of the
syringe type. This implement allows convenient transfer and approx-
imate volumetric measurement of some viscous liquids.
2. Class I Biological Safety Cabinet (glovebox) suitable for handling
chemical carcinogens. The cabinet should have an interchange panel for
introducing materials, a retaining tray to catch spills, and a static
pressure gauge {Kewaunee, Inc. - Model SH-3704-MS-X, or equivalent).
3. Water bath - Heated, with concentric ring cover, capable of temperature
control (±2°C). The bath should be used in a hood.
4. Balance - Analytical, capable of accurately weighing 0.0001 g.
5. Gas chromatograph - An analytical system complete with chromatographi.c
column suitable for on-column injection and all required accessories
Including syringes, analytical columns, gases, electron capture detector,
and strip-chart recorder. A data system is recommended for measuring
peak areas.
5.1 Column 1. Supelcoport (100/120 mesh) coated with 1.5% SP-2250,
1.95% SP-2401, packed 1n a 1.8 m x 4-mm I.D. Pyrex glass column. Use
argon 95%/methane 5%, carrier gas at a flow rate of 60 ml/min.
Column temperature Isothermal at 200*C. This column was used to
deveiop cne method performancs stataments "Ms tad
-------
5.2 Column 2. 1.8 m long x 4 mm I.D. glass columns, packed with 3? OV-1
on Supelcoport (100/120 mesh) or equivalent.
5.3 Column Conditioning. Proper thermal conditioning is essential to
minimize column bleed and to provide acceptable gas chroma tographic
analysis. A number of procedures may be used for this purpose such
as the following. Install the packed column in the oven. Do not
connect the column to the detector. However, gas flow through the
detector should be maintained. This can be done using the diluent
gas line or, in dual column ovens, by connecting an unpacked column
to the detector. Heat the oven to near the maximum recommended tem-
perature for the liquid phase without gas flow for 2 hours. Reduce
the oven temperature to approximately 4Q°C below the maximum recom-
mended temperature and allow temperature to equilibrate for a
minimum of 30 minutes without flow. Then adjust the carrier gas
flow to about 50 ml/min for a 6.4-mm (1/4-inch) column and about 25
ml/min for a 3.2-mrn (1/8-inch) column (Caution: NOTE 1). After 1
hour, increase the temperature to about 20°C above normal operating
temperature for 24 to 48 hours while maintaining the gas flow. (Do
not exceed the maximum recommended operating temperature.) Cool
down and connect the column to the detector system, then raise it to
the normal operating temperature. Columns prepared and conditioned
1n this manner should yield good chromatograms with no further
treatment.
NOTE 1: Caution - Bleed-off of liquid phase will occur if the
liquid phase is not fully temperature-equilibrated.
6. Gas Chromatographic Detectors
6.1 Electron Capture Detector (ECD). The electron capture detector is
capable of responding to picogram quantities of chlorinated hydro-
carbons, but the practical sensitivity of the detector is only 30
to 50 times greater than microcoulometry. An ECD was used to
develop the method performance statements provided in Tables 9 and
10 of Subsection J.2.2.
6.2 Microcoulometric Detector (NOTE 2). The microcoulometric titri-
metric detection system is the most specific detector for chlori-
nated hydrocarbons in general use. This titration-cell detector is
capable of responding to low nanogram quantitities for most of these
materials under oxidative conditions, while almost entirely Insensi-
tive to other organics. Under optimum conditions it is capable of
measuring 5 to 20 ng in a reproducible manner, depending on the
chlorine content of the individual compound and its Chromatographic
qualities. However, relatively large samples must be processed in
order to approach the sensitivity of the electron capture detector
described above (Subsection E.I).
NOTE 2: electrolytic conauc-civrcy jetectors that are 2 *:o 3
mor« sensitive than microcoulometric detectors, but less selective,
may be used as a substitute.
III-141
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7. Gas Chromatograph/Mass Spectrometer, Finnigan 3200 and INCOS 2300 Data
System.
8. Glassware
8.1 Separatory funnels - 2,000 ml, 500 ml, and 250 ml, with Teflon stopcock,
8.2 Drying column - Chromatographlc column, approximately 400 mm long x
19 mm I.D., with coarse frit.
8.3 Chromatographlc column - Pyrex, 400 mm long x 22 mm I.D., with coarse
fritted plate and Teflon stopcock (Kontes K-42054 or equivalent).
9. Kuderna-Danish concentrator fitted with graduated evaporative concen-
trator tube. Available from the Kontes Glass Co., each component bearing
the following stock numbers:
9.1 Flask, 250 ml, Stock No. K-570001
9.2 Snyder column, 3-ball, Stock No. K-503000
9.3 Steel springs, 1/2 1n., Stock No. K-662750
9.4 Concentrator tubes, 10 ml, Size 1025, Stock No. K-570050
10. Desiccator.
11. Crucibles, porcelain, squat form, Size 2.
12. Omni or Sorvall mixer with chamber of approximately 400 ml.
13. Filter tube, 180 mm x 25 mm.
14. Pans, approximately 35 cm x 25 cm x 6 cm.
15. Oven, drying.
16. Muffle furnace.
17. Pyrex glass wool, pre-extracted with methylene chloride in a Soxhlet
extractor.
18. Disposable pipets.
19. Magnetic Stirrer and 5/8-1n. Teflon-coated stirring bars.
20. Graduated centrifuge tubes, 15 ml with glass stoppers.
21. tfoiumeiric rlasxs.
22. Micro-syringes, 10 ui for GLC injection and 100 ul for preparation of
standard solutions.
ni-142
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23. Graduated plpets, 2 and 10 ml.
24. Heating plate.
25. Vortex genie.
26. Boiling chips, approximately 10/40 mesh. Heat to 400eC for 30 minutes or
Soxhlet-extract with methylene chloride.
27. Spatula. Stainless steel or Teflon. Fisher Scientific, Catalog No.
14-375-10, or equivalent.
28. Vials, specimen, Teflon-lined screw cap, approximately 20 ml. Cali-
brate at 10 ml by pipetting 10 ml of solvent into the tube and marking
the bottom of the meniscus.
29. Beakers, 400 ml.
30. Biichner Funnels, 9 cm.
31. Filter Paper, Whatman No. 41, ashless.
32. Vacuum filtration apparatus (Fisher 9-788) or 500-ml suction filtration
flasks.
33. Drying column, 25 mm x 200 mm packed with 7 to 10 cm sodium sulfate and
4 cm glass wool.
34. Vials, 2 ml.
35. Sonicator Cell Disrupter, Model W-375 both high gain, 3/4-in. probe
(Heat Systems - Ultrasonics, Inc. or equivalent).
36. Food Processor (Hobart Model 8181D or equivalent).
37. Rotary vacuum evaporator.
38. Centrifuge Tubes, graduated, 12 to 15 ml.
39. N-evap apparatus (or equivalent) for evaporation of solvent under a
gentle nitrogen stream.
40. Extractor, Soxhlet, 125, 250, or 500 ml.
41. Cleanup microcolumn, Chromaflex column, size 22, 20 cm x 7 mm (Kontes)
or equivalent.
42. Pasteur pipettes (23 cmT.
F. REAGENTS
1. Reagent watsr - Reagent *ater is defined as water in which an interferent
III-143
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is not observed at the method detection limit of each parameter of
interest.
2. Sodium hydroxide solution (ION) - (ACS). Dissolve 40 g NaOH in reagent
water and dilute to 100 ml.
3. Sodium thiosulfate - (ACS). Granular.
4. Sulfuric acid solution (1+1) - (ACS). Slowly, add 50 ml ^$04 (sp.
gr. 1.84) to 50 ml of reagent water.
5. Acetone, pesticide quality or equivalent.
6. Hexane, pesticide quality or equivalent.
7. Isooctane, pesticide quality or equivalent.
8. Methylene chloride, pesticide quality or equivalent.
9. Acetonitrile.
10. Benzene.
11. Petroleum ether, pesticide quality or equivalent.
12. Acetone-hexane, 1:1.
13. Methylene chloride - hexane, 15% v/v.
14. Ethyl ether, pesticide quality or equivalent, redistilled in glass, if
necessary.
14.1 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).
14.2 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.
15. Sodium sulfate (ACS) granular, anhydrous. Purify by heating at 400*C for
4 hours in a shallow tray. Pre-rinse or Soxhlet extract with methylene
chloride.
16. Sodium sulfate solution, saturated.
17. Florist! - PR grade (60/100 mesh); purchase activated at 1250*F and store
1n glass containers with glass stoppers or foil-lined screw caps. Before
use. activate each batch at least 16 hours at 130"C in a foil-covered
glass container.
-------
17.1 Florlsil from different batches or sources may vary in adsorptive
capacity. To standardize the amount of Florisil which is used, the
use of lauric acid valued 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 col-
umn is calculated by dividing this factor into 110 and multiplying
by 20 grams.
17.2 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and demonstrate the absence of interferences from
the reagents.
18. Neutral alumina, Hoelm, activity Grade I deactivated with 5 percent water.
19. Alumina, basic, 60 mesh, Alfa Products, or equivalent. Adjust to
Brockmann activity IV by adding 6 percent (w/w) distilled water to the
adsorbent in the flask, stoppering, and shaking well. Allow to equili-
brate for at least 15 hours before use. Discard after 2 weeks.
20. Nitrogen gas, dry, purified.
21. Polyurethane foam plugs for air sampling.
21.1 Cutting of Air Sampling Cartridges
Polyurethane foam (PUF) plugs are cut from 3-in. (7.6 cm) sheet
stock of upholstery material (polyether type, density 0.0225
Q/cm3) using a 24-mm U.D.) stainless steel cutting die. This die
is turned in a drill-press while a stream of water is directed on it
to provide cooling.
21.2 Cleanup of Air Sampling Cartridges
PUF plugs are pre-cleaned by Soxhlet extraction, either singly or in
batches, as desired.
21.2.1 Extract with 5 percent diethyl ether in ji-hexane (glass-
distilled, pesticide quality or equivalent) for 100 cycles.
21.2.2 Analyze an extract from a representative sample of each batch
of plugs using procedures in Subsection J.5.1.5. Soxhlet
extract the plugs twice for 50 cycles each, concentrate the
combined extracts, and analyze for possible background con-
tamination.
21.2.3 Dry the plugs under vacuum at 758C.
21.3 Loading of Air Sampling Cartridges
21.3.1 Using forceps, carefully place the °UF plug into the
hexane-rinsed glass sampling cartridge.
III-145
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NOTE 3: It is Important not to touch the cleaned PUF with
bare hands.
21.3.2 Wrap the sampling cartridge in hexane-rinsed aluminum foil
and store in glass jars until ready for.use.
22. Mercury, triple distilled.
23. Copper powder, activated.
24. Stock standard solutions (1.00 ug/ul) - Stock standard solutions can be
prepared from pure standard materials or purchased as certified
solutions.
25. Standards, analytically pure, obtainable from the Pesticides and Indus-
trial Chemicals Repository, U.S. Environmental Protection Agency, MD-8,
Research Triangle Park, North Carolina 27711. Telephone (919) 541-3951.
25.1 Prepare stock standard solutions by accurately weighing 0.0100 grams
of pure material. Dissolve the material in 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 co
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 ati independent source.
25.2 Transfer the stock standard soiutions 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 stan-
dards 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 Moni-
toring and Support Laboratory, in Cincinnati.
25.3 Stock standard solutions must be replaced after 6 months, or sooner
1f comparison with check standards indicates a problem.
G. QUALITY CONTROL
1. The minimum requirements of a formal quality control program consist of
an initial demonstration of laboratory capability and the analysis of
spiked samples as a continuing check on performance. The laboratory
should maintain performance records to define the quality of data that
are generated.
1.1 Before performing any analyses, the analyst must demonstrate the
ability to generate dcceptaole accuracy and precision with this
method. This ability is established as described in Subsection G.2.
III-146
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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 Subsection G.2.
1.3 The laboratory must spike and analyze a minimum of 10 percent of all
samples to monitor laboratory performance. This procedure is
described in Subsection G.4. Five percent (1 of 20) or each time a
set of samples is prepared (whichever is more frequent), a blank of
the reagents, water, and solvents should be prepared and analyzed.
Contaminants shall be below the detection limits based on a 1-gram
(1-ml) sample aliquot.
2. The ability to generate data of acceptable accuracy and precision must be
demonstrated by performing the following operations:
2,1 For each compound to be measured, select a spike concentration
representative of the expected levels in the samples. Using stock
standard solutions, prepare a quality control check sample con-
centrate in acetone 1,000 times more concentrated than the selected
concentrations. Quality control check sample concentrates, appro-
priate for use with this method, are available from the U.S.
Environmental °~otection Agency, Environmental Monitoring and
Support Laooratory, Cincinnati, Ohio 45268.
2.2 Using a pipet, add 1.00 ml of the check sample concentrate to each
of a minimum of four 1,000-ml aliquots of reagent water. A repre-
sentative wastewater may be-used in place of the reagent water, but
one or inore additional diiquots must oe analyzed to determine b2c!;-
ground levels, and the spike level must exceed twice the background
level for the test to be valid. Analyze the aliquots according to
the appropriate method in Subsection J.
2.3 Calculate the average percent recovery, (R), and the standard devia-
tion of the percent recovery(s). Wastewater background corrections
must be made before R and s calculations are performed.
2.4 Using the appropriate data from Table 2, determine the recovery and
single-operator precision expected for the method and compare these
results to the values calculated in Subsection G.2.3. If the data
are not comparable, the analyst must review potential problem areas
and repeat the test.
3. The analyst must calculate method performance criteria and define the
analytical performance for each spike concentration and compound being
measured.
III-147
-------
TABLE 2. SINGLE-OPERATOR ACCURACY AND PRECISION FOR
PESTICIDES AND PCBs ANALYZED BY 6C/ECD
;a=============================================================================
Parameter
AT dri n
Alpha-BHC
Beta-BHC
Delta-BHC
Gamma-BHC
Chlordane
4,4'-DDD
4,4'-DDE
4,4'-DDT
Dieldrin
Endosulfan !
Endosuifan II
Endosulfan Sulfate
Endrin
Endrin aldehyde
Heptachlor
rieptacnior Lpoxiae
Toxaphene
PCB-1016
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
Average
Percent
Recovery
89
89
88
86
97
93
92
89
92
95
96
37
99
95
87
8&
93
95
94
96
88
92
90
92
91
Standard
Deviation
%
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
Spike
Range
(ug/1)
2.0
1.0
2.0
2.0
1.0
20
6.0
3.0
8.0
3.0
3.0
5.0
15
5.0
12
1.0
2.0
200
25
55-100
110
28-56
40
40
80
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
3
3
aaaaaaasaaaaasaaaaaaaaasBaaaaaaaaaaasaaaaaaaBaaaaaaaaaaaaaaaaaaaaaaaaaaaaasaaa
3.1 Calculate upper and lower control limits for method performance:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCD * R - 3 s
where R and s are calculated as detailed in Subsection G.2. The
UCL and LCL can be used to construct control charts15 that are
useful in observing trends in performance.
III-148
-------
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 state-
ment should be developed by the analysis of four aliquots of waste-
water as described in Subsection G.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 con-
trol in Subsection G.4. The accuracy statements should be updated
regularly.15
4. The laboratory is required to collect a portion of their samples in
duplicate to monitor spike recoveries. The frequency of spike-sample
analysis must be at least 10 percent of all samples, or one sample per
month, whichever is greater. One aliquot of the sample must be spiked
and analyzed as described in Subsection G.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
Subsection M.5. The laboratory should monitor the frequency of data so
qualified to ensure that it remains at or below 5 percent.
5. Before processing any samples, the analyst should demonstrate, through
the analysis of a 1-liter aliquot of reagent water, that all glassware
and <"«?aqent interferences are under control. Each time a set of samples
is extracted or there 1s a change in reagents, a lacoratory reagent oianK
should be processed as a safeguard against laboratory contamination.
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 laooratory ana che ,iatjre of >tne
samples. Field duplicates may be analyzed to monitor the precision of
the sampling technique. When doubt exists over the identification of a
particular peak in a chromatogram, independent confirmatory techniques
such as gas chromatography using a minimum of two columns of different
polarity, specific element detectors, or mass spectrometry must be used.
Whenever possible, the laboratory should perform analysis of standard
reference materials and participate in relevant performance evaluation
studies.
H. CALIBRATION
1. Establish gas chromatographic operating parameters which produce reten-
tion times equivalent to those indicated 1n Table 3. The gas chromato-
graphic system may be calibrated using the external standard technique
(Subsection H.2.) or the internal standard technique (Subsection H.3.).
2. External standard calibration procedure:
III-149
-------
TABLE 3. CHROMATOGRAPHIC RETENTION TIMES AND METHOD DETECTION
LIMITS FOR SELECTED PESTICIDES AND PCBs
Parameter
Retention Time
(min)
Column 1
Column 2
Method
Detection Limit
ug/l
Alpha-BHC
Gamma-BHC
Beta-BHC
Heptachlor
Delta-BHC
Aldrin
Heptachlor epoxide
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
4,4'-ODD
Endosusfan II
4,4'-DDT
Endrin aldehyde
Endosulfan sulfate
Chlordane
Toxaphene
PCB-101S
PCB-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
1.35
.70
1.90
2.00
2.15
40
50
50
13
45
1,
2.
82
13
7.
Q
6.55
.83
.00
9.40
11.82
14.22
mr
Tir
mr
mr
mr
mr
mr
mr
mr
1.97
3.35
2.20
4.10
5.00
6.20
7.15
7.23
8.10
9.08
R.28
11.75
9.30
10.70
mr
mr
mr
mr
mr
mr
mr
mr
mr
0.003
0.004
0.006
0.003
0.009
0.004
0.083
0.014
0.004
0.002
0.006
0.011
0.004
0.012
0.023
0.066
0.014
0.24
nd
nd
nd
0.065
nd
nd
nd
==========
Column 1 conditions: Supelcoport (100/120 mesh) coated with 1.5% SP-2250/
1.95% SP-2401 packed in a 1.8-m x 4-mm I.D. glass column with 5%
methane/95% argon carrier gas at a flow rate of 60 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 35 OV-1 packed
in a 1.8-m long x 4-mm I.D. glass column with 5% methane/95% argon
carrier gas at a flow rate of 60 ml/min. Column temperature, iso-
thermal at 200eC, for th« pesticides; 140*C for PCB-1221 and 1232;
170eC for PCB-1016 and 1242 to 1268.
mr - Multiple peak response. See Figures 2 thru 10.
nd - Not determined.
-------
Column: 1.5% SP-2250 +
1.95% SP-2401 on Supeicoport
Temperature: 200° C.
Detector: Electron Capture
Retention Time - Minutes
Figure 2. Gas chromatogram of chlordane.
III-151
-------
Column: 1.5% SP-2250 -
1.95% SP-2401 on Supelcoport
Temperature: 200° C.
Detector: Electron Capture
i i i i
6 10
i I
14
i
18
^ I i T
22 26
Retention Time • Minutes
Figure 3. Gas chromatogram of toxaphene.
III-152
-------
Column: 1.5% SP-2250 + 1.95% SP-2401 on Supelcoport
Temperature: 160° C.
Detector: Electron Capture
• i • i * • • J *
2 6 10 14 18
Retention Time - Minutes
22
Figure 4. Gas chromatogram of PCB-1016.
III-153
-------
Column: 1.5% SP-2250 + 1.95% SP-2401 on Supelcoport
Temperature: 160° C.
Detector: Electron Capture
I I
I I I I I I 1 I
10 . 14 18 22
Retention Time • Minutes
Figure 5. Gas chromatogram of PCB-1221.
Ai.i-J.3t
-------
Column: 1.5% SP-2250 + 1.95% SP-2401 on Supelcoport
Temperature: 160° C.
Detector: Electron Capture
10
til
14
I T
18
22
Retention Time • Minutes
Figure 6. Gas chromatogram of PCB-1232.
i i
24
III-155
-------
Column: 1.5% SP-2250 + 1.95% SP-2401 on Supelcoport
Temperature: 160°C.
Detector: Electron Capture
I I I I I I I
6 10 14 18
Retention Time • Minutes
22
Figure 7. Gas chromatogram of PCB-1242.
III-156
-------
Column: 1.5% SP-2250 + 1.95% SP-2401 on Supelcoport
Temperature. 160°C.
Detector: Electron Capture
Retention Time - Minutes
Figure 8. Gas chromatogram of PCS-1248.
26
III-157
-------
Column: 1.5% SP-2250 + 1.95% SP-2401 on Supelcoport
Temperature: 200° C.
Detector: Electron Capture
« I • 1 i I i T ' I '
2 6 10 14 18 22
Retention Time - Minutes
Figure 9. Gas chromatogram of PCB-1254.
III-158
-------
Column: 1.5% SP-2250 + 1.95% SP-2401 on Supelcoport
Temperature: 200° C.
Detector: Electron Capture
i
2
6
i
10
I
14 18
Retention Time • Minutes
22
I T
26
Figure 10. Gas chromatogram of PCB-1260.
III-159
-------
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 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.
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 com-
pound. 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.
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 pre-
dicted 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 ccmoound.
Internal Standard Calibration Procedure.
3.1 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 tnat the mea-
surement of the internal standard is not affected by method or
matrix interferences. In addition, the Internal standard should
elute close to the analyte under analysis (±6 min). Because of
these limitations, no internal standard can be suggested that is
applicable to all samples.
3.2 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 stan-
dards, and dilute to volume with isooctane. One of the 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 1n real samples or should define the
working range of the detector.
3.3 Using Injections of 2 to 5 ul of each calibration standard, tabulate
peak height or area responses against concentration for each com-
pound and Internal standard, and calculate response factors (RF) for
«ach comoound using Eauation 1:
RF « (ASC«SV(A1SCS) Ea.
III-160
-------
where:
As = response for the parameter to be measured
Ais = response for the Internal standard
C-(S = concentration of the internal standard, (ug/1)
Cs * concentration of the parameter to be measured,
(ug/1).
If the RF value over the working range is a constant (<1Q% 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. Cs.
3.4 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 ±10%, the test must be repeated using a fresh calibration
standard. Alternatively, a new calibration curve must be prepared
for that compound.
I. DAILY GC/MS PERFORMANCE TESTS
At the beginning of each day, the mass calibration of the GC/MS system
must be checked and adjusted, if necessary, ;o meet DFTPP specifications.
Additionally, each day pesticides or PCBs are to be analyzed, the column
performance specifications with benzidine must be met. DFTPP can be mixed in
solution w.ith benzidine to complete the two performance tests with one
injection, if desired.
•
Evaluate the analytical system performance each day that it is to be used
for the analyses of samples or blanks by examining the mass spectrum of DFTPP.
The following instrumental conditions are required to perform the mass cali-
bration test of a GC/MS system:
Electron Energy - 70 volts (nominal)
Mass Range - 35-450 atnu
Scan Time - 7 seconds or less
Source Temperature - 280-300*C
Inject a solution containing 50 ng DFTPP and check to ensure that established
performance criteria listed in Table 4 have been satisfied. If the system
performance criteria are not met, the analyst must retune the spectrometer and
repeat the performance check.
The performance criteria must be met before any samples or standards may
be analyzed.
Column performance is evaluated by injecting 100 ng of benzidine into the
Instrument. The tailing factor for the resultant peak, as calculated in
Figure ii, must oe less cnan ^ r'or ;ne per-onnance k.o be considered accsnt-
able.
III-161
-------
TABLE 4. DFTPP KEY IONS AND ION ABUNDANCE CRITERIA
Mass Ion Abundance Criteria
51 30 to 60 percent of mass 198
68 Less than 2 percent of mass 69
70 Less than 2 percent of mass 69
127 40 to 60 percent of mass 198
197 Less than 1 percent of mass 198
198' Base peak, 100 percent relative abundance
139 3 :s } percen* of mass 198
275 10 to 30 percent of mass 198
365 Greater than 1 percent of mass 198
441 Present but less than mass 443
442 Greater than 40 percent of mass 198
443 17 to 23 percent of mass 442
III-162
-------
BC
Tailing Factor =- ——
AB
Example Calculation:
Peak Height = DE = 100 mm
10% Peak Height = BD = 10 mm
Peak Width at 10% Peak Height = AC = 23 mm
AB = 11 mm
BC = 12 mm
Therefore: Tailing Factor = — = 1.1
Figure 11. Tailing factor calculation.
III-163
-------
J. ANALYTICAL PROCEDURES
1.1 Analysis of Hazardous Waste Samples for Pesticides
Analytical Procedure: available
Sample Preparation: available
1.1.1 Reference/Title
U.S. Environmental Protection Agency, "Method for Preparation
of Medium Concentration Hazardous Waste Samples," U.S. EPA,
Region IV, Athens, Georgia. (1981).16
1.1.2 Method Summary
Approximately 1-gram aliquots of soil, solid, aqueous liquid,
or non-aqueous liquid are transferred to viails inside a
chemical carcinogen glovebox. The samples are then removed
from the enclosure for dilution with hexane. Half of the
hexane extract is cleaned up for GC/EC identification and
quantification. The other half of the solution can be used
for TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin).
1.1.3 Applicability
This procedure is designed for the safe handling and prepara-
tion of potentially hazardous samples from hazardous waste
sites. The method is directed to contaminated soil samples
and waste samples that may be solid, aqueous liquid, or non-
aqueous i-'quid, and suspected to contain l«»s?. *han 10 percent
of any one organic chemical component. The procedure has an
approximate detection limit of 0.1 ppm for pesticides.
1.1.4 Precision and Accuracy
These extraction and preparation procedures were developed
for rapid and safe handling of hazardous samples. The design
of the methods thus did not stress efficient recoveries of
all components. Rather, the procedures were designed for
moderate recovery of a broad spectrum of organic chemicals.
The results of the analyses thus may sometimes reflect only
the minimum of the amount present in the sample.16
The procedure has been used in one laboratory. Pesticide
recovery data expressed as percent recovery and standard
deviations are summarized in Table 5.
1.1.5 Sample Preparation
Place the original sample container into the glovebox.
Additional •'terns that should be 1n the rjlovebox -include (l\
calibrated and tared 20-ml vials with caps, (2) a spatula,
III-164
-------
TABLE 5. RECOVERY DATA FROM SOIL BY REGION IV MEDIUM '
CONCENTRATION HAZARDOUS WASTE METHOD«
===============================================================================
Pesticide Fraction
Dieldrin
p.p'-DDT
p.p'-DDE
p.p'-DDD
Alpha-endosulfan
Beta-endosulfan
Endosulfan sulfate
Endrin
Heptachlor
Heptachlor epoxide
ilnha-BHC
Gamma-BHC
Beta-BHC
Delta-BHC
Cone.
ug/g
0.04
0.10
0.04
0.10
0.04
0.08
0.20
0.04
0.04
0.04
0.02
0.04
0.04
0.04
Avg.
% Rec.
99
97
99
98
99
98
97
97
101
98
102
100
101
101
Std.
Dev.*
1
1.5
0
0
0.5
0
1
1
i.5
1
1
1.5
1.5
1.5
S=======;=========================================================
* - ± one standard deviation based on two trials.
(3) a balance, (4) a capped vial containing 10 ml of
Interference-free methanol, (5) a vial containing distilled
water, and (6) a medicine dropper. The vial of methanol is
to be used as a method blank. (One method blank should be
run for each batch of 20 samples or less.) .Open the sample
transportation can and remove the sample vial. Note and
record the physical state and appearance of the sample. If
the sample bottle is broken, immediately repackage the sample
and terminate the analysis.
!!1-165
-------
Open the sample vial and mix the sample. .If the sample is a
liquid, transfer one drop to a vial containing water to
determine whether the sample is aqueous or non-aqueous.
Record the result.
Transfer approximately 1 gram (or 1 ml) of the sample to each
of the calibrated vials. Wipe the mouth of the vials with
tissue to remove any sample material. Cap the vials. Record
the exact weights of sample taken. Reseal the sample and
replace it in the original packaging.
Proceed with a hexane extraction of the pesticide-containing
sample based on the miscibility of the original sample with
water. Follow paragraph (a) for an aqueous sample, paragraph
(b) for a non-aqueous sample, and paragraph (c) for a soil or
solid-phase sample.
a. If the sample was determined to be aqueous, add 10 ml of
hexane to the sample vial. Cap and shake for 2 minutes.
Add 2 grams of anhydrous sodium sulfate to each vial to
adsorb all water. Shake the sample.
b. If the sample was determined to be non-aqueous, dilute
the sample to a final volume of 10 .Til with hexane. ^dd 1
gram of anhydrous sodium sulfate to adsorb any water in
the sample. Shake the sample.
c. If the sample is a solid matrix such as soil, sediment or
sludge, add 10 ml of hexane. Cao the samole and shake
for 1 hour on a wrist action shaker. Add 1 gram of
anhydrous sodium sulfate to the sample and thoroughly
mix.
1.1.6 Extract Analysis
Process 5 ml of the hexane solution through one of the
cleanup techniques specified in Method 608,17 such as
acetonitrile partitioning (Subsection J.I.2.7, p. III-175).
Perform GC/EC analysis as specified.
Retain the other 5 ml of hexane solution for TCDD analysis
following Method 613 (Section 8).I7
III-166
-------
1.2 Analysis of Transformer Fluid and Waste Oil for Polychlorinated
Biphenyls
Analytical Procedure: available
Sample Preparation: available
1.2.1 Reference
U.S. Environmental Protection Agency, "The Analysis of
Polychlorinated Biphenyls in Transformer Fluid and Waste
Oil," U.S. EPA, Office of Research and Development,
Environmental Monitoring and Support Laboratory, Cincinnati,
Ohio. 36 p. February 1981.4
1.2.2 Method Summary
The sample is diluted on a weight/volume basis with hexane
so that the concentration of each PCB isomer is within the
analytical limits of the gas chromatographic (GO system
(0.01 to 10 ng/nl). The diluted sample is injected into a GC
for separation of the PCB isomers. Measurement is accom-
plished with a halogen-specific detector that maximizes
baseline stability and minimizes interferences normally
encountered with other detectors. The electron capture
detector (ECD) can normally be substituted for the halogen-
specific detector when samples contain dichloro- through
decachlorobiphenyls, or when the sample matrix does not
interfere with PCB analysis. A mass spectrometer operating
*n the selected ion monitoring mode of data acquisition may
also be used as an alternative detector -*hen ''CS levels ire
sufficiently high and the PCS m/e ranges are free from
interferences. Several cleanup techniques (acid cleanup,
Fluorisil cleanup, alumina cleanup, silica gel cleanup, gel
permeation cleanup, acetonitrile partitioning) are provided
for samples containing interferences. However, even
exhaustive use of these procedures may not remove all inter-
ferences from all waste oil samples.
The concentrations of PCBs are calculated on a mg/kg basis
using commerical mixtures of PCBs as standards. The analysis
time, not including data reduction, is approximately 35
minutes per sample.
1.2.3 Applicability
This 1s a gas chromatography (GC) method applicable to the
determination of commercial mixtures of polychlorinated bi-
phenyls (PCBs) in .transformer fluids and certain other
hydrocarbon-based waste oils. The method can be used to
analyze waste oils for Individual PCB isomers or complex
mixtures of chlonnateo oiphenyls from •nonochlorobipheny'1
through decachlorobiohenyl-only if they have been previously
111-167
-------
Identified by other methods1^ or by knowledge of the sample
history. The method detection limits are dependent upon the
complexity of the sample matrix and the ability of the
analyst to properly maintain the analytical system. Using a
carefully optimized instrument, this method has been shown to
be useful for the determination of commercial PCB mixtures
spiked into transformer fluid over a range of 5.0 to 500
mg/kg. Based upon a statistical calculation at 5 mg/kg for a
simple oil matrix, the method detection limit for Aroclors
1221, 1242, 1254, and 1260 is 1 mg/kg. The method detection
limit (MOL) is defined as the minimum concentration of a
substance that can be measured and reported with 99 percent
confidence that the value is above zero.
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.
1.2.4 Precision and Accuracy
Separate accuracy statements of laboratory performance should
be maintained for the analysis of waste oil and similar sam-
ple matrices. Single-operator precision and recovery data
ror this method are presented in Tables 6 through 3.
1.2.5 Sample Preparation
Because of the unique sample matrix, PCB concentrations are
determined by diluting the sample with solvent and analyzing
the resultant mixture directly, in order co ansurs cnai cne
diluted sample is within the analytical range of the instru-
ment, the approximate PCB concentration of the sample may be
determined by x-ray fluorescence, microcoulometry, density
measurements, or by analyzing a very dilute mixture of the
sample (10,000:1) according to paragraph 1.2.6.
Based on the estimated PCB concentration of the sample,
continue processing the sample according to either (a), (b),
(c), or (d), as appropriate.
a. For samples in the 0- to 100-mg/kg range, dilute
100:1 with hexane. Pipet 1.0 ml of sample into a
100-ml volumetric flask using a 1.0-ml Mohr pipet.
For viscous samples, 1t may be necessary to cut the
capillary tip off the pipet. Dilute the sample to
volume with hexane. Stopper and mix. Analyze
according to paragraph 1.2.6.
Using the same pipet, deliver 1.0 ml of sample into
a tared beaker weighed to ±0.001 g. Reweigh the
beaker to sO.OOI g co determine cne mjignt of
used ^n preparing the diluted sample.
III-168
-------
TABLE 6. ACCURACY AND PRECISION DATA FOR THE GC ANALYSIS OF PCBs
IN SPIKED MOTOR OIL SAMPLES4
Detector*
HED
ECD
HED
ECD
HED
ECD
HED
ECD
HED
ECD
HED
ECD
MED
ECD
HED
ECD
HED
^CD
HED
ECD
HED
ECD
HED
ECD
HED
ECD
HED
Method
Cleanup
None
None
None
None
1.2.7.1
1.2.7.1
1.2.7.1
1.2.7.1
1.2.7.2
1.2.7.2
1.2.7.2
1.2.7.2
" ,2.7.2
i'.i'j'.i
1.2.7,3
1.2.7.3
1.2.7.4
1 2 7,4
1.2.7.4
1.2.7.4
1.2.7.5
1.2.7.5
1.2.7.5
1.2.7.5
1.2.7.6
1.2.7.6
1.2.7.6
ECD 1.2.7.6
Spike
mg/kg
30.3
30.3
31.1
31.1
30.3
30.3
31.1
31.1
30.3
30.3
31.1
31.1
30.3
30.3
31.1
31.1
30.3
30.3
31.1
31.1
30.3
30.3
31.1
31.1
30.3
31.1
30.3
31.1
«HED = Hall Electrolytic
ECD * Electron Cai
Aroclor
Spike
1242
1242
1260
1260
1242
1242
1260
1260
1242
1242
1260
1260
1242
1242
1260
1260
1242
1242
1260
1260
1242
1242
1260
1260
1242
1242
1260
1260
35SSS«E3»SS3
Detector
Avg.
Cone.
Found
mg/kg
28.2
26. 7b
27.2
23.9
28.4
25. 4&
28.1
24.3
• 30.7
27. 3b
30.9
31.0
30.3
28.913
29.8
30.8
29.4
26. 4b
29.4
23.6
31.9
23. 4b
33.6
30.9
34.4
23. 4b
29.1
(Precision)
Rel. Std.
Deviation
%
4.2
5.7
2.0
2.2
11.5
6.1
8.0
7.8
2.4
10.2
3.6
8.6
8,6
5.0
4.7
6.5
5.8
5.3
5.2
4.5
8.5
3.0
9.2
5.5
3.8
4.4
4.2
27.0 4.6
(Accuracy)
Percent
Recovered
93.1
88.1
87.5
76.8
93.7
83.8
90.3
78.1
101
90.1
99.4
99.7
100
95.4
95.8
99.0
97.0
87.1
94.5
75.9
105
77.2
108
99.4
107
77.2
96.7
Number
of
Dilutions
5
3
5
3
3
3
3
3
4
4
4
4
3
3
3
3
3
3
3
3
3
2
3
3
4
4
4
86.7 4
iture Detector
t>Severe interference problems in elution area of 1242. Measurement based
upon only 3 of the 10 normally resolved major peaks. Cleanup techniques
1n paragraph 1.2.7 did not Improve the quality of the 1242 chromatogram.
If this were an unknown sample, it would be impossible to qualitatively
Identify the presence of Aroclor 1242 using ECD. The HED provided an
-------
TABLE 7. ACCURACY AND PRECISION DATA FOR THE GC ANALYSIS OF PCBs
IN WASTE TRANSFORMER FLUID SAMPLES*
(Precision)
Method 1260 Avg.D Rei . std. (Accuracy) Number
Dilution Clean- Spike Cone. Deviation Percent of
Sample Ratio Detector* up mg/kg Found % Recovered Dilutions
A
A
A
A
A
A
A
A
A
A
- A
A
A
. A
B
B
B
B
C
C
C
C
100:1 ECD None
100:1 HED None
100:1 ECD 1.2.7.1 ~
100:1 HED 1.2.7.1
100:1 ECD 1.2 7.2 —
100:1 HED 1.2.7.2 —
100:1 ECD 1.2.7.3 —
100:1 HED 1.2.7.3 —
100:1 ECD 1.2.7.4 --
100:1 HED 1.2.7.4 «
1.00:1 ECD 1.2,7.5
100:1 HED 1.2.7.5
100:1 ECD None 27.0
100:1 HED None 27.0
1000:1 ECD None
1000:1 HED None
1000:1 ECD None 455
1000:1 HED None 455
1000:1 ECD None
1000:1 HED None
1000:1 ECD None 300
1000:1 HED None 300
22.6
27.0
22.8
29.7
22.4
28.2
22.7
27.8
20.9
30.2
23.8
28.6
45.0
55.2
452
471
875
916
284
300
607
686
3.6
1.7
2.5
1.4
1.0
2.2
1.3
2.8
--
...
0.3
4.1
3.3
1.5
0.8
1.2
0.5
2.0
1.2
1.4
3.6
3.9
7E
7E
7E
7
3E
3^
3^
3^
1
1
7E
7t
91 7E
102 7E
7E
-- 7
96 7E
99 7E
7
7
104 7E
114 7
==============================================================::==================
* HED *
ECD =
A - dark
Hall Electrolytic Detector
Electron Capture Detector
waste oil
B - black waste oil with suspended solids
C - clear waste oil
D - all
E - dupl
samples contained Aroclor 1260
icate analyses made at each dil
ution
III-170
-------
TABLE 8. ACCURACY AND PRECISION AND LIMIT OF DETECTION DATA
RESULTS OF ANALYSES OF SHELL TRANSFORMER FLUID
SPIKED WITH PCB AT 5.0 AND 27 mg/kg4
Aroclor
Spike
(rag/kg)
Number of
Analyses
Avg.
(mg/kg)
Standard
Deviation
% Recovery
MDL*
(mg/kg)
1221
1242
1254
1260
1221
1242
1254
1260
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
Electron Capture Method
(100:1 Dilution)
7
14
7
14
6
7
6
7
7.5
3.8
4.1
4.7
0.43
0.18
0.08
0.18
Hall Method
(100:1 Dilution)
7.5
5.9
5.8
5.4
0.23
0.17
0.16
0.10
150
76
82
94
150
118
116
108
1.4
0.5
0.2
0.5
0.7
0.5
0.5
0.3
Shell Transformer Oil +27 ppm Aroclor 1260
(100:1 Dilution)
Rel. Std.
Method
(mg/kg)
vuir.ber of
Analyses
avq.
(mg/kg)
Deviation
% Recovery
Electron Capture 27
Hall 700-A 27
14
7
24.0
28.3
.70
2.1
89
105
===============================================================================
3MDL = Method Detection Limit at 99 percent confidence that the value is
above zero.
NOTE: At these values it would be impossible to identify Aroclor
patterns with any degree of confidence. 1 mg/kg appears to be a
reasonable MDL.
where:
MDL
t(n-1..99)
t(n-l,.99)
the method detection limit
the students' t value appropriate for a 99 percent
confidence level and a standard deviation estimate
with i-1. degrees of freedom
standard deviation of the rephcate analyses.
III-171
-------
b. Alternatively, weigh approximately 1.0 g of sample
±0.001 g into a volumetric flask and dilute to
100 ml in hexane. Store the diluted sample in a
narrow-mouth bottle with a Teflon-lined screw cap.
Analyze according to paragraph 1.2.6.
c. For samples above 100 mg/kg PCS, dilute 1000:1 with
hexane. Pipet 0.10 ml of sample into a 100-ml vol-
umetric flask using a 0.10-ml Mohr pipette. Dilute
to volume with hexane, stopper and mix. Analyze
according to paragraph 1.2.6.
Using the same pi pet, transfer 0.10 ml of sample
Into a tared beaker weighed to ± 0.001 g. Reweigh
the beaker to determine the weight of sample used in
preparing the diluted sample.
d. Alternatively, weigh approximately 0.1 ± 0.0001 g of
sample into a 100-ml volumetric flask and dilute to
volume with hexane. Store the diluted sample in a
narrow-mouth bottle with a Teflon-lined screw cap.
Analyze the diluted sample according to paragraph
1.2.6.
1.2.6 Sample Analyses
Analyze the sample by injecting the hexane mixture into the
GC using.auto injectors or the solvent flush technique.19
The 2C should be tssnoerature-orogrammable, eauiooed -for
on-column injection, capable of accepting 6.4 mm 0.0. glass
columns, and equipped with one of the following detectors:
(a) A halogen-specific detector to eliminate interferences
causing misidentification or false-positive values due to
non-organohalides that commonly coelute with the PCBs.
Electrolytic conductivity detectors such as the Hall
Model 700-A have been shown to provide the necessary
sensitivity and stability. Other halogen-specific detec-
tors, including older model electrolytic conductivity
detectors and microcoulometric titration, may be used.
However, the stability, sensitivity, and response time of
these detectors may raise the method detection limit and
adversely affect peak resolution.
(b) Semi-specific detectors such as ECD may be substituted
for halogen-specific detectors when sample chromato-
graphic patterns closely match those of the standards.
Either acid cleanup or Florisil slurry (paragraph 1.2.7)
cleanup techniques should be routinely incorporated into
the analytical scheme orior to sample injection when
these detectors are used.
III-172
-------
The GC columns and conditions listed below are recom-
mended for the analysis of PCB mixtures in oil. If these
columns and conditions are not adequate, the analyst may
vary the column parameters to improve separations. The
columns and conditions selected must be capable of ade-
quately resolving the PCBs in the various Aroclor mix-
tures so that each Aroclor is identifiable through isomer
pattern recognition (Figures 4 through 10).
Capillary columns and the required specialized injection
techniques are acceptable alternatives to the recommended
packed columns. Due to problems associated with the use
of capillary columns the analyst must demonstrate that
the entire system will produce acceptable results by
performing the operations described in Subsection G.
Recommended primary analytical column: Glass, 6.4 mm
O.D. (2 mm I. D.), 6 ft (180 cm) long, packed with Gas-
Chrom Q (100/120 mesh) coated with 3% OV-1.
Carrier gas: 40 to 60 ml/min (helium, nitrogen or 10%
methane in argon).
Temperature program: 120"C isothermal for 2 minutes,
6'C/minute to 220JC, and noid at 220"C until all
compounds elute.
Isothermal Operation:
Aroclor 1221, 1232, or Cl]_ chrough 014 isomers - 140
to 150'C.
Aroclor 1016, 1242, 1254, 1260, 1262, 1268, or C13
through Clio isomers 170 to 200°C.
Recommended confirmatory column: Glass tubing 6.4 mm O.D.,
2 mm I.D., 6 ft (180 cm) long, packed with Gas Chrom Q
100/120 mesh coated with 1.5% OV-17 + 1.95% OV-210.
Carrier gas: 40 to 60 ml/min (helium, nitrogen or 10%
methane in argon).
Column temperatures: Aroclor 1221, 1232, or Clj
through Cl4 Isomers - 170 to 180°C.
Aroclor 1016, 1242, 1254, 1260, 1262, 1268, or C13
through Clio isomers - 200*C.
If the resulting chromatograms show evidence of column
flooding or non-linear responses due to excess sample,
dilute tne sample as necessary (paragraph 1.2.5) using
hexane as solvent.
III-173
-------
Determine whether or not PCBs are present in the sample
by comparing the sample chromatogram to that obtained
with a PCB locator mixture. Proceed as indicated below:
(a) If a series of peaks in the sample match some of the
retention times of PCBs in the PCB locator mixture,
attempt to identify the source by comparing chromat-
ograms of each standard prepared from commercial
mixtures of PCBs.
(b) If a 1000:1 or higher dilution ratio sample was
analyzed and no measurable PCB peaks were detected/
analyze an aliquot of sample diluted 100:1.
(c) Samples diluted 100:1 and analyzed by electron
capture GC consistently produce results that are 10
to 20 percent lower than the true value due to
quenching of the detector response by high-boiling
hydrocarbons coeluting with the PCBs. The degree of
error is matrix dependent and is not predictable for
samples of unknown origin. As the PCB concen-
tration approaches 20 percent of a control level,
e.g. 50 mg/kg, the analyst must routinely reanalyze
a duplicate 'spiked sample to determine the Actual
recovery. Spike the duplicate or diluted sample at
twice.tne apparent concentration of the original
sample and correct the results accordingly.
(d) If PCB interference problems are encountered or **
PCB ratios do not match the standards, proceed to
paragraph 1.2.7 to clean up the sample prior to
further analysis, analyze the samples using alter-
nate columns, or use GC/MS analytical techniques to
verify whether or not the non-representative
patterns are due to PCBs.
Quantitative GC/MS techniques may be used in place of GC
analysis; the recommended approach is selected ion moni-
toring. The GC/MS system must have a program that
supports this method of data acquisition. The program
must be capable of monitoring a minimum of eight ions,
and it is desirable for the system to have the ability to
change the ions monitored as a function of time. For PCB
measurements, several sets of ions may be used depending
on the objectives of the study and the data system capa-
bilities. The alternatives are as follows:
Single ions for 154, 188, 222,
sensitivity 256, 292, 326,
360, 394
III-174
-------
Short mass ranges which 154-156, 188-192,
may give enhanced sensi- 222-226, 256-260,
tivity depending on the 290-295, 322-328,
data system capabilities 356-364, 390-398
Single ions that give 190, 224, 260, 294,
decreased sensitivity 330, 362, 394
but are selective for
levels of chlorinationl?
The data system must have the capability of integrating
the abundances of the selected ions between specified
limits, and relating integrated abundances to concentra-
tions using the calibration procedures described in this
method.
1.2.7 Sample Cleanup Procedures
Several tested cleanup techniques are described below.
Depending on the past experience of the analyst with the
particular sample matrix being analyzed and the complexity of
the sample, one or all of the following techniques may be
required to seoarate any PCBs that may be present from the
interferences.
1.2.7.1 Acid Cleanup
(a) Place 5,0 ml of concentrated sulfuric acid into a
40-ml narrow-moutn screw cap Don!a. Add 10.0 ml of
the diluted sample. Seal the bottle with a Teflon-
lined screw cap and shake for 1 minute.
(b) Allow the phases to separate, transfer the sample
(upper phase) to a clean narrow-mouth screw-cap
bottle. Seal with a Teflon-lined cap. Analyze
according to paragraph 1.2.6.
(c) If the sample is highly contaminated, a second or
third acid cleanup may be employed.
NOTE 4: This cleanup technique was tested over a
period of about 6 months using both electron capture
and electrolytic conductivity detectors. Care was
taken to exclude any samples that formed an emulsion
with the acid. The sample was withdrawn well above the
sample-acid interface. Under these conditions no
adverse effects associated with column performance and
detector sensitivity to PCBs were noted. This
cleanuo technique could adversely affect the chromato-
graphic column performance for future ^c*d-degradable
samel 9$.
III-175
-------
1.2.7.2 norfsll Column Cleanup
(a) Variances between batches of Florisll may affect the
elution volume of the various PCBs. For this reason,
the volume of solvent required to completely elute all
of the PCBs must be verified by the analyst,, The
weight of Florisil can then be adjusted accordingly.
(b) Place a 20.0-g charge of Florisil, activated at 130°C,
into a Chromaflex column. Settle the Florisil by
tapping the column. Add about 1 cm of anhydrous
sodium sulfate to the top of the Florisil. Pre-elute
the column with 70 to 80 ml of hexane. Just before
the exposure of the sodium sulfate layer to air, stop
the flow. Discard the eluate. Add 2.0 ml of the
undiluted sample to the column with a 2-ml Mohr pipet.
(For viscous samples, cut the capillary tip off the
pipet.) Add 225 ml of hexane to the column.
(c) Carefully wash down the inner wall of the column with
a small amount of the hexane prior to adding the total
volume. Collect and discard the first 25.0 ml.
(d) Tollect exactly 200 ml of hexane eluate in a 200-ml
volumetric flask. All of cne PCBs should be in :nis
fraction. Using the same pipet as in (b), deliver
2.0 ml of sample into a tared 10 ml oeaker weighed to
±0.001 g. Reweigh the beaker to determine the weight
of the sample diluted to 200 ml.
(e) Analyze the sample according to paragraph 1.2.6.
1.2.7.3 Alumina Column Cleanup
(a) Adjust the activity of the alumina by heating to 200°C
for 2 to 4 hours. When cool, add 3% water (wt:wt) and
mix until uniform. Store in a tightly sealed bottle.
Allow the deactivated alumina to equilibrate with room
air at least 1/2 hour before use. Reactivate weekly.
(b) Variances between batches of alumina may affect the
elution volume of the various PCBs. For this reason,
the volume of solvent required to completely elute all
of the PCBs must be verified by the analyst. The
weight of alumina can then be adjusted accordingly.
(c) Place a 50,0-g charge of alumina Into a Chromaflex
column. Settle the alumina by tapping. Add about
1 cm of anhydrous sodium sulfate. Pre-elute the
column with 70 to 80 ml of hexane. Just before
III-176
-------
exposure of the sodium sulfate layer to air, stop the
flow. Discard the eluate.
(d) Add 2.5 ml of the undiluted sample to the column with
a 5-ml Mohr pipet. (For viscous samples, cut the
capillary end off the pipet.) Add 300 ml of hexane to
the column. Carefully wash down the inner walls of
the column with a small volume of hexane prior to
adding the total volume. Collect and discard the 0-
to 50-ml fraction.
(e) Collect exactly 250 ml of the hexane in a 250-ml vol-
umetric flask. All of the PCBs should be in this
fraction. Using the same pipet as in (d), deliver 2.5
ml of sample into a tared 10-ml beaker weighed to
±0.001 g. Reweigh the beaker to determine weight of
sample diluted to 250 ml. Analyze the sample
according to paragraph 1.2.6.
1.2.7.4 Silica Gel Column Cleanup
(a) Activate silica gel at 135°C overnight. Variances
between batches of silica gel may affect the elution
volume of the various PCBs. For this reason, tne
volume of solvent required to comoletely elute all of
the PCBs must be verified by the analyst. The weight
of silica gel can then be adjusted accordingly.
(b) Place a 25-g charge of activated silica gel into a
Chromaflex column. Settle the silica gel by tapping
the column. Add about 1 cm of anhydrous sodium
sulfate to the top of the silica gel.
(c) Pre-elute the column with about 70 to 80 ml of hexane.
Discard the eluate. Just before the exposure of the
sodium sulfate layer to air, stop the flow.
(d) Add 2.0 ml of the undiluted sample to the column with
a 2-ml. Mohr pipet. (For viscous samples, cut the
capillary tip off the pipet.)
(e) Wash down the inner wall of the column with 5 ml of
hexane. Elute the PCBs with 195 ml of 10 percent (V/V)
diethyl ether in hexane. Collect exactly 200 ml of the
eluate in a 200-ml volumetric flask. All of the PCBs
should be in this fraction.
(f) Using the same pi pet as in (d), deliver 2.0 ml of
sample Into * tared 10-ml beaker (±0.001 g). Reweigh
to determine the weignt of sample diluted co 200 ,nl.
Analyze the sample according to oaragraoh 1.2.6.
TII-17?
-------
1.2.7.5 Gel Permeation Cleanup
(a) Set up and calibrate the gel permeation cnromatograph
with an SX-3 column according to the instruction
manual. Use 15 percent (V/V) methylene chloride in
cyclohexane as the mobil phase. Place 1.0 ml of sample
into a 100-ml volumetric flask, using a 1-ml Mohr
pipet. (For viscous samples, cut the capillary tip
off the pipet.)
(b) Dilute the sample to volume, using 15 percent (V/V)
methylene chloride in cyclohexane.
(c) Using the same pipet as in (a) deliver 1.0 m'l of
sample into a tared 10-ml beaker weighed to ±0.001 g.
Reweigh the beaker to determine the weight of sample
used in (a).
(d) As an alternative to (a) and (b), weigh approximately
1 gram ±0.001 g of sample and dilute to 100.0 ml in
15 percent (V/V) methylene chloride in cyclohexane.
(e) Inject 5.0 ml of the diluted sample into the instru-
ment. Col'ect the fraction containing the C'h
through Cl IQ PCBs (see operator's manual) in a K-0
flask equipped with a 10-ml ampule.
(f) Concentrate the (PCB) fraction down to less'than 5 ml.
using K-D evaporative concentration techniaues.
(g) Dilute to 5.0 ml with hexane, then analyze according
to paragraph 1.2.6. Be sure to use 100 ml as the
dilution volume for the final calculation.
1.2.7.6 Acetonitrile Partitioning
(a) Place 10.0 ml of the previously diluted sample into a
125-ml separatory funnel with enough hexane to bring
the final volume to 15 ml. Extract the sample four
times by shaking vigorously for 1 minute with 30-ml
portions of hexane-saturated acetonltrile.
(b) Combine and transfer the acetonltrile phases to a
1-Hter separatory funnel and add 650 ml of distilled
water and 40 ml of saturated sodium chloride solution.
Mix thoroughly for 30 to 35 seconds. Extract with two
100-ml portions of hexane by vigorously shaking about
15 seconds.
(c) Combine the hexane extracts in a 1-1 separatory funnel
and wash with two iOO-mi portions of disclllea *dier.
III-178
-------
Discard the water layer and pour the hexane layer
through a 7 to 10 cm anhydrous sodium sulfate column
into a 500-ml K-D flask equipped with a 10-ml ampule.
Rinse the separatory funnel and column with three 10-
ml portions of hexane.
(d) Concentrate the extracts to 6 to 10 ml in the K-D
evaporator in a hot water bath, then adjust the volume
to 10.0 ml. Be sure to use the correct dilution vol-
ume for the final calculation.
(e) Analyze according to paragraph 1.2.6.
1.2.7.7 Florisil Slurry Cleanup
(a) Place 10 ml of the diluted sample into a 20-ml
narrow-mouth screw cap container. Add 0.25 g of Flor-
isil. Seal with a Teflon-lined screw cap and shake
for 1 minute.
(b) Allow the Florisil to settle, then decant the treated
solution into a second container. Analyze according
to paragrapn i.2.6.
III-179
-------
2.1 Analysis of Water Samples for Pesticides/Polychlorinated Biphenyls
Analytical Procedure: evaluated
Sample Preparation: available
2.1.1 Reference/Title
"Organochlorine Pesticides and PCB's - Method 608," Federal
Register, Vol. 44, No. 233. 69501-69509, December 3,
1979.20
2.1.2 Method Summary
A measured volume of sample, approximately 1 liter, is
solvent-extracted with methylene chloride using a separatory
funnel. The methylene chloride extract is dried, exchanged
to hexane, and concentrated to a final volume of 10 ml or
less. Gas chromatographic conditions are described which
permit the separation, identification, and quantification of
the chemical compounds in the extract by electron capture gas
chromatography.21
A Florisil column cleanup procedure and an elemental sulfur
removal procedure are also described to aid in the elimina-
tion of interferences that may be encountared.
2.1.3 • Applicability
This method is suitable for the determination of the com-
pouncs "-istcd in Tab1es 1 and 2 when present in municipal and
industrial discharges. The method detection limits are diso
presented in Tables 1 and 3. However, actual detection
limits will vary with the sample size chosen, the extent of
extract concentration, the types of interferences present,
and the nature of the original sample matrix.
This method should only be used 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 Subsection
G.2.
2.1.4 Precision and Accuracy
This procedure was used by a single laboratory to analyze
triplicate spiked wastewater samples on separate days. The
average recovery and calculated standard deviation for each
compound is presented in Table 2 (p. 111-148).
This method "ias been tested *or "Hnearly of sm'ke recovery
from reagent water and has been demonstrated to be appiicaole
over ;he concentration range rrom l x MOL 'jp to 1000 x MDL
III-180
-------
with the following exceptions: chlordane recovery at 4 x MDL
was low (60%); toxaphene recovery was demonstrated linear
over the range of 10 x MDL to 1000 x MDL.21
NOTE: The Environmental Protection Agency 1s 1n the process
of conducting an Inter!aboratory study to fully define the
performance of this method (1982).
2.1.5 Sample Preparation
Mark the water meniscus on the side of the sample bottle for
later determination of sample volume. Pour the entire sample
Into a 2-1 Her separatory funnel.
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 2 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 mechan-
ical 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 meth-
ylene chloride extract 1n a 250-ml Erlenmeyer flask.
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.
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 1f the requirements of Subsection G.2. are
met.
Pour the combined extract through a drying column containing
about 10 cm of anhydrous sodium sulfate, and collect the
extract 1n the K-D concentrator. Rinse the Erlenmeyer flask
and column with 20 to 30 ml of methylene chloride to complete
the quantitative transfer.
Add 1 or 2 clean boiling chips to the evaporative flask and
attach a three-baTl Snyder column. Pre-wet the Snyder column
by adding about 1 ml methylene chloride to the top. Place
the K-D apparatus on a hot *ater bath !60 to 55*C) «o *,hat
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
III-181
-------
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.
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. Pre-wet
the column by adding about 1 ml of hexane to the top. Con-
centrate the solvent extract as before. The elapsed time of
concentration 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.
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. If the
sample extract requires no further cleanup, proceed with gas
chromatographic analysis. If the sample requires cleanup,
proceed to paragraph 2.1.6.
ns the or^'gi"3.! isnol^ "oluins bv *"9f'''''ir'|n ths
bottle to the mark and transferring the liquid to a 1,000-ml
graduated cylinder. Record the sample volume to the nearest
5 ml.
2.1.6 Sample Cleanup and Separation
2.1.6.1 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 cir-
cumstances demand the use of an alternative cleanup procedure,
the analyst must determine the elution profile and demon-
strate that the recovery of each compound of interest is no
less than 85 percent. The Florisil column allows for a select
fractionation 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.
2.1.6.2 Florisil Column Cleanup
(a) Add a weight of Florisil (nominally 21 g) predeter-
mined by calibration (Subsection H.4. and H.5.K to
III-182
-------
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 chromatography column.
Discard the eluate.
(b) Adjust the sample extract volume to 10 ml 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.
(c) 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 layer 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 IS% ethyl ether in hexane (v/v)
(Fraction 2), into a second K-D flask. Perform the
;hird aiution using 200 ml of SOS ethyl ether 1-n
hexane (v/v) (Fraction 3). The distribution patterns
for the pesticides and ?CBs are shown in Table 9.
(d) Concentrate the eluates by standard K-D techniques
Subsection J.2.1.5), substituting hexane for the
glassware rinses and increasing the water batn to aoout
85*C. Adjust final volume to 10 ml with hexane,
Analyze by gas chromatography.
2.1.6.3 Elemental sulfur will usually elute entirely in Fraction 1
of the Florisil column cleanup. To remove sulfur inter-
ference from this fraction or the original extract, pipet
1.00 ml of the concentrated extract into a clean concen-
trator tube or Teflon-sealed vial. Add 1 to 3 drops of
mercury and seal.*4 Agitate the contents of the vial for
15 to 30 seconds. Prolonged shaking (2 hours) may be re-
quired. If so, this may be accomplished with a reciprocal
shaker. Alternatively, activated copper powder may be used
for sulfur removal.22 Analyze by gas chromatography.
2.1.7 Sample Analysis by Gas Chromatography
Table 3 summarizes the recommended operating conditions for
the gas chromatograph. This table includes retention times
and method detection limits that were obtained under these
conditions. Ixamcl^s cf the seoarations achieved bv column 1
III-183
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TABLE 9. DISTRIBUTION OF CHLORINATED PESTICIDES AND PCBs
INTO FLORISIL COLUMN FRACTIONS20
======3===============================================::=======
Percent Recovery by Fraction
Parameter
Fraction 1
Fraction 2
Fraction 3
Aldrin
Alpha-BHC
Beta-BHC
Delta-BHC
Gamma- BHC
Chlordane
4, 4 '-ODD
4, 4 '-DDE
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
^ndn'n aldehyde
Heptachlor
Heptachlcr epoxide
Toxaphene
PCB-1016
°CS-1221
PCB-1232
PCB-1242
PCB-1248
PCB-1254
PCB-1260
100
100
97
98
100
100
99
98
100
0 100
37 64
0 7
0 0
4 96
0 68
100
100
96
97
97
95
97
103
90
95
91
106
26
:============== =====
====:==
Eluant composition by fraction:
Fraction 1-6% ethyl ether in hexane
Fraction 2 - 15% ethyl ether 1n hexane
Fraction 3 - 50% ethyl ether In hexane
are shown in Figures 2 to 10. Other packed columns, chro-
ma tographic conditions, or detectors may be used if the
requirements of Subsection G.2. are met. Capillary (open-
tubular) columns may also be used if the relative standard
deviations of responses for replicate injections are demon-
strated to be less than 6% and the requirements of Subsection
G.2. are met.
Calibrate the system daily as described in Subsection H.
III-184
-------
If the internal standard approach 1s bei/ig used, the Internal
standard must be added to the sample extract and mixed thor-
oughly immediately before injection into the instrument.
Inject 2 to 5 ul of the sample extract using the solvent-
flush technique. Smaller (1.0 pi) volumes can be injected if
automatic devices are emp^yed. Record the volume Injected
to the nearest 0.05 pi, tne total extract volume, and the
resulting peak size in area or peak height units.
The width of the retention time window used to make Identi-
fications should be based upon measurements of actual reten-
tion 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.
If the response for the peak exceeds the working range of the
system, dilute the extract and re-analyze.
If the measurement of the peak response is prevented by the
presence of interferences, further cleanup is required using
one of the methods presented in Subsection J.I.2.7.
Calculate the concentrations of identified sample constitu-
ents as indicated in Subsection K.
III-185
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2.2 Analysis of Water Samples for Organochlorlne Pesticides/
PolychloMnated Biphenyls
Analytical Procedure: evaluated
Sample Preparation: available
2.2.1 Reference
American Society for Testing and Materials, "Tentative Method
of Test for Organochlorine in Water." Method D 3086-72T, D-19
Water pp. 519-534 (1980), American Society for Testing and
Materials, Philadelphia, Pennsylvania.1^
2.2.2 Method Summary
The method offers several analytical alternatives, dependent
on the nature and extent of Interferences and the complexity
of the pesticide mixtures found. It is recommended for use
only by experienced residue chemists or under the close
supervision of such qualified persons. Specifically, the
procedure describes the use of an effective co-solvent for
efficient sample extraction and provides, through use of
thin-layer and column chromatography, methods for the
elimination of non-pesticide Interferences and the pre-
separation of pesticide mixtures. Identification is by
selective gas chromatographic separations chrougn the use of
two or more unlike columns. Detection and measurement are
accomplished by electron capture and microcoulometric or
electrolytic conductivity gas chromatography. Results are
reported in nanograms per liter without correction for
recovery data but such data should be included wr;h the data,
2.2.3 Applicability
This method covers the determination of various chlorinated
hydrocarbon pesticides, Including some pesticidal degradation
products and related compounds, in water. Such compounds are
composed of carbon, hydrogen, and chlorine, but may also
contain oxygen, sulfur, phosphorus, or nitrogen.
The following compounds may be determined Individually by
this method; BHC, lindane, heptachlor, aldrin, heptachlor
epoxide, dleldrin, endrin, Perthane, ODE, ODD, DDT, meth-
oxychlor, endosulfan, gamma-chlordane and Sulphenone. Under
ideal circumstances, Strobane, toxaphene, Kelthane, chlordane
(technical grade), and others may also be determined.
When chlorinated hydrocarbons exist as complex mixtures, the
individual compounds may be difficult to distinguish. High,
low, or otherwise unreliable results may be obtained through
misidentification, or one compound obscuring another of
III-186
-------
lesser concentration, or both. Provisions Incorporated 1n
this method are Intended to minimize the occurrence of such
Interferences.
2.2.4 Precision and Accuracy
The precision of the method in paragraph 2.2 within the desig-
nated range varies with the determined concentration as shown
1n Table 10. Additional data on the recoveries observed with
this method are presented in Table 11.
2.2.5 Sample Extraction
The size of the sample taken for extraction is dependent on
the type of sample, the detection system employed, and the
sensitivity required. Background information on the pesti-
cide levels previously detected at a given sampling site will
help to determine the sample size required as well as the
final volume to which the extract needs to be concentrated.
The extract should not be concentrated further than required
to meet the sensitivity dictated by the purpose for the anal-
ysis. Each time a set of samples is extracted, an aliquot of
solvent equivalent to that used for extraction is carried
through tne entire procedure to provide a method blank. To
assist in interpretation of results, the pH of the sample is
taken prior to extraction. When the volume of the sample
permits, one set of duplicates and one dosed sample should
also be analyzed as a quality control check.
•
If the extract Is to be analyzed by microcoulometric tecn-
niques, follow paragraph (a). If the extract is to be
analyzed by electron capture analysis, follow the instruc-
tions in paragraph (b).
(a) Microcoulometric Detection:
1. Place 3 liters of sample 1n a 4-liter separatory
funnel, equipped with a TFE-fluorocarbon stop-
cock, and extract with 150 ml of a mixture of ethyl
ether 1n hexane (15+85) by shaking vigorously for 2
minutes. Rinse the sample container with each
aliquot of extracting solvent prior to extraction of
the sample.
III-187
-------
TABLE 10. PRECISION OF METHOD FOR DETERMINATION OF
ORGANOCHLORINE PESTICIDES IN NATURAL WATERSlO
Pesticide
Aldrin
Lindane
Dieldrin
DDT
Pretreatment
no cleanup
cleanup^
no cleanup
cleanupb
no cleanup
cleanupb
flo cleanup
cleanupb
Mean
Recovery
ng/liter
10.42
79.00
17.00
64.54
9.67
72.91
14.04
59.08
21.54
105.83
17.52
84.29
40.30
154.87
35.54
132.08
Precision
ng/liter*
Sr So
4.86 2.59
32.06 20.19
9.13 3.48
27.16 8.02
5.28 3.47
26.23 11.49
8.73 5.20
27.49 7.75
18.16 17.92
30.41 21.84
10.44 5.10
34.45 16.79
15.96 13.42
38.80 24.02
22.62 22.50
49.83 25.31
aS.. = overall orecision, and .
S0 = single-operator precision.
bllse of Florisil column cleanup prior to analysis,
2. Allow the mixed solvent to separate from the water,
and draw off the water into the original container
(1f it is of approximate size of the sample being
extracted) or into a second 4-liter separatory fun-
nel. Pass the organic layer through a small column
of anhydrous sodium sulfate topped with a pledget of
cotton (previously rinsed with hexane) and collect
in a 600-ml tall-form beaker. Repeat the extraction
and treat the solvent as above. Add approximately
100 ml of sodium-sulfate-saturated water to the
sample and complete a third extraction with 150 ml
of hexane (not hexane-ethyl ether). Pass this sol-
vent, too, after separation, through the column of
sodium sulfate. Rinse the column with several small
portions of hexane. Blow out this solvent and recover
*n the collection beaker containing the combined
extracts. Partially evaporate tne contents of tfie
II1-188
-------
TABLE 11. RECOVERY OF ORGANOCHLORINE PESTICIDES FROM NATURAL WATERS10
SBasassssaaasaaaaaaasasassaaaaaaaasaaaaaaasaaasaaBassaasssssaaaasaasaazaasaaaaar.
Pesticide
Aldrin
Llndane
Dleldrln
DDT
Added
Level
ng/liter
15
110
10
100
20
125
40
200
=============
Recovery,
without
Cleanup,
percent
69
72
97
73
108
85
101
77
Added
Level
ng/l1ter
25
100
15
85
25
130
30
185
Recovery,
with
Cleanup,
percent
68
65
94
70
70
65
118
71
beaker to aoout 300 ml In a *ater bath 3t 70°C,
applying no air or vacuum, and transfer to a 500-ml
Kuderna-Danish (K-D) evaporator equipped with a
10-ml receiver ampule.
Hdjust che ;"'inal. axtrsct /olunje f^r tsmplss ?f Mgh
pesticide content (for example, pesticide plant waste
water samples), as necessary. Concentrate samples
containing small quantities of pesticides (low nano-
gram amounts, for example, most surface water samples)
to 1 ml.
Mount a three-ball Snyder column to the top of the
flask and evaporate solvent in a steam bath. After
no more solvent actively distills, remove the
assembled K-D evaporator from the bath and allow to
cool. Disconnect the ampule and concentrate the
volume of the extract to 1 ml in a warm water bath
(70*C) with a gentle draft of clean dry air. Make
an Initial gas chromatographic analysis on this
volume, as described in paragraph 2.2.7. If Insuf-
ficient pesticide Is present for quantitation at
this volume and greater sensitivity is required,
concentrate the extract further 1n accordance with
paragraph 2.2.7.
III-189
-------
5. Interferences in the form of distinct peaks or high
background, or both, in the Initial gas chromato-
graphic analysis, along with the physical character-
istics of the extract (color, cloudiness, and
viscosity) will indicate whether cleanup is re-
quired. When these conditions interfere with measure-
ment of the pesticides, proceed in accordance with
paragraph 2.2.6. Whether required for quantitative
analysis or not, all extracts should be subjected to
these procedures, subsequent to the initial analysis,
and rechromatographed for qualitative corroboration of
the results.
(b) Electron Capture Detection:
1. Drain 1 liter of sample into a 2-liter separatory
funnel, equipped with a TFE-fluorocarbon stopcock,
and extract with 60 ml of a mixture of ethyl ether
and hexane (15+85) by shaking vigorously for 2
minutes. Rinse the sample container with each
aliquot of extracting solvent prior to extraction of
the sample.
2. Allow the mixed solvent to saoarate from the water
and draw the water into the original sample
container (if 1t is of the aoproximate si^e of the
sample" being extracted) or into a second 1-liter
separatory funnel. Pass the organic layer through a
small column of anhydrous sodium suTfate topped with
a plug- of cotton (previously rinsea witn nexane; and
collect in a- 250-ml beaker. Repeat the extraction
and treat the solvent as above. Add approximately
35 ml of sodium-sulfate-saturated water to the
sample and complete a third extraction with 60 ml of
hexane (not hexane-ethyl ether). Pass this solvent
through the sodium sulfate column and collect in the
beaker. Rinse the column with several small portions
of hexane, blow out the solvent and recover it in the
collection beaker containing the combined extracts.
3. Adjust the final extract volume for samples of high
pesticide content (for example, pesticide plant waste
water samples), as necessary. Concentrate samples
containing small quantities of pesticides (low nano-
gram amounts, for example, most surface water samples)
to 1 ml.
4. Mount a three-ball Snyder column to the top of the
flask and evaporate solvent in a steam bath. After
no more solvent actively distills, remove the
assemolea K-0 evaporator rrom ;ne oath ana allow :o
III-190
-------
cool. Disconnect the ampule and concentrate the.
volume of the extract to 1 ml 1n a warm water bath
(70eC) with a gentle draft of clean dry air. Make
an Initial gas chromatographic analysis of this
volume as described in paragraph 2.2.7. If insuf-
ficient pesticide is present for quantisation at
this volume and/or greater sensitivity is required,
concentrate the extract further in accordance with
paragraph 2.2.7.
5. Interferences 1n the form of distinct peaks or high
background, or both, 1n the initial gas chromato-
graphic analyses, along with the physical character-
istics of the extract (color, cloudiness, and
viscosity) will Indicate whether cleanup is re-
quired. When these factors Interfere with measure-
ment of the pesticides, proceed 1n accordance with
paragraph 2.2.6. Whether required for quantitative
analysis or not, all extracts should be subjected to
these procedures, subsequent to the initial anal-
ysis, and rechromatographed for qualitative corrob-
oration of the results.
2.2.6 Sample Cleanup and Separation Procedures
A sample that contains interferences such as oil or wax-like
materials and other organic matter may be treated in several
ways:, column chromatography23, thin-layer chromatog-
rapny^.-S^ or Coiumn cnromatography followed by thin-i?yer
Chromatography. These procedures not only remove many of
the oily and waxy substances that prevent distinct and
specific gas chromatograms, but pre-separate mixtures of
pesticides, thereby aiding the analyst in interpretation of
subsequent gas chromatograms.
(a) Florisll Column Adsorption Chromatography:
1. Dilute the sample extract previously concentrated
to 1 ml to 10 ml with hexane. Place 15 g of
activated Florisil, that has been stored in an
airtight container at 130*C, in a column over a
small layer [13 mm (1/2 in.)] of anhydrous gran-
ular sodium sulfate. After tapping the Florisil
Into the column, add a 2-cm (3/4-1n) layer of gran-
ular sodium sulfate to the top. After cooling, pre-
elute the column with approximately 75 ml of
hexane. .Discard the pre-eluate, and just prior to
exposure of the sulfate layer to air, quantitatively
transfer the sample extract into the column by
aecamcation and subsequent hexane washings. *d.iust
the elution rate to-approximately 5 ml/min for two
eluates collected separately in the 300-iiil X-D
III-191
-------
apparatus equipped with 10-ml ampules. Perform the
first elutlon with 200 ml of a mixture of ethyl
ether 1n hexane (6+94) and the second elutlon with
200 ml of a mixture of ethyl ether 1n hexane
(15+85). Connect the K-D apparatus containing the
eluates to three-ball Snyder columns and evaporate
the solvents in accordance with paragraph 2.2.5.D.4.
2. Eluate Composition. If the Florisil has been prop-
erly activated23 and stored, and if the reagents
have been carefully prepared, the following eluate
compositions will be obtained when the pesticides
are present. The first eluate (ethyl ether in
hexane (6+94)) will contain lindane, BHC, Kelthane,
aldrin, heptachlor, DDE, ODD, DDT, Perthane, hep-
tachlor epoxide, methoxychlor, toxaphene, Strobane,
chlordane (gamma and tech), and endosulfan I. The
second eluate (ethyl ether in hexane (15+85)) will
contain dieldrin, endrin, endosulfan II, lindane,
and Kelthane.
3. Standard pesticide mixtures should be used fre-
quently to demonstrate the effectiveness of the
r'on'sil to characterize the aluate composition and
provide quantitative recovery. The concentrated
extract may be analyzed directly by injecting
suitable aliquots from the K-D ampule into the gas
chromatograph. If the residues are high in total
ornanics, further cleanup prior to gas chromato-
graphic analysis may oe necessary.
(b) Thin-Layer Chromatography (TLC):
1. The sample extract [paragraph 2.2.5(a) or 2.2.5 (b)]
or the Florisll treated sample extracts [paragraph
2.2.6 (a)] may be further purified by TLC to separate
pesticides and remove interfering substances.
2. Layer Preparation - Prepare layers of silica gel
0.25 mm thick on 20-cm x 20-cm (8 x 8-1n) glass
plates. Prepare a homogeneous slurry by shaking 30 g
of silica gel in approximately 60 ml of water.
With the aid of a variable thickness applicator,
immediately spread the slurry over five plates held
on a mounting board. Allow the layers to stand for
5 minutes then activate them in an oven for 60
minutes at 110'C and store 1n a desiccator for
future use. Reactivate layers stored longer than 1
week before use. Just prior to use, make marks 15
and 115 mm above the bottom edge of the layer to
define che spotting ".ine and the point at .rtiich the
solvent front has moved 100 mm.
III-192
-------
3. Developing Solvent - Add the developing solvent,
carbon tetrachloride, to the developing chamber to a
depth of 10 mm. Place two filter paper wicks, one
on each side of the chamber, so that one end con-
tacts the solvent. After the I1d is in place, allow
the chamber to equilibrate for 1 hour. It is
Important that the chamber be protected from drafts
and large temperature changes.
4. Sample Spotting - Carefully evaporate the sample
extracts contained 1n K-0 ampules [paragraph
2.2.6 (a)3, or extracts retained [paragraph 2.2.5 (a)
or 2.2.5 (b)], 1n a bath of warm water at 40"C with a
fine draft of clean air to an appropriate volume for
spotting (not less than 200 ul) (NOTE 5). Adjust
the final sample volume so that no more than 100 ul
must be spotted 1n order to maintain adequate gas
chromatographlc sensitivity when analyzing the
subsequent eluates. Never concentrate the eluates
below 100 ul. Very carefully wash down the Internal
wall of the ampule with small portions of ethyl
ether and evaporate by the same technique to
approximately 100 yl. Carry out this step three
times. Then adjust the volume
-------
the layer 1s in contact with the developing solvent,
and replace the lid. When the developing solvent
reaches the upper reference line (100 mm),, remove
the layer from the chamber and allow it to air dry
at room temperature.
7. Visualization and Sectioning of Layer - After
development, evenly spray the portion of the layer
containing the standards (from 10 to 20 ug) with a
fairly heavy coat of Rhodamine B (0.1 mg/ml in
ethanol). Allow the sprayed area to thoroughly dry
(approximately 5 minutes) and then expose it to and
view 1t under a short-wave UY light. The pesticides
appear as quenched areas (dark) on a fluorescent
background. Mark the location of each pesticide.
8. The distance of travel for pesticides present in the
unknown samples and recovery test standards will be
the same as those of the standards. From this
Information, divide the vertical zone for each
sample into five horizontal sections. Identify the
sections with Roman numerals.
Examoles of respective Rf and Rr values for various
pesticides are listed in Facie 12.
9. Pesticide Removal from the TLC Plates - Using tne
spotting template as a ruler and with the aid of a
sharp pointed object, individually rule off the
silica gel sections or interest. Kith tne JIG of a
mild vacuum draw the silica gel, first from the
periphery of the section and then from the center of
the section, Into a medicine dropper that is plugged
close to the tip with filtering-grade glass wool.
Quantitatively elute the pesticides adsorbed on this
silica gel into a 10-ml K-D ampule with successive
small washes of ethyl ether-petroleum ether (1+1) to
a total volume of 5 to 10 ml. Plug the ampule with
a glass stopper and retain the contents for the
determinative steps 1n paragraph 2.2.7.
2.2.7 Sample Analysis
Obtain reasonable positive Identification of a pesticide by
corroborating the results using a minimum of two chromato-
graphlc columns of different polarity. Columns recommended
for this purpose and their approximate operating temperatures
are: 5 percent OV-17 on Gas Chrom Q (80 to 100 mesh)-210eC,
5 percent QF-1 plus 3 percent DC-200 on Gas Chrom Q (80 to
100 mesh)-185°C, 3 percent OY-101 on Gas Chrom Q (80 to 100
mesn)-i90'iC, ana 3 percent-«V-£10 on 3as Chrom Q (3G to 100
III-194
-------
TABLE 12. SOME Rf AND Rr VALUES OF PESTICIDES DEVELOPED WITH CCL4
ON SILICA GEL THIN LAYER PLATE
a=a=================z======z==========================================
Pesticides and Metabolites Rf Value9 Rr Value5 Section
Dieldrin
Endrin
Heptachlor epoxide
Lindane
ODD
Gamma-Chlordane
Heptachlor
DDT
DDE
Al dri n
0.17
0.20
0.29
0.37
0.54
0.55
0.67
0.68
0.72
0.73
0.33
0.37
0.52
0.69
1.00
1.02
1.24
1.26
1.33
1.35 .
II
III
IV
B==============================================================================
aRr - Distance traveled V/ the ^omcound divided by the distance traveled
by the solvent front.
^R - Distance traveled by the compound divided by the distance traveled
by the distance traveled by standard p,p'-DDD.
mesh)-185°C. The analyst must determine the optimum condi-
tions to obtain the best results for compounds under study
with the available equipment. Chromatograph standard mix-
tures of interest to determine the required separation with
maximum resolution. Evaluate parameters such as gas flow,
temperature, column length and diameter, as well as detector
performance and alter as required to achieve the desired
results.
Final Extract Concentration
If necessary to meet the sensitivity requirements for the
purpose at hand, concentrate the sample extract contained in
a graduated K-D receiver ampule from the appropriate extrac-
tion or purification step [paragraph 2.2.5 (a), 2.2.5 (b), or
2-2.5 ?b)l *o ""sss than 1 ml 1n a beaker of warm water (60eC).
Continue to carefully evaporate the solvent with a fine
stream of dried and filtered air to no less than 0,3 ml.
in-195
-------
Intermittently wash down the internal wall of the tube with
hexane during this final concentration step and adjust the
volume with hexane to an appropriate level (from 0.3 ml to
0.5 ml for micro-coulometric determination or from 0.5 to 1.0
ml for electron capture determination).
If more exhaustive evaporation of the extract is required to
achieve the necessary sensitivity for microcoulometric
detection, the use of "keeper" is strongly recommended.
Place 2 mg of "keeper" in the concentrated extract by syringe
addition of 100 ul of 20 ug/ul light mineral oil in hexane.
This "keeper" will not interfere with microcoulometric detec-
tion and will prevent major residue losses in this exhaustive
evaporation step.
Sample Analysis - Note the volume of the sample extracts and
analyze suitable aliquots (from 20 to 100 ul for microcoulo-
metric detection and from 5 to 10 ul for electron capture
detection) by gas chromatography, employing at least two
columns for identification and quantification. Frequently
inject standards to ensure optimum operating conditions. Gas
chromatograms of several standard pesticides are shown in
Figures 12, 13, 14, and 15. The elution order as well as
elution ratios for various pesticides are provided in Table
13 us u guide only. It Is the responsibility of the analyst
to develop his own identification keys to fit the chosen
operating conditions of the instrument. Calculate the
concentration of identified sample constituents as indicated
in Subsection K.
III-196
-------
Linda ne
Heptachlor
[ Heptachlor
Epoxide
Aldrin
p.p'-DDE
UU
p.p'-DDT
I
4
I
6
I
10
I
12
I
14
Retention Time in Minutes
(Chart speed one-half inch per minute)
Column Packing- 3% QC-200 + 5% Qf-1 on Gas Chrom Q (80/100 Mesh)
Carrier Gas- Nitrogen at 80 ml/min
Column Temperature- 200°C
Figure 12. Electron capture gas chromatogram of pesticide standards
III-197
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1 —
0
1
2
1
4
1
6
1
8
1
10
1
12
Retention Time in Minutes
(Chart speed one-half inch per minute)
Column Packing- 3% OV-101 on Gas Chrom Q (80/100 Mesh)
Carrier Gas- Nitrogen at 80 ml/min
Column Temperature- 175°C
Figure 13. Electron capture gas chromatogram of pesticide standards,
III-198
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Lindane
Heptachlor
jHeptachlor
Epoxide
p.p-DDT
0 2 46 8 10 12 14 16
[Retention Time in Minutes]
Chart speed one-half inch per minute
Column Packing-5% OV/17 on Gas Chrom Q [60/80 Mesh]
Carrier Gas-Nitrogen at 80 ml/min.
Column Temperature 200°C
18
Figure 14. Electron capture gas chromatogram of pesticide standards,
III-199
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Lindan* • H«pt»chlor
Hepuchlor
I Atdrin Epoxide
p.p'-DDT
I
4
I
6
8
I
10
I
12
I
14
Retention Time in Minutes
(Chart speed one-half inch per minute)
Column Packing- 3% OV-210 on Gas Chrom B (80/100 Mesh)
Carrier Gas- Nitrogen at 80 ml/min
Column Temperature- 180°C
Figure 15. Electron capture gas chromatogram of pesticide standards.
III-200
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TABLE 13. RETENTION TIMES OF ORGANOCHLORINE PESTICIDES RELATIVE TO ALDRIN
Liquid
Phase3
Column
Temperature
==========================
5 Percent
DC-200 +
3 Percent 5 Percent 3 Percent 3 Percent
QF-1 OV-17 OV-101 OV-210
200 °C
200'C
175*C
160°C
Relative
Sensitivity
to EC
Detectorb
Pesticide
absolute)
RRtc
RRtc
Alpha-BHC
Lindane
Heptachlor
Aldrin
-------
3.1 Analysis of Sediment Samples for Pesticides/Polychlorinated
Biphenyls
Analytical Procedure: available
Sample Preparation: available
3.1.1 Reference/Title
Thompson, J. F., "Analysis of Pesticide Residues in Human and
Environmental Samples," U.S. Environmental Protection Agency
Pesticide and Toxic Substances Effects Laboratory. Research
Triangle Park, North Carolina. 197426
3.1.2 Method Summary
A sediment is partially dried and extracted by column elution
with a mixture of 1:1 acetone/hexane. The extract 1s washed
with water to remove the acetone and then the pesticides are
extracted from the water with 15 percent methylene chloride
1n hexane. The extract is dehydrated, concentrated to a
suitable volume, subjected to Florisil partitioning, desul-
furized, if necessary, and analyzed by gas chromatography.
3.1.3 Applicability
3.1.4 Precision and Accuracy
Many pesticides and PCBs can be recovered at the 85-104 per-
cent level based on duplicate analyses. However, compounds
iucn as r.eptacr.ior, vhlorcbenz'ilate, and arganophosphates are
degraded by the sulfur cleanup procedure and effectively
lost.26
3.1.5 Sample Preparation
Decant and discard the water layer over the sediment. Mix
the sediment to obtain as homogeneous a sample as possible
and transfer to a pan to partially air dry for about 3 days
at ambient temperatures.
NOTE 6: Drying time varies considerably depending on soil
type ana drying conditions. Sandy soil will sufficiently dry
in 1 day, whereas muck requires, at least, 3 days. Silt and
muck sediments are sufficiently dry when the surface starts
to split, but there should be no dry spots. Moisture content
will be 50 to 80 percent at this point.
Weigh 50 g of the partially dried sample Into a 400-ml Omni
mixer chamber. Add 50 g of anhydrous sodium sulfate and mix
well with a large spatula. Allow to stand with occasional
Erring "or -ipprox-! mats'* y \ sour.
> V t i
III-202
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NOTE 7: If the final calculations will be made on a dry-
weight basis, it is necessary at this point to initiate the
test for percent total solids on the sample being extracted
for pesticide evaluation. Immediately after weighing the
50-g sample for extraction, weigh approximately 5 g of the
partially dried sediment into a tared crucible. Determine
the percent solids by placing the sample in a drying oven
overnight at 103 to 105°C. Cool the sample, desiccate, and
weigh the residue. Repeat the process until a constant
weight is obtained.
Attach the 400-ml chamber to an Omni or Sorvall mixer and
blend for about 20 seconds. The sample should be fairly
free-flowing at this point.
Carefully transfer the sample to a chromatographic column.
Rinse the mixer chamber with small portions of hexane, adding
the rinsings to the column.
Elute the column with 250 ml of 1:1 acetone-hexane at a flow
rate of 3 to 5 ml/min into a 400-ml beaker.
Concentrate tne sample extract to about 100 ml under a
nitrogen stream and at a temperature no higher than 55°C.
Transfer to a 500-ml separatory funnel containing 300 ml of
distilled water and 25 ml of saturated sodium sulfate sol-
ution. Shake the separatory funnel for 2 minutes.
Drain the water layer into a clean beaker and the hexane
layer into a clean, 250-ml separatory funnel.
Transfer the water layer back into the 500-ml separatory
funnel and re-extract with 20 ml of 25 percent methylene
chloride in hexane, again shaking the separatory funnel for 2
minutes. Allow the layers to separate. Discard the water
layer and combine the solvent extracts in the 250-ml separ-
atory funnel.
Wash the combined solvent extract by shaking with 100 ml of
distilled water for 30 seconds. Discard the wash water and
rewash the extract with an additional 10 ml of distilled
water, again discarding the wash water.
Attach a 10-ml evaporator concentrator tube to a 250-ml
Kuderna-Danish flask and place under a filter comprised of a
small wad of glass wool and approximately 1.2 cm of anhy-
drous sodium sulfate in a filter tube.
Pass che solvent extract through the drying filter into the
K-0 flask and rinse with three portions of approximately
5 ml each of nexane.
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Attach a Snyder column to the top joint of a K-D flask,
immerse tube in an 80"C water bath, and concentrate extract
to 5 ml.
Remove the tube and rinse the joint with a small volume of
hexane. The sample is now ready for Florisil partitioning.
3.1.6 Sample Cleanup with Florisil Columns
Prepare a Florisil chromatographic column containing 10 cm
(after settling) of activated Florisil topped with 1 cm of
anhydrous, granular sodium sulfate. A small wad of glass
wool, pre-extracted with hexane, is placed at the bottom of
the column to retain the Florisil.
NOTE 8: If the oven is of sufficient size, the columns may
be prepacked and stored 1n the oven, for withdrawal of
columns a few minutes before use.
The amount of Florisil needed for proper elution should be
determined for each lot of Florisil.
Place a 500-ml Erlenmeyer flask under the column and prewet
the packing with hexane (40 to 50 ml, or a sufficient volume
to completely cover the sodium sulfate layer).
NOTE 9: From this point and through the elution process, the
solvent level should never be allowed to go below the top of
tr.e :oaium sulfats layer. If air Is 'ntrcducsd, channel :nc
may occur that results in reduced column efficiency.
Assemble two more K-D apparatus but with 500-ml flasks and
position the flask of one assembly under the Florisil column.
However, at this point, use 25-ml graduated evaporator con-
centrator tubes instead of the 10-ml size used previously.
Using a 5-ml Mohr or a long disposable pipet, immediately
transfer the extract from the evaporator tube onto the column
and permit 1t to percolate through. Rinse tube with two
successive 5-ml portions of hexane, carefully transferring
each portion to the column with the pipet.
NOTE 10: Use of the Mohr or disposable pipet to deliver the
extract directly onto the column precludes the need to rinse
down sides of the column.
Commence elution with 200 ml of 6 percent diethyl ether in
petroleum ether (Fraction 1). The elution rate should be
approximately 5 ml per minute. When the last of the eluting
jolvent reaches 3 point spproxlmatelv 3 im *rom t.he too
of the sodium sulfate layer, place the second 500-ml Kuderna-
Oanisn assemoly under the column and continue slutlon with
III-204
-------
200 ml of 15 percent diethyl ether In petroleum ether (Frac-
tion 2). Place both Kuderna-Dam'sh evaporator assemblies in
a water bath and concentrate extracts to approximately 20 ml.
NOTE 11: If there is reason to suspect the presence of
malathion in the sample, have a third 500-ml K-D assembly
ready. At the end of the 15 percent fraction elution, add
200 ml of 50 percent diethyl ether in petroleum ether
(Fraction 3), evaporating the eluate in the same manner.
Remove K-D assemblies from the bath, cool, and rinse the
T-joint between the tube and flask with a little petroleum
. ether. Finally, dilute both extracts to exactly 25 ml and
proceed with the GLC determinative step.
3.1.7 Sample Analysis by Gas Chromatography
Inject 5 ul of each fraction extract into the gas chromato-
graph (electron capture mode) primarily to determine whether
the extracts will require further adjustment by dilution or
concentration.
When aoDrooria-te dilution adjustments have been made in the
extracts and the column oven ~.z set it trie <-equired tempera-
ture, the relative retention values of the peaks on the
chromatograms should be calculated. Wien these values are .
compared with the values in Table 3 for the appropriate
column, the operator should be able to make tentative com-
pouna identifications, ''icrocoulcmetry ard/or "H.C may be
required for positive confirmation of some of the tentatively
identified chlorinated compounds, whereas flame photometric
detectors (FPD) may be utilized for confirmation of the
organophosphate compounds.
An analytical problem that must be considered when sediment
samples are analyzed for chlorinated hydrocarbon pesticides
is sulfur interference. Elemental sulfur is encountered in
most sediment samples, marine algae, and some industrial
wastes. The solubility of sulfur in various solvents is very
similar to the organochlorine and organophosphate pesticides;
therefore, the sulfur interference follows along with the
pesticides through the normal extraction and cleanup tech-
niques. The sulfur will be quite evident in gas chromato-
grams obtained from electron capture detectors, flame photo-
metric detectors operated in the sulfur or phosphorus mode,
and Coulson electrolytic conductivity detectors. If the gas
chromatograph is operated at the normal conditions for
pesticide analysis, the sulfur interference can completely
mask the region from the solvent peak through aldrin.
One techniaue eliminates sulfur by the formation of copper
sulfide on the surface of the copper. There ?re two critical
II1-205
-------
steps that must be followed to remove all the sulfur: (a)
the copper must be highly reactive; therefore, all oxides
must be removed so that the copper has a shiny, bright
appearance; and (b) the sample extract must be vigorously
agitated with the reactive copper for at least 1 minute.
It will probably be necessary to treat both the 6 and
15 percent Florisil eluates with copper if sulfur crystal-
lizes out upon concentration of the 6 percent eluate.
Certain pesticides such as the organophosphates, chloro-
benzilate, and heptachlor can be degraded by this technique.
However, these pesticides are not likely to be found in
routine sediment samples because they are readily degraded in
the aquatic environment.
If the presence of sulfur is indicated by an exploratory
injection of the final extract concentrate (presumably 5 ul)
into the gas chromatograph, proceed with removal as follows:
(a) Under a nitrogen stream at ambient temperature, con-
centrate the extract in the concentrator tube to exactly
1.0 ml.
(b) If" the sulfur concentration is sucn that crystalization
occurs, carefully transfer, by syringe, 500 ul of the
supernatant extract (or a lesser volume if tne sulfur
deposit is too heavy) into a glass-stoppered, 12 ml
graduated conical centrifuge tube. Add 500 ul of iso-
octane. AUQ approximate"!,/ 2 g of bright copper powder,
stopper, and mix vigorously 1 minute on a vortex genie
mixer.
MOTE 12: The copper powder, as received from the
supplier, must be treated for removal of surface oxides
with 6 N[ HN03. After about 30 seconds of exposure,
decant acid and rinse several times with distilled water
and finally with acetone. Dry under a nitrogen stream.
(c) Carefully transfer 500 ul of the supernatant-treated
extract into a 10-ml graduated concentrator tube. An
exploratory injection into the gas chromatograph at this
point will provide Information as to whether further
quantitative dilution of the extract 1s required.
II1-206
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3.2 Analysis of Sediment Samples for Pesticides/Polychlorinated
Biphenyls
Analytical Procedure: evaluated
Sample Preparation: available
3.2.1 Reference/Title
Environment Canada, "Analytical Methods Manual," Inland
Waters Directorate, Water Quality Branch, Ottawa, Ontario,
Canada. (1974).27
3.2.2 Method Summary
Sediment is extracted with acetonitrile and the chlorinated
hydrocarbons are partitioned into petroleum ether. The ether
extract is cleaned up on a Florisil column and separated into
four fractions for subsequent GLC analysis.
3.2.3 Applicability
This method has been used to quantify the following chlor-
inated hydrocarbons and PCBs in sediment samples. The values
1n parentheses are the lowest level of detection (in ppm).
Lindane (0.001) tndrin (O.C1)
Heptachlor (0.001) p,p'.-Methoxychlor (0.05)
Heptachlor epoxide (0.001) Alpha-endosulfan (0.01)
Aldrin (0.001) Beta-endosulfan (0.01)
Disldr-'n (0.001) Cis-chlordane (0.005)
p,p'-ODD iO.OOl) • Trans-chlcrdane (C.OOS)
p.p'-DDT (0.001) Aroclor 1248 (0.100)
p.p'-ODE (0.001) Aroclor 1254 (0.100)
o,p'-DDT (0.001) Aroclor 1260 (0.100)
3.2.4 Precision and Accuracy
Recovery studies with the method demonstrated over 95% recovery
for all compounds listed in paragraph 3.2.3 in the 10-ng
range. The overall recovery including Florisil column frac-
tionation is approximately 90% in the 10 to 50 ng range. The
accuracy of the procedure has yet to be determined.28
3.2.5 Sample Preparation
Transfer a 10-g dry-weight equivalent of sediment into the
glass jar of a Waring blender with a Bakelite top. (Do not
use a rubber or p-lastic top.) Add 120 ml of acetonitrile and
blend at medium-high speed for 15 minutes. Allow solid
particles to settle somewhat. Pour the acetonitrile extract,
which may contain some suspended particles, into an All inn
filter tube containing prewashed celite covering the win
glass,
II1-207
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NOTE 13: If the residue In the Allihn filter tube becomes
excessive, it should be scraped out with a spoon-type spatula
and combined with the material in the blender before the
second blending and extraction.
(a) To the residue in the blender, add another 120 ml of
acetonitrile and 40 ml of distilled water and blend for
10 minutes. Filter as before.
(b) Pour 60 ml of acetonitrile into the blender and blend
the homogenate for 10 minutes. Transfer all the resi-
due, if necessary, with 2 to 20 ml acetonitrile, into
the Allihn tube and filter. Apply sufficient; suction to
recover as much solvent as possible.
(c) Quantitatively transfer the acetonitrile extract to a
1-1 funnel with several petroleum ether washes of the
filter flask. Adjust the aqueous content of the extract
to approximately 20 percent with distilled water.
Extract the resulting mixture with 150 ml of petroleum
ether and twice with 75 ml petroleum ether.
(d) Wash the combined petroleum ether extracts with approx-
imately 200 ml distilled water. Discard water washing
and pass the organic extract, under suction or witn air
pressure, through an anhydrous sodium sulfate (10 to 15
g) column using a 500-ml round-bottomed flask; as a
receiver.
(e) In a rotary evaporator, concentrate the contents in tne
500-ml flask to 2 or 3 ml. (Do not let contents go dry
and do not use a water bath temperature over 40°C;
otherwise, there may be a possible loss of pesticides
and PCBs.)
3.2.6 Sample Extract Cleanup
Transfer the concentrated petroleum ether extract with a
clean disposable pipet onto a 30-g column (to determine the
exact amount of Florisll required, follow the guidance pro-
vided with the water analysis procedure) with 1 cm of anhy-
drous sodium sulfate on the top of the Florisll. Use a 300-ml
round-bottomed flask as a receiver.
Allow the extract to enter the Florisil column just to the
• sodium sulfate layer. Rinse the round-bottomed flask with 2
or 3 ml of petroleum ether and transfer the rinsing with the
same disposable pipet onto the column. Let the rinsing
solvent again drain just to the sodium sulfate layer. Rinse
the "ound-bottomed flask again with 2 or 3 ml of petroleum
ether ana transfer the rinsing onto tne column.
II1-208
-------
Again rinse the round-bottomed flask, this time with 20 to
30 ml petroleum ether. Carefully pour the petroleum ether
onto the column so that the sodium sulfate layer is not dis-
turbed. Elute the column with a total of 200 ml (including
the above rinsings) of petroleum ether.
Concentrate eluate with a rotary evaporator to 1 to 2 ml and
transfer, with benzene rinsings, to a 10-ml volumetric flask.
Make up to 10 ml with benzene for GLC examination (Frac-
tion 1).
Change the receiver and elute column with 200 ml of 6-percent
diethyl ether containing 2 percent ethanol. Concentrate
eluate to 1 to 2 ml on a rotary evaporator. Transfer to a
10-ml volumetric flask. Rinse round-bottomed flask with
benzene and add to volumetric flask. Dilute to volume with
benzene. This fraction, Fraction 2, is now ready for GLC
analysis.
With a third 300-ml round-bottomed flask as receiver, elute
the column with 200 ml of 15-percent ether in petroleum
ether. Concentrate to 10 to 20 mi witn a rotary evaporator.
Add 50 to 60 ml of benzene and concentrate to 1 to 2 ml.
Make up to 10 ml with benzene in a volumetric flask
(Fraction 3).
Elute the column with 200 ml chloroform or 200 ml 50-percent
diethyl ether in petroleum ether. Collect th^ eluate in a-
round-bottomed flasic and concentrate to 2 to 3 ml on a rotary
evaporator. Add 50- to 60-ml portion of benzene and evaporate
to 2 to 3 ml. Add a second 50- to 60-ml portion of benzene
and evaporate to 2 to 3 ml. Transfer the concentrate to a
10-ml volumetric flask. Rinse the round-bottomed flask with
benzene and add the rinsing to the volumetric flask. This
fraction is now ready for GLC analysis (Fraction 4).
3.2.7 Sample Analysis
The petroleum ether fraction (Fraction l) contains PCBs,
heptachlor, aldrin, p,p'-ODE, and alpha-BHC.
The 6-percent diethyl ether petroleum ether fraction
(Fraction 2) contains lindane, heptachlor epoxide, p,p'-DDT,
p,p'-DDD, methoxychlor, o,p'-DDT, cis-chlordanes, and
trans-chlordanes.
The 15-percent diethyl ether petroleum fraction (Fraction 3)
contains dieldrin, alpha-endosulfan, and endrin.
The last fraction (4) contains beta-andosulfan.
-------
Extracts may have to be cleaned up for sulfur interference.
Follow procedures given In Sediment Subsection J.3.1.6.
Analyze extracts by gas chromatography using suggested
instrument operating conditions. Further concentration
and dilution may be necessary to produce on-scale GLC peaks.
Procedures for confirmation of identity are the same as for
water extracts (Subsection K).
II1-210
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4.1 Analysis of Biological Tissue for Pesticides/Polychlorinated
Biphenyls
Analytical Procedure: available
Sample Preparation: available
4.1.1 Reference/Title
U.S. Environmental Protection Agency, "Extraction and Analysis
of Priority Pollutants in Biological Tissue," U.S. EPA, S&A
Division, Region IV, Laboratory Services Branch, Athens,
Georgia. Method PPB 10/80. 7 p., 1980.8
4.1.2 Method Summary
A 10-gram aliquot of homogenized fish tissue is mixed with
40 grams of anhydrous sodium sulfate, and extracted with
petroleum ether using an ultrasonic probe. The samples are
filtered, concentrated to 10 ml or less, cleaned up with
acetonitrile partitioning and concentrated to 1 ml. The
extract is analyzed by gas chromatography.
4.1.3 Applicability
The limit of detection for this method is usually dependent
upon the level of interferences rather than instrumental
limitations. Where interferences are not a problem, the
limit of detection for most compounds analyzed by GC/MS is 2
mg/kg (wet weight basis).
This method is recommended for use only by experienced residue
analysts or under the close supervision of such qualified persons.
4.1.4 Precision and Accuracy
Information is not presently available.
4.1.5 Sample Preparation
Blend equal amounts of fish tissue and dry ice. The pre-
ferred procedure is to use a blender but a food processor or
meat grinder may be more appropriate with large samples.
Weigh 10 g of homogenized sample into a 400-ml beaker. Mix
with 40 g anhydrous sodium sulfate until the sample is
thoroughly mixed.
Add 100 ml petrol.eum ether to the sample. Using an ultra-
sonic probe, sonicate the sample at 50% pulse for 3 minutes.
Allow the layers to separate and decant the solvent through a
Bucnner funnel filtration system.
Add a second 100-ml portion of petroleum ether to the tissue
III-211
-------
residue in the beaker. Repeat the extraction process and add
the filtered solvent layer to the first extract.
Extract the sample with a third 100-ml portion of solvent.
Filter the entire sample through the Buchner funnel filtra-
tion system and combine the petroleum ether extracts.
Quantitatively transfer the extract to a K-D flask equipped
with a 10-ml concentrator tube. Add a boiling chip to the
flask and attach a three-ball Snyder column. Place the K-D
apparatus on the water bath and concentrate the extract to
10 ml.
4.1.6 Sample Extract Cleanup (Acetonitrile Partitioning)
This procedure is used to isolate fats and oils from the
sample extracts. It should be noted that not all pesticides
are quantitatively recovered by this procedure. The analyst
must be aware of this and demonstrate the efficiency of the
partitioning for specific pesticides.
Quantitatively transfer the previously concentrated extract
to a 125-ml separatory funnel with enough hexane to bring the
final volume to 15 ml. Extract the sample four Vnes Ky
shaking vigorously for 1 minute with 30-ml portions of
hexane-saturated acatonitrile.
Combine and transfer the acetonitrile phases to a 1-liter
saparatory c'jnne! and add 650 ml of distilled wate- *nd 40 nl
of saturated sodium chloride solution. Mix thoroughly for 30
to 45 seconds. Extract with two 100-ml portions of hexane by
vigorously shaking about 15 seconds.
Combine the hexane extracts in a 1-liter separatory funnel
and wash with two 100-ml portions of distilled water.
Discard the water layer and pour the hexane layer through a
drying column containing 7 to 10 cm sodium sulfate and 2 cm
glass wool into a 500-ml K-D flask equipped with a 100-ml
ampule. Rinse the separatory funnel and column with three
10-ml portions of hexane.
Concentrate the extracts to 6 to 10 ml in the K-D evaporator
'in a hot water bath. Concentrate the extract to 1 ml using
the nitrogen blowdown technique. Transfer the extract to a
GC vial and label. The extract is ready for analysis.
4.1.7 Gas Chromatography/Electron Capture Analysis of Pesticide
Extracts
\ 1:50 iilutlon will provide adeauate detection limits for
most samples. Calculate the concentration of sample constitu-
ents as indicated ii Subsection K.
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5.1 Sampling and Analysis of Air for Polychlorinated Biphenyls
Analytical Procedure: available
Sample Preparation: available
5.1.1 Reference
Lewis, R. G., "Procedures for Sampling and Analysis of
Polychlorinated Biphenyls in the Vicinities of Hazardous
Waste Disposal Sites," Advanced Analysis Techniques Branch,
Environmental Monitoring Systems Laboratory, Research
Triangle Park, North Carolina. 14 p. March 16, 1982.3
5.1.2 Method Summary
Polyurethane foam plugs from air samplers are Soxhlet-extracted
with a mixed solvent consisting of 5 percent ether in hexane.
The extract is reduced in volume and passed through an alumina
cleanup column. PCBs in the extract are quantified using
GC/ECD. Analyte confirmation is conducted by GC/MS analysis of
composited or selected samples.
5.1.3 Applicability
Several different parameters involved in both the sampling
and analytical steps of this method collectively determine
tne sensitivity with which each PCS isomer is detected. As
the volume of air sample collected is increased, the
sensitivity increases proportionately within limits set by:
(.a) *,!".e ~-?t3nticn «f*5).
Sample recoveries for individual PCB mixtures generally fall
within the range of 90 to 110% but recoveries ranging from 75
to 115% are considered acceptable. More volatile components
give lower recoveries such as 40 to 60% to the early eluted
components of Aroclor 1242. Overall recoveries for Aroclors
1242, 1254 and 1260 have been found to be 96%, 95% and 109%,
respectively.
5.1.5 Sample Extraction -
After sampling, the foam plug in the glass cartridge must be
wrapped ^n hexane-rinsed aluminum foil until analysis.
III-213
-------
(a) Remove the foam plug with forceps and place into
Soxhlet extractors.
(b) Extract with an appropriate volume of 5 percent
ether in hexane for 50 to 100 cycles in the extractor.
(c) Remove the boiling flask to a rotary evaporator and
reduce the solvent volume to approximately 5 ml.
(CAUTION: Do not let sample go dry.)
(d) Transfer the concentrate to a graduated centrifuge tube
with rinsing and adjust the volume to exactly 10 ml.
Reduce the volume in the centrifuge tube to just below 1 ml
by careful evaporation under a gentle stream of nitrogen at
room temperature.
5.1.6 Alumina Cleanup
Place a small plug of pre-extracted glass wool in the
Chromaflex column and wash with 10 ml of hexane, which is
then discarded.
Pack the column with 10 cm of Woelm activity grade IV
alumina.
Transfer the sample from the centrifuge tube to the top of
the column; rinse the tube three times with successive 1-ml
•*or*::ons of ^-Hexane. adding and eluting «>ach rinse into the
column. ~
Add 15 ml of n-hexane and elute the column, collecting the
eluate in a 15~-ml centrifuge tube.
Adjust the final volume of the eluate by nitrogen evaporation
to 10 ml for gas chromatographic analysis.
5.1.7 GC Analysis
Determine PCBs on 180-cm x 4-mrn I.D. glass columns packed
with 1.5 percent OV-17/1.95 percent OV-210 or 4 percent
SE-30/6 percent OV-210. The nitrogen flow should be 65 to
85 ml/minute; column temperature, 200°C.
Inject 5 ul, or other appropriate volume, of the sample
extract or cleanup column eluate into the gas chromatograph.
Record chromatograms and measure retention times relative to
p,p'-DDE or other suitable reference standard.
III-214
-------
Compare the retention time of each major GC peak against
those of the corresponding primary Arochlor standard.
Quantify PCB mixtures by comparison of the total heights or
areas of GC peaks with the corresponding peaks in the
best-matching standard. Use Aroclor 1242 for early-eluting
and either Aroclor 1254 or Aroclor 1260, as appropriate, for
late-el uting PCBs.
K. CALCULATIONS
1. The concentration of pesticides and PCBs in a sample 1s determined by
calculating peak areas for a sample extract, either by disk integrator,
planimeter, triangulation, or digital Integrators, and comparing the area
to a standard calibration curve. The standard curve 1s a plot of area
response produced by known quantities of the Individual pesticides or
PCBs under identical analytical conditions versus amount or concentration
of the compound. The calibration curve should be prepared daily.
Pesticide and PCB concentrations in the sample extracts may also be
determined by direct comparison to a single standard when the injection
volume and peak area are very close to that of the sample.
2. The concentration of individual pesticides and/or PCBs in the original
sample matrix is calculated using tne appropriate equation identified
below:
2.1 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 3ubsecticn H.C 2. "^e
concentration in the sample can be calculated from Equation ?:
(A) (Vt)
Concentration, yg/1 = - Eq. 2
(V) (V)
where:
A = Amount of material Injected, in nanograms
V-j * Volume of extract Injected (ul)
Vt * Volume of total extract (ul)
Vs = Volume of water extracted (ml).
2.2 If the internal standard calibration procedure was used, calculate
the concentration in the sample using the response factor (RF)
determined in Subsection H.3.2 and Equation 3.
(As (Is)
Concentration, ug/1 - - Eq. 3
III-215
-------
where:
As * Response for the parameter to be measured
A-js = Response for the internal standard
Is = Amount of Internal standard added to each extract
(ug)
Ys = Volume of water extracted, in liters.
When it is apparent that two or more PCS (Aroclor) mixtures are
present, the Webb and McCall procedure^ may be used to identify
and quantify the Aroclors.
2.3 The chlorinated hydrocarbon pesticide or PCB concentration of
sediment can be calculated as:
(A) (B) (C)
Chlorinated hydrocarbons ug/kg (wet weight)
Chlorinated hydrocarbons ug/kg (dry weight)
(E) (F) (G)
(A) (B) (C)
(E) (F) (G) (K)
where:
A = nanograms standard injected into GC
B = peak height (or area) produced by -sample Injection
C = rinal volume of sample extract, ml
E = peak height (or area) produced by standard injection
A
F = wet weight of sediment sample initially extracted, g
G » volume of extract injected to produce B, ml
IS = sediment percent solids as a decimal fraction.
2. For multicomponent mixtures (chlordane, toxaphene and PCBs), match
retention times of peaks in the standards with peaks in the sample.
Quantitate every identifiable peak unless interferences with individual
peaks persist after cleanup. Add peak height or peak area of each
identified peak in the chromatogram. Calculate as total response in the
sample versus total response in the standard.
3. 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.
4. For samples processed as part of a set where the laboratory spiked sample
recovery falls outside of the control limits in Subsection G.3., data for
the affected parameters must be labeled as suspect.
TII-216
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If a sample is reported as negative for a given pesticide, also report
the minimum method detectable limit for that compound. Report sample
response of less than three times the detector noise level (N) as neg-
ative. For sample response at three times the detector noise level, list
the result as presumptive. Quantify responses of greater than 3 N if
possible. In cases of questionable identification, qualify the reported
result to ensure that subsequent misinterpretation will not occur.
III-217
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REFERENCES
1. U.S. Environmental Protection Agency. "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. (1979).
2. U.S. Environmental Protection Agency. "Preservation and Maximum Holding
Time for the Priority Pollutants." U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio. (In
Preparation).
3. Lewis, R. G. "Procedures for Sampling and Analysis of Polychlorinated
Biphenyls in the Vicinities of Hazardous Waste Disposal Sites." Advanced
Analysis Techniques Branch, Environmental Monitoring Systems Laboratory,
Research Triangle Park, North Carolina. 14 p. March 16, 1982.
4. U.S. Environmental Protection Agency. "The Analysis of Polychlorinated
Biphenyls in Transformer Fluid and Waste Oil." U.S. Environmental
Protection Agency, Office of Research and Development, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio. (1981).
5. American Society for Testing and Materials. "Standard Practice for
Preparation of Sample Containers and for Preservation." ASTM Annual Book
of Standards, Part 31, D3694. American Society for Testing and
Materials, Philadelphia, Pennsylvania. 679 p. .(1980).
6. Giam, C. S., H. S. Chan and G. S. Nef. "Sensitive Method for Determina-
tion of Phthalate Ester Plasticizers in Open-Ocean Biota Samples."
Analytical Chemistry 47_:2225 (1975).
7. Giam, C. S. and H. S. Chan. "Control of Blanks in the Analysis of
Phthalates in Air and Ocean Biota Samples." U.S. National Bureau of
Standards, Special Publication 442:701-708 (1976).
8. U.S. Environmental Protection Agency. "Extraction and Analysis of
Priority Pollutants in Biological Tissue." U.S. Environmental Protection
Agency, SAA Division, Region IV, Laboratory Services Branch, Athens,
Georgia. Method PPb 10/80. 7 p. (1980).
9. U.S. Environmental Protection Agency. "Analytically Determined Method
Detection Limits for Priority Pollutant Methodology as Method Performance
Criteria." U.S. Environmental Protection Agency, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio. (In Preparation).
10. American Society for Testing and Materials. "Tentative Method of Test
for Organochlorine in Water." Method D 3086-72T, D-19 Water, pp. 519-534,
{1980). ^me^can Society *or Testing and Materials, Philadelohia,
Pennsylvania.
III-218
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11. "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.
12. "OSHA Safety and Health Standards, General Industry;" (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206 (Revised,
January 1976).
13. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
14. Mills, P. A. "Variation of Florisil Activity: Simple Method for
Measuring Absorbent Capacity and its use in Standardizing Florisil
Columns," Journal of the Association of Official Analytical Chemists,
51:29 (196W-;
15. Webb, R. G. and A. C. McCall. "Quantitative PCB Standards for Electron
Capture Gas Chromatography." Journal of Chromatographic Science 11:366
(1973). ~
16. U.S. Environmental Protection Agency. "Method for Preparation of Medium
Concentration Hazardous Vaste Samoles." U.S. Environmental Protection
Agency, Region IV, Athens, Georgia. (1981).
17. U.S. Environmental Protection Agency. "Guidelines Establishing Test
Procedures for the Analysis of Pollutants; Proposed Regulations."
Federal Regirrtar ^ No, ?33:69*64-69575. Decembers, 1979.
18. Eichelberger, J. W., L. E. Harris and W. L. Budde. "Analysis of the
Polychlorinated Biphenyl Problem. Application of Gas Chromatography-
Mass Spectrometry with Computer Controlled Repetitive Data Acquisition
from Selected Specific Ions." Anal. Chem 46:227-232(1974).
19. White, L. D., D. G. Taylor, P. A. Mauer, and R. E. Kupel. "Convenient
Optimized Method for the Analysis of Selected Solvent Vapors in the
Industrial Atmosphere." AIHA Journal 3jh pp. 225-232 (1970).
20. U.S. Environmental Protection Agency. "Organochlorine Pesticides and
PCB's - Method 608." Federal Register 44 No. 233:69501-69509.
December 3, 1979.
21. "Development and Application of Test Procedures for Specific Organic
Toxic Substances in Wastewaters. Category 10-Pesticides and PCB's."
Report for EPA Contract 68-03-2606 (In Preparation).
22. "Manual of Analytical Methods for the Analysis of Pesticides in Human and
Environmental Samples." U.S. EPA, Health Effects Research Laboratory,
Research Wangle ''ark. North Carolina, EPA Report 600/8-80-038, Section
11, B, p. 6. (1980).
III-219
-------
23. "Official Methods of Analysis of the Association of Official Agricultural
Chemists," Association of Official Agricultural Chemists, Washington,
D. C. 20044, 10th Edition, 1965, p. 382.
24. Smith, D. and J. W. Eichelberger. JWPFA, Vol. 37, 1965, p. 77.
25. Breidenbach, A. W., et al. "The Identification and"Measurement of
Chlorinated Hydrocarbon Pesticides in Surface Waters," Publication WP-22,
U.S. Department of the Interior, Federal Water Pollution Control
'Administration, Washington, D. C. 20242, 1966.
26. Thompson, J. F. "Analysis of Pesticide Residues in Human and Environ-
mental Samples." U.S. Environmental Protection Agency, Pesticide and
Toxic Substances Effects Laboratory. Research Triangle Park, North
Carolina. (1974).
27. Environment Canada. "Analytical Methods Manual." Inland Waters
Directorate, Water Quality Branch. Ottawa, Ontario, Canada (1974).
III-220
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SECTION 5
METHODS FOR THE DETERMINATION OF ORGANOPHOSPHORUS PESTICIDES
A. SCOPE
The analytical procedures provided in Subsection H of this Section cover
the determination of organophosphorus (OP) pesticides in pesticide formulations
(hazardous waste) (Subsection H.I.), water (Subsection H.2.), soil (Subsection
H.3.), biological tissues (Subsection H.4.), vegetable and fruit tissues (Sub-
section H.4.) and air (Subsection H.5.). All of the determinations are based
on gas chromatography (GC). Several do not require extensive cleanup because
of the selectivity of the flame photometric detector. The analytical method
for human tissues determines alkyl phosphate metabolites rather than the parent
pesticides, which are usually not present in these samples in significant
amounts.
B. SAMPLE HANDLING AND STORAGE
1. Sampling
See Section 4, Subsection B of this Chapter for general information c:i
pesticide sampling that is applicable to OP compounds. General and spec-
ific sampling procedures have been discussed in detail for human and
environmental samples in Chapter 8 of the EPA Quality Control Manual! and
for food samples in Sections 140-142 of the U.S. Food and Drug Administra-
tion Pesticide Analytical Manual (FDA PAM).2
Analytical results can be no more valid than the samples or sampling scheme
used. Samples should truly represent the component being examined and
should be compatible with the goals of the analysis. The location of sam-
pling sites, the sampling technique, the number of samples, and the frequency
and duration of sampling should be such that the analytical results can be
evaluated in a statistically satisfactory manner. The size and number of
samples should enable replication of analyses and confirmation of any
residues found.3
OP pesticides are collected from air using high volume air samplers.
Collection media have included polyurethane foam-granular sorbent combina-
tion filter pads, glass fiber-Poropak composite pads, and polyurethane
foam plugs. The use of polyurethane cartridges for collection of PCBs from
air? *- iiscusse-i *n Section 4, Subsection B of this Chapter. Section 8A
of the EPA PAM4 contains methods and equipment for anwnent
-------
Most recovery studies to date have been made for organochlorines and PCBs
present in air.
Acceptable methods for sampling liquids, emulsions, suspensions, wettable
powders, and dusts are described in detail in Official Methods of Analysis
of the Association of Official Analytical Chemists, 13th Edition (1980).
2. Sample Handling
Water samples containing high concentrations of suspended matter or sedi-
ment may require special handling such as decantation, filtration, or
centrifugation, after which the water and sediment can be analyzed sep-
arately, if so desired.
Plant or animal samples, either frozen or unfrozen, are frequently ground
with sodium sulfate to bind the tissue moisture before extraction. Appro-
priate manuals or publications should be consulted for specific details
for preparing plant or animal tissues.1.2,4
It is generally undesirable to dry soil samples beyond the air-dry state
before extraction because of possible decomposition or irreversible adsorp-
tion of pesticide residues on soil surfaces. However, soil samples should
not be too wet because of particle aggregation, which can result in
extraction aiffaculties. ~hey should be in a damn, friable condition
before extraction, with a moisture content of about 5 to 15 percent,for
sandy soils and 10 to 30 percent for loams. Satisfactory extraction from
muck soils can be made with moisture levels as high as 85 percent. Mois-
ture content should be determined on a separate portion of soil so that
results -ran be reported on a dry-weight basis.
Sediment samples should be in the same condition as soils prior to
extraction. Excess water should be removed by decantation, filtration,
or centrifugation, and air-drying, if necessary, until the sediment is
friable.
3. Storage
3.1 Temperature
Samples other than water should ordinarily be stored in a freezer,
preferably below 0*C. Even then, physical and chemical changes may
occur 1n either the sample or in the residues sought. Extended
storage in freezers can cause moisture to migrate to the surface of
the sample and then to the freezer coils, slowly desiccating the
sample. This effect may be of importance if water content affects
the subsequent analysis and can affect the calculated residue concen-
tration. Water samples should be stored slightly above freezing to
avoid rupture of the containers as a result of freezing.
3.2 Tfme
Samples should be analyzed as quickly as possible after collection,
III-222
-------
before physical and chemical changes occur. If prolonged storage is
required, it may be preferable to extract the samples, and store the
extracts at a low temperature.
When feasible, studies of the stability of residues in samples or ex-
tracts, with time and temperature of storage, should be carried out
with representative pesticides and substrates. When there is doubt
about the stability of residues in storage, spiked control samples or
extracts should be held under the same conditions as the samples or
extracts. OP pesticides degrade more quickly than organochlorine
pesticides and should be analyzed within 4 days of sampling.
3.2 Light
Light degrades many pesticides; therefore it is advisable to protect
the samples and any solutions or extracts from needless exposure. In
the case of air sampling, the collection apparatus should be shielded
from light during sample collection.
3.3 Containers
Avoid plastic containers, or plastic-lined caps, unless made of Teflon
or other inert plastic which does not interfere with the analytical
method. Aluminum foi1 liners for caos may tear; hence it is preferable
to use Teflon sheeting or liners. If polyethylene or other plastic
storage containers are used, tests should be made to determine whether
there is any analytical interference due to the containers being used;
laboratories fre-quently have experienced such interferences.
If cans are used, they should first be cneckea to demtnctrats ths ab-
sence of materials, such as oil films, lacquers, or rosin from sol-
dered joints, that could interfere with analyses.
Glass containers should be used for water or liquid samples and. should
be thoroughly cleaned and rinsed with one or more suitable solvents
such as acetone, Isopropanol, or hexane, and dried before use. Pesti-
cides can migrate to the walls of a container and be adsorbed; hence
even a glass container, after the water sample is poured out, should
be rinsed with solvent if the extraction is not made in the container
Itself.
In summary, any type of container or wrapping material should be checked
before use for possible interferences in the analytical method and at
the limit of detection employed in the analysis. Sample handling and
storage information is summarized in Figure 1.
C. INTERFERENCES
Solvents and reagents should be free of substances that interfere with
analysis •sr -leqrade the samole. To obtain low background levels and avoid
spurious peaks arising from solvent impurities, it is usually necessary to
employ ipecially purified or distilled-in-glass solvents. Solvents should be
111-223
-------
I
ro
ro
-p.
>*»rw««
IMitCI
|
•~-
C |
Pm
,..«,
— "
*—
1M
l><
km
*n*
•Cl/
»MI
'
«r>*
fra*tt*w*««
IP
E Kit Ml
1
Figure 1. Handling and sample sto>age infomu,tion for organophosphorus compounds.
-------
checked by using them in the amounts and manner described in the method without
a sample being present, concentrating them as described in the method, and then
testing with the particular detector prescribed. Similar precautions must be
taken with reagents to make sure that they will not cause undue interference.
Cleanup procedures described in the various methods will usually eliminate
Interferences from the samples. Use of the phosphorus-selective GC detector
such as the flame photometric or alkali flame ionization detector should reduce
problems caused by interferences and reduce the amount of extract cleanup
required.
D. SAFETY
Precaution when using diazopentane reagent: Because of the demonstrated
carcinogenicity and skin irritating characteristics, do not allow the nitroso-
gyanidine or the diazoalkane to come in contact with the skin. Wear dispos-
able vinyl gloves and safety goggles while handling.Avoid breathing vapors.
Working inside a glove box, if possible, is strongly recommended. Do not use
ground-glass-stoppered bottles or bottles with visible interior etching. Avoid
strong light.
E. APPARATUS
1. Gas chromatograph aquipped with thermal conductivity or flame ionization
detector and recorder for formulation analysis. The roil owing parameters
are typical: glass column, 4 ft (1.2 meters) x 3-4 mm I.O., 3-10 percent
liquid phase, coated on 80/100 mesh acid-washed and silanized diatomite
(e.g., Gas Chrom Q, Anabrom ABS), injector temperature 190°C, helium
(carrier qas^ flow rate 80 ml/min, detector temperature 175°C.
2. Erlenmeyer flask, 50 ml.
3. Bottle, 4 oz, with Vinylite-lined screw top.
4. Chromatographic-column, glass, 400 mm x 20 mm.
5. Volumetric flask, 50ml.
6. Blender, high speed.
7. Gas chromatograph fitted with a flame photometric detector with 526 nm
phosphorus filter (thermionic detection may be substituted for the FPD)
for residue analysis. GC columns, borosilicate glass, 1.8 m x 4 mm I.D.,
packed with 1.5% OV-17/1.95% OV-210, and 5% OV-210, both coated on Gas-
Chrom Q, 80/100 mesh. The following are typical operating parameters
used: column, 165 to 200°C; nitrogen carrier gas flow, 70 to 80 ml/min;
hydrogen flow, 50 to 100 ml/min; oxygen content of air flow, 0.2 to 0.4
of the hydrogen flow; total air flow, 1.5 times the hydrogen flow;
injector block, 225eC; FPD detector 175 to 225°C (thermionic detector,
250°Ch transfer line, 235°C. A switching valve between the column and
FPD allows solvent venting and prevents flame oiowout when Injectors ire
made. Other columns and conditions that have been used for determination
of OP pesticide residues with the FPD detector include: a) 1.2 m x
111-225
-------
2 mm I.D. 2% DECS column, 180°C, 60 ml/min helium carrier gas flow; b)
1.2 m x 2 mm I.D. 2% OV-101 column, 200'C, 30 to 60 ml/miri He flow; and c)
76.2 x 2 mm I.D., 4% SE-30/6.5% OV-210 column, 200eC, 60 ml/min He flow.
8. Chromaflex columns, size 22, 7 mm I.D. x 200 mm, Kontes 420100 or
equivalent.
9. Chromaflex column, size 241, 22 mm I.D. x 300 mm, Kontes 420530 or
equivalent.
10. Rinco evaporator, rotating, such as Scientific Glass Apparatus Co. E-5500,
E-5500-1 or equivalent, with appropriate stand.
11. Variac or comparable voltage control regulator.
12. Water bath for operation at 35'C.
13. Vacuum source of 125 mm Hg, optimally.
14. Kuderna-Danish evaporators, 250 ml, 500 ml.
15. Centrifuge tubes, conical, 15 ml, graduated, Corning No. 8082 with Teflon-
lined plastic screw caps, thread finish 415-15, Corning 9998 or equivalent.
16. Tubes, culture, screw caps with Teflon liner, 16 x 125 mm, Corning 9326 or
equivalent.
17. Evaporative concentrator tubes, 10 ml, graduated from 0.1 to 10.0 ml, size
1D25 iit.h outer joint T 19/22.
-------
26. IEC centrifuge, Model EXD, explosion proof, or equivalent, suitable for
operation at 2,000 rpm.
27. Culture tubes, glass, 16 ran x 150 tart.
28. Pipet, 0.1 ml" capacity, graduated in 0.01 ml units.
29. Pipets, assorted capacities, to be used in combination with appropriate
volumetric flasks for preparation of standard solutions.
30. Bottles, reagent, narrow mouth, 1 oz. capacity, with polyseal screw caps
(A. H. Thomas 2203-C bottles and 2849-E caps or equivalent).
31. Nitrogen evaporator with water bath maintained at 40°C (Organomation
Associates N-Evap or equivalent).
32. Exhaust hood with minimum draft of 150 linear feet per minute.
33. Extractor, Soxhlet, 1000, 500, and 250 ml.
34. Separatory funnel, 500 ml, and 1 1, with Teflon stopcocks.
35. Suchner filtration apparatus.
36. Chromatoflo chromatography column, 25 cm x 9 mm I.D. , Pierce No. 29020, or
equivalent, equipped with a Teflon mesh support membrane, Pierce No. 29268
or equivalent, lower end plate, adapter, and 500-ml solvent reservoir
;Ace '!o. 5321-10 ^
F. REAGENTS
(Solvents should be distilled-in-glass, pesticide grade.)
1. Acetone.
2. Acetonitrile.
3. Benzene.
4. Contaminant- free water. To 1500 ml of distilled water 1n a 2-1 separatory
funnel add 100 ml methylene chloride, stopper, and shake vigorously for 2 •
minutes. Allow the phases to separate, discard the solvent layer, and
repeat the extraction with another 100-ml portion of methylene chloride.
Drain the double-extracted water Into a glass stoppered bottle for storage,
withdrawing 500 ml to serve as a reagent blank with each set of samples.
5. Diethyl ether, containing 2 percent ethanol .
6. Hexane.
7. Hydrochloric acid, reagent grade, approximately 37 percent.
8. Isopropanol.
111-227
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9. Keeper solution, 1 percent paraffin oil, USP grade, in hexane.
10. Keeper solution, polyethylene glycol-acetone (5/95 v/v).
11. Methanol.
12. Methylene chloride.
13. Anion exchange resin, Amberlite CG-400 AR or equivalent, 100/200 mesh
(available from Mallinckrodt 3345 and other vendors), in the chloride
form, or BioRad AG1 x 8 or equivalent, 100/200 mesh (available from
BioRad Laboratories, Richmond, California and other vendors in the
chloride form).
14. Carborundum chips, fine. Purify by Soxhlet extracting with methylene
chloride for ca. 60 discharge cycles.
15. Diazopentane reagent - Preparation:
a. Dissolve 2.3 g KOH in 2.3 ml distilled water in a 125 ml Erlenmeyer
flask. When solution is complete, cool in a freezer for 30 minutes.
b. Add 25 ml cold diethyl ether, cover flask mouth with foil, and
cool in a -13°C freezer for 15 -ninutss.
c. In a very high draft hood, add 2.1 g N-amyl-fT-nitrp-N-nitroso-
guanidine to the flask in small portions over a perio'd" of a few
minutes, swirling the flask vigorously after each addition.
d. Decant the ether layer into a 1-oz. reagent bottle fitted with a
Teflon-lined screw cap. This may be stored at -20°C for periods up
to a week.
16. Glass wool, preextracted with methanol, acetone, and methylene chloride to
remove any contaminants.
17. N-amyl-N'-nitro-N-nitrosoguanidine (available from Aldrich Chemical Co.).
18. Pesticide reference standards, analytical grade.
19. Petroleum ether.
20. Potassium hydroxide, pellets, AR grade.
21. Silica gel, Woelm, activity grade I (available from ICN Pharmaceuticals,
Inc. and other vendors), activated at 130°C for 48 hours and stored 1n a
desiccator.
22. Silica gel, Woelm, deactivated. Activate for 48 hours at 175*C before
usa. "rppare *inal deactivated material by adding 1.0 ml of water to
5.0 g silica gel 1n a vial with a Teflon-lined screw cap. Cap cigntiy a
III-228
-------
mix on the Roto-Rack for 2 hours at ca 50 rpm. Discard deactivated silica
gel after 5 days.
NOTE: It 1s recommended that the amount of silica gel activated at 175°C
be restricted to the quantity needed for Immediate deactivation.
23. Sodium sulfate, granular, anhydrous. Purify by Soxhlet extracting with
methylene chloride for car 60 discharge cycles.
G. CALIBRATION
Calibration or linearity plots are obtained on varying ratios of the
pesticide primary standard and the internal standard. The mixtures (four are
sufficient) are prepared so that they contain a constant amount of internal
standard and the range of the pesticide concentration is varied so that it
covers one-half to two times that which is to be used for the sample analysis.
To be more explicit, the linearity range should cover 10 to 50 ug of pesticide
per injected volume and the concentration of pesticide in a sample solution
prepared for analysis should contain 5 to 10 mg pesticide per milliliter of
solution. Linearity curves are plots of peak area ratio versus concentration
in micrograms per volume injected. The analysis of formulation samples is per-
formed by the addition of the same amount of internal standard to a specified
volume of sample solution (obtained after dilution or extraction of the formu-
lation with a suitable solvent) and the concentration determined either by
comparing the peak height to a standard curve or by comparing the peak height
ratio of the analyte to the internal standard in the sample to the analyte/
Sterna! standard ratio in the calibration standard.
The use of a calibration curve for quantitation is not very satisfactory
when high precision is required. Calibration curves having the identical
slope cannot be reproduced exactly from day to day and often not even within
an 8-hour work day. However, high precision and accuracy can be achieved by
using a pesticide standard solution and replication of solution injections for
both the standard and sample solutions according to the injection sequence
described in the following example:
The formulation solution or extract is diluted with the appropriate sol-
vent containing a known concentration of the internal standard to give a 1-
percent solution (10 mg/ml or 10 pg/ul) of the pesticide. Simultaneously, two
solutions are prepared containing 5 mg/ml and 10 mg/ml of the pesticide
standard and the exact same amount of Internal standard.
Identify these as standard solution A and standard solution B, respec-
tively. Set the gas chromatographic conditions as predetermined for the
specific pesticide, and using standard solution B, determine the appropriate
attenuation setting and injection portion to yield a minimum peak height for
the internal standard of 50 percent full-scale recorder deflection. Inject
aoorooriate portions of standard solution B until a consistent response is
obtained (three consecutive injections giv'ng -esocnse -.itios Mithin ? oercent
of each other). The response ratio for each injection is calculated as
follows:
III-229
-------
peak height (area) of pesticide
response ratio = —
peak height (area) of internal standard
The average of the last three response ratios is used to calculate the
standard response factor in the formula below and the pesticide content of the
first three samples of a series. Three portions of standard solution A are
injected and the response ratios are calculated and also must be within 2
percent. The response factor for each standard is calculated by the following
formulas:
concentration (mg/ml) x purity of standard
response factor standard A = —
average response ratio standard A
concentration (mg/ml) x purity of standard
response factor standard B =
average response ratio standard B
The response factors should be within 2 percent of each other. If they
are not, new standard solutions should be prepared and the response factor
again determined. Continued discrepancies of the standard response factors
indicate a lack of linearity, and this condition must be resolved before
meaningful and valid analysis of samples can be conducted.
""^o portions ;f each sarcole^extract ire then •injected. These response' "
ratios should also be within 2 percent of eacn otner. If cnis precision
limit is not met, two more portions of the solution are -injected. Failure
to meet the 2 percent specification with the second pair of injections
indicates instrumental difficulties which must be corrected before proceeding
with the analysis.
After every six samples, standard solution B is reinjected in duplicate.
The average of these response ratios should be within 2 percent of the
preceding average response ratio obtained with this standard solution.
Failure to meet these specifications indicates instrument drift which must
either be corrected or compensated for by more frequent measurement of
standard solution B than specified above. The frequency of standard measure-
ment should be adjusted depending on the degree of drift observed.
III-230
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H. ANALYTICAL PROCEDURES
1. Determination of Organophosphorus Pesticides in Hazardous Wastes
(Pesticide Formulations)
Analytical Procedure: available
Sample Preparation: available
1.1 Reference
Zweig, G. and J. Sherma, "Gas Chromatographic Analysis," Vol. 6 of
"Analytical Methods for Pesticides and Plant Growth Regulators."
Ch. 4, p. 107, Academic Press, New York (1972).6
1.2 Method Summary
The sample is dissolved in or extracted with an appropriate solvent,
internal standard is added, and the analyte is determined by gas
chromatography with thermal conductivity or flame ionization
detection.
1.3 Applicability
The method is ami •'cable to serosols, solid samples, liquid formula-
tions, and liquid solutions containing OP pesticides.
1.4 Precision and Accuracy
Accuracy ind sreclsion (relative standard deviation) values within
±1 to 2 percent are possible using internal standardisation arvd j
reference standard of known purity.
1.5 Sample Preparation
Typical examples of product formulations include aerosols, baits,
dust concentrates, wettable powders, granules, emulsifiable
concentrates, ultralow-volume concentrates, technical materials,
water-miscible liquids, water-soluble concentrates, oil solutions,
emulsions, and suspensions. Some general procedures for sample
preparation prior to gas chromatography, which include separation of
the pesticide from the formulation ingredients or merely a dilution
of the sample with a suitable solvent, are discussed in this section.
These procedures are intended to be used only as guides and may not
be applicable in all cases.
1.5.1 Aerosols
Weigh the container and contents, cool in a dry-ice chamber or
other satisfactory cooler such as a refrigerator freezing
compartment, for approximately 0.5 hour. Punch a very small
hole 1n the top of the container, and allow ;•£ to s-;and ;n i
hood at room temperature while the propellant gas escapes.
HI-231
-------
After the propel!ant has volatilized, carefully cut off the
top of the container and gently warm near a steam bath to
boil off the remainder of the volatile solvents. Finally,
warm on the steam bath until all the solvent is expelled.
Cool to room temperature, and weigh the container with the
nonvolatile material. Transfer the nonvolatile material to
a suitable container and retain for analysis. Rinse the
aerosol container with ether, dry, and weigh. The difference
between these weights represents the weight of nonvolatile
material in the aerosol. Weigh the top of the container
which had previously been removed, add this weight to that
of the empty and dried container, and subtract their combined
weight from the original gross weight of the aerosol to
obtain the net content. Calculate the percentage of nonvol-
atile residue obtained from the aerosol.
Accurately weigh a portion of the nonvolatile, well mixed
residual concentrate equivalent to 0.500 gm of pesticide into
a 50-ml flask (final concentration to be 10 mg/ml), add the
internal standard to the flask, and dilute to volume with
solvent.
1.5.2 Wettable Powders, Dust Concentrates, Baits, and Granules
The clays and carriers used in these formulation types are
hignly sorptive and as such have a tendency to bind or retain
the' pesticides so that their complete removal may be a prob-
lem. Although a pesticide may be equally soluble in a
numner sf solvents, it does not mean that ?H the solvents
will extract the pesticide quantitatively from"the highly
sorptive carriers. The Inability to efficiently extract the
pesticide from the carrier with any one solvent is more
evident on aged formulation samples. A solvent that will
penetrate or wet the carrier will generally be the most
efficient for this purpose. For example, acetonitrile or
methanol are used to extract organophosphate pesticides from
fresh and aged formulations more efficiently than hexane or
Isooctane, although this group of compounds shows good
solubility in all of the solvents.
Extraction of pesticides from carriers is generally performed
1n a number of ways. They are all quite simple, give compar-
able results, and require little actual work time, and the
main difference lies 1n the time necessary to complete the
extraction. In all cases, a sample equivalent to 2.0 gm of
the pesticide 1s taken for analysis. In the first method of
extraction, the sample to be extracted 1s placed into a 4-
ounce screw cap bottle with a Yinyllte liner, 50 ml of the
appropriate solvent 1s added, the cap Is screwed tightly,
and *he samole 1s shaken on a mechanical shaker for 30 to
60 minutes. After filtration, a portion of the filtrate
necessary to produce z final concentration of '.0 -ng/ml is
III-232
-------
.taken and mixed with the appropriate predetermined volume of
internal standard.
In the second method of extraction, the same formulation sample
size (equivalent to 2.00 gm of pesticide) is transferred to
an empty 20-mm x 400-mm chromatographic column, and the extrac-
tion solvent is percolated through the column at a drop rate
of 1 to 2 drops per second until 100 ml of the effluent
is collected. A portion of the effluent to give a final
concentration of 10 mg/ml is taken and mixed with the appro-
priate predetermined volume of internal standard as described
earlier.
Some analysts prefer to use a Soxhlet extraction. This re-
quires a much longer extraction period (minimum of 4 hours)
and the use of heat, which may cause destruction of heat-
sensitive compounds. Sample size, volume of extraction, and
dilutions are the same as described for the chromatographic
column method in the previous paragraph (second method).
1.5.3 Emulsifiable Concentrates, Oil Solutions, Ultra-low Volume
Concentrates, Water-Miscible Liquids, and Water-Soluble
Concentrates.
These types of formulations are the easiest to prepare for
gas chromatographic analysis. A sample size equivalent to
0.500 gm of the pesticide is transferred to a 50-ml volumetric
^Msk '-^'lal concentration to be 10 mq/ml K the aoorooriate
volume of internal standard is added to tne flasx, ana cr,e *
sample diluted to volume with the proper solvent. The
solvent may be the same as that used for solid formulations
analysis. However, it should be completely miscible with
the formulation ingredients, eluted rapidly from the gas
chromatographic column, and the instrument should return to
a flat baseline before the peaks to be measured elute.
Acetonitrile is a very good solvent for emulsifiable concen-
trates, water concentrates, and water-miscible concentrates.
Hexane is preferable for oil solutions.
1.5.4- Suspensions and Emulsions
These mixtures are generally obtained by the dilution of
wettable powders or emulsifiable concentrates with water and
contain low concentrations of the pesticide (below 1 percent)
1n contrast to the preceding formulations which generally
have very high concentrations (5-95 percent). A much larger
sample size is necessary, larger volumes of water-immiscible
solvents are required for the extraction, and a concentration
step is necessary. Susoensions are either filtered or centri-
fuged to remove the solids whicn are then extracted as described
above, and the aqueous phase is extracted several times with a
water-immiscible solvent (one volume of aqueous phase to two
-------
volumes of solvent). The solvent extracts for both the aque-
ous and solid phase are combined, a keeper added (3 drops of
a 5-percent solution of polyethylene glycol in acetone) and
the solution is taken to dryness with a rotary vacuum evapo-
rator. The appropriate amount of internal standard is added
and the solution diluted with the predetermined solvent to
give a concentration of 5 to 10 mg/ml pesticide.
Emulsions are diluted with a small volume of concentrated
salt solution (10- to 20-percent solution) and extracted with
two volumes of a water-immiscible solvent using gentle to
moderate shaking. Concentration and final sample prepara-
tion are accomplished as described above for suspensions.
1.6 Sample Analysis
Inject the sample and standards using conditions that will elute the
analyte and internal standard with similar retention times, but with
peaks that are narrow and completely resolved. For complex samples,
the selective flame photometric or thermionic detector might be more
advantageous than the thermionic or flame ionization detector, which
is generally used for formulation analysis. Examples of internal
standards and GC columns and temperatures for some OP pesticides are
shown below:
Column Conditions
Pesticide
Retention
Time
(minutes)
Internal
Standard
Retention
Time
(minutes)
Temperature
Substrate (°C)
Dimethoate 2.9
Oisyston 3.4
Fenitrothion 2.8
Malathion 7.0
Malathion 2.5
Methyl parathion 1.9
Methyl-parathion 2.17
Parathion 3.9
Parathion 3.17
Phorate 2.5
Phorate 2.5
Trithion 3.6
Dibutyl sebacate 5.9
Aldrin 5.8
Dibutyl sebacate 5.5
Dimethoate 3.4
Dibutyl sebacate 4.5
Dieldrin 5.1
Dibutyl sebacate 5.2
Dieldrin' 7.0
Dibutyl sebacate 5.5
Benzyl benzoate 3.5
Lindane 3.5
Heptachlor 1.0
5% DC-550 170
10% SE-30 205
5% DC-550 165
5% OV-22 180
0.25% DC-200 160
10% SE-30 208
0.25% DC-550 160
10% SE-30 195
0.25% DC-550 160
5% DC--550 165
5% DC-550 155
10% SE-30 170
Calculate organophosphorus concentrations as indicated in Sub-
section L
III-234
-------
Determination of Organophosphorus Pesticides in Water
Analytical Procedure: available
Sample Preparation: available
2.1 Reference
Sherma, J. and M. Beroza, "Manual of Analytical Methods for the
Analysis of Pesticides in Humans and Environmental Samples,"
EPA-600/8-80-038, Section IDA (June, 1980).*
2.2 Method Summary
Compounds are extracted from water with methylene chloride, and
the extract volume is reduced at low pressure and temperature in an
evaporative concentrator. Compounds are separated into groups on
a column of deactivated silica gel by elution with solvents of
increasing polarity. Figure 2 shows that OP pesticides appear in
Fractions II, III and IV. These compounds are determined by GC with
a P-selective flame photometric detector (FPD).
2.3 Applicability
Water samples were analyzed for the thirty-eight OP pesticides listed
in Table 1 at concentrations ranging from i.6 to 400 ppb.
2.4. Precision and Accuracy
Thirty-one ?.f the 38 organophosphorus compounds were recovered in
the 80+ percent range ana six between ou ana 79 percsnt [~s?. Tab! =
1). Reproducible and satisfactory recoveries were not achieved for
carbophenoxon, disulfoton, methamidophos, monocrotophos, and
oxydemeton methyl. Of these five compounds, excellent extraction
efficiency was observed for carbophenoxon and disulfoton, but
complete loss was experienced on the silica gel column. Six com-
pounds were partially recovered in the 0 to 60 percent range. Of
the 7 OP compounds yielding total recoveries of less than 80 per-
cent, six of these gave over 90 percent extraction recovery, but
losses occurred during silica gel chromatography (see Table 1).
2.5 Sample Extraction and Concentration
Transfer 500 ml of water to a 1-liter separatory funnel and add 10 g
of anhydrous sodium sulfate and 50 ml of methylene chloride.
Shake vigorously for 2 minutes and allow a sufficient length of
time for complete phase separation.
NOTE 1. If the expected pesticide concentration is extremely low,
i.e., under Q.(M ug/1 , it may be advisable to increase the initial
sample size to 1000 to 2000 ml. in this case, the volume of T.ethvlene
chloride should be •'ncreased to 75 ml and the separatory funnel size
to 2 or 3 1.
III-235
-------
500 ml Water
Discard <
AQ
xtract
with 2x50 ml
MeCl
Percolate through
anhydrous granular
N32S04
Concentrate to
^ 4 ml
Concentrate to
Pass Through
silica gel column
1 gm deactivated
with 20% water
Add hexane
10 ml
hexane
Fraction I
16 OGC's
PCB's
15 ml
60% benzene
in hexane
Fraction II
23
16
2
OGC's
OGP's
Carbamates
(partial )
15 ml
5% CH3CN
in benzene
Fraction III
8 OGC's
12 OGP's
7 Carbamates
15 ml
25% acetone
in MeCl?
Fraction IV
7 OGP's
Figure 2. Scheme for water analysis.
III-236
-------
TABLE 1. RECOVERIES OF 38 ORGANOPHOSPHORUS COMPOUNDS FROM WATER
===============================================================================
Recoveries in Percent
Compound
Azinphos methyl (Guthion)
Carbophenothion (Trithion)
Carbophenoxon
Chlorpyrifos (Dursban)
Cruf ornate (Ruelene)
DEF
D1az1non
Dlazoxon
Dichlofenthion (YC-13)
Dicrotophos (Bidrin)
Dfmethoate (Cygon)
Dioxathion (Delnav)
Disulfoton (Di-Syston)
EPN
Ethlon
Ethoprop (Prophos)
Fenltrothion (Sumithion) *
Fenthion (Baytex)
"onofoc !Dyfonats)
Leptophos (Phosvel)
Malaoxon
Ma lath ion
Methamidophos (Monitor)
Mevlnphos (Phosdrin)
Monocrotophos (Azodrin)
Maled (Dibrom)
Oxydemeton methyl
(Metasysotox R)
Paraoxon ethyl
Paraoxon methyl
Parathion ethyl
Parathion methyl
Phencapthon
Phorate (Thimet)
Phosalone (Zolone)
Phosmet (Imidan)
Phosphamidon (Dlmecron)
Ronnel
Ronnoxon
Cone.
(pob)
320
48
80
4
90
24
20
10
1.6
120
24
28
2.6
60
20
2
12
12
20
200
80
4
200
6
72
56
300
40
36
16
16
60
1.3
400
220
. 80
4
120
======
Extraction
Only
78
99
94
99
80
102
108
92
102
17
40
103
92
99
100
97
99
93
98
107
104
100
5
69
0
92
67
99
98
101
99
99
98
102
82
43
100
94
===============
Silica Ge
Elution
I II
93
87
102
72
96
94
84
76
78
91
99
93
98
56
91
96
1 Partition!
Fraction
III IV
88
58
90
104
72
15
60
17
96
50
78
32 33
45
90
93
85
43
92
:===== =================
ng only
Total
88
93
0
87
58
90
104
72
102
15
60
89
0
96
94
96
84
76
78
•31
50
78
0
65
0
45
0
90
93
99
93
98
56
91
85
43
96
92
III-237
-------
NOTE 2. To avoid troublesome caking of the sodium sulfate at the
bottom of the funnel, shaking should be conducted instantly after
adding the sodium sulfate.
NOTE 3. A reagent blank of 500 ml of the preextracted water should
be carried through all procedural steps in exactly the same manner
as the sample(s).
Place a small wad of glass wool at the bottom of a 25 x 300 mm
Chromaflex column and add a 2 inch depth of anhydrous sodium sulfate.
Position the tip of the column over a Kuderna-Danish assembly con-
sisting of a 250 ml K-D flask attached to a 10 ml evaporative concen-
trator tube containing two or three carborundum chips and 5 to 10
drops of keeper solution.
Drain the lower layer (methylene chloride phase) from the separatory
funnel through the sodium sulfate column, taking care to avoid the
transfer of any of the aqueous phase.
Add 50 ml more of methylene chloride to the aqueous phase in the
funnel. Stopper and repeat the 2-minute shaking, phase separation,
and draining of the organic layer through the sodium sulfate column
into the K-D flask.
NOTE 4. It is not uncommon with highly contaminated water samples
to encounter persistent and sometimes severe emulsion problems at the
methylene chloride-water interface. When this occurs, for example,
in the extraction of some wastewater samples containing high surfac-
tant concantrations, "'t is inadvisable to oass the methylene chloride
phases through the sodium sulfate because the aqueous emulsion tends
to clog the column and make filtration difficult. A good way to cope
with an emulsion is to pack a filter tube (A. H. Thomas 4797-N15 or
equivalent) with a 25 mm thick pre-washed glass wool pad and pass the
extract containing the emulsion through this filter into a 400 ml
beaker, applying air pressure if necessary. If the emulsion persists
on the second methylene chloride extraction, this treatment is re-
peated. The glass wool pad is then rinsed with 25 ml of methylene
chloride, collecting the extract and the washing on the surface of
the filtrate. Repeat the process with a second glass wool filter.
Connect the K-D flask to the rotary evaporator and incline the
assembly to an angle approximately 20° from the vertical, with the
concentrator tube about half immersed in a water bath previously
adjusted to 35°C. Turn on the rotator, adjusting the speed to a slow
spin. Switch off the bath heat and apply vacuum to the evaporator
at a pressure of ca. 125 mm of Hg.
NOTE 5. The recommended adjustments of temperature, vacuum, and the
pitch of the assembly should result in a steady boiling action with
no bumping. The -Ditch should be such that no extract condensate
collects in the lower part of the K-D flask (paragraph 2.8, NOTE i).
111-238
-------
Continue evaporation until the extract is condensed to ca. 4 ml,
remove the assembly from the water bath, and rinse down the walls
of the flask with 4 ml of hexane delivered with a disposable
pi pet.
Disconnect the concentrator tube from the K-D flask, rinsing the
joint with ca. 2 ml of hexane delivered with a disposable pipet.
Place the tube under a gentle stream of nitrogen at ambient tempera-
ture and concentrate the extract to ca. 0.5 ml.
NOTE 6. Under no circumstances should air be used for the blowdown
as certain organophosphorus compounds may not survive the oxidative
effects.
2.6 Silica Gel Fractionation and Cleanup
Before starting the following steps, place 10 drops of the paraffin
oil-hexane keeper solution in the two 15-ml centrifuge tubes intended
as the receivers for the eluates of Fractions III and IV.
Prepare a silica gel column as follows:
a. Lightly plug a size 22 Chromaflex column with a smal"1 wad of
preextracted glass wool.
b. Add 1.0 g of deactivated silica gel, tapping firmly to settle,
then too with 1 inch of anhydrous sodium sulfate and again tap
firmly.
c. Pass 10 ml of hexane through the column as a prewash, discarding
the eluate.
When the last of the prewash hexane just reaches the top surface of
the sodium sulfate, quickly place a 15-ml conical centrifuge tube
under the column, and using a disposable pipet, carefully transfer
the 0.5 ml of sample extract to the column. After the extract has
entered the column, rinse the walls of the centrifuge tube with 1.0
ml of hexane, and, using the same disposable pipet, transfer this
washing increment to the column. Repeat this 1.0-ml hexane wash
twice more and finally add 6.5 ml hexane to the column. The result-
ing 10 ml total effluent is Fraction I.
NOTE 7. There must be no interruption of the procedure during this
step. Extreme care should be taken to apply the sample to the column
at the precise moment the last of the hexane prewash reaches the top
surface of the column.
NOTE 8. Faultless technique is required in this step to avoid any
losses, particuiariy auring the transfer of *he 0.5 ml concentrated
extract and the first rinse.—All the pesticide extracted from tne
original sample is concentrated in this very minisc-jle extract. The
III-239
-------
loss of one drop may Introduce a recovery error of 20 percent or
more.
Immediately position another 15-ml centrifuge tube under the column
and pass 15 ml of the benzene/hexane (60/40 v/v) elutlng solution
through the column. This 1s the Fraction II eluate.
Elute the column a third time using 25 ml of acetonitrile/benzene
solution (5/95 v/v). This eluate is Fraction III.
A fourth elution fraction is necessary if there is reason to suspect
the presence of crufomate, dicrotophos, dimethoate, mevinphos,
phosphamidon, or the oxygen analogs of diazlnon and malathion. The
elution solution is 15 ml of acetone/methylene chloride (25/75 v/v).
This is Fraction IV.
Place the eluates under a gentle nitrogen stream at ambient
temperature and concentrate as follows:
a. Concentrate Fraction I and II to ca. 3.0 ml, rinse down the tube
sidewalls with ca. 1.5 ml hexane, and adjust the volume to exactly
5.0 ml with hexane. Cap the tubes tightly and mix on the Vortex
mixer for 1 minute.
b. Concentrate Fractions III and IV to 0.3 ml, rinse tube sidewalls
with hexane, and dilute back to exactly 5.0 ml with hexane.
NOTE 9. Fractions III and IV contain eluant solvents that may inter-
fere in the GC determination, whereas those solvents in Fractions I
and II would create no sucn prooiems. for em's reason, "-actions *:!
and IV are reduced to a lower volume to increase the removal of the
original solvents.
Fractions II and III may contain carbamates as well as organophos-
phorus compounds. Gas chromatography of organophosphorus compounds
by flame photometric detection 1s conducted on the eluates adjusted
to 5.0 ml.
2.7 Sample Analysis
For multlresidue analysis of samples with unknown pesticidal
contamination, two GC columns yielding divergent compound elution
patterns will aid confirmation. Two such columns are 5 percent
OV-210 and 1.5 percent OV-17/1.95 percent OV-210. Sensitivity levels
for the FPD detector should be carefully established before starting
chromatographic determination. The majority of water samples will
contain extremely low pesticide concentrations, and, therefore, an
insensitive GC system will severely handicap the analysis. When flow
and temperature parameters are optimum, baseline noise should not
exceed 2.5 percent of full scale and injection of 2.5 ug of parathion
should result in a peax of at least 50 percent if *un seal a.
II1-240
-------
The majority of the halogenated pesticides will be found in Frac-
tions I and II, with a few of the more polar compounds in Fraction
III. Most of the organophosphorus compounds will be in Fractions II
and III, none in Fraction I, and a very few in Fraction IV. Carba-
mates are eluted in Fractions II and III (Table 1).
A number of organophosphorus compounds chromatographed with the FPD
detector require considerable column preconditioning by repetitive
injection of standards of relatively high concentrtion before
attempting quantification. Failure to carefully monitor linearity
of response may result in erroneous quantitative values.
A typiical gas chromatogram of silica gel column Fraction II is
shown in Figure 3.
2.8 Miscellaneous Notes
1. The recommended operation of the concentrator is unusual for
pesticide analysis. Customarily, solvent evaporation is achieved
by immersing the concentrator tube in a water bath at a higher
temperature .than the boiling point of the solvent, or the flask is
attached to a conventional rotary evaporator. The system used
achieves two ^oortant objectives-, the extract is exposed to a
maximum temperature of less than 35"C to minimize degradation if
heat labile compounds; and the concentrated extract is confined to
one,container, thereby eliminating need for a transfer. Using the
temperature and vacuum levels specified, 100 ml of methylene chlo-
-*de extract can be reduced to 5 ml in ca. 20 minutes in this
apparatus.
2. The activity and performance of deactivated silica gel changes
in a matter of days. It is desirable to deactivate only the amount
required for a 2- or 3-day period. Continuous storage of activated
silica gel at 175°C may result in a shift of the compound elution
pattern of deactivated columns prepared from this adsorbent. The
quantity of silica gel activated should be limited to a 1-week
supply.
3. Recoveries of OP pesticides were found, in general, to be far
better when methylene chloride-extracted water rather than unextrac-
ted distilled water was used as the spiking substrate to evaluate
this procedure. Therefore, unextracted distilled water was used for
all recovery studies. As a further test, a sample of water was
obtained a few hundred yards downstream from the outfall of a large
chemical manufacturing plant and was fortified with a mixture of
pesticides and analyzed using the extraction and silica gel fractiona-
tion steps. No extraneous peaks were observed with the flame photo-
metric detector, indicating applicability of the method to aqueous
environmental water samples.
TII-241
-------
I
ro
ro
32
40
Figure 3. Four organophosphorus compounds eluted in Fraction II,
OV-17/1.95% OV-210.
GC column 1.5%
-------
3. Determination of Organophosphorus Pesticides 1n Soil
Analytical Procedure: available
Sample Preparation: available
3.1 Reference
Carey, A.E., J. A. Gowen, H. Tai, W. 6. Mitchell and 6. B. Wiersma,
"Pesticide Residue Levels in Soils and Crops from 37 States, 1972 -
National Soils Monitoring Program (IV)." Pest. Monit. J. 12(4):
209 (1979).7
3.2 Method Summary
Pesticides are extracted from soil with hexane-isopropanol, and
extracts are analyzed without cleanup by GC with a phosphorus-
selective detector.
3.3 Applicability
DEF, diazinon, malathion, ethyl parathion, methyl parathion, trith-
ion, and ronnel were determined in cropland soils at minimum detec-
table levels of 0.01-0.03 ppm. The method should be applicable to
other OP pesticides with similar polarities.
3.4 Accuracy, and Precision
Pesticide recovery from soil was close to 100 percent and ranged from
30 to 100 percent.
3.5 Sample Preparation
Moisten a 100-g subsample of soil from a thoroughly mixed field
sample with 25 ml distilled water. Extract with 200 ml of hexane/
isopropanol (3/1 v/v) by shaking for 4 hours on a mechanical mixer or
shaker. Remove the isopropanol with three distilled water washes.
Dry the hexane extract through a small column of anhydrous sodium
sulfate. Store the sample extract at low temperature prior to GC
analysis.
3.6 Sample Analysis
Analyze samples on a gas chromatograph equipped with a flame photo-
metric or thermionic detector. Identify compounds based on elution
characteristics on at least two columns with differing polarity.
Confirm residues by use of alternate selective detectors (e.g.,
Dohrman microcoulometric or electrolytic conductivity) or by GC/MS.1
Carry standards through the analytical procedure to monitor recovery.
Residue concentrations can be corrected for recovery if desired.
Kesults of joil sample analyses are usually converted to a dry-weight
basis. To determine moisture content, weigh a separate portion of soi i
taicen for extraction, heat in an even overnight, at 105 to 110'C. cool
in a desiccator, and reweigh.
i11-243
-------
4. Determination of Organophosphorus Pesticides in Fruits and Vegetables
Analytical Procedure: available
Sample Preparation: available
4.1 References
Luke, M. A., J. E. Froberg and H. T. Masumoto, -"Extraction and
Cleanup of Organochlorine, Organophosphate, Organonitrogen,
and Hydrocarbon Pesticides in Produce for Determination by Gas-
Liquid Chromatography." J. Assoc. Off. Anal. Chem. 55(5):1020
(1975).H
Luke, M. A., J. E. Froberg, 6. M. Doose and H. T. Masumoto,
"Improved Multiresidue Gas Chromatographic Determination of
Organophosphorus, Organonitrogen, and Organohalogen Pesticides
in Produce Using Flame Photometric and Electrolytic Conductivity
Detectors." J. Assoc. Off. Anal. Chem. 64(5):1187 (1981).12
4.2 Method Summary
Samples are extracted with acetone and partitioned with methyl-
ene chloride and petroleum ether to remove water. The methylene
chloride is removed with a Kuderna-Danish evaporator and the
resultant petroleum ether extract is analyzed by GC using an
FPD detector.
4.3 Applicability
The method is applicable to nonionlc OP pesticides and metabol-
ites present in fruit or vegetaoie matrices at concantratlcri
levels of ca 0.01 to 5 ppm.
4.4 Precision and Accuracy
Table 2 lists recovery data for 44 OP compounds from a variety
of produce samples. Recoveries are 1n the range from 88 to
118 percent. ReproducibiHty of the method has not been reported.
4.5 Sample Preparation
Chop or blend fruits and vegetables and mix thoroughly. Weigh
100 g of chopped or blended sample into a high-speed blender
jar, add 200 ml acetone, and blend for 2 minutes at high speed.
Do not add Celite. Filter with suction through a 12 cm Biichner
funnel fitted with sharkskin paper, and collect the extract in
a 500 ml suction flask.
Place 80 ml of sample in a 1-1 Her separatory funnel,, add 100
ml petroleum ether and 100 ml methylene chloride, and shake for
1 minute. Transfer the lower aqueous phase into a second 1-liter
separator/ funnel. Dry the upper organic layer 1n *,he f
-------
TABLE 2
Compound
RECOVERIES OF OP PESTICIDES
asaaaaa:
Recovery
====================================================
Fortification
Level (ppm)
Sampl e
Azinphos-ethyl
Carbophenothion sulfone
Chlorfenvinphos
Chlorpyrifos
Chlorthiophos
DDVP
OEF
Demeton-S-sulfone
Dial if or
Dicrotophos
Dimethoate oxygen analog
EPN
Fenamiphos
Fenitrothion
Fensulfothion
Fenthion
Fonofos
Leptophos
Malathion oxygen analog
Mephosfolan
Methidathion
Methyl carbophenothion
Naled
Oxydemeton-methyl
Oxydemeton-methyl sulfone
Parathion oxygen analog
Phenthoate
Phorate sulfone
Phorate sulf oxide
Phosalone
Phosmet
Phoxim
Phoxim oxygen analog
Profenofos
Prometryn
Pyrazophos
Ronnel
Sulprofos
Sulprofos sulfone
Sulprofos sulf oxide
Tetrachlorvinphos
Thionazin
Triazoohos
Trichlorfon
-:== ===-=::aaaa = a=aasasasaaasBa
1.00
1.94
0.324
0.10
0.0918
0.105
0.69
5.7
1.32
0.094
1.51
1.05
0.436
1.00
1.0
0.14
1.00
0.102
1.52
0.17
0.862
0.56
3.79
0.12
0.113
1.50
0.0117
0.105
0.114
1.80
0.25
0.10
0.10
0.10
0.108
0.66
0.10
0.105
0.10
0.105
1.08
0.608
0.134
0.110
============
102
116
97
99
95
90
105
115
115
105
90
105
97
88
107
97
92
113
112
106
93
118
97
88
103
105
110
93
116
92
108
110
105
102
104
107
104
105
105
106
113
100
106
no
===================
tomato
cucumber
bell pepper
green beans
tomato
tomato
lettuce
pepper
potato
green beans
grapes
green beans
bell pepper
blueberry
rutabaga
green beans
parsley
potato
potato
. tomato
orange
tomato
strawberry
grapes
grapes
tomato
tomato
lettuce
lettuce
grapes
tomato
tomato
tomato
tomato
cucumber
tomato
pears
green beans
green beans
green beans
green beans
tomato
tomato
orange
IH-245
-------
by passing through a 4-cm layer of Na2SC>4 supported on washed
glass wool in a 10-cm funnel and collect the solution in a 500-ml
Kuderna-Danish concentrator. To the separatory funnel with the
aqueous phase, add 7 g NaCl and shake for 30 seconds until most of
the salt is dissolved. Add 100 ml of methylene chloride, shake for
1 minute, and dry the lower organic phase through the sane
column. Extract the aqueous phase with an additional 100 ml
methylene chloride and dry as above. Rinse the N32S04 with ca.
50 ml of methylene chloride.
Attach a Snyder column to the K-D concentrator and start evapo-
ration slowly by placing only the receiver tube into steam.
After 100 to 150 ml have evaporated, expose the concentrator to
more steam. Concentrate to 1 ml, add 100 ml of petroleum
ether to the concentrator, and reconcentrate the solution to
1 ml. Repeat the reconcentration with 50 ml of petroleum ether
and then with 20 ml of acetone.
4.6 Sample Analysis
Cool and adjust the volume of the concentrated extract to 7 ml.
Inject ca. 2 ul into a gas chromatograph equipped with an FPD and
a 2-percent DEGS, 2-percent OV-101, or 4-percent SE30/6.5-percent
OV-"10 column.
111-246
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5. Determination of Organophosphorus Pesticides in Air
Analytical Procedure: available
Sample Preparation: available
5.1 Reference
Sherma, J. and M. Beroza, "Manual of Analytical Methods for the
Analysis of Humans and Environmental Samples." EPA-600/ 8-80-038,
Section 8B (June, 1980).4
5.2 Method Summary
The sampling medium, such as polyurethane foam or composite filter
pad, is Soxhlet extracted with hexane/diethyl ether (95/5 v/v). OP
pesticides are measured by direct GC with an FPO detector.
5.3 Applicability
The method is suitable for quantifying OP pesticides in ambient air
at ultratrace levels (ng/m3 and pg/m3). The analytical scheme pre-
supposes collection of. samples by a high-volume air sampler on poly-
urethane foam or on Tenax GC sorbent.
5.4 Precision and Accuracy
The collection efficiencies of OP pesticides on polyurethane/granular
sorbent combination media are shown in Table 3. Essentially no
differences are observed among sorbent systems for the pesticides
studied. Collection efficiencies using the IRCO high -clurre :anpler
.are shown in Table 4 for filter pads and in Table 5 for a tandem pair
of polyurethane foam plugs.
5.5 Extraction of the Sampling Module
Place the sampling medium in a Soxhlet extractor, handling with
forceps rather than hands.
NOTE 10. After sampling, glass fiber filters and foam plugs should
have been wrapped in aluminum foil until analysis. Use plugs and
filters carried to the field along with those employed for sampling
as controls.
Extract with an appropriate volume of n-hexane/acetone/diethyl ether
(47/47/6 v/v) for 16 to 24 hours at 4 cycles per hour for the large
Soxhlets and 8 to 12 hours at 8 cycles per hour for the smaller
Soxhlets.
NOTE 11. As examples, extract large foam plugs in 1000-ml Soxhlet
extractors with a total of 300 to 750 ml of solvent, and smaller
plugs and filters in 500 ml Soxhlets *ith 20fi to 350 ml.
111-247
-------
TABLE 3. HIGH-VOLUME COLLECTION EFFICIENCIES OF PESTICIDES ON
FOAM/GRANULAR SORBENT COMBINATIONS
% Collection on Foam/Sorbent Combinations
After 24 hours at 225 1/minute
Calc. Air Chromosorb
Cone. Foam 102 Porapak R XAD-20 Tenax GC Florisil
Pesticide (ng/m3) Alone (20/40) (50/80) (16/20) (60/80) (16/30)
Diazinon
Methyl
Parathion
Ethyl
Parathion
Malathion
3.0-30.0
1.8-18.0
3.6-36
0.9-9.3
===========
63
91
96
37
========
72
82
85
88
=======
59
72
72
78
71
80
81
89
76
87
86
91
72
83
83
81
TABLE 4. ERCO SAMPLER COLLECTION EFFICIENCIES AT
183 1/MINUTE FOR 2 HOURS
===============================================================================
Calculated Air Concentration Collection Efficiency
Compound (ug/m3) (%)
Methyl parathion 0.02 - 150 105
Diazinon 0.11 - 2.2 93
ChlorpyHfos . 0.02 - 0.22 77
:=========::======== = = :
111-248
-------
TABLE 5. AVERAGE COLLECTION EFFICIENCIES ON POLYURETHANE FOAM FOR
ORGANOPHOSPHORUS PESTICIDES AT 225 1/MINUTE AND 184 1/MINUTE
===================
Pesticide
Diazinon
Methyl parathion
Malathion
Parathion
:==========================================:
Air Calculated
Volume Air Concentration
(m3) (ng/m3) % Collected
326
265
265
326
265
265
326
265
265
326
265
255
30.7
18.9
3.8
18.4
11.3
2.3
36.8
22.6
4.5
9.2
5.7
1.1
70.4
91.0
75.5
73.6
73.3
71.9
87.2
76.6
81.2
84.8
70.3
65.8
Statistical Data
n
5
6
6
5
5
4
5
5
4
5
5
4
sigma
3.97
16.19
14.40
4.56
6.52
4.12
33.40
12.47
14.68
4.15
4.70
3.75
==============================================================================
Remove the boiling flask to a rotary evaporator and reduce the
solvent volume to approximately 5 ml.
Transfer the concentrate to a 15-ml graduated centrifuge tube with
rinsing.
5.6 Sample Analysis
Adjust the final volume in the centrifuge tube as required.
Inject 5 ul directly without cleanup into the gas chromatograph
equipped with an FPD detector. Typical GC conditions for determina-
tion are as follows: 183 cm x 4 mm I.D. glass column packed with 1.5
percent OV-17/1.95 percent OV-210 and/or 4 percent SE-30/6 percent
OV-210 on 80 to 100 mesh Gas Chrom Q; column, 200°C; injection port,
215°C; nitrogen carrier gas, 60 to 85 ml/minute; P-mode FPD, 200°C.
Record chromatograms under the above parameters and measure retention
times relative to aldrin or another suitable reference standard.
Compare the relative retention time of each component of interest
against those of the corresponding primary standard.
Quantify peaks in the usual way, i.e., by measuring pea* neignts to
the nearest HOT when the basa width ss
-------
Confirm results as required by combined GC/MS or some other
appropriate procedure.
I. CALCULATIONS
1. Extract Quantification
The organophosphorus pesticide concentration of the sample extract is
calculated based on the method of standardization.
la. Internal Standardization
By adding a constant known amount of internal standard (C-js in yg)
to every sample extract, the concentrtion of contaminant (C0) in
ug/1 in the extract is calculated as:
(As)(C1s)
Co -
(A1s) (RF)
where As = response for the parameter being measured
C-jS = concentration of the internal standard
Ais = response for the internal standard
RF = calculated response factor.
Ib. External Standardization
The concentration of the unknown in the extract is calculated from
the slope and intercept of the calibration curve. The extract con-
centration is calculated as:
(A)(Vt)
(Vi)(Vs)
where C0 = extract concentration
A = mass of compound from the calibration curve (ng)
Vi = volume of extract injected (pi)
Vt - volume of total extract (yl)
Vs * volume of water extracted (ml).
Report ^11 results in ug/l to two significant figures without
correction for recovery data. When duplicate and spiked samples
are analyzed, all data obtained should be reoorted.
III-250
-------
2. Sample Concentration
From the concentration of pesticide in the extract, calculate the con-
centration in the original sample.
2.1 Formulations
(A)(B)
Concentration (mg/g) =
(C)
A * concentration in extract (mg/ml)
B = volume of extract (ml)
C * sample mass (g).
2.2 Water Samples
Concentration (mg/liter) =
(C)
A = concentration in extract (wg/ml)
B * volume of extract (ml)
C * sample volume (ml).
1.3 Soi'i Dumpies
2.3.1 Dry Basis
(A)(B)(100-D)
Concentration (ng/g) =
(C) (100)
A - concentration in hexane extract (ng/ml)
B = final volume of hexane extract (ml)
C * wet mass of sample (g)
D = percent moisture of sample.
2.3.2 Wet Basis
(A)(B)
Concentration (ng//g) =
(C)
A = concentration in hexane extract
B = final volume of hexane extract
C * sample mass (wet).
III-251
-------
2.4 Air Samples
Concentration (ng/m3) =
(0(1000)
A » concentration in extract (ng/1)
B = extract volume (ml)
C = sample volume (m3).
2.5 Fruits and Vegetables
(A)(B)
Concentration (ng/g) =
(C)
A = concentration in final extract (yg/ml)
B = final volume of extract (ml)
C = sample mass (g).
III-252
-------
REFERENCES
1. Sherma, J. "Manual of Analytical Quality Control for Pesticides 1n Human
and Environmental Samples." EPA-600/2-81-059 (April, 1981).
2. McMahon, B. M. and L. D. Sayer, editors. "Pesticide Analytical Manual -
Volume I: Methods Which Detect Multiple Residues." United States FDA
(September, 1982).
3. "Guidelines on Sampling and Statistical Methodologies for Ambient Pesticide
Monitoring." Federal Working Group on Pest Management, Washington, D.C.
(October, 1974).
4. Sherma, J. and M. Beroza. "Manual of Analytical Methods for the Analysis
of Pesticides in Humans and Environmental Samples." EPA-600/8-80-038
(June, 1980).
5. Lewis, R. G. "Procedures for Sampling and Analysis of PCBs in the Vicinities
of Hazardous Waste Disposal Sites." Advanced Analysis Techniques Branch,
Environmental Monitoring Systems Laboratory, Research Triangle Park, North
Carolina. 14 p. (March 16, 1982).
6. Zweig, G. and J. Sherma. "Gas Chromatographic Analysis," Volume 6 of
"Analytical Methods for Pesticides and Plant lirowtn Regulators." Chapter
4, p. 107, Academic Press, New York (1972).
7. Carey, A. £., -j. A. aowen, n. 7
-------
12. Luke, M. A., J. E. Froberg, G. M. Doose and H. T. Masumoto. "Improved
Multiresidue Gas Chromatographic Determination of Organophosphorus,
Organonitrogen, and Organohalogen Pesticides in Produce, Using Flame
Photometric and Electrolytic Conductivity Detectors." J. Assoc. Off.
Anal. Chem. 64(5):1187 (1981).
III-254
-------
SECTION 6
METHODS FOR THE DETERMINATION OF ORGANONITROGEN PESTICIDES
A. SCOPE
The analytical procedures provided in Subsection I of this section cover
the determination of carbamate, urea, and related pesticides in water (Sub-
section I.2.). soil (Subsection I.3.), vegetable and fruit tissues (Subsection
1.4.) and air (Subsection I.5.). The water and crop analyses are based on high-
performance liquid chromatography with UV and fluorescence detection, respec-
tively. Gas chromatography with N-mode electrolytic conductivity detection is
the determinative procedure for soil and air analyses, and HPLC is also used for
soil analysis. All analyses involve solvent extraction of residues from the
sample, followed in certain cases by solvent partitioning and column chromato-
nraohy cleanuo steps and derivatization prior to detection and determination.
Compounds covered in this section are all broadly classified as organo-
nitrogen pesticides.
«J. SAMPLE HANDLING AND STORAGE
See Subsection B of Section 5 (Organophosphorus Pesticides) in this Chap-
ter for general information on pesticide sampling and storage that is appli-
cable to carbamate and related compounds.
Sampling techniques and sample preservation are important criteria in any
monitoring program. Although sampling and analysis are distinct parts of the
program, they are interdependent since each controls certain aspects of the
other. An analytical result can be no more valid than the samples or sampling
scheme used. Good sampling procedures should be practiced, and the following
precautions should be taken to ensure that the samples received and analyzed
represent the field situation.
Samples should be analyzed as soon as possible after collection to
avoid any biological or chemical alteration of the pesticides; in the interim
the sample should be frozen, or, in the case of water, stored at a low tempera-
ture (about 4°C). Since many of the N-methylcarbamates are prone to hydrolysis
at the pH of natural waters, the addition of acid (to about pH 2) is recom-
mended. Since photochemical changes are also possible, the samples should be
stored in the dark to minimize these possibilities.
Frequently, samples cannot be analyzed as quickly as desired. Hence, :t
may be desirable to extract the samoles upon receipt (or in the field) and
store the extracts pending analysis. All samples must be extracted within
111-255
-------
7 days and completely analyzed within 40 days of extraction. The stabil-
ity of a pesticide during any type of storage is questionable, and it is often
beneficial to examine the stability of a standard or field-incurred residue in
a sample or extract Under various conditions of time, temperature, and in differ-
ent substrates. The stability of carbofuran and carbaryl appears to be more
dependent on the substrate than on the storage temperature. On the other hand,
methomyl stability is related to the storage temperature, and it appears to be
most stable under freezing conditions. Ethylenebisdithiocarbamate (EBDC) fun-
gicides tend to decompose immediately after maceration of substrates, and
storage by freezing is preferred over refrigeration. Chloropropham (CIPC) and
propham (IPC) show significant losses during storage.
Clean glass bottles with Teflon- or aluminum-lined tops should be used
for water samples. Polyethylene bags or glass jars are recommended for other
substrates. The bottle must not be prerinsed with the water sample before
collection. Automatic sampling equipment must be as free as possible of plas-
tic and other potential sources of contamination. When composite water samples
are collected, it is advisable to refrigerate the sample during the compositing
period.
Phenylurea herbicides are relatively stable and do not undergo rapid
chemical or biological degradation. Thus, water and sediment samples collected
from field sites can be shipped in glass containers without special treatment.
On arrival at cne iaooratory, water samples T,ay be filtersd -hrough jlass '-wol
to remove solid material and then stored in a refrigerator at 4"ct If the
analysis cannot be carried out within 2 weeks, the sample should be frozen at
-20"C. Sediment samples should be frozen at -20*C on arrival at the labora-
tory, and prior to analysis should be thawed, filtered to remove excess water,
ana a'ir-ur'iea.
Plant and animal samples are stored at -40°C until analysis. A storage
stability study on the ground or chopped sample is recommended by fortifying
untreated check samples.
C. INTERFERENCES
Method interferences may be caused by contaminants in solvents, reagents,
glassware, and other sample processing apparatus that lead to discrete arti-
facts or elevated baselines in chromatograms. All reagents and apparatus must
be routinely demonstrated to be free from interferences under the conditions of
the analysis by running laboratory reagent blanks.
Clean all glassware* as soon as possible after use by thoroughly rinsing
with the last solvent used in it. Follow by washing with hot water and deter-
gent and thorough rinsing with tap and reagent-grade water. Drain dry, and
heat 1n an oven or muffle furnace at 400°C for 15 to 30 minutes. Do not heat
volumetric glassware. Thermally stable materials such as PCBs might not be
eliminated by this treatment. Thorough rinsing with acetone and pesticide-
quality hexane may be substituted for the heating. After drying and cooling,
seal ana store glassware *n 3 clean environment to prevent accumulation o* dust
or other contaminants. Store inverted or capped with aluminum foil.
III-256
-------
Matrix interferences may be caused by contaminants that are coextracted
from the sample, and will vary in extent depending on the nature of the
sample and its source. The cleanup procedures in the methods will usually
overcome these interferences, but unique samples may require additional cleanup
approaches.
0. SAFETY
The toxicity or carcinogenicity of each reagent used in the methods in
this section has not been completely defined. However, each chemical compound
must be treated as a potential health hazard. Exposure to chemicals must be
reduced to the lowest possible level by all means available. Each laboratory
1s responsible for maintaining a current awareness file of OSHA regulations
regarding the safe handling of all chemicals specified in the methods.2t3
E. APPARATUS
1. Equipment for discrete or composite sampling of water:
1.1 Grab-sample bottle, amber borosilicate or flint glass, 1 quart or
1 liter volume, fitted with screw caps lined with TFE fluorocarbon,
or aluminum foil if the sample is not corrosive. If amber bottles
are not available, protect samples from light. Wash the container
ana cap iliner, nnse *ith acstone Dr methylene chloride, ?nd Hry
before use to minimize contamination.
» 1.2 Automatic sampler (optional), incorporating glass sample containers
for-collection of a minimum of 250 ml. Refrigerate (4*C) and protect
sample containers "rein "ight d'jf'rig tempos i^ng. !* the samoler uses
a peristaltic pump, a minimum length of compressible silicone rubber
tubing may be used. Before use, rinse the tubing thoroughly with
methanol, followed by repeated rinses with reagent-grade water to
minimize the potential for contamination of the sample. An integra-
ting flow meter is required to collect flow-proportional composites.
2. Analytical balance, capable of weighing to ±0.0001 g.
3. Water bath, heated, with concentric cover, capable of temperature
control (±2*C).
4. Beakers, 2000 ml.
5. Gas chromatograph, Tracer Model 560 or equivalent, equipped with a
1inearized63Ni electron capture detector, or a Model 700 Hal.l electrol-
ytic conductivity detector operated in the reductive mode with a nickel
wire catalyst (Ventron), strontium hydroxide scrubber, and isopropanol/
water (15/85 v/v) conductivity solvent. Detector used depends on the
requirements of the method.
6. GC column, 1.3 m x 2 m !.D., glass,
-------
with normal carrier gas flow. Alternative columns include 1 percent
OV-17 and the mixed phase 0.5 percent OV-210 + 0.65 percent OV-17, both
on Ultra-Bond. A temperature program of 115 to 1758C at lO'/minute can be
used for complex samples, in place of Isothermal operation at 170°C. This
column is to be used when the Hall electrolytic conductivity detector is
specified.
7. GC column, 1.8 m x 4 mm I.D., glass, containing 3 percent (w/w) OV-225
on 80 to 100 mesh Chromosorb W(HP) or 3.6 percent (w/w) OV-101/ 5.5
percent (w/w) OV-210 on acid-washed, dimethyldichlorosilane-treated
Chromosorb W. This column is to be used when the electron capture detec-
tor is specified.
8. Liquid chromatograph, high performance analytical system complete with
high pressure syringes or sample injection loop, analytical column,
gradient elution system, detector, and strip-chart recorder or mini-
computer data-handling system. The Installation of a guard column is
also recommended. Typical modules Include an Altex Model 322 MP
programmable gradient system, Valco Model 16 AS-7000 automatic sampler
with 10-ul injection loop, Spectra Physics Model 4000 Microprocessor/
printer plotter, and 7 cm x 2.1 mm I.D. guard column containing 25 to
37 urn Co-Pell ODS (Whatman).
*?. MPLC column, ^0 ~-n x 4 mm !.0., stainless steel, packed with u-Bondaoak
Cis (10 urn) (Waters Associates); 30 cm x 4.6 mm I.O., stainless steel,
packed *1th Spherisorb ODS (5 '^m) (Phase Separations Ltd.); or 25 cm
x 4.6 mm.I.D., stainless steel, containing Zorbax C-8 (6 urn) (DuPont).
All of these are chemically bonded reversed-phase columns.
10. HPLC fluorescence detector, Perkin-Elmer Moaei s5Q-iQLC or equivalent,
with 20-iil cell. This detector requires the following apparatus:
10.1 Carbamate hydrolysis chamber - column bath (18x18x13 cm) from
Model 5360 Barber-Colman gas chromatograph with Model 700-113
proportional temperature controller (RFL Industries, Inc., Boonton,
NJ) containing 3 m x 0.48 mm I.D. No. 321 stainless steel tubing
(Tubesales, Forest Park, GA).
10.2 Sodium hydroxide and reaction solution reservoirs - 60 cm x 25 mm
I.D. glass columns with Teflon fittings (Glenco Scientific, Inc.,
Houston, TX). Pressurize reservoirs with nitrogen. Connect 6 m
x 0.5 mm Teflon restriction coil from reservoir to 15 cm x 0.18 mm
I.D. ss tubing. Connect ss tubing to 0.74 mm I.D. ss reaction tee
(No. ZVT-062, Valco).
10.3 Connecting tubing - No. 304 ss tubing to connect Injector, columns,
and first tee.
11. HPLC UV detector, capable of monitoring absorbance at 240 nm, 254 nm, and
280 nm.
III-258
-------
12. Chromatography. columns, 400 mm x 19 mm I.D., with coarse fritted disc at
the bottom and TFE-fluorocarbon stopcock (Kontes K-420540-0224).
13. Cleanup columns, Chromaflex size 22, 20 cm x 7 mm (Kontes K-420100-
0022), and Chromaflex 30 cm x 22 mm I.D. (size 233) with coarse porosity
frit and Yaribor stopcock (size 2) (Kontes K-420540-9042).
14. Desiccator.
15. Rotary evaporator.
16. Extractors, Soxhlet, 1000, 500, and 250 ml.
17. Swinny filter holder, 13 mm filter size (Millipore No. XX3001200).
18. Filter paper, Whatman No. 1 and No. 42, and S&S 597.
19. Miltex filters, 5 urn, 13 mm diameter, white, plain (Millipore No.
LSWP 01300).
20. Filtration apparatus, as needed to filter solvents prior to HPLC.
21. Flasks, Stanmeyer, 500 11! and 1000 ml.
22. Flasks, 2000 art, 1000 ml, 500 ml, and 250 ml round bottom, with I 24/40
joints.
:2. ""isks, volumetric. !0 Til. 1000 ml, and 25 ml (actinic). "
24. Separatory funnels, 2000 ml, 500 ml, and 250 ml, with TFE-fluorocarbon
stopcock and ground glass or TFE stoppers.
25. Homogenlzer, Polytron Model PT 10-35 or equivalent, with PT 35K generator
containing knives (Brlnkmann Instruments) and four-sided glass quart jar
(Tropicana Products, Inc., Bradenton, FL).
26. Pipets, volumetric, assorted sizes.
27. Wrist-action shaker.
28. Centrifuge tube, 15 ml.
29. Vacuum adapter I 24/40.
30. Vacuum filtration apparatus, with 12-cm Buchner funnel and 500-ml
filter flask.
31. Vials, 10 to 15 ml, amber glass, with TFE-fluorocarbon lined screw caps.
!!!-259
-------
F. REAGENTS
(Solvents should be pesticide quality, distilled-in-glass or HPLC grade
for mobile phases.)
1. Glacial acetic acid.
2. Acetone
3. Acetonitrile
4. Benzene
5. Carbon-Celite chromatography mixture. Mix Nuchar S-N and silanized
Celite (4/1 w/w). Test the adsorbent with a freshly prepared mixed
carbamate solution (carbaryl, methiocarb, methiocarb sulfoxide, methomyl )
prepared in methanol at 5 yg of each compound per ml. Pipet 5 ml of this
solution into a 250-ml round-bottom flask and 5 ml into a 25-ml actinic
volumetric flask. Dilute the solution in the volumetric flask to 25 ml
with methanol and use as HPLC reference standard. Evaporate the standard
solution in the round-bottom flask to.dryness with a vacuum rotary evap-
orator as described in Subsection H.4.1.5. Dissolve the carbamate residue
in 10 ml of methylene chloride. Transfer the solution to a prepared
adsorbent column and •elute as described in Subsection H.4.1.7. After evap-
oration of the eluate in the round-bottom flask, dissolve the residue in
25 Til of methanol. Filter 5 to 8 ml of this solution through a Swinny
filter holder, as described in Subsection H.4.1.7. Quantify recovery of the
carbamates using HPLC. Nuchar: S-N is satisfactory if recovery is >^9
6. Silanized Celite 545. Slurry 150 g of Celite 545 (Johns-Manville) *itn
1 liter of 6 M hydrochloric acid in a 2-liter beaker, cover with a watch
glass, and stir magnetically while boiling it for 10 minutes. Cool,
slurry, filter, and wash with ultrapure water until the filtrate is
neutral. Wash Celite with 500 ml of methanol followed by 500 ml of
methylene chloride, then air dry in a hood on a watch glass to remove
solvent. Transfer Celite to a 1-liter Erlenmeyer flask with ground-glass
joint. Heat in the unstoppered flask at 120°C overnight, and then cool
the flask in a desiccator. Place the flask in a hood and carefully pipet
3 ml of dichlorodimethylsilane (Pierce Chemical Co.) onto the Celite.
Stopper, mix well, and allow to remain at room temperature for 4 hours.
Add 500 ml of methanol to the flask, mix, and let stand 15 minutes.
Filter the silanized Celite and wash with isopropanol until neutral. Air
dry in a hood to remove isopropanol. Dry the silanized Celite at 105°C
for 2 hours, cool in a desiccator, and store in a glass-stoppered con-
tainer. Test Celite for total silanization by placing 1 g in 50 ml of
water and 1 g in 20 ml of toluene saturated with methyl red/toluene solu-
tion. If Celite neither floats on water nor appears yellow with the
methyl red solution, repeat the silanization to cover active sites.
7. Charcoal, Nuchar S-N. Slurry 100 g of Nucnar 5-N vFisner} *ith 70C oil
of HC1 , ".ove** with a watch glass, and boil for one hour with constant
III-260
-------
stirring. Add 700 ml of distilled water, stir, and boil for 30 minutes.
Cool, slurry, filter, and wash with ultrapure water until neutral. Wash
with 500 ml of methanol followed by 500 ml of methylene chloride and air
dry in a hood to remove solvent. Dry at 120'C for 4 hours, cool in a
desiccator, and store in a glass-stoppered container.
8. Ethyl ether, free of peroxides as indicated by EM Quant strips (Scien-
tific Products Co. No. P1126-8). Procedures recommended for removal of
peroxides are provided with the test strips. After cleanup, 20 ml of
ethanol preservative must be added to each liter of ether.
9. Florisil-PR grade (60/100 mesh). Purchase Florisil activated at 1250°F
and store it in the dark in a glass container with a ground-glass stopper
or foil-lined screw cap. Before use, activate each batch at least 16
hours at 130°C in a foil-covered glass container.
10. Hexane
11. Hydrochloric acid aqueous solution, 6M.
12. Isooctane
13. Isopropanol
14. Methylene chloride
15. Methanol
•
16. Nitrogen gas, ary, pun flea.
'17. Pentafluorobenzyl (PFB) bromide reagent, 1 percent (v/v); prepare by
dissolving 1 ml of reagent (Pierce Chemical Co., Rockford, IL, No. 58220)
or alpha-bromo-2,3,4,5,6-pentafluoroto1uene (Aldrich Chemical Co.,
Milwaukee, WI) in 100 ml of acetone in a low actinic volumetric flask.
Prepare fresh every 2 to 3 weeks. Caution: the reagent is a strong
lachrymator.
18. Petroleum ether
19. pH indicator paper.
20. Potassium carbonate, analytical reagent grade.
21. Potassium hydroxide, ACS reagent grade.
22. Reaction solution for HPLC fluorescence detection of carbamate insecti-
cides. Weigh 500 mg of £-phthalaldehyde, transfer to a 1-liter volumetric
flask, add 10 ml of methanol, and swirl to dissolve. Add ca. 500 ml of
0,05 M sodium tetraborate solution and 1.0 ml of 2-mercaptoethanol and
dilute to volume with 0.05 M sodium teiraborate solution.
-------
23. Silica gel, grade 950 (Davison Chemical Co., Baltimore, MD), deactivated
by adding 1.5 percent (w/w) distilled water and mixing for 2 hours. Store
in a tightly-stoppered container in a desiccator.
24. Sodium chloride, ACS reagent grade.
25. Sodium hydrogen carbonate, ACS reagent grade.
26. Sodium hydroxide solution, 0.05 M, prepared in degassed ultrapure water.
27. Sodium sulfate, ACS, granular, anhydrous. Heat-treat in a shallow tray at
400*C for at least 4 hours to remove phthalates and other interfering
organic substances. Alternatively, heat for 16 hours at 450 to 5008C in a
shallow tray, or Soxhlet-extract with methylene chloride for 48 hours.
28. Sodium tetraborate solution, 0.05 M, prepared in degassed ultrapure water.
29. Sulfuric acid, concentrated, ACS reagent grade.
30. Analytical standards. Dilute stock standards with methanol or
acetonitrile to give 1 ug/ml or as needed. Store solutions in actinic
glassware, and in the refrigerator. Most carbamate standards stored in
this manner are stable for several months. However, methiocarb sulfone
and sulfoxide degrade within hours and days, respectively, even *ith these
precautions.
31. Stock standard solutions, 1.00 ug/ul. Accurately weigh ca. 0.0100 g
of pure material, dissolve in pesticide-quality acetonitrile or
..•.atnancl, ..nti ri'uts 'o -clime ~'r, ~ ^O-^l "olumetr^'c f1ask. '..aroer
volumes may be used, if necessary. If the compound purity is certified
at 96 percent or greater, the weight can be used without correction to
calculate solution concentrations. Commercial stock standards certified
by the manufacturer can also be used. Transfer the solutions into
TFE-fluorocarbon-sealed screw-cap vials and store at 4°C protected from
light. Frequently check stock standard solutions for degradation or
evaporation, especially just prior to preparing calibration standards
from them. Replace stock standards after 6 months, or sooner if
comparison with check standards indicates a change in concentration.
32. Tetradecane
33. Toluene
34. Reagent-grade water, tested for the absence of interferences at the
method detection limit of each compound of interest.
35. Water, ultrapure, prepared using the Milli-Q water purification system
(Millipore Corp.). Water and solvents used for HPLC mobile phases are
degassed by placing them in a glass bottle, applying vacuum and slowly
stirring with i magnetic stirrer for 5 minutes.
III-262
-------
G. QUALITY CONTROL
1. The minimum requirements of this program consist of an initial demonstra-
tion of laboratory capability and the analysis of spiked samples as a
continuing check on performance. The laboratory should maintain perform-
ance records to define the quality of data that are generated.
Before performing any analyses, the analyst must demonstrate the ability
to generate data of acceptable accuracy and precision with this method.
This ability is established as described in Subsection G.2. Each time
the analytical method is modified, in response to state-of-the-art ad-
vances, the analyst is required to repeat the procedure in Subsection G.2.
The laboratory should spike and analyze a minimum of 10 percent of all
samples to monitor continuing laboratory performance as described in
Subsection G.4.
2. To establish the ability to generate data of acceptable accuracy and
precision, the analyst must perform the following operations:
Select a representative spike concentration for each compound to be meas-
ured. Using stock standards, prepare a quality control check sample
concentrate in acetomtriie or methanol 1000 times more concentratea than
the selected concentrations.
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
waszewaxer may -e ussa ,r, piacs of ;ne reagent .vaisr-, jut jne jr nore
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 with
Subsection 1.2.1.5.
Calculate the average percent recovery (R), and the standard deviation of
the percent recovery (s), for the results. Wastewater background correc-
tions must be made before R and s calculations are performed.
Table 2 (p. III-270) provides single-operator recovery and precision data
for most of the carbamate and urea pesticides. Similar results should be
expected from reagent water for all compounds listed with the method.
Compare these results to the values calculated. If the data are not
comparable, review potential problem areas and repeat the test.
3. The analyst should calculate method performance criteria and define the
performance of the laboratory for each spike concentration and compound
being measured.
Calculate upper and lower control limits for method performance as
foi1ows:
Upper Control Limit (UCL) = -R-+ 3 s
Lower Control Limit (LCL) « R - 3 s
III-263
-------
where R and s are calculated as above. The UCL and LCL can be used to
construct control charts' that are useful in observing trends in per-
formance.
Separate accuracy statements of laboratory performance should be developed
and maintained 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 Subsection
6.2, followed by the calculation of R and s. Alternatively, the analyst
may use four wastewater data points gathered through the requirement for
continuing quality control. The accuracy statements should be updated
regularly.?
4. The laboratory is required to collect in duplicate a portion of their
samples to monitor spike recoveries. The frequency of spiked-sample
analysis should be at least 10 percent of all samples or one spiked sample
per month, whichever is greater. One aliquot of the sample must be spiked
and analyzed as described in Subsection G.2 If the recovery for a par-
ticular parameter does not fall within the control limits for method
performance, the results reported for that compound in all samples pro-
cessed as part of the same set must be qualified as described in Sub-
section J. The laboratory should monitor the frequency of data so
qualified to ensure that it remains at or below 5 percent.
5. Before processing any samples, the analyst must demonstrate through the
analysis of a^l-liter aliquot of reagent water that ail 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
mus": oe processed as d safeguard against: laooratory ^an^umir.aL'ion.
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 chromatography with a
dissimilar column, or ratio of absorbance at two or more wavelengths may
be used. Whenever possible, the laboratory should perform analysis of
quality control materials and participate in relevant performance evalu-
ation studies.
H. CALIBRATION
Establish HPLC operating parameters equivalent to those indicated in
Table 1 (p. III-269). The HPLC system may be calibrated using either, the
external standard technique or the internal standard technique.
1. External Standard Calibration Procedure
For eacn compound of interest, prepare calibration standards it i minimum
of three concentration levels by adding accurately measured volumes of one
or more stocx standards to a volumetric f'ask and diluting to volume with
III-264
-------
acetonitrile or methanol. One of the external standards should be repre-
sentative of a concentration near, but above, the method detection limit.
The other concentrations should correspond to the range of concentrations
expected 1n the sample concentrates or should define the working range of
the detector.
Using Injections of 10 pi 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, the
ratio of the response to the mass injected, defined as the calibration
factor (CF), may be calculated for each compound at each standard concen-
tration. If the relative standard deviation of the calibration factor is
less than 10 percent over the working range, the average calibration fac-
tor can be used in place of a calibration curve. The working calibration
curve or calibration factor must be verified on each working shift by the
measurement of one or more calibration standards. If the response for any
compound varies from the predicted response by more than ±10 percent, the
test must be repeated using a fresh calibration standard. Alternatively,
a new calibration curve or calibration factor must be prepared for that
compound.
Internal Standard Calibration Procedure
To use this approach, the analyst must select one or more internal stand-
ards 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. Due to these
V* mi tat"! ens, n •'iter^al standard annl1'cable to all samples can be sug-
gested.
Prepare calibration standards at a minimum of three concentration levels
for each compound 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 or methanol. One of the standards should be
representative of a concentration near, but above, the method detection
limit. The other concentrations should correspond to the range of con-
centrations expected in the sample concentrates or should define the
working range of the detector.
Using Injections of 10 ul of each calibration standard, tabulate the peak
height or area responses against the concentration for each compound and
Internal standard. Calculate response factors (RF) for each compound as
fol1ows:
RF = (AsC1s)/(A1s Cs)
where:
As = response for the compound to be measured
AiS - response for the Internal standard
III-255
-------
C-js = concentration of the Internal standard in ug/1
Cs = concentration of the compound to be measured in ug/1.
If the RF value over the working range is constant, less than 10 percent
relative standard deviation, the RF can be assumed to be invariant, and
the average RF may be used for calculations. Alternatively, the results
may be used to plot a calibration curve of response ratios, As/A-js,
against RF.
The working calibration curve or'RF must be verified on each working shift
by the measurement of one or more calibration standards. If the response
for any parameter varies from the predicted response by more than ±10
percent, the test must be repeated using a fresh calibration standard.
Alternatively, a new calibration curve must be prepared for that compound.
III-266
-------
I. ANALYTICAL PROCEDURES
1.1 Determination of Carbamates and Urea Pesticides in Hazardous Waste
Samples. Reserved.
m-267
-------
2.1 Determination of Carbamate and Urea Pesticides in Industrial and Municipal
Wastewater
Analytical Procedure: available
Sample Preparation: available
2.1.1 Reference
Pressley, T. A. and J. E. Longbottom, "The Determination of Carba-
mate and Urea Pesticides in Industrial and Municipal Wastewater -
Method 632." Report No. EPA-600/4-82-014, Environmental Monitoring
and Support Laboratory, Office of Research and Development, U.S.
EPA, Cincinnati, Ohio (February, 1982).4
" 2.1.2 Method Summary
A measured volume of sample, approximately 1 liter, is solvent-
extracted with methylene chloride in a separatory funnel. The
extract is dried and concentrated to a volume of 10 ml or less. If
necessary, the extract is cleaned up on a Florisili column. The
extract or column eluate is analyzed by reversed-phase HPLC with UV
detection.
2.1.3 Applicabilty
The method covers the determination of the compounds listed in
Table 1 plus the Bellowing: aminocarb, carbofuran, fenuron,
fenuron-trichloroacetate (fenuron-TCA), monuron-trichloro-
acetate (monuron-TCA), Siduron, and Swep. Neither monuron and
Ticnuron-TCA nor fenuron and fenuron-TCA are distinguished by the
method; results ror tnese pairs are reported as monuron ana renurcn,
respectively. Detection limits range from 0.003 to 11 ug/1
(Table 1). The detection limit is defined as the lowest concen-
tration of pesticide that can be measured and reported with 99
percent confidence that the value is above zero. The values
listed in Table 1 were obtained using reagent or river water.
2.1.4 Precision and Accuracy
Single-laboratory recovery and precision (standard deviation) data
are listed in Table 2 for 15 pesticides that were determined with
this method using water from five sources.
2.1.5 Sample Extraction
Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
2-liter separatory funnel.
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 funnei ana extract cne sample by shaking the runnel ffor
III-268
-------
TABLE 1. CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION LIMITS
==========================
Parameter
Mobile
Phase*
Retention
Time (Min)
UV
Wavelength
(nm)
Method
Detection
Limit (ng/1)
Mexacarbate
Propoxur
Monuron
Carbaryl
Propham
Di uron
Linuron
Methiocarb
Chlorpropham
Barban
Neburon
Me thorny!
Carbaryl
Diuron
Linuron
Propoxur
Carbofuran
Fl uorometuron
Oxamyl
A
A
A
A
A
A
A"
A
A
A
A
3
B
8
B
B
C
C
8.7
14.3
14.3
17.0
17.2
19.5
21.0
21.4
21.8
22.3
24.3
2.0
6.5
14.1
15.5
17.9
i. 7
3.5
3.6
3.2
254
280
254
280
254
254
254
254
254
254
254
280
254
280
254
254
230
280
254
254
0.52
0.11
0.003
0.02
0.07
0.
0.
0.
.009
.009
.02
0.03
0.05
0.012
0.11
6.9
O.D2
0.009
0.009
3.2
11.1
9.2
aaa===========================================================================
*Mobile Phase:
A = Methanol /I percent acetic acid, programmed linearly from 5 to 95 percent
methanol at 2.0 ml/min flow rate and at ambient temperature.
B = Acetonitrile/water, orogrammed linearly from 10 to 100 percent acetonitrile
1n 30 min at a flow rate of 2.0 ml/min.
C * 50 percent acetonitrile in water at a flow rate of 2.0 ml/min.
D « 35 percent methanol 1n water at a flow rate of 2.0 ml/min.
Column: u Bondapak Cia (10 urn) packed in a 30 cm long x 4 mm I.D. stainless
steel column, with a Whatmann Co. PELL ODS (30-38 urn) guard column, 7 cm
long x 4 mm I.D.
-------
TABLE 2. SINGLE-OPERATOR
Sample Spike
Parameter Type* (ug/1)
Fl uorometuron
Propoxur
Oxamyl
Me thorny!
Diuron
4
Linuron
•
Carbofuran
Barban
Carbaryl
Chlorpropham
Methiocarb
Mexacarbate
Monuron
Neburon
Propham
====================
* » Sample type
1 * Reagent water
1
2
4
1
3
4
5
1
2
2
1
3
2
2
1
*
2
2
5
1
2
2
2
5
1
4
5
5
5
5
5
c
5
5
50
50
1724
550
2200
550
0.5
100
53
1080
100
30660
100
1960
10
SCO
10
400
0.05
10
1000
10
210
0.05
37
148
0.3
0.1
0.2
0.2
4.0
0.05
0.05
0.3
ACCURACY AND PRECISION
=======================
Average
No. of Percent
Analyses Recovery
7
7
7
7
3
7
5
7
7
7
4
4
7
7
4
4
7
7
5
4
d
7
7
5
7
7
5
5
5
5
5
5
5
5
93 9
80.0
99
94.5
105
87.2
93
87
84.9
89.8
74.4
48.2
91.8
94.4
89.8
<6.1
90.6
35.7
98"
95.0
?2.2
93.0
103
99
87.8
99.3
98
101
95
95
96
97
96
88
= = = = = === = ===== = = = = =.= = = = = = = = = = = = = = = =====s = :s = =
1
Standard
Deviation
(I)
7.0
7.2
11.6
1.7
3.0
7.3
6.0
8.4
5.5
2.7
2.4
2.8
2.8
1.9
1.0
5.0
2.5
3.2
4.7
3.4
5.1
1.5
4.6 '
4.7
2.7
1.4
4.1
4.1
3.9
2.6
3.5
1.7
6.6
5.9
==============
2 * Municipal wastewater
3 = Industrial process water, pesticide
4 = Industrial wastewater,
5 - 31ver yatsr
manufacturing
pesticide manufacturing
III-270
-------
2 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 opti-
mum 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.
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.
It is necessary to exchange the extract solvent to hexane if the
Florisil cleanup procedure is to be used. For direct HPLC analysis,
the extract solvent must be exchanged to a solvent (either methanol
or acetonitrile) that is compatible with the mobile phase. The
analyst should only exchange a portion of the extract to the HPLC
solvent if there is a possibility that cleanup may be necessary.
Pass a measured fraction or all of the combined extract through a
drying column containing about 10 cm of anhydrous sodium sulfate
and collect the extract in a 500-mi rouna-oottom fiasK. rvinse :ne
. Er'enmeyer flask and column with 20 to 30 ml of methylene chloride
to complete the quantitative transfer.
Attach the 500-ml round-bottom flask containing the extract to the
rotary evaporator ana parviai'y immerse ,n che 50°C *aisr jawh.
Concentrate the extract to approximately 5 ml in the rotary evap-
orator at a temperature of 50°C. Other concentration techniques
may oe used if che accuracy and precision requirement in the
Quality Control Subsection are met.
Add 50 ml of hexane, methanol, or acetonitrile to the round-bottom
flask and concentrate the solvent extract as before. When the
apparent volume of liquid reaches approximately 5 ml, remove the
500-ml round-bottom flask from the rotary evaporator and transfer
the concentrated extract to a 10-ml volumetric flask. Wash the
round-bottom flask with 2 ml of solvent and add the wasmngs to
the extract. Dilute the extract to volume with solvent.
Stopper the volumetric flask and store it at 4°C if further pro-
cessing will not be performed immediately. If the extracts will
be stored longer than 2 days, they should be transferred to
TFE-f1uorocarbon-sealed screw-cap bottles.
Determine the original sample volume by refilling the sample bot-
tle to the mane ana transrerring tne water ;o d *000-ini ^r
cylinder. °ecord the •samole volume to the nearest 5 ml.
III-271
-------
2.1.6 Cleanup and Separation
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 industrial and municipal
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 for the cleanup procedure is no less than 85 percent.
The following Florisi 1-column cleanup procedure has been demon-
strated to be applicable to the five pesticides listed in Table 3.
It should also be applicable to the cleanup of extracts for the
other carbamate and urea pesticides listed in paragraph 2.1.3.
Add a weight of Florisil (nominally 20 g), predetermined by
lauric acid calibration (see below), to a chromatographic column.
Settle the Florisil by tapping the column. Add anhydrous 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
chromatography column. Discard the eluate.
Adjust the sample extract volume to 10 mi with nexane ana transfer
it from the volumetric flask to the Florisil column. Rinse the
flask twice with 1 to 2 ml hexane, adding each rinse to tne column.
Drain the column until the sodium sulfate layer is nearly exposed.
tlute tne column wren £00 .ni of ^cnyi etner/hexane ^20/50 .n]
(Fraction 1) using a drip rate of ca. 5 ml/minute. Place a 500-rnl
round bottom flask under the column. Elute the column again with
200 ml of acetone/hexane (6/94 v/v) (Fraction 2) into a second
flask. Perform a third elution using acetone/hexane (15/85 v/v)
(Fraction 3), and a final elution with 200 ml of acetone/hexane
(50/50 v/v) (Fraction 4), into separate flasks. The elution
patterns for five of the pesticides are shown in Table 3. Con-
centrate the eluates to 10 ml with a rotary evaporator, exchanging
the solvent to acetonitrile or methanol as required.
Florisil from.different batches or sources may vary in adsorptive
capacity. To standardize the amount of Flonsil tnat is used, the
use of the lauric acid value is suggested. With this procedure5,
the adsorption from hexane solution of lauric acid, in mg per g of
Florisil is determined. The amount of Florisil to be used for
each column is calculated by dividing this factor into 110 and
multiplying by 20.g. 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 inter-
ference from the reagents.
III-272
-------
TABLE 3. RECOVERY FROM COLUMN CLEANUP PROCEDURE
Percent Recovery by Fraction
Parameter No. 1 No. 2 No. 3 No. 4
Diuron
Linuron
Met homy!
Oxamyl
Propachlor
Florisil eluate compos i ton by
0
0
0
0
0
fraction
0
13
0
0
94
24
82
0
92
0
58
0
84
0
0
Fraction 1 - 200 ml of 20 percent ethyl ether in hexane
Fraction 2 - 200 ml of 6 percent acetone in hexane
Fraction 3 - 200 ml of 15 percent acetone in hexane
Fraction 4 - 200 ml of 50 percent acetone in hexane
2.1.7 Sample Analysis by HPLC
In Table 1, the recommended operating conditions for the liquid
cnrcmaiograpn are jummar'zed. Included in tm's taole ire estimated
-etsntion times and method detection limits that can be achieved by
chis metnoa. An example of the 3epar2tions achieved by *his column
is shown in Figure 1. Other HPLC columns, chromatographic condi-
tions, or detectors may be used if the quality control requirements
Calibrate the system daily as described below. The standards and
extracts must be dissolved in a solvent (acetonitrile or methanol)
compatible with the mobile ohase.
If the internal standard approach is being used, add the internal
standard to sample extracts immediately before injection into the
instrument. Mix thoroughly.
Inject 10 yl of the sample extract. Record the volume of the
extract injected to the nearest 0.05 vl , and the resulting peak
size in area or peak height units.
The width of the retention time window used to make identifications
should be based upon measurements of actual retention time varia-
tions of standards over the course of a day. Three times the
standard deviation of a retention time can be used to calculate a
suggested window size for a compound. However, the experience of
the analyst should weigh heavily in the interpretation of chro-
matograms.
If the response for the peak exceeds the working range of the
system, dilute cne dxtracz ana raanalyre.
iiI-273
-------
0 6 10 15 20
Retention Time (minutes)
c*g'jre l. Mould rthromatoaram of urea herbicides on Column 1. Compounds are:
1) methomy!; 2) diuron; 3) linuron. r'or conditions, see 7dDie A.
III-274
-------
If the measurement of the peak response 1s prevented by the presence
of Interferences, further cleanup 1s required. Calculate results
according to Subsection J.
2.1.8 Confirmation of Residues
When this method Is used to analyze unfamiliar samples for any or
all of the compounds to which it is applicable, compound identi-
fication should be supported by at least one additional qualitative
technique, such as GC/MS.
A similar procedure for the determination of phenylureas 1n water
was published by Farrington et a!.6 The sample 1s extracted with
methylene chloride, and determination is by HPLC on a 5-um Cjg
reversed phase column developed with methanol/0.6 percent aqueous
ammonia (60/40 v/v), with UV detection at 240 nm. Recoveries in
excess of 95 percent and detection limits of 0.01 ug/ml were
reported for residues of chlorbromuron, chloroxuron, diuron,
linuron, metobromuron, monolinuron and monuron.
III-275
-------
3.1 Determination of Carbamate Pesticides in Soil
Analytical Procedure: available
Sample Preparation: available
3.1.1 Reference
Hall, R. C. and D. E. Harris, "Direct Gas Chromatographic
Determination of Carbamate Pesticides Using Carbowax 20M-
Modified Supports and the Electrolytic Conductivity Detector."
0. Chromatogr. 169:245(1979).8
3.1.2 Method Summary
A sample is extracted with acetone/aqueous sodium chloride/
aqueous sodium hydrogen carbonate solution and the extract is
partitioned with benzene. The solution is concentrated,
cleaned up on a deactivated Florisil column, and the pesti-
cides in the column eluate are determined by GC using a
Carbowax 20M column and Hall electrolytic conductivity
detector.
3.1.3 Applicability
The 22 carbamate pesticides shown in Table 4 were determined
.at 0.1 and 1.0 ppm levels.
3.1.4 Precision and Accuracy
As seen in Table 4, recoveries at ooth concentration ;eveis
ranged from 66 to 112 percent with an average of 39 percent,
excluding 2-chloroallyl diethyldithiocarbamate (CDEC) and
carbaryl at 0.1 ppm. The former compound was recovered to the
extent of 33 percent, and the latter could not be determined
because of low response. Standard deviations for three trials
ranged from 0 to 20 percent, with an average of 5 percent.
3.1.5 Sample Preparation
Air dry a 50-g soil sample and pass it through a 20-mesh sieve.
Thoroughly mix the soil and extract with 100 ml of extraction
solution on a wrist-action shaker for 10 minutes. The extraction
solution is acetone/aqueous 2 percent sodium chloride + 1 percent
sodium hydrogen carbonate (80/20 v/v). Filter the extract through
Whatman No. 1 filter paper, and rinse the filter cake with 100 ml
of extraction solution. Dilute the filtrate with an additional
100 ml of extraction solution and extract three times with 50 ml
of benzene. Combine the extracts and dry on a sodium sulfate
column (40g). Elute any residual pesticide with an additional
25 ml of benzene. Add 1 ml of a 2-oercent tetradecane solution
in benzene to the dried extract 10 serve as a keeper". Evaporate
the 3xtr;ict *.o ca. 15 ml on 3 '•otary avaoorator, To determine
recovery with this method, fortify samples at concentrations
IIT-276
-------
TABLE 4.
RECOVERY OF CARBAMATE PESTICIDES
FROM FORTIFIED SOIL
Compounds
Recovery {%)
0.1 pm 1.0 ppm
Aminocarb
Benthiocarb
Butyl ate
Bux
Carbaryl
Carbofuran
CDEC
Chlorpropham
Dimetilan
EPTC
2,3,5-Landrin
3,4,5-Landrin
Meobal
Methiocarb
Mexacarbate
Pebulate
Propham
Pyramate
SWEP
Terbutcl
Triallate
Vernolate
85 ± 7*
82 ± 20
95 ± 3
88 ± 6
— **
85 ± 4
92 ± 5
86 ± 2
99 ± 5
85 ± 0
92 ± 4
33 ± 5
92 ± 9
83 ± 3
75 ± 5
112 i 8
96 ± 8
87 ± 2
84 ± 3
86 ± 7
105 ± 7
101 * i
99 ± 5
85 i 8
QO t 6
92 ± 9
87 ± 2
saaaasaassaa:
86 ± 5
66 ± 3
80 ± 5
71 ± 2
87 ± 3
96 ± 4
98 ± 4
92 ± 5
—
85 ± 1
94 ± 3
04 ^ a
92 ± 5
82 ± 6
83 t 6
75 ± 6
90 ± 4
:aaaaas=aa=a=
* Standard deviations for three detenrnnations.
** Determination precluded by insufficient reponse.
of 0.1 and/or 1.0 ppm by addition of 2 ml of a benzene solution
of the pesticides.
Transfer the solution quantitatively to a ?5-ml Kuderna-Oanish
concentration tube, and reduce the volume to 0.75-1.0 ml on a
rotary evaporator. Transfer the concentrated sample quanti-
tatively to a 200-mm x 9-mm column containing 1.5 g of Florisil
(that has been deactivated by adding 10 percent water) and a top
layer of 1 gram of sodium sulfate. Elute with 25 ml of diethyl
ether/benzene (25/75 v/v).
3.1.6 Sample Analysis
Concentrate the column eluate to an appropriate volume (e.g.,
2.5 mi for 0.1 ppm of pesticide) and 'nject ca. S ul onto +.he
III-277
-------
GC column. Compare sample chromatograms with those of standards
obtained under Identical chromatographic conditions. Represent-
ative chromatograms of soil extracts on the 3-percent OY-101/
Carbowax 20 M column are shown in Figures 2 and 3. Extraneous
peaks were not observed in the 1-ppm samples, but impurity peaks
were present at 0.1 ppm. These impurities interfered somewhat with
the determination of butyl ate, pebulate, EPIC, mexacarbate, and
vernolate on the OV-101 column. Use of one of the alternative
columns or alternate temperature programs might improve analysis
of these pesticides. Calculate results acording to Subsection J.
3.1.7 Confirmation of Residues
Residues can be confirmed by GC/CI-MS under the following
conditions:
A Finnigan Model 3200 gas chromatograph-mass spectrometer
equipped with a chemical-ionization source and a Model 6100
data system is used with isobutane as the reaction gas.
Silanized glass columns (1.5 m x 2 mm I.D.) are operated with
isobutane as the carrier gas. The carrier gas also serves as
the reaction gas. Source pressure is maintained at 550 urn.
Column, source, transfer and separator temperatures are 170°,
60*, 190*; ?nd ?2Q°, •"esDectivelv. The electron energy is 82
eV, and the emission current is *.Ji ..TA.
III-278
-------
>
-s <
« I
E I
(a)
-All
(b)
I
0 I 4 0 5 4
Retention Time (minutes)
Figure 2. Chromatograms of soil extract of carbamate pesticides separated
on a 3-percent OY-101 on Ultra-Bond column. Compounds are:
1) pebulate, 2) 2,3,5-landrin, 3) 3,4,5-Landrin, 4) aminocarb
5) benthiocarb. a) 10rng standard; b) extract of fortified soil.
Operating conditions: Helium at 25 ml/min is the carrier gas and
hydrogen at 30 -nl/rain *s the ^action qas. The temperatures are:
170*C, column; 180*C, Inlet; 200"C, transrer tine; ema /20*C, rurnace.
111-279
-------
1.CH
2
(b)
024 024
Retention Time (minutes)
Figure 3. Chromatograms of soil extract of carbamate pesticides separated
on a 3-percent OY-101 on Ultra-Bond column. Compounds are:
1) propham, 2) pyramate, 3) mexacarbate.
*) 10-nq standard: b) extract of fortified soil.
Operating conditions ire -nven under F
III-280
-------
3.2 Determination of Urea Herbicides in Soil
Analytical Procedure: available
Sample Preparation: available
3.2.1 Reference
Farrington, D. S., R. G. Hopkins, and 0. H. A. Ruzicka,
"Determination of Residues of Substituted Phenylurea Herbicides
in Grain, Soil, and River Water by Use of Liquid Chromatography."
Analyst 102:377 (1977).6
3.2.2 Method Summary
The phenylurea herbicides are extracted from soil with meth-
anol and are determined by HPLC on microparticulate Cjg-banded
silica using a mixture of methanol, water, and ammonia as the
mobile phase, and a UV absorption detector.
3.2.3 Applicability
Residues of chlorbromuron, chlortoluron, chloroxuron, diuron,
linuron, metobromuron, monolinuron, and monuron were determined
{n soil 2t a-concentration of 2 rag/kg fpom). The lower limit.
of detection was estimated co oe Q.I ppm.
3.2.4 Precision and Accuracy
The recoveries obtained with the method are shown in Table 5.
The precision or cne .netncci ,i ^ncr.cn'cc-a -,y ;r,e -inges :f-
recovery for five determinations of aach compound. The
.recovery for all compounds averaged 99.8 percent.
3.2.5 Sample Preparation
Air dry a soil sample and transfer 50 g into a 500-ml
Erlenmeyer flask. Add 100 ml of methanol and shake on a
wrist-action shaker for 1 hour. Filter the resulting slurry
through Whatman No. 1 filter paper at reduced pressure. Wash
the flask with 50 ml of methanol and add the washings to the
filter funnel, leave for 4 minutes, and then apply vacuum.
Repeat the wasning procedure with another JO ,m of aietnanoi.
Combine the extract and washings and remove the methanol on
a rotary evaporator with a water bath at 55°C. Dissolve the
residue in methylene chloride, using a total volume of 50 ml,
and pass the methylene chloride extract through a column of
anhydrous sodium sulfate (50 g). Wash the sodium sulfate with
50 ml of methylene chloride, combine the extract and washings,
and evaporate to dryness at 55°C in a rotary evaporator.
Cooi the flasK ana dda £.0 ,nl of rnethanol, jwirl *o dissolve
the residue, and filter through Whatman No. 42 filter paper.
TII-281
-------
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-------
o
1
JD
<
0.01
unit
0.01
unit
(a)
0 10 20 30 40 0 10 20 30
Retention Time (minutes)
40
Figure 4. Typical chromatograms obtained from 5-iil Injections of son
extracts. Compounds are.: 1) monuron, 2) monolinuron, 3) metobromuron,
4) chlortoluron, 5) diuron, 6) Unuron, 7) chlorbromuron, and
8) chloroxuron. (a) unfortified; and (bj fortified with
\iron herbicides ».t ? mq kq-1.
Permaphase ODS column, 240 nm detection wavelength.
III-283
-------
4.1 Determination of Carbamate Pesticides in Fruits and Vegetables
Analytical Procedure: evaluated
Sample Preparation: available
4.1.1 References
Krause, R. T., "Multiresidue Method for Determining
N-Methylcarbamate Insecticides in Crops, Using High Perfor-
mance Liquid Chromatography." J. Assoc. Off. Anal. Chem.
63(5):1114 (1980).13
Krause, R. T. and M. August, "Applicability of a Carbamate
Insecticide Multiresidue Method of Determining Additional
Types of Pesticides in Fruits and Vegetables." J. Assoc.
Off. Anal. Chem. 66(2):234 (1980)."
4.1.2 Method Summary
Residues are extracted from crops using methanol. Coextrac-
tants are removed by solvent partitioning and Chromatography.
The carbamate residues are separated on a reversed-phase HPLC
column eluted witn an acetonitrile/water gradient and detected
using an in-line post-column fluorometric labeling technique.
4.1.3 ApplicaDility
The method is applicaole to the seven caroamates and four
related carbamate. metabolites shown in Table 6, at concentra-
tion levels of 0.05 ppm or greater. ' The method was recently
sxtsnaea tcj induce pesticiues or otner clas3C-!: and -'afyir.rj
polarity.1'*
4.1.4 Precision and Accuracy
Except for aldicarb sulfoxide, pesticide recovery from forti-
fied samples averaged 99 percent at both 0.050 and 1.0 ppm,
with standard deviations of 5.0 percent (n = 86) and 5.6
percent (n = 87), respectively. The average recoveries for
the polar metabolite aldicarb sulfoxide were 55 and 57 at the
respective concentration levels (Tables 6 and 7). An inter-
laboratory study of the method was conducted (Table 8). The
average recovery was 95 percent irange 37-101 percent) for
duplicate analyses of grapes and potatoes fortified with
carbaryl, methiocarb, and methomyl at levels ranging from
0.050-15 ppm.
4.1.5 Sample Extraction.
Select the appropriate method of sample extraction based on
the water content of the original sample. For high-moisture-
content .samples roi'iow paragraph i and "or "*ow-Tioisture-
content sameles follow oaragraph b.
III-284
-------
TABLE 6. CARBAMATE INSECTICIOE AND Mi TABOLIT£ i;LCOVERIES (PERCENT) THROUGH METHOD
AT 0.05 PPM iORTIFICATlON LEVEL3
(rop
Appl es
Carrots
Green beans
Green peppers
Lettuce
Oranges
Soybeans
£ Strawberries
VTomutoes
00
^Av.
SO
Grand av.
Grand SO
Ald1-
carb
100
100
99
96
100
100
102
97
94
99
2.5
99e
5.0e
Aldi-
carb
sulfone
91
98
101
96
108
94
97
6.5
Oxamyl
94
93
96
93
97
92
95
98
91
94
2.4
a ^.Inijle determinations.
b Not jsed 1n
c Interference
av. or
due to
SD.
apparent
methomyl
1n crop
d Apparent crop coextractlve Interference.
e Aldicarb sul
foxide
not Included.
-------
TABLE 7. CARBAMATE INSECTICIDE AND METABOLITE KECOVERIES (PERCENT) THROUGH METHOD
AT 1.0 PPM lORTIFICATiON LEVEL*
1-4
1— 1
1
ro
ro
Aldi-
Crop carb
Apples 95
Carrots 100
Green beans 94
Green peppers 97
Lettuce 92
Onngt.s 98
So /beans 108
Strawberries 105
Tomatoes 97
Av. 98
SD 5.2
Grdmd av. 99e
Grand SD 5.6«
==-=======
Aldi-
carb
sul f one
91
97
92
98
99
85
95
107
96
96
5.7
Aldi-
carb
sulf-
oxide
54
62
53
63
68
45
50
62
56
57
7.3
Bufen-
carb
93
99
102
97
99
98
105
109
98
100
4.7
Carb-
o-
fUi an
y'j
102
101
;)9
11)1
97
106
lil
100
Iu2
4.7
3-Hy-
droxy
Carbo-
furan
95
100
101
100
90
102
107
108
98
102
5.6
Methio-
carb
96
102
97
98
97
d
108
106
98
100
4.6
=========
Methio-
carb
sulf-
oxide
104
97
97
101
105
89
94
110
98
99
6.3
Meth-
omyl
92
99
b
102
100
85
95
106
95
97
6.5
Oxamyl
93
92
90
94
76C
89
95
103
94
94
4.3
a Si.igle determinations.
" Interference due to
c Nol used in av. or
apparent
sn.
methomyl
in ci
0,,.
d Apparent crop coextractive interference.
e Aldicarb sul f oxide
not included.
-------
TABLE 8. INTERLABORATORY COMPARISON OF HPLC METHOD FOR CARBAMATES
===============================================================================
Grapes Potatoes
Percent
Recovery
Carbamate/
metabolite
Carbaryl
Carbofuran
3-Hydroxy
carbofuran
Methiocarb
Met homy!
=====8=================
3 Interference present
"rfded,
ppm
8.0
1.0
0.05
0.05
0.05
=============
•
1
94
87
93
94
87
2
96
94
96
95
93
Added,
ppm
0.05
0.05
1.0
5.0
15.0
Percent
Recovery
1
a
101
99
99
94
=======
2
a
96
95
93
96
========
a. High-Moisture Products (greater than 75 percent water) - Add
150 g of chopped sample and 300 ml of .nethanol to a homcgenirer jar
and homogenize for 30 seconds at one-half speed (Polytron setting
7) and cnen for oO ^ecanas ^t ~ur ;peed. '-'acuu^-'ilter -.he
hcmogenate through 3 12-cm nerforsted Buchner funnel containing
SharKSKin or 597 535 filter paper, jollacting the filtrate 'i \
500-ml filter flask. Transfer a portion of filtrate equivalent to
100 g sample to a 2-liter I 24/40 round-bottom flask.
NOTE: Volume of 100 g sample equals mi water 'n 100 g samole, plus
200 ml methanol, minus 10 ml contraction factor.
Add ultrapure water to the round-bottom flask to give a total of
100 ml of water.
Proceed to paragraph 4.1.6.
b. Dry or Low Moisture Products (e.g., grains) - Add 75 g of
ground sample and 300 ml of methanol to a homogenizer jar. Homog-
enize and filter as above in (a). Transfer a portion of the
filtrate equivalent :o 50 9 of jampla ;o a 2-t'ter 5 24/*0 round-
bottom flask.
NOTE: Volume of 50 g sample equals ml of water in 50 g sample plus
200 ml of methanol.
Add ultrapure water to the round-bottom flask to give a total of
100 ml of water. Add a small star magnetic stirrer to the flask.
.'lacs a 250-inl ~ tfr/~Q trap en the "-liter -ryjnd-bottom *lask and
attach to a rotary evaporator. Apply vacuum slowly to minimize
III-287
-------
frothing. After full vacuum is applied, slowly place the flask
in a 35°C water bath. Concentrate the extract to 75 ml.
Proceed to Subsection 4.1.6.
4.1.6 Cleanup of Extract by Solvent Partitioning
Transfer the concentrated extract from the round-bottom flask
to a 500-ml separatory funnel containing 15 g of sodium chloride.
Shake the funnel until the salt is dissolved. Wash the flask
with three 25-ml portions of acetonitrile, transferring each
to the separatory funnel, shake 30 seconds, and let the layers
separate for 5 minutes. Drain the aqueous phase into a 250-ml
separatory funnel containing 50 ml of acetonitrile. Shake 20
seconds, let the layers separate, and discard the aqueous
layer.
Add 25 ml of 20-percent aqueous sodium chloride solution to
the acetonitrile in the 500-ml separatory funnel, shake 20
seconds, let the layers separate, and transfer the .solution
to the 250-ml separatory funnel. Shake this funnel for 20
seconds, let the layers separate, and discard the aqueous
layers.
Add 100 ml of petroleum ether to the 500-ml separatory
funnel; shake 20 seconds, ''at *.fte Bayers separate, ;md oral n
the.acetonitrile layer into a second 500-ml separatory funnel.
Transfer the acetonitrile in the 250-mV funnel to the first
^Or-Til ^'jr.ns! *hat tontairs rstr^leum ether-, shake 20 seconds.
let the layers separate, ana transfer the acetomtrile to zne
second 500-ml separatory funnel. Add 10 ml of acetonitrile
to the first funnel; shake, let the layers separate, and trans-
fer the acetonitrile to the second 500-ml funnel. Discard the
petroleum ether.
Add 50 ml of 2-percent aqueous sodium chloride solution to the
acetonitrile in the second 500-ml separatory funnel. Extract
the mixture successively with 100-, 25-, and 25-ml portions of
methylene chloride, shaking each mixture for 20 seconds (shake
the 25 ml portions gently). Drain the lower methylene chloride/
ac3toni*ri1-3 "* ayers through a 22-mrn T.D. column containing
ca. 5 cm of anhydrous granular sodium sulfate. Collect the
eluate in a 1-liter 5 24/40 round-bottom flask and evaporate the
solution to dryness with a rotary evaporator as described
earlier. Remove the flask from the evaporator immediately after
the last traces of solution have evaporated and then add 10 ml of
methylene chloride to the flask.
4.1.7 Chromatographic Cleanup
Fit a 1-hole No. 5 ruboer stopper into the tip of & cnromatog-
r-aphic tube dth "an'bor stopcock, idd "5 ? ?4/^0 side arm
III-288
-------
vacuum adaptor and 500 ml f 24/40 round-bottom flask, open the
stopcock, and connect the apparatus to a vacuum line. Place
0.5 g of silanized Celite 545 in the tube, tamp, add 5 g of
Nuchar S-N/silanized Celite 545 (1/4) mixture, and tamp again.
Place a 1 to 2 cm glass wool plug on top of the adsorbent.
Prewash the column with 50 ml of toluene/acetonitrile (1:3,
v/v), closing the stopcock when the solution is ca. 5 mm from
the top of the glass wool. Disconnect the vacuum, discard the
eluate in the round-bottom flask, and reconnect the flask.
Transfer the solution from paragraph 4.1.6 in 10 ml methylene
chloride to the column and elute at 5 ml/min. Wash the 1-liter
round-bottom flask with 10 ml of methylene chloride and then with
25 ml of the eluting solution. Transfer each separately to the
column and elute each to the top of the glass wool before
adding the next solution. Add 100 ml of eluting solution to
the column and elute at 5 ml/min. Turn off the stopcock when
the top of the eluting solution reaches the top of the glass
wool.
Evaporate the solution in the 500-ml round-bottom -Mask just
to dryness on the vacuum evaporator as before. Remove the
"""asx ~rom :r,s evaporator :nmealately -ntar ~Ti :he ""auid has
evaporated. Immediately pipet 5 ml of methanol into tne flask
to dissolve the ^asidue. Pour the metnanol into a 10~-ni alass
'syringe containing a Swinny filter holder with a 5-pm filter.
Push the methanol solution through the filter with the plunger,
•rT! acting the 'Citrate n'n a.lO-ml centrifuqe tube or other
suitable container. Approximately 4.5 .711 or fi"crate JHOUIO
be recovered. If the solution requires dilution, piper an
aliquot into another container and dilute to volun.e as needed.
4.1.8 Sample Analysis
Inject 10 vl of the methanol sample solution onto the HPLC col-
umn using the following parameters: Adjust the mobile phase
flow rate to 1.50 ± 0.02 ml/minute at acetonitrile/water (1/1
v/v). Equilibrate the system at acetonitrile/water (12/88
v/v) for 10 minutes, inject the sample, and begin a 30-minute
linear gradient to acetonitrile/water (70/30 v/v), Adjust the
flow rate of the 0.05 M sodium hydroxide and reaction solution
to 0.050 ± 0.02 ml/minute each. Operate the column oven at 35°C
and the hydrolysis chamber at 100°C. Set the fluorescence
detector excitation and emission wavelengths to 340 and 455
nm, respectively, with slit widths 15 and 12 nm, respectively.
Set the detector gain to "low" and time constant to 1 second.
Adjust sensitivity so that 1 ng of carbofuran gives 50 percent
full scale deflection on the printer plotter set at attenu-
ation •:.
III-289
-------
NOTE: If the system will not be used for several days, re-
place water mobile phase with methanol and pump through the
system, drain sodium hydroxide and reaction solutions from
reservoirs, and wash reservoirs and associated tubing first
with water and then methanol. When starting up the system,
change methanol mobile phase to water, and wash reaction
reservoirs and associated tubing with water before adding
reaction solutions.
Tentatively identify residue peaks based on retention times
(Table 9). Measure peak areas or heights and determine
residue amounts by comparison to areas or heights obtained
from known amounts of appropriate reference material(s).
Calculate results according to Subsection J. To ensure valid
measurement of residue amounts, sample and standard peaks
should aqree within ±25 percent. Chromatograph reference
material is) Immediately after samples. Figure 5 Illustrates
the separation of the carbamates by HPLC.
TABLE 9. RETENTION DATA FOR 7 CARBAMATES AND * CARBAMATE METABOLITES,
USING ZORBAX C-8 HPLC COLUMN
Retention time relative
Carbamate/metabol ite y.s -arfrofuran
Aldicarb sulfoxide 0.33
^l-Hcarb sulfone . 0.40
Oxamyl J.-+4
Methorny! 0.46
3-Hydroxy carbofuran 0.60
Methiocarb sulfoxide 0.64
Aldicarb 0.83
Carbofuran 1.00
Carbaryl 1.06
Methiocarb 1.26
Bufencarba 1.44b
====================3=3==3=3===================================================
aMixture of 1-methyl butyl and 1-ethylpropyl phenyl N-methylcarbamates (3/1)
with 70 percent meta, 20 percent para, and 4 percent ortho isomers.
bMajor peak.
4.1.9 Confirmation of Residues
An HPLC procedure reported by Lawrence1^ can serve to confirm
the identity and amount of carbamate residues in food crops
at 0.1 +o 0.3 nrm. The carbamates are extracted from samoles
with acetone and then partwonea into nexane/metnyiene
III-290
-------
10 15 20
Retention Time (minutes)
25
30
Figure 5. Chromatogram of carbamates and carbamate metabolites at 10 ng,
using Zorbax C-8 column. Compounds are: 1) aldicarb sulfoxide;
?) ildicarb «ulfone; 3) rsxamyl: 4^ niethomyl: 5) 3-hvdroxy carbofuran;
6) methiocarb sulfoxide; 7) alaicaro; 3} w'aroofuran; 3} carbaryi,
'.Q) -^ethiocarb: M) bufencarb.
111-291
-------
chloride/water. The organic extract is cleaned up by Florisil
chromatography (acetone/hexane, 15/85 v/v eluent) and the
eluate analyzed by HPLC with a silica gel column and UV absorp-
tion detector (254 nm). The use of a different cleanup column,
different mode of separation (adsorption rather than reversed
phase), and a different type of detector assures that the re-
sults will be truly independent from those from the primary
method, a requirement for reliable confirmation.
A similar HPLC method for urea herbicides in vegetables was
also reported by Lawrence.16 Samples are extracted with acetone,
and the filtrate is partitioned with hexane/methylene chloride.
The organic phase is dried and concentrated for Florisil
column chromatography using acetone/hexane (15/85 and 50/50 v/v)
to elute the areas. The column fractions are evaporated to
dryness and redissolved in isooctane for HPLC on a silica gel
column with isopropanol/isooctane (20/80 or 15/85 v/v) mobile
phase. The compounds are detected by UV absorption at 254 nm.
Linuron, monuron, diuron, chlorbromuron, fluometuron, chloro-
xuron, and fenuron were determined in cabbage, corn, potato,
turnip, and wheat at 0.01-1.0 ppm with recoveries greater than
80 percent in most cases.
III-292
-------
5.1 Determination of Carbamate Pesticides in Air
Analytical Procedure: available
Sample Preparation: available
5.1.1 Reference
Sherma, J. and M. Beroza, "Manual of Analytical Methods for
the Analysis of Pesticides in Humans and Environmental
Samples." EPA-600/8-80-038, Sections 8A and 8B (June, 1980).9
5.1.2 Method Summary
The sampling medium, such as polyurethane foam or composite
filter pad, is Soxhlet-extracted with hexane/diethyl ether
(95/5 v/v). Carbamate pesticides are measured by direct
GC with an N-selective electrolytic conductivity detector
or by electron capture GC after chemical derivatization
with alpha-bromo-2,3,4,5,6-pentafluorotoluene.
5.1.3 Applicabiity
The method is suitable for quantifying carbamate pesticides
•:i Ambient air at trace ''svels 'nq/r.3 '.nd -q/nv^. The anal-
ytical scneme presupposes collection OT samples oy use of
30!yursthane foam oiugs or Tenax-oC adsorbent.^
5.1.4 Precision and Accuracy
almost no rsports on tr.e coi'idcv^n =f-:u:3rcy ~T .aroamata
pesticides in air nave oeen puoiisned. A glass-fiber filter
was found to have a collection efficiency of 100 percent for
carbaryl at levels of 2 to 13 mg/m3 of air.iri
Fenitrothion and aminocarb were collected on Tenax-GC adsor-
bentH, and fourteen urea, carbamate, and thiocarbamate
pesticides on acetylcellulose or perch!orovinyl filter papers
(aerosols) and on silica gel or glass wool or in traps con-
taining acetone (vapors).12 The accuracy and precision of the
method will depend mainly upon the collection efficiency of the
sampling medium *or the pesticide(s) of interest.
5.1.5 Extraction of the Sampling Module
Place the sampling medium in a Soxhlet extractor, handling
with forceps rather than hands.
NOTE: After sampling, the glass-fiber filters and foam plugs
should have been wrapped in aluminum foil until analysis.
Use oluqs and filters carried to the field along with those
employed for sampling as controls.
-------
Extract with an appropriate volume of _n-hexane/acetone/
diethyl ether (47/47/6 v/v) for 16 to 24 hours at 4 cycles per
hour for the large Soxhlets and 8 to 12 hours at 8 cycles per
hour for the smaller Soxhlets.
NOTE: As examples, extract large foam plugs in 1000 ml Soxhlet
extractors with a total of 300 to 750 ml of solvent, and smaller
plugs and filters in 500-ml Soxhlets with 200 to 350 ml.
Attach the boiling flask to a rotary evaporator and reduce
the solvent volume to approximately 5 ml.
Transfer the concentrate to a 15-ml graduated centrifuge
tube. Rinse the boiling flask with solvent and add rinsing to
concentrate.
Adjust the final volume in the centrifuge tube as required.
5.1.6 Sample Analysis
Inject yl of extract into the gas chromatograph containing a
3-percent OV-101/Ultra Bond 20M. column and alectrolytic conduc-
tivity detector. Compare the relative retention time for each
•:cmponent of :ntsr*st against '•.hose of the ccr*-?SDonding orimary
standard. Quantify peaks in the usual way, i.e., oy measuring
peak heights to tne learest
-------
Transfer the hydrolysis solution, by washing with 50 to 60 ml
of distilled water, into a 500-ml separatory funnel and add
50 ml of methylene chloride.
Shake briefly and discard the methylene chloride.
Acidify to pH 2 or less with 0.3 to 0.5 ml of 50-percent
sulfuric acid.
Extract the hydrolysis solution with two 50-ml portions of
benzene, and dry the benzene by suction filtration through a
10-gram sodium sulfate column into a 250-ml round-bottom
flask.
Evaporate the benzene to 1 to 2 ml on a rotary evaporator with
a water bath at 40°C.
Transfer to a 15-ml centrifuge tube by rinsing with 5 to 6 ml
of acetone.
Add 25 ul of 5-percent aqueous potassium carbonate and 100 yl
of 1-percent PFB bromide solution to the centrifuge tube.
Stopper, sna
-------
NOTE: Determine the elution pattern of the PFB ether derivatives
of the carbamates of interest on the silica gel column under local
laboratory conditions. A typical elution pattern is as follows:
PFB ether
derivative
Recovery, Percent8
Fraction I
Fraction II
Fraction III
Propoxur
Carbofuran
3-Ketocarbofuran
Metmercapturon
Carbaryl
Mobam
84-89
97-100
96-99
93-97
94-97
12-15
0-2
0-3
2-5
2-4
96-98
aObtained by comparing the peak areas of 5 samples passed through
the silica gel columns with 3 samples not fractionated.
Concentrate the eluate fractions as needed and analyze by EC-6C on
the 3-percent OV-225 or 3.6-percent CV-1Q1/5.5-percent OV-210
column. Relative retention times of PFB ether derivatives on the
two tslumns ire is *o11ows:
Derivative
RRT
OY-101/OV-210*
RRT
PrQDOXUr
Carbofuran
3-Ketocarbofuran
Metmercapturon
Carbaryl
Mobam
0.43
0.64
1.15
1.26
1.38
1.48
0.41
0.63
1.13
1.28
1.31
1.31
^Relative to aldrin 2.7 minutes
^Relative to aldrin 7.9 minutes
Operating conditions: injector temperature, ?05"C; column, 190°C;
detector, 280*C; 5-percent methane-argon carrier gas flow rate, 50
ml/minute + 20 ml/minute purge for the OV-101/ OV-210 column, 30
ml/minute + 30 ml purge for OV-225; EC detector in pulsed mode with
electrometer settings of 55V, 90 jisec pulse rate,, 8 usec pulse
width, and 6.4 to 1.6 x 10~9 amp full-scale attenuation.
Quantify by comparison of peak areas against chromatograms of
derivatized standard carbamate phenols. The standard derivatives
synthesized as follows:
III-296
-------
React each carbamate phenol with a tenfold molar excess of PFB
bromide in acetone and a tenfold molar excess of methanolic
potassium hydroxide.
Reflux for 2 to 3 hours, cool, and remove the solvent on a
rotary evaporator.
Dissolve the product in benzene and wash the benzene twice
with equal volumes of 0.1 M potassium carbonate.
Dry the benzene, using suction, by passing it through a 10 to
20 gram column of anhydrous sodium sulfate.
Remove the benzene on a rotary evaporator and recrystallize
from hexane or methanol.
Inject 5 ul or another appropriate volume of the sample extract
into the gas chromatograph.
Record chromatograms and measure retention times relative to
aldrin or another suitable reference standard. Calculate results
according to Subsection J.
0. CALCULATIONS
Determine the concentration of individual compounds *n the simole. If
the external standard calibration procedure is usea, calculate the amount of
material injected 'from the peak response using the calibration curve or cali-
bration factor in Subsection H. The concentration in the sample can be
1. Liquid Samples
'A)(Vt>
Concentration, yg/1 =
(VO(VS)
where:
A = Amount of material injected, in ng
V-i * Volume of extract injected in yl
Vt = Volume of total extract in yl
Vs = Volume of water extracted in ml.
If the internal standard calibration procedure is used, calculate the
concentration in the sample using the response factor (RF) determined in
Subsection H.2. as follows:
•(As)ds)
Concentration, yg/1 -
III-297
-------
where:
As
Ais
Is
Response for the parameter to be measured
Response for the internal standard
Amount of internal standard added to each extract in yg
Volume of water extracted, in liters.
2. Solid and Tissue Samples
Concentration, yg/kg (wet weight)
(A)(Vt) 1000
where:
Concentration, yg/kg(dry weight) =
(A)(Vt) 1000
A =
Vt =
3 :
%s =
3. Air Samples
amount of material injected (from Calibration Curve), ng
volume of extract injected, yl
total volume of extract, ul
,-ret -veigni of campie extracted, u
percent solids in sample as a decimal fraction.
Concentration., mg/nP =
where:
A = amount of material injected (from Calibration Curve), ng
V-j = volume of extract injected, yl
Vt = total volume of extract, ul
V = volume of air filtered, m^.
Report results in appropriate units without correction for recovery
data. When duplicate and spiked samples are analyzed, report all data
obtained with the sample results.
For samples processed as part of a set where the laboratory spiked sample
recovery falls outside of the control limits in Subsection G, data
for the affected parameters must be labeled as suspect.
III-298
-------
K. CONFIRMATION
Detailed discussion of general and specific aspects of residue confirm-
ation is available.I? In many cases, particularly where a high concentration of
contaminant is apparent, residues should or must be confirmed. The most gener-
ally accepted method of confirmation of pesticide identity is on two or more GC
columns of different polarity, and/or with different detectors if heteroatoms
are present, or with different derivatives. GC/MS of pesticide residues is one
of the best techniques for confirming identity. TLC confirmation, once used
more extensively prior to the introduction of routine mass spectrometers, may
also be used, as may confirmation by jj-values, polarography, spectrometry (IR,
UV, NMR), or via chemical derivatization. In general, pesticide residues are
confirmed by analysis under different analytical conditions (i.e., different
columns, detectors, derivatives, or methodologies) or by ancillary techniques.
Numerous reports of analytical methods for single residues or small
numbers of compounds have been published recently. Some of these are
listed as references 18 to 30 at the end of this section. Readers may find
these methods useful for determinations of carbamates and related compounds
in samples other than those covered in this section or for confirmation of
results obtained by these methods. References 21 and 24 are especially
pertinent because they provide retention data on various HPLC columns and
detection wavelengths and sensitivities for over 40 carbamate compounds.
III-299
-------
REFERENCES
American Society for Testing and Materials. "Standard Practice for
Preparation of Sample Containers and for "Preservation." American Society
for Testing and Materials Annual Book of Standards, Part 31, D3694.
American Society for Testing and Materials, Philadelphia, Pennsylvania.
p. 679 (1980).
Occupational Safety and Health Administration Safety. "OSHA and Health
Standards, General Industry." (29 CFR 1910), Occupational Safety and
Health Administration, OSHA 2206. (Revised, January 1976).
American Chemical Society. "Safety in Academic Chemical Laboratories."
American Chemical Society Publication, Committee on Chemical Safety, 3rd
edition. (1979).
Press! ey, T. A. and J. E. Longbottom. "The Determination of Carbamate
and Urea Pesticides in Municipal and Industrial Wastewater - Method 632."
Report No. EPA-600/4-82-014 (February, 1982).
American Society for Testing and Materials. "Standardization of Florisil
Column by Weight Adjustment Based on Adsorption of Laurie Acid." ASTM
book of Standards, Part 31, D3086, Appendix X3. American Society for
Testing and Materials, Philaaelpnia, Pennsylvania, p. ^55 J
6. Farrington, D. S. , R. G. Hopkins, ana J. H. A, Ruzicka. "Determination
of Residues of Substituted Phenylurea Herbicides in Grain, Soil, and
River Water by Use of Liquid Chromatography." Analyst _10j!:377 (1977).
7. 'J.3. Environmental Protection Agency. "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 <*5268. 'March. 1979).
8. Hall, R. C. and D. E. Harris. "Direct Gas Chromatographic Determination
of Carbamate Pesticides Using Carbowax 20M-Modified Supports and the
Electrolytic Conductivity Detector." J. Chromatogr. Jj>9:245 (1979).
i
9. Sherma, J. and M. Beroza. "Manual of Analytical Methods for the Analysis
of Pesticides in Humans and Environmental Samples." EPA-600/8-80-038,
Sections 8A ana 3B. (June, 1980).
10. U.S. Department of Health, Education, and Welfare. "Carbaryl". Method
S273, NIOSH Manual of Analytical Methods, 2nd Edition, Volume 3, Center
for Disease Control, National Institute of Occupational Safety and Health,
Cincinnati, Ohio. (April, 1977).
11. Krzymien, M. E. "Measurement of Atmospheric Fenitrothion and Aminocarb
Concentrations Near the Spray Area." Int. J. Environ. Anal. Chem. 13:69
II1-300
-------
12. Aleksandrova, L. G. and M. A. KHsenko. "Identification and Determination
of some Urea, Carbamate, and Thiocarbamate Derivatives in Air." J.
Chromatogr. 247^255 (1982).
13. Krause, R. T. "Multiresidue Method for Determining N-Methylcarbamate
Insecticides in Crops, Using High Performance Liquid Chromatography."
J. Assoc. Off. Anal. Chem. 63(5):1114 (1980).
14. Krause, R. T. and M. August. "Applicability of a Carbamate Insecticide
Multiresidue Method for Determining Additional Types of Pesticides in
Fruits and Vegetables." J. Assoc. Off. Anal. Chem. 66(2):234 (1983).
15, Lawrence, J. F. "Direct Analysis of Some Carbamate Pesticides in Foods by
High Pressure Liquid Chromatography." J. Agr. Food Chem. 25:211 (1977).
16. Lawrence, J. F. "High Pressure Liquid Chromatographic Analysis of Urea
Herbicides in Foods." J. Assoc. Off. Anal. Chem. 59:1066 (1976).
17. Sherma, J. "Manual of Analytical Quality Control for Pesticides in Human
and Environmental Samples." EPA-600/2-81-059 (April, 1981).
13. Kirk!and, •]. J. "Method for High-Speed Liauid Chromatograohic Analysis
of Benomyl and/or Metabolite Residues in Cow rfil'rt, Urine, races, ana
T1ssue£.:1 J. Agr. food Chem. 21_:i7i (1972;.
19. Kirkland, J. J., R. F. Holt, and H. L. Pease. "Determination of Benomyl
Residues *n SoiU and Plant Tissues by High-Speed Cation Exchange Liquid
Chromatograpny. ' J. Agr. Fooa Chem. £l_.~56 ,197C).
20. Ernst, G. F., S. 0. Roder, G. H. Tjan, and J. T. A. Jansen. "Thin Layer
Chromatographic Detection and Indirect Gas Chromatographic Determination
of Three Carbamate Pesticides." J. Assoc. Off. Anal. Chem. 58:1015
(1975). ~~
21. Sparacino, C. M. and J. W. Hines. "High Performance Liquid Chromatography
of Carbamate Pesticides." J. Chromatogr. Sci. ^4_:549 (1976).
22. Lawrence, J. F. "Confirmation of Some Organonitrogen Herbicides and Fun-
gicides by Chemical Derivatization and Gas Chromatography." J. Agr. Food
Chem. 24:1236 (1976).
23. Thean, J. E., W. G. Fong, D. R. Lorenz, and T. L. Stephens. "High Pressure
Liquid Chromatographic Determination of Methomyl and Oxamyl on Vegetable
Crops." J. Assoc. Off. Anal. Chem. 61_:15 (1978).
24. Lawrence, J. F. and D. Turton. "High Performance Liquid Chromatographic
Data for 166 Pesticides." J. Chromatogr. J.59_:207 (1978).
25. Galoux, M., J.-C. Van Damme, A. Semes, and ^. Potvin. 'Gas-Liquid
Chromatographic Determination of Aldicarb, Aldicarb Sulfoxide, and Aldicarb|
Sulfone in Soils and Water Using a Hall tleciroiytic Cpnaucfivity Detec-cr."
J. Chromatogr. 177_:245 (1979).
III-301
-------
26. Blaicher, G., W. Pfannhauser and H. Woidlch. "Problems Encountered with
the Routine Application of HPLC to the Analysis of Carbamate Pesticides."
Chromatographia 13:438 (1980).
27. Wehner, T. A. and J. N. Seiber. "Analysis of N-Methylcarbamate Insecticides
and Related Compounds by Capillary Gas Chromatography." J. High Resolut.
Chromatogr. Chromatogr. Commun. £:348 (1981).
28. Mayer, W. J. and M. S. Greenberg. "Determination of Carbamate Pesticides
by High Performance Liquid Chromatography with Electrochemical Detection."
J. Chromatogr. 208_:295 (1981).
29. Gustafsson, K. H. and R. A. Thompson. "High Pressure Liquid Chromatographic
Determination of Fungicidal Dithiocarbamates." J. Agr. Food Chem. 29:729
(1981).
30. Grou, E., V. Radulescu, and A. Csuma. "Direct Determination of Some
Carbamate Pesticides in Water and Soil by High Performance Liquid
Chromatography." J. Chromatogr. 260:502 (1983).
II1-302
-------
SECTION 7
METHODS FOR THE DETERMINATION OF CHLORINATED PHENOXY ACID HERBICIDES
A. SCOPE
Chlorophenoxyacetic adds such as 2,4-dichlorophenoxyacetic acid (2,4-D),
2,4,5-trichlorophenoxyacetic acid (2,4,5-T), and silvex [2-(2.4,5-trichloro-
phenoxyjpropionic acid] are herbicides used for weed control.1 Each com-
pound may exist as a free acid or an ester. In addition, the ester of each
herbicide is less stable than the free acid and may hydrolyze in aquatic
environments.2
The analytical procedure for these compounds consists of three steps.1»3
Residues are extracted into an organic solvent and esterifisd using boron
trifluoride (BF3). The methyl esters are then extracted into benzene and
quantified using gas cnromatography.
B. SAMPLE HANDLING AND STORAGE
Water samples should be collected in an all-glass system. The sample
snouid oe acidified *itn jui'-jr-'': ic*!d 'H?S04; *o "H <2 *™ediately after
collection and stored at 4"C In the dar^, Extraction of the samples snouio
begin within 12 hours of collection as the degradation of 2,4-D is rapid in
aqueous systems.3
Sediment samples should be stored in glass containers. Immediate extrac-
tion of samples is recommended to minimize the effects of sampTe degradation.
However, when necessary, sample freezing at -20°C has been shown to prolong
the stability of 2,4-D.4
All sample containers should be sealed with Teflon-lined screw caps.l An
alternate method is to use pre-cleaned, heavy-duty aluminum foil to prevent the
sample from coming in contact with plastic caps and associated glue lining.
The aluminum foil may be cleaned by washing in acetone, followed by rinsing
with pesticide-grade hexane.3
A flowchart for the processing of samples to be analyzed for chlorinated
phenoxy acid residues is presented in Figure 1.
C. INTERFERENCES
txtraneous .natter, especially in highly colored water samoles, is a ooten-
*1a1 interference. The cleanup procedure described here will usually eliminate
this source of interference. Many organic compounds Jan -interfere with *he
III-303
-------
SAmi AIR
wmii
FILTER
PAH* OR
HUM
CMTRIOGC
SAWIE
PROCESSING
D1GES1
1
ANALYZt
Purpose Total
Cone.
In Air
SMVLE
CONTAINEII
SMVLE 1
PRESERVATIVE
STORAGE
TIME
SAMPLE
SUE
1 WATER OR SlUOtt. SOI
1 UACHAH
1
11
FILTER MET STORAGE I
NO till AN 1
MEIO
EEP TOIICIU
RESERVE
1 fP EITRACT
| STORE lOIICITY
EITRACT 1
I ,
I EXTRACT iITRACT
KHALI tt [ ANALYZE
I ANALYZE ma.ni
Total Dissolved' Ti
Cone; Cone. In Nobility Kibtllty In
In Hater Hater at pH S at pH S
6 C 6 G
•2*04 to >H 1 KjSOi to pH I 4'C 4*C
4*C 4*C
i< nmrt u hours <1H
-------
analysis, however. Boron trifluoride/methanol reagent is used because 1t
reacts specifically with carboxylic acids, whereas diazomethane may react with
phenols and other organics with relatively active hydrogens. All reagents must
be thoroughly checked and any interferences from this source eliminated.
In natural sediment samples, benzene may elute humic substances which,
upon GLC Injection, remain in the glass sleeve liner due to their nonvolatile
nature.
Where emulsions form at the solvent/water interface, the emulsion should
remain with the aqueous phase. This allows the emulsion to be extracted
further with methylene chloride and prevents the sodium sulfate in the funnel
from becoming saturated with water. Any amount of water in the extract could
inhibit esterification and result in decreased recoveries.
Any compounds which are co-extracted from the sample and chromatograph
similarly to the compounds of interest are possible sources of interference.
Certain esters of the chlorophenoxy acids may cause mutual interference.
Phenols and chlorophenols may also Interfere.*
Because the herbicides react readily with alkaline substances, loss may
occur if there is contact with alkaline substances at any time except in the
controlled alkaline hydrolysis step. Glassware and glass wool should be acid
rinsed to mimimize this possibility.A
D. APPARATUS
*!" :las3war«? Ttust be washed in chromic acid, rinsed with dilute hydro- . _.
chloric acid, followed by distiTiea *ater ana ir\er. .--nsea *f*h icstcne :nd
hexane. Heat treatment is carried out at 300°^ on all glassware sxceot volu-
metric flasks and pipettes. Care must be taken to ensure that the glassware
is not alkaline. Considerable loss at low herbicide concentrations can occur
due to the alkalinity of the glassware.
1. Graduated centrifuge tubes, 15 ml, with ground-glass stoppers.
2. Volumetric flasks (1.0 ml, 2.0 ml, 10 ml, 100 ml, 250 ml, and 2 1).
3. Round-bottom flask, 300 ml.
4. Erlenmeyer flask, 250 ml.
5. Separatory funnels, 60 ml, 500 ml, and 21. With TFE-fluorocarbon
stopcocks and tapered ground-glass stoppers, Kontes or equivalent.
6. Beakers, 100 ml, 200 ml.
7. Flasks, 500 ml, flat-bottomed.
8. Flasks, suction with ground-glass joints.
9. Funnels, coarse, sintered glass with grouna-giass joints.
-------
10. Funnels, powder, glass.
11. Graduated cylinders, 50 ml, and 1 liter.
12. Watch glass.
13. Pipettes, Pasteur, disposable, 140 mm long x 5 mm I.D., glass.
14. Microsyringes, Hamilton, 10 ul for injections.
15. Evaporator concentrator, Kuderna-Danish, 250-ml flask and 5-ml volumetric
receiver, Kontes or equivalent.
16. Snyder columns, three-ball macro, one-ball micro.
17. Rotary evaporator.
18. Gas chromatograph such as a Varian 2800, Microtec 220, or equivalent. It
should be equipped with an electron capture detector, a glass-lined
injection port, and a recorder. The following operating conditions are
recommended: injection temperature, 215°C; oven temperature, 185°C;
column temperature, 185*C; and a carrier gas flow of 70 ml/min in a 6.4
mrn-O.D. column.
Alternative operating conditions chat nave deen used are: column temper-
ature, 200"C; injection port, 230°C: ana ietector temoerature. 340°C.
Use 5 percent methane and 95 percent argon for both earner gas flow (40
ml/min) and make-up gas flow (20 ml/min).
19. Chromatograpm'c coiumn: the use of ;;vo :aiumns s .uggestaa "or 'asnfi-
fication and confirmation. One coiumn is packed.with a mixture of 1.5
percent OV-17 and 1.95 percent QF-1 on a 100/120-mesh Gas Chrom Q. The
second column is packed with 5 percent OV-210 on a 100/120-mesh Gas
Chrom Q.
Additional column packings that have been shown to be useful for separ-
ating and quantifying chlorinated phenoxy acetic acids and herbicides
are:
11 percent OV-17 + QF-1, mixed phase by weight, on 80/100-mesh Gas Chrom
Q, available from Applied Science.
3 percent OY-17 on Chromosorb W, HP 80/100-mesh, available from Applied
Science.
20. Chromatograph column, glass U-tube, 2 m by 3.5 mm O.D.
21. Column: chromatographic (10 mm I.D. x 300 mm) with coarse frit and
stopcock. Reservoir at top (28 mm I.D. x 150 mm).
22. Sana oath, fiuidizea iTeCam or equivalent) or water bath.
III-306
-------
23. Oven, capable of maintaining 300°C.
24. Ultrasonic homogenizer, such as the Sonicator Cell Disrupter Model W-375
with a solid disrupter form (No. 280-0.75). This is available from Heat
Systems-Ultrasonic, Inc., 38 East Mall, Plainview, Long Island.
25. Centrifuge tube heater, such as the Kontest Tube Heater block set at
40°C combined with a gentle stream of pure nitrogen gas for controlled
evaporation.
26. Sep-Pak, Cj8« reversed phase, Waters Association.
27. Steam bath.
28. High-pressure liquid chromatograph (HPLC).
28.1 Dual mobile phase.
28.2 Gradient elution.
28.3 5,000 psi back pressure.
28.4 UV absorption detector, 284 nm.
28.5 Recorder or integrating recorder.
29. Mechanical shaker.
All solvents and reagents must be of pesticide quality and checked before
use for purity by the gas chromatographic procedure. Much time and effort can
be saved by selecting high-quality reagents that do not require extensive pur-
ification. Some purification of reagents may oe necessary as outlined below.
If more rigorous treatment is indicated, the reagent should be obtained from
an alternative source.
1. Benzene
2. Chloroform
3. Methanol
4. Ethyl ether, reagent grade. Redistill in glass after refluxing over
granulated sodium-lead alloy for 4 hours.
5. Hexane
6. Methylene chloride
7. Acetone
I I 1-307
-------
8. 1:1 Acetone:hexane
9. Aceton1tr1le
10. Acetic add, glacial
11. Sodium chloride, reagent grade
12. Ethanol, USP or absolute
13. Potassium hydroxide, reagent grade.
14. Potassium hydroxide solution: dissolve 37 g KOH 1n distilled water and
dilute to 100 ml.
15. Hydrochloric acid, analyzed reagent grade or better.
16. Sulfuric acid, cone., analyzed reagent grade or better.
17. Sulfuric add, 9 N.
18. Boron trifluoride methanol reagent, 14 percent boron trifluoride by
weight (available from Analabs).
19. Florisil adsorbent - 60/100 mesn, ractory-dctivatea at 650°C. The
F Tonsil 1s heated to 130°C for 1 hour and stored in a desiccator prior
to use. Each oaten must be cneckea ror activity and for contamination.
20. Sodium sulfate - ACS grade or better, anhydrous. The heat-treated
material is aiviaea, ana one part :s "uoei'.iG 'ieutr.il ;oaiura ^ul "ate"
and stored at 130"C. The other part is slurried with snough 3ther to
cover the crystals and acidified to pH 4 by adding a few drops of
sulfuric acid.
To determine the pH, a small quantity of slurry is removed, the ether
evaporated, water is added to cover the crystals, and the pH 1s measured
with a pH meter. The ether 1s removed by vacuum from the acidified
sodium sulfate. This fraction 1s labelled "acidified sodium sulfate" and
stored at 130*C.
21. Sodium sulfate solution: dissolve 50 ml anhydrous sodium sulfate 1n
distilled water and dilute to 1 liter.
22. Acidified organic-free water: add hexane (50 ml) to distilled water
(5 1) stir for 4 hours with a magnetic stirrer at maximum speed.
Transfer to a large separatory funnel and remove the water layer Into
storage bottles. Add cone. HC1 (2 ml/I).
23. Celite filter aid: keep silica gel overnight at 650*C, homogenize with
5 percent organic-free water for 2 hours prior to use.
?4. Glass *ool: filtering qrade, acid washed.
III-308
-------
25. Analytical standards: 2,4,5-T; 2,4-D; 2,4,5-T methyl ester; 2,4-D methyl
ester; si 1 vex; si 1 vex methyl ester; all at least 98+ percent pure
(available from Dow Chemical, Polyscience, or as EPA reference stan-
dards).
26. Stock herbicide solution: dissolve 100 mg herbicide or methyl ester in
60 ml ethyl ether; dilute to 100 ml in a volumetric flask with hexane.
1.00 ml = 1.00 mg.
27. Intermediate herbicide solution: dilute 1.0 ml stock solution to 100 ml
1n a volumetric flask with a mixture of equal volumes of ethyl ether and
benzene. 1.00 ml = 10.0 ug.
28. Standard solution for chromatography: prepare final concentration of
methyl ester standards in benzene solution according to the detector
sensitivity and linearity.
29. GLC columns and conditions.
a) McKinley and McCully's column3, 4% SE-30 and 6% QF-1 on 100/120
mesh size Chromosorb W, acid-washed and DMCS treated.
b) 3% Dexil 300 GC on Chromosorb W, acid-washed, DMCS treated, 100/
120 mesh size.
c] 2% OV-1 on Chromosorb W, acid-washed, DMCS treated, 100/120 mesh
size.
d) Chau-Wilkinson Column3, 4% OV-101/6% OV-210 on Chromosorb W, acid-
*dsn8d, HOME treated, SO ts 100 ?,esh «1*s.
Column: Glass 1.8 m x 4-mm I.D.; packed with one of the aoove
packing materials:
Column Temperature:
Injection Temperature:
Detector Temperature:
Attenuation:
Chart Speed:
Flow Rate:
Detector:
Recorder:
195'C
250°C
275*C
16
1/2 inch/min
60 ml N2/min
Ni63 electron capture
1 millivolt strip chart
111-309
-------
F. ANALYTICAL PROCEDURES
1.1 Analysis of Solid Waste Samples for Chlorinated Herbicides
by High Performance Liquid Chromatography
Analytical Procedure: available
Sample Preparation: available
1.1.1 Reference
Stephens, R. D., J. Nakao, and B. P. Simmons, "Methods for the
Analysis of Hazardous Wastes." California Department of
Health Services, Berkeley, California. (1981).5
1.1.2 Method Summary
Samples are acidified and extracted with dichloromethane. In
the presence of interfering substances, samples are hydro!-
yzed in alcoholic potassium hydroxide, diluted with water,
and washed with dichloromethane prior to acidification and
extraction with dichloromethane. The dried extracts are then
diluted in methanol and injected into an HPLC equipped with a
reverse-phase column and UY detector at 285 nm.
1.1.3 Apolicaoili'y
This method is aopropr-ste for non-aqueous liquid industrial
wastes and agricultural chemicals.
!.1.4 Recovery studies in which nine chloroohenoxv herbicides were
determined in extracts or tftrse separate soil samples snowed
an average recovery of 92% (82.3-98.?%) with an average co-
efficient of variation of 9.2%. The herbicides used in this
study were 2,4-D, 2,4,5-T acid, 2,4-DB acid, silvex, 2,4-D
isopropyl ester, 2,4-0 outyi ester, 2,4-0 outoxyethanol
ester, 2,4-D propylene glycol butyl ether ester, and 2,4-D
ethyl hexyl ester.
1.1.5 Sample Preparation
Weigh 20.0 g sample into a 250-ml beaker. Add 50 ml 0.2 M
methanolic potassium hydroxide to the beaker. Cover the
beaker with a watch glass and place on a steam bath (reflux
temp.) for at least 1 hour. Mix periodically to assure good
surface contact. Proceed to paragraph 1.1.6.
1.1.6 Sample Hydrolysis
Remove the beaker from the steam bath, allow to cool, and
transfer the sample (total volume of sample plus washings not
to exceed 50 ml) into a 500-ml seoaratorv funnel containing
250 ml water and 60 mi sodium cnionae-saturatea water.
T.xtract the :amols *hn?e times with 50 ml dichloromethane
III-310
-------
each. Discard this extract (bottom layer). Acidify the
sample (pH <2) and extract three times again with 50.ml
dichloromethane each. Combine the extracts from the acidi-
fied sample and evaporate to dryness under a hood. To speed
the drying process, evaporate on the edge of a steam bath.
1.1.7 Extract Cleanup
It is essential that all non-soluble particulate matter above
1.0 u be removed since the HPLC column is composed of 5 u
particles and is easily plugged. Apply one of the following
techniques for particulate removal.
1.1.7.1 Dissolve the dried sample in 2.0 ml of methanol. Filter
according to conventional methods such as Sweny-type
filter.
1.1.7.2 Transfer the dried sample with 1.0 ml methanol onto a
reversed phase Sep-Pak and elute with methanol into a
2-ml volumetric flask.
1.1.8 Sample Analysis
Inject 10 nl sample :nto HPLC equilibrated ander the
following conditions:
Column: C^, 5 u, 15 cm, reversed phase.
Mooile pnase: CC% icstonitr''!^ :roqr"rnmed *o "''ncrease to
30* acetonitr-'le over 3 minutes and 70*
dilute acetic acid (1%) programmed to
decrease to 20%.
1.0 minutes
8.0 minutes
2.0 ml/minutes
284 nm
1.0
0.5 cm/minutes
Initial hold
Final hold
Flow rate
Wavelength
Attenuation
Chart speed
1.1.9 Data Interpretation
Tentatively identify peaks based on retention time and quan-
titate by peak height or peak area. Compare the peak height
or peak area to a standard which is known to be in the linear
range of the instrument (Subsection H).
III-311
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2.1 Analysis of Water Samples for Chlorinated Phenoxy Acid Herbicides
by Chloroform Extraction
Analytical Procedure: evaluated
Sample Preparation: available
2.1.1 Reference
Environment Canada, "Analytical Methods Manual." Inland
Waters Directorate, Water Quality Branch, Ottawa, Ontario,
Canada, 1974.3
2.1.2 Method Summary
The water sample is acidified to pH 2 or less and extracted
with chloroform. The solvent is dried, concentrated, and
replaced with a small amount of methanol. The herbicide
residues are converted to their methyl esters using boron
trifluoride/methane! reagent. The methyl esters are
extracted into benzene; the extract is cleaned up and
analyzed by gas liquid chromatography.
2.1.3 Applicability
This method includes the qualitative and quantitative gas
cnromatograpnic aetenm nation of 2.+-0, si"!vex, 2,4,5-7, and
other phenoxy acid herbicides.
The practical limits of measurement utilizing electron
capture detection are listed below for some of these herbi-
cides as netnyi estar-; ' n a ",-' ;ter «atar :-ample,
1. 2,4-D (0.010 ppb)
2. 2,4,5-T (0.010 ppb)
3. Silvex (0.010 pob)
4. MCPA (0.050 ppb)
The absolute lower limit of detection will vary with size and
characteristics of the sample as well as analytical condi-
tions.
Strict attention to protocol is required on the part of the
analyst to obtain reproducible and satisfactory recovery. In
the steps where solvents are evaporated, extreme care must be
exercised, especially when working with the methyl esters. The
extracts should never be taken to dryness as the esters are
extremely volatile.
2.1.4 Precision and Accuracy
Recovery studies at the 0.05-ppb level showed that the
recoveries "or 2,+-?> *ere consistently 35 oercent or better.
III-312
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The recoveries for 2,4,5-T and si 1 vex were better than 90
percent. At the 1.0-ppb level recoveries of all the herbicides
were better than 95 percent.
2.1.5 Sample Extraction
Acidify the water sample {1,000 ml) to approximately pH 2.0
with concentrated sulfuric acid and transfer quantitatively
to a 2,000-ml separatory funnel.
Add 50 ml chloroform to the separatory funnel and shake
the mixture thoroughly for 1 minute. Occasionally emulsions
are formed, but they can usually be broken by adding small
portions of 2-propanol, acetone or a saturated sodium chlor-
ide solution.
Allow at least 5 minutes for complete separation of the
layers and draw off the bottom chloroform layer into a clean
500-ml separatory funnel.
Extract the sample twice more and combine the extracts in the
500-ml separatory funnel. The chloroform extracts are then
washed with 100 ml slightly scldic class-distilled xatar and
the aqueous layer is removed.
The combined chloroform extracts are dried "over acidified
anhydrous sodium sulfate for about 10 minutes. Caution: The
axtract ihculd "ot "W.ain
-------
About 5 ml 5% aqueous sodium sulfate solution is added to
the tube and extraction of the methyl esters is carried out
with two 2-ml portions of hexane. The hexane extract is
concentrated to 1 ml under a stream of dry nitrogen.
The hexane phase containing the methyl esters of the phenoxy
acid herbicides is passed through a small column, prepared by
plugging a disposable pipette with glass wool and packing
with 2 cm neutral anhydrous sodium sulfate over 2 cm
Florisil. The herbicide esters are eluted with 10 ml of
benzene.
The benzene solution is concentrated to 0.5 ml under a stream
of dry nitrogen, quantitatively transferred to a 1-ml volu-
metric flask and made up to volume. This solution is now
ready for GLC determination.
2.1.7 Sample Analysis
Preliminary identification is achieved via electron capture
GLC in which at least two different stationary phases of
different polarity are employed (Subsection F). The identity
of the herbicide is based on the retention time relative to
aldrin (Subsection H).
III-314
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2.2 Analysis of Water Samples for Chlorinated Phenoxy Acid Herbicides
by Ethyl Ether Extraction
Analytical Procedure: available
Sample Preparation: available
2.2.1 Reference
American Public Health Association, "Standard Methods for the
Examination of Water and Wastewater." 15th Ed. American
Public Health Association, New York, New York. 1,134 p.
(1980).
2.2.2 Method Summary
Chlorinated phenoxy acids and their esters are extracted from
an acidified water sample with ethyl ether. The extracts are
hydrolyzed and extraneous material is removed by a solvent
wash. The acids resulting from the hydrolysis are converted
to methyl esters, and the extract is cleaned up on a micro-
adsorption column. The methyl esters are quantified using
gas chromatography.
2.2.3 Applicability
This method is suitable for quantifying chlorophenoxy acid
herbicides in aqueous samples. The detection ~! unit will
depend on the initial sample size, degree of extract concen-
tration, and the presence of interferring substances. If the
extract from a t-Mter samnle *s concentrated to 2.00 ml and
5.0 ul of concentrate is injected into a gas cnromatograpn
equipped with an electron capture detector, reliable mea-
surements of 50 ng/1 2,4-D, 10 ng/1 Si! vex, and 10 ng/1
2,4,5-T are oossible. Concentrating the extract to 0.5 ml
permits the detection of approximately 10 ng/1 2,4-0, 2 ng/1
Silvex, and 2 ng/1 2,4,5-T.
2.2.4 Precision and Accuracy
Information is not presently available.
2. 2. "5 Sample Extraction
Acidify 1 liter of aqueous sample to pH 2 with concentrated
sulfuric acid and transfer to a 2-liter separatory funnel.
Add 150 ml ethyl ether to the separatory funnel and shake
vigorously for 1 minute. Let the phases separate for 10
minutes. If emulsions form, drain off the aqueous layer,
invert the funnel, and shake rapidly.
1: Vent *he *unne1 freauentlv to orevent excessive
pressure buildup.
III-315
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Collect the extract in a 250-ml ground-glass stoppered
Erlenmeyer flask containing 2 ml KOH solution. Repeat the
extraction with two 50-ml portions of ethyl ether. Combine
the extracts in the Erlenmeyer flask.
2.2.6 Extract Hydrolysis
Add 15 ml distilled water and a small boiling stone to the
flask. Attach a three-ball Snyder column. Remove the ether
on a steam bath and continue heating for a total of 60
minutes.
Transfer the concentrate to a 60-ml separatory funnel. Ex-
tract the sample with 20 ml ethyl ether and discard the ether
layer. Repeat the ether extraction and again discard the
ether layer. The herbicides are retained in the aqueous
phase.
Acidify with 2 ml cold (4°C) 9M sulfuric acid. Extract once
with 20 ml ethyl ether and twice with 10 ml ethyl ether.
Collect the extracts in a 125-ml Erlenmeyer flask containing
0.5 g acidified anhydrous sodium sulfate. Let the extract
remain in contact wtih the sodium sulfate for at least 2
lours.
2.2.7 Esterification
Fit a Kuderna-Danish apparatus with a 5-ml volumetric re-
ceiver. Transfer the ether extract to the Kuderna-Danish •
apparatus through a runnei plugged with 'jiass too;, jse
liberal washing of ether. Crush any hardened sodium julfate
with a glass rod. Before concentrating, add 0.5 ml benzene.
Reduce the volume co
-------
stopper and shake vigorously for about 1 minute. Let stand
for 3 minutes to facilitate phase separation.
NOTE 3: Care must be taken to ensure that the tubes are
tightly capped and remain so after introduction of the boron
tri fluoride methanol reagent. The temperature should be
about 50°C for good yields. The methylation is a very crit-
ical step in the procedure.
2.2.8 Sample Cleanup and Analysis
Pi pet the solvent layer from the receiver to the top of a
small column prepared by plugging a disposable Pasteur pi pet
with glass wool and packing with 2 cm sodium sulfate over
1.5 cm Florisil adsorbent. Collect the eluate in a 2.5-ml
graduated centrifuge tube. Complete the transfer by repeat-
edly rinsing the volumetric receiver with small quantities of
benzene until a final volume of 2.0 ml of eluate is obtained.
Check calibration of centrifuge tubes to ensure that the
graduations are correct.
Analyze the benzene extract by gas chromatography using at
least two columns. Injections of 5 to 10 pi should be
sufficient for this puroose.
Inject methyl «ster standards freauently to ensure optimum
operating conditions. Always inject the --ame volume. Adjust
the volume of sample extract with benzene, 1f necessary,, so
that the heights of the peaks obtained are close to those of
tne standards. (If 3 portion of the extract solution was
concentrated, the dilution factor 0 is iess cnan », ~ r: *ds
diluted, the dilution factor exceeds 1.)
Confirmation of residue identity can be achieved by trans-
esterificationS, thin layer chromatography^, or mass
spectroscopy.
III-317
-------
3.1 Analysis of Sediment Samples for Chlorinated Pheno^y Acid
Herbicides by Acetone-Hexane Extraction
Analytical Procedure: available
Sample Preparation: available
3.1.1 References
Walton, A., "Ocean Dumping Report 1. Methods for Sampling
and Analysis of Marine Sediments and Dredged Materials."
Department of Fisheries and Environment, Ottawa, Ontario,
Canada. 74 p. (1978).6
Peake, A. A., and H. S. Lesick, "Procedure for the Analysis
of Phenoxy Acid Herbicides in Sediments." Water Quality
Branch, Inland Waters Directorate, Calgary, Alberta, Canada
9 p. (No date).7
3.1.2 Method Summary
A 25-g sample of homogenized sediment is extracted with 1:1
acetone/hexane under acidified conditions. The extract is
dried, exchanged into benzene, and esterified with boron
trifluoride/methanol. Extract cleanup is achieved with a
3:!*ca nel column and ina'vsis *s comoletsd 'jslng a gas
chromatograph equipped with an electron capture detector.
3.1.3 Applicability
The orocedure is suitable for use with sediment, dredged
material, and soil samples, r.ie oetscticn ,-.mit or ;ne
procedure will be influenced z>y sample 51 ze, extraction
efficiency, extract concentration, esterification efficiency,
and the presence of interferences.
3.1.4 Precision and Accuracy
The procedure has been shown to produce greater than 90%
recoveries with known standards.' Recoveries from natural
sediments with high organic matter and high sulfur content
ranged from 68 to 106%. The presence of organic matter can
have a negative effect on the recovery of chlorophenoxy acetic
acids.
3.1.5 Sample Extraction
Weigh out a 25-g dry-weight equivalent of homogenized
sediment sample. -Transfer the sample to a 250-ml beaker and
add 10 ml acidified, organic-free water. The resultant
slurry should be approximately 20 to 30% water,
Thoroughiy mix cne sediment jiurry and carefully uc-.tiify ;he
samele with 4 ml concentrated hydrochloric acid. Add the
III-318
-------
acid slowly with constant mixing to prevent mechanical loss
due to gas evolution. Allow the mixture to stand 20 minutes
with occasional stirring.
Add 5 ml 1:1 acetone:hexane mixture to the acidified sediment
slurry. Place the disruptor horn of an ultrasonic homog-
enizer approximately 2 cm into the sample. Activate the
disruptor for 2 minutes in the pulsed mode at 35% duty cycle
with maximum output. Allow the sediment to settle.
Prepare a slurry consisting of 1:1 acetone:hexane and celite.
Pour the slurry into a sintered glass funnel which is con-
nected to a suction flask. Remove the solvent from the
celite filter bed by suction and discard the acetone/hexane.
Decant the supernatant solvent from the sample extraction
into the funnel and apply a vacuum to collect the extract in
the suction flask. Retain the solids for a second extrac-
tion.
Add 75 ml 1:1 acetone:hexane to the original sediment sample.
Mix with the ultrasonic homogenizer, allow the sediment to
settle, ind '•i1tsr throuqh the celite ""Star bed as before.
Transfer the combined extract to a 500-ml separator^ funnel.
Add 100 ml acidified, organic-free water and shake for 1
minute. Release the pressure frequently. Allow the layers
to seoarate and transfer the aqueous layer back to the
suction flask.
Slowly pour the solvent layer through a glass powder funnel
plugged with glass wool and containing approximately 2 cm of
acidic sodium sulfate. Collect the solvent in d 500-ml flat-
bottomed flask.
Return the aqueous layer from the suction flask to the sep-
aratory funnel. Rinse the suction flask with 75 ml methylene
chloride and add the rinses to the aqueous phase in the
separatory funnel. Shake for 1 minute and allow the layers
to separate. If an emulsion persists, leave it with the
aqueous layer. Pass the lower solvent layer through the
sodium sulfate funnel and collect in the 500-ml flat-bottomed
flask with the acetone:hexane extract.
Extract the aqueous phase with a second 75-ml portion of
methylene chloride. Filter the methylene chloride phase
through the acidified Na2$04 funnel and combine with
previous extracts. If necessary pour the combined extract
through a second dryinq column 'acidified Na^SOa) as
water can interfere with tne estenfication reaction ana
result •>, "low -scoveries.
-------
3.1.6 Extract Esterification
Transfer the extract to a Bu'chi evaporator and reduce the
volume to 2 to 5 ml. Transfer the residue to a 15-ml graduated
centrifuge tube and evaporate to 0.5 ml.
Add 1 ml of benzene to the sample extract 1n the graduated
centrifuge tube and shake. Reduce the volume to 0.5 ml.
Repeat the process of adding 1 ml of benzene and reducing the
volume to 0.5 ml until the extracted residue has been
exchanged from methylene chloride to benzene.
Add 0.2 ml 14-percent boron trifluoride methanol esterifica-
tion reagent and shake for 1 minute. Seal tightly and place
the tube in a water bath at 50°C for 30 minutes. (Care must
be taken to ensure that the tubes are tightly capped and
remain so after the introduction of the boron trifluoride/
methanol reagent. The temperature should be about 50"C for
good yields. The methylation process is a very critical step
in the procedure).
Cool to room temperature and add 5 ml 5-percent sodium
sulfate solution. Shake for 1 minute and allow the layers to
separate. Withdraw the top solvent layer into a clean cen-
triruge tuoe using a Pasteur pipet.
Add 1 ml benzene and jhaice. Allow the layers to separate and
transfer the top benzene layer to a clean centrifuge tube.
Repeat the benzene extraction a second and a third time.
' Comoine ;he oenzene extncts.
Evaporate the final benzene extract to a volume of 0.5 ml.
Add 1 ml hexane, shake, and reduce the volume to 0.5 ml.
Repeat this process a second and a third time to exchange the
sample residue into hexane.
Prepare a cleanup column by adding pre-heated silica gel to a
height of 75 mm in a disposable pipet. Tap the column while
packing. Add 1 cm of neutral anhydrous sodium sulfate to the
top of the column. Elute the column with approximately 30 ml
of hexane and discard the eluant.
Quantitatively transfer the 0.5-ml hexane concentrate to the
cleanup column. Rinse the centrifuge tube with three 1-ml
portions of hexane and add each rinsing to the cleanup col-
umn. Allow the column to elute until the hexane layer just
recedes into the top of the sodium sulfate layer.
Elute the column with 90 ml hexane. Discard the eluant.
Elute the column with 100 ml benzene. Collect the solvent
in j 500-nl Hat-bottomed "ask. ^lute *.he ralumn with i
second 100-ml portion of benzene and combine with tne first
III-320
-------
eluant. Reduce the volume to approximately 5 ml on a Buchi
evaporator. Transfer the concentrate to a 10-ml volumetric
flask and dilute to volume with benzene.
3.1.7 Sample Analysis
Preliminary identification is achieved via electron capture
GLC in which at least two different stationary phases of
different polarity are employed. The identity of the herbi-
cide is based on the retention time relative to aldrin.
Calculate the concentration of sample constituents as
indicated in Subsection H. Methyl ester standards should be
injected frequently to ensure optimum instrument operating
conditions.
Confirmation of identity can be achieved by transesterifica-
thin layer chromatographyS, or mass spectroscopy.
III-321
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3.2 Analysis .of Soil Samples for Chlorinated Phenoxy Acid Herbicides by
High Performance Liquid Chromatography
Analytical Procedure: available
Sample Preparation: available
3.2.1 Reference
Stephens, R. D., J. Nakao and B. P. Simmons, "Methods for the
Analysis of Hazardous Wastes." California Department of
Health Services. Berkeley, California. (1981).5
3.2.2 Method Summary
Samples are acidified and extracted with dlchloromethane. In
order to Isolate the chlorophenoxy herbicides from possible
Interfering substances, sample extracts are hydrolyzed with
alcoholic potassium hydroxide, diluted with water, and washed
with dlchloromethane. Herbicide residues are acidified and
extracted Into dlchloromethane. The extracts are dried,
diluted with methanol, and analyzed by HPLC using a reversed-
phase column and a UV detector at 285 nm.
3.2.3 Applicability
This method 1s suitable for quantitying chioropnenoxy add
heroicides In soiid-onase \soi';) samples. The method detec-
tion limit will depend on Initial sample size, degree of
extract concentration, and the presence of Interfering
substances.
3.2.4 Precision ana Accuracy
Recovery studies 1n which nine chlorophenoxy herbicides were
determined in extracts of three separate soil samples showed
an average recovery of 92% (82.3-98.7%) with an average
coefficient of variation of 9.2%. The herbicides included in
the study were 2,4-D add, 2,4,5-T add, 2,4-DB acid, silvex,
2,4-D isopropyl ester, 2,4-D butyl ester, 2,4-D butoxyethanol
ester, 2,4-D propylene glycol butyl ether ester, 2,4-D ethyl-
hexyl ester.
3.2.5 Sample Extraction
Weigh 20.0 g homogenized sample Into a 250-ml Erlenmeyer
flask, acidify with 5 ml 24 M sulfuric acid, and extract with
50 ml dlchloromethane for at least 4 hours on a mechanical
shaker. Decant the dlchloromethane into a 125-ml beaker.
Use an additional 10 to 15 ml dichloromethane to rinse the
flask and sample. Add the wash to the Initial extract. Allow
dichloromethane to evaporate at room temperature to dryness
unaer a nooa. To speea the drying process, evaporate on :he
-------
interfering substances (pigments, oils, lipids, etc.), proceed
directly to paragraph 3.2.7. If the sample contains interfering
substances, continue with the hydrolysis step, paragraph 3.2.6.
3.2.6 Extract Hydrolysis
This step is designed to convert the chlorophenoxy esters to
the sodium salts of their respective acids. Since the acids
are soluble in water, interfering organic compounds can then
be removed with dichloromethane. When using the hydrolysis
step, both the quantitative and qualitative information on
the chlorophenoxy esters is lost and the data must be
reported as total chlorophenoxy acids.
Add 50 ml 0.2 M methanolic potassium hydroxide to the beaker
containing the sample residue. Cover the beaker with a watch
glass, place on a steam bath, and allow to reflux for at
least 1 hour.
Remove the beaker from the steam bath, allow to cool, and
transfer the sample .{total volume of sample plus washings not
to exceed 50 ml) into a 500-ml separatory funnel containing
250 *ni jisfi'lsd -rater ind 30 ?.l sodium thloride-saturated
water. Extract the sample three times witn 50-ini portions of
dichloromethane. Discard the organic extracts.
Acidify the aqueous phase in the separatory funnel to pH <2.
" I/.trict *he •amnle with three 50-ml oortions of dichloro-
methane. Combine tne sxtracts r'rom cne uciav.;"ea sample -ind
evaporate to dryness in a nood. Place the extract on the
edge of a steam bath to speed the drying process.
3.2.7 Extract Cleanup
It is essential that all non-soluble particulate matter above
1.0 u be removed since the HPLC column is composed of 5 u
particles and is easily plugged. Select one of the following
techniques for particulate removal.
a. Dissolve the -iried residue in 2.0 ml methanol. Filter
according to conventional methods such as a 3weny-type
filter. Dilute the filtered sample to 2.0 ml.
b. Transfer the dried residue with 1.0 ml methanol on to
reversed phase Sep-Pak and elute with methanol into a
2-ml volumetric flask. Dilute to volume with methanol.
3.2.8 Sample Analysis
Inject 10 ul of standara or sampie .nto d HPLC that "las been
equilibrated :nder the *3T!owinq conditions:
-------
Column: CIQ, 5 u, 15 cm, reversed-phase, ultrasphere
Mobile Phase: 30% acetonitrile programmed to Increase to 80%
acetonitrlle over 9 minutes and 70% dilute acetic acid (1%)
programmed to decrease to 20%.
.Initial hold * 1.0 minutes
Final hold - 8.0 minutes
Flow rate * 2.0 ml /minutes
Wavelength = 285 nm
Attenuation » 1.0
Chart speed = 0.5 cm/minutes
3.2.9 Data Interpretation
Tentatively identify peaks based on retention time and
quantltate by peak height or peak area. Compare the peak
height or peak area to a standard which is known to be in the
linear range of the instrument.
III-324
-------
G. CALCULATIONS
Compare the peak height of a standard to the peak height of the sample to
determine the amount of the herbicide injected.
Calculate the concentration of herbicides in liquid samples as follows:
A x B x C x D
P(ng/l)
E x F x G
where
P *. concentration of chlorinated phenoxy acid herbicides
A * weight of herbicide standard injected
B * peak height of sample, mm
C - final extract volume, pi
D = dilution factor
E * peak height of standard, mm
F s extract volume injected, pi
~ - volume of samole extracted, ml.
7he concentration of chlorinated phenoxy acid herbicides in sediment
samples can be calculated as:
? [wet weight)
P (dry weight)
E x F x H
A x B x C
E x F x H x IS
where
o = concentration of chlorinated phenoxy acid herbicides, pg/kg
A = weight in picograms of standard
B » peak height (or area) of sample
C = final volume of sample extract, ml
E s peak height (or area) of standard
F * volume of extract required to produce B, pi
H * wet weight of sediment initially extracted, g
IS » percent solids in sediment sample (expressed as a decimal
fraction).
111-325
-------
REFERENCES
1. American Public Health Association. "Standard Methods for the Examination
of Water and Wastewater Including Bottom Sediments and Sludges." 15th
Edition: American Public Health Association, New York, Mew York. 1,134
p. (1980).
2. Junk, 6. A., J. J. Richard, J. S. Fritz and H. J. Svec. "Resin Sorption
Methods for Monitoring Selected Contaminants in Water." In: "Identifica-
tion and Analysis of Organic Pollutants in Water. L. H. Keith (Ed.).
Ann Arbor Science Publishers; Ann Arbor, Michigan, pp. 135-153 (1976).
3. Environment Canada. "Analytical Methods Manual." Inland Waters
Directorate, Water Quality Branch; Ottawa, Ontario, Canada (1974).
4. Bristol, D. "Effects of Storage Conditions on Residues of 2,4-D and
2,4-DCP in Potatoes." In: "Accuracy in Trace Analysis: Sampling,
Sample Handling, and Analysis." National Bureau of Standards Special
Publication 422. pp. 737-745 (1976).
5. Stephens, R. D., J. Nakao and B. P. Simmons. "Methods for the Analysis
of Hazardous Wastes." California Department of Health Services,
Berkeley, California. (1981).
6, Walton, A. "Ocean Dumoinq Report 1. Methods for Sampling and Analysis
:f Marine Pediments and j-rsaged Material 3. ' Department of Fisheries and
Environment; Ottawa, Ontario, Canada. 74 p. (1978).
, V &„ -»r,d H. S. '.ssick. ""-ocedurs for the ^nalvsis of Phenoxy
Acirt Heroiciaes in Sediments.*" n'atar Guaiity orancn, 4,iiana Waters
Directorate; Calgary, Alberta, Canada. 9 p. iNo aate).
8. Yip, G. "Confirmation of Chlorophenoxy Acid Herbicide Residues by
Transesterification." J. Assn. Gffic. Anal. Chem. 54:343-34 (1971).
9. Chau, A. S. Y. "Analysis of Chloriniated Hydrocarbon Pesticides in
Waters and Wastewaters. Methods in Use in Water Quality Division Labora-
tories." Department of the Environment, Inland Waters Directorate;
Ottawa, Ontario, Canada (1972).
III-326
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SECTION 8
METHODS FOR THE DETERMINATION OF DIOXIN (TCDD)
A. SCOPE
This Section provides methods for the isolation and identification of
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in wastes and environmental
samples. The general procedure consists of methylene chloride extraction,
extract cleanup, solvent-exchange into hexane, and quantification using GC/MS
methodology.
Due to the highly toxic nature of this compound, the analyst is urged to
exercise extreme caution when handling the pure compound (standards) and
samples known or suspected to contain this material. General safety guidance
and protocols are provided in Subsection D.
B. SAMPLE HANDLING AND STORAGE
Aqueous samples should be collected in amber glass containers and sealed
with' foil- or Teflon-lined screwcaps. The container should not be prewashed '
vit^ :amol^ prior to collection. Also, all samojinq eouioment must be as free
as possible of Tygon and other potential sources or jomannnation.
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, 80 mg/1 sodium thiosulfate snould be added to the sample. Field
test kits are available to determine the need for use of a thiosulfate
preservative. Aqueous samples should be extracted within 7 days of collection
and analyzed within 40 days of extraction.1
Soil and sediment samples should be collected in glass containers and
refrigerated until processing. The maximum storage period is not known;
therefore, it is recommended that these samples be orocessed within the same
time frame as water samples.
Biological tissue samples should be wrapped in solvent-rinsed foil.
Samples should be frozen immediately after collection and remain frozen until
analysis is initiated.
This information is summarized in Figure 1.
III-327
-------
82C-III
•o
Ol
Q.
A
<-»•
O
Cat
U3
(T
O
01
9 ••*• (">W> V
rt f? tst II 5
•i- '| ,-,4 si l
1ft
3
?
3
?
3
-4
i!
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i =
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I = S
-------
C. INTERFERENCES
Method interferences caused by contaminants in solvents, reagents, glass-
ware, and other hardware used in sample processing may lead to discrete arti-
facts and/or elevated backgrounds for the ions being monitored. All of these
materials must be routinely demonstrated to be free from interferences under
the conditions of the analysis by running laboratory reagent blanks.
All glassware must be scrupulously cleaned2 after each use. Glassware
should be rinsed with the last solvent used in it, followed by detergent-wash-
ing with hot water and rinsing with tap water and distilled water. Glassware
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 elimi-
nated by this treatment.) Solvent rinses with acetone and pesticide-quality
hexane may be substituted for the muffle furnace heating. Volumetric glassware
should not be heated in a muffle furnace. After drying and cooling, glassware
should be sealed or capped with aluminum foil and stored inverted in a clean
environment to prevent any accumulation of dust or other contaminants.
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.
Matrix -'nt2rferenc3s ^av be caused by contaminants that are coextracted
from the sample. The extent of matrix interferences «nr< t'ary cans', aeraoiy
from. sample to sample, (ieoenainq on ;he nature and Jiversity cf *he sample.
TCDD is often associated with interfering chlorinated compounds wmcn are
present at concentrations several magnitudes higher than TCDD. The suggested
:' ;=nuD orocsdures can overcome many of these interferences, but unique samples
may reauire additional cleanup-"0 co eliminate ;ai':s ;os".""-vss :nc Achieve ",he
method detection limit.
Some other tetrachlorodibenzo-p-dioxin isomers may also interfere with the
measurement of 2,3,7,8-TCDD.3-6 Capillary column 933 chromatography can be
used to achieve separation, but identification of some Isomers may not be
possible because of virtually identical mass fragmentation patterns.
D. SAFETY
The toxidty or carcinogenldty of each reagent used in this method has
not teen precisely defined. Benzene and TCDD have been identified as
suspected human or mammalian carcinogens and the otner reagents snouid be
treated as potential health hazards. From this viewpoint, exposure to these
chemicals must be reduced to the lowest possible level.
A strict laboratory safety program for the handling of TCDD samples
should be developed that includes the following practices:
1. Potential contamination of the laboratory should be minimized by con-
"lucfinq ill samole manioulations in a hood, glovebox, or secondary
containment device.
III-329
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2. The effluents of sample splitters for the gas chromatograph 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.
3. Liquid wastes 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). When TCDD can no longer be
detected, dispose of waste solutions as appropriate.
Dow Chemical USA has issued the following precautions (11/78) for the
safe handling of TCDD in the laboratory. They are as complete as possible
based on available toxicological information but necessarily general in nature
since detailed, specific recommendations can only be made for the particular
exposure and circumstances of each Individual case. Inquiries about specific
operations or uses may be addressed to the Dow Chemical Company, Midland,
Michigan.
1. Protective equipment including disposable plastic gloves, apron or lab
coat, safety glasses, and a lab hood adequate for radioactive work should
always be used.
2. Workers must be trained in the proper method of removing contaminated
gloves and clothing without contacting the exterior surfaces.
3. Thorough washing of hands ana rorearms after eacn inampuiation ana oefore
oreans (coffee, iuncn. end of snift; are .nanaatory.
4. The work area should be posted with signs and isolated. All work
surfaces should be covered with clastic-backed absorbent paper. Separate
glassware and toois snouiu oe prcviaea -cr ;ne drs&.
5. Waste cans should be lined with plastic bags. Janitors must be trained
in safe handling of potentially hazardous wastes (one accidental case of
chloracne resulted from handling laboratory wastes .n a routine manner).
It is a good practice to minimize the volume of contaminated wastes when
possible.
6. The following suggestions are offered for the disposal of TCDD related
wastes: TCDD decomposes above 8008C. Low-level waste such as absorbent
paper, tissues, animal remains, and plastic gloves may be burned in a
.good incinerator. Gross Quantities (milligrams) of TCDD 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 placed in a disposable
container and allowed to evaporate in a hood. Residues can then be
handled as described above.
7. In the event that contamination occurs, the following decontamination
procedures are recommended.
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7.1 Decontaminate personnel with any mild soap and plenty of scrubbing
action.
7.2 Satisfactory cleaning of glassware, tools, and surfaces may be
accomplished by rinsing with Chlorothene (Dow Chemical-Company),
then washing with any detergent and water. Dish water may be
discharged to the sewer. It is prudent to minimize solvent wastes
that may require special disposal through expensive commercial
services.
7.3 Lab coats or other clothing worn in TCDD work may be laundered.
Clothing should be collected in plastic bags. Persons who convey
the bags and launder the clothing should be advised of the potential
hazard and trained in proper handling of the clothes. 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.
8. A useful method of determining the cleanliness of work surfaces and tools
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
micrograms per wipe. This procedure is provided in Subsection L. Less
inan 1 ^9 T-DO per <«ioe indicates accactable cleanliness; anything higher
warrants further cleanup. More tnan 10 ug fCDD per *ipe indicate
unacceptable work practices have oeen employed in the past and that an
acute hazard exists that requires prompt attention before further use of
the equipment or work space is acceptable.
?. *ny proceaure tnat may producs aircorne .antamination lust be ^cne in ^n
area with good ventilation. However, yross .esses ;o 2 ventilation system
must not be allowed. Handling of dilute solutions normally used in
analytical and animal work presents no inhalation hazard except in the
case of an accident.
10. When accidents occur, remove the contaminated clothing immediately while
taking precautions not to contaminate additional skin surfaces or other
articles. Wash exposed skin vigorously and repeatedly until medical
attention is obtained.
For clinical advice, contact B. B. Holder, M. D.» Medical Director, Dow
Chemical USA, Midland, Michigan 48640 (Telephone (517) 636-2109). For detailed
safe handling precautions for specific procedures, contact L. G. Silverstein,
Industrial Hygiene Laboratory, Dow Chemical USA, Midland, Michigan 48640
(Telephone (517) 636-1688).
E. APPARATUS
1. Sampling
1.1 Grab sample bottle, amoer giass, i-liter or .-quart volume, with
foil- or Tef on-lined screwcaDS (foil should not be used if the
sample is corrosive). If amber Potties are not availaoie, samples
-------
must be protected from light. Containers must be washed, rinsed with
acetone or methylene chloride, and dried before use.
1.2 Automatic sampler (optional). Must incorporate glass sample con-
tainers 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, the length of
compressible tubing (Tygon or silicone rubber) used should be mini-
mized. Before use, the compressible tubing should be thoroughly
rinsed with methanol, followed by repeated rinsings with distilled
water to minimize potential contamination of the sample. An integra-
ting flow meter is required to collect flow proportional composites.
1.3 Freezer, for tissue samples. Wipe tests should be performed before
and after use to eliminate risk of cross-contamination.
1.4 Ice chest or refrigerator, for water and soil/sediment samples.
2. Safety
2.1 Plastic gloves. Inner gloves of Viton or nitrile rubber, covered
by disposable surgical gloves, have been used.
2.2 Aprons, polyethylene or Saranex-laminated (Durafab P2110 or
equivalent),
2.3 Safety glasses, equipped with side-shields.
2.4 Ultraviolet lamp, with wavelength greater than 290 nm.
3. Samoie Preparation and Analysis
3.1 pH paper, wide range.
3.2 Funnel, glass.
3.3 Erlenmeyer flasks, 250 ml and 125 ml.
3.4 Round-bottom flasks, 500 ml and 100 ml.
3.5 Chromatographlc column, glass, 1 cm I.D. x 50 cm.
3.6 Distillation receiver, 12 ml.
3.7 Oven, capable of maintaining 200eC.
3.8 Desiccator.
3.9 Graduated Chromaflex sample tube, 2 ml (for water samples).
III-332
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3.10 Centrifuge (for soil/sediment and tissue samples), with bottles and
tubes.
3.11 Extraction Apparatus
3.11.1 Extraction jar, 250 ml or larger; shaker or stirrer; filter
paper (Whatman No. 4 or equivalent), or:
3.11.2 Soxhlet extraction apparatus, with thimbles, rotary evapor-
ator, N-Evap analytical concentrator.
3.11.3 Chromatographic column (1 cm I.D. x 10 cm) and conical mini-
vials, 2 ml, with Teflon-lined septa, or:
3.11.4 Glass macro-column, 2 cm O.D. x 23 cm, tapered 1 cm, and
5 ml disposable pipet.
3.11.5 Blender, high-speed.
3.12 Glass tubing, 3 mm I.D. x 7 mm.
3.13 Mortar and pestle, glass.
3.14 uiass wooi, ii^dmzsa.
.3.15 Separatory funnels, 125 ml and 2000 Til, -yitn Teflon stopcocks.
3.16 Concentrator tube, Kuderna-Danish, 10 ml, graduated (Kontes
K-370050-1025, ;-r sqir •'.t-'-nt}. ^jl-'br^tion "lust be checked at the
volumes employed •" the **st. A nrouna gidss stopper is necessary
to prevent evaporation of extracts.
3.17 Evaporative *lssk, Kuderna-Oanish, 500 ml (Kontes 570001-0500 or
equivalent). Attach to concentrator tube with springs.
3.18 Snyder column, Kuderna-Danish, three-ball macro (Kontes K-503000-
0121 or equivalent).
3.19 Snyder column, Kuderna-Danish, two-ball micro (Kontes K-569001-0219
or equivalent).
3.20 Vials, amber glass, 10 to 15 ml capacity, with Teflon-lined
screwcaps.
3.21 Chromatography column, 30 cm x 1 cm I.D., with coarse fritted disc
at bottom and Teflon stopcock.
3.22 Chromatography column, 40 cm x 1 cm I.D., with coarse fritted disc
at bottom and Teflon stopcock.
*?.23 Boiling chips, 10/40 mesh. Prior to use, neat to 4t)0"C for 50
minutes or ioxniet-^xtract *i c,*i .Tiethylene "hlor-'de.
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3.24 Water bath, heated, with concentric ring cover. The bath should be
used 1n a hood and capable of ±2°C temperature control.
3.25 Gas chromatograph/mass spectrometer analytical system.
3.25.1 Gas chromatograph. An analytical system complete with all re-
quired accessories Including syringes and gases. The injection
port must be designed for capillary columns. Either split or
splitless Injection 1s acceptable.
3.25.2 Capillary column, 50 m x 0.25 mm I.D. glass coated with SILAR-
10C or equivalent). An equivalent column must resolve 2,3,7,8-
TCDD from the other 21 TCDD Isomers.
3.25.3 Mass spectrometer. Equipped with a 70 volt (nominal) ion source
and capable of acquiring 1on abundance data 1n real time for
groups of four or more Ions (Selected Ion Monitoring or SIM).
3.25.4 GC/MS interface. Any GC-to-MS interface that gives acceptable
calibration points for 50 ng of TCDD per injection may be used.
GC-to-MS interfaces constructed of all glass or glass-lined
materials are recommended. Glass surfaces can be deactivated by
silanizing with dichlorodimethylsilane.
2.2S.5 Oata :ystsm. \ computer system ~5ust be *nterfacsd to the
spectrometer that allows continuous acquisition and storage on
machine-readaDle media of al1 SIM data obtained throughout the
duration of the chromatographic program. This output is defined
as the Selected Ion Current Profile (SICP). The computer must
^av*> software available that allows slotting the SICP and inte-
grating the abundance or any ;or. ,n ;ne ilC? oetween specified
time or scan number limits. The capaoility for real -time analog
output of the SICP 1s useful but not required.
3.26 Balance, analytical. Capable of accurately weighing 0.0001 g.
F. REAGENTS
1. Reagent water (defined as Interference-free at the method detection
limit of TCDD).
2. Sodium hydroxide solution, 10 N. Dissolve 400 g NaOH in reagent water
and dilute to 1 liter. Wash the solution with methylene chloride and
hexane before use.
3. Sodium thiosulfate, granular.
4. Sulfuric add, concentrated.
5. Methylene chloride, pesticide quality or equivalent.
6. Hexane, pesticide quality or equivalent.
II 1-334
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7. Benzene, pesticide quality or equivalent.
8. • Tetradecane, pesticide quality or equivalent.
9. Sodium sulfate, anhydrous, granular (ACS). Purify by heating at 400°C
for 4 hours in a shallow tray.
10. Alumina, neutral, 80/200 mesh (Fisher Scientific Co., No. A-540 or
'equivalent). Activate for 24 hours at 130°C in a foil-covered glass
container before use.
11. Silica gel, 100/120 mesh, high purity grade. (Fisher Scientific Co.,
No. 5-679 or equivalent).
12. Stock standard solutions (1.00 pg/yl). Stock standard solutions can be
prepared from materials of known purity or purchased as certified
solutions.
12.1 Prepare stock standard solutions of 2,3.7,8-TCDD (mol. wt. 322),
and either 37C14TCDD (mol. wt. 328) or I3C12-TCDD (mol. wt. 332)
in a glove box 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 *lask. If comoound ourity
is certified at 96% or greater, cne weignt can oe used witnout
correction to calculate the concentration of tha itcck standard.
Commercially prepared stock standards can oe used at any concen-
tration if they are certified by the manufacturer or an independent
source.
12.2 Transfer the stock standard solutions into Teflon-sealed :crew-cap
bottles. Store in a glove box protected from light. Stock stand-
ard solutions should be checked frequently for signs of degradation
or evaporation, especially just prior to preparing calibration
standards or spiking solutions from them.
12.3 Stock standard solutions must be replaced after six months, or
sooner if comparison with check standards indicates a problem.
13. Internal standard spiking solutions (25 ng/ml). Using stock standard
solution, prepare a spjkinq solution by the appropriate dilution of
either 13C12-TCDD or J/C14-TCDD in acetone.
14. Sodium carbonate, anhydrous, powder.
6. QUALITY CONTROL
The minimum requirements'of a quality control program consist of an
initial demonstration of laboratory capability and the analysis of spiked
sameles as a continuing check on performance. Performance records should
be maintained to aeiine che quality jf generated aata.
-------
Before performing any analyses, the analyst should demonstrate the ability
to generate acceptable accuracy and precision with this method. This can
be accomplished by performing the following operations:
1. The analyst should demonstrate, through the analysis of a 1-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 to monitor pos-
sible laboratory contamination.
2. Using stock standard solution, prepare a quality control check sample
concentrate containing TCDD at a concentration of 15 ng/ml . Using a
pipet, add 1.00 ml of the check sample concentrate to each of a minimum of
four 1,000-ml aliquots of reagent water. A representative waste water 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 given in Subsection J.2.
3. Calculate the average percent recovery, (R), and the standard deviation
of the percent recovery, (s), for the replicate analyses. Vlastewater
background corrections must be made before R and s calculations are
performed.
4. Acceptable single-operator precision and accuracy data for the procedure
ara:
Spike Average Percent ______ Standard Deviation
TCDD 0.015 ' 83.7 5.1
TCDD 0.150 82.2 4.2
If the calculated R and s values are not comparable to those provided
above, the analyst must review potential problem areas and repeat the
tests.
5. The analyst must calculate method performance criteria and define the
performance of the laboratory for each spike concentration. Calculate
upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCD = R - 3 s
where R and s are the values calculated in paragraph 3 above. The UCL
and the LCL can be used to construct control charts7 that are useful in
observing performance trends.
6. The laboratory should develop and maintain separate accuracy statements
of 1aboratory oerfomancs *or »ach samole matrix. An accuracy statement
for the method is defined as R ± s and snoula oe developed cased on the
III-336
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analysis of four aliquots as described in paragraphs 2 and 3. These
accuracy statements should be updated regularly.7
7. Spiked samples should be prepared and analyzed at a frequency of 10% of
the total sample loading, or one sample per month, whichever percentage
is greater. The spiked samples must be analyzed as indicated in paragraphs
2 and 3. If the TCDD recovery does not fall within the control limits for
method performance, the TCDD results reported for all samples processed as
part of the same set must be qualified as suspect. The frequency of
generation of suspect data should be monitored and corrective action
should be taken if the frequency exceeds 5 percent.
Additional quality assurance practices should be adopted by the laboratory
as needed. Examples are the analysis of field duplicates to monitor the pre-
cision of the sampling techniques, the analysis of standard reference materials,
or participation in relevant performance evaluation studies.
In recognition of the rapid advances that are occurring in chromatography,
it is realized that procedural modifications may be implemented to improve
separations or lower analytical costs. Each time analytical methods are modi-
fied, the ability to generate data of acceptable quality must be demonstrated.
H. CALIBRATION
Establish gas chromatoqraphic conditions ror the GC/MS system equivalant
to those indicated 'n ^abla 1. The GC/MS system ,-nust be calibrated using the
.internal standard technique. By injecting calibration stanaaras, establish ion
response factors for TCDD vs. an internal standard (either ^C^-TCDD or
-;C'.tt-"C3D), !.?e Tubsecticn -1.2*1,7 for GC/MS Specific ion monitoring condi-
tions for both mgh-resolution and iow-resoiuc'ion ,,iass ^pecirometr'
1. Using stock standards, prepare GC/MS calibration standard solutions in
hexane or tetradecane that will allow measurement of relative response
factors of at least three concentration ratios of "TCDD to -internal
standard. Each solution must be prepared to contain the internal
standard at a concentration of 25 ng/ml. If- significant interferences
are contributed by the internal standard at m/e 320 and 322, the
concentration should be reduced in the calibration standards and in the
internal standard spiking solution (Subsection F.13.). One of the
calibration standard solutions should be prepared to contain TCDD
representing a concsntration near, but above, the method detection limit.
The other TCDD concentrations should cover tne range of concentrations
expected in the samples to be analyzed.
2. Using injections of 2 to 5 yl, tabulate m/e area responses against the
concentration of TCDD and internal standard in each calibration standard,
and calculate response factors (RF) for TCDD using Equation 1.
(As) (C1s)
^F = Eq. 1
III-337
-------
where
As = SIM response for TCDO m/e 320
A-jS = SIM response for internal standard
("C1?-TCDD at m/e 332 or
37CTj-TCDD at m/e 328)
(3
Cis = concentration of the internal standard, yg/1
Cs = concentration of TCDD, yg/1.
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 factors, As/AiS vs. Cs.
I. DAILY PERFORMANCE TESTS
The working calibration curve or RF must be verified on each working day
by the measurement of one or more TCDD calibration standards. If the response
for TCDD varies from the predicted response by more than 10 percent, the test
must be reoeated using a fresh calibration standard. Alternatively, a new
on curve .r.ust oa preparad.
oefore using any cleanup procedure, the inalyst must process a series of
calibration standards through the procedure to validate elution patterns and
the absence of interferences from the reagents.
TABLE 1. CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION LIMIT
(Water Samples)8
===================================== 3=^============ ===========================
Retention Time Detection Limit
Method (min) (yg/D
Glass Capillary Column 34.5 0.002
= = = ss ==z = s = = ===3: = ==3==:5=== »= ==== ==== = === = = = === === = = = = === = = = = = = = = = = = = = = = = = = = = = = =
Column conditions: SILAR-10C coated on a 50 m x 0,25 mm I.D. glass column
vor equivalent), with helium carrier gas at 30 cm/sec l-'near velocity, split-
less injector. Column temperature programmed: isothermal, 100°C for 3
minutes, then 20°C/min to 180°C, and 2°C/min to 250°C.
111-338
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J. ANALYTICAL PROCEDURES
1.1 Analysis of Hazardous Waste Samples for Dioxin. Reserved.
111-339
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2.1 Analysis of Water Samples for Dioxin
Analytical Procedure: available
Sample Preparation: available
2.1.1 Reference
U.S. Environmental Protection Agency, "2,3,7,8-Tetrachloro-
dibenzp-p-dioxin—Method 613," Methods for Organic Chemical
Analysis of Municipal and Industrial Wastewater. U.S. EPA,
EPA-600/4-82-057, 1982.
2.1.2 Method Summary
A 1-liter sample of water is spiked with an internal standard
of labeled TCDD. The spiked sample is then extracted with
methylene chloride using separatory funnel techniques. The
extract is concentrated, solvent-exchanged into hexane and
concentrated to a volume of 1.0 ml or less.
Capillary column GC/MS conditions are described that allow
for the separation and measurement of TCDD in the extract.
2.1.3 Applicability
This is a gas cnromatograpmc/mass spectrometnc »3C/MSJ
method applicable to. the determination of TCDD *n municipal
and industrial discharges. The range of.the procedure Can 5e
modified"by adjusting the sample size and/or the degree of
extract concentration.
This metnod shoulc be restricted co use only by or under the
supervision of analysts experienced in the use of gas
chromatograph/mass spectrometers and skilled in the interpreta-
tion of mass spectra. Each analyst should demonstrate the ability
to generate acceptable results with this method using the quality
assurance procedures in Subsection G.
2.1.4 Precision and Accuracy
The method detection limit (MDL) for TCDD using this procedure
is 0.002 ug/1 (MDL is the minimum concentration that can be
measured and reported witn 99% confidence tnat the value is
greater than zero). The reported MDL was determined on a
secondarily-treated sewage effluent. This value may vary
with sample matrix.
Single-operator recoveries from reagent water fortified at 0.005
ug/1, industrial wastewater fortified at 0.005 ug/1, and munic-
ipal wastewater fortified at 0.025 ug/1 were 95.4 ± 10.2%, 85.8
± 6.6%, and 92.4 ± 18.7%, respectively (R ±o).8
III-340
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2.1.5 Sample Extraction
CAUTION: It is recommended that all of the following
operations be performed in a limited access laboratory with
the analyst wearing full protective covering for all exposed
skin surfaces.
Depending on the sample bottle, either mark the liquid level
on the bottle for later volume determination or pour nearly
1 liter of sample into a 1-liter graduated cylinder, recording
the sample volume. Pour the sample into a 2-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. Spike each sample with 20 nanograms of internal
standard.
Rinse the sample bottle or graduated cylinder with 60 ml
methylene chloride and add the solvent to the sample in the
separatory funnel. Extract the sample by shaking the funnel
for 2 minutes with periodic venting to release vapor 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 size of the solvent layer, the analyst
•nust employ mechanical techniaues *,o romolete the phase sepa-
ration. The optimum tecnnique depends upon cne sample, uut may
include stirring, filtration of the emulsion through -jluss wool,
or centrifugation. Collect the metnylene cnioride extract in a
'• ; 250-ml Erlenmeyer flask.
Ado a seccna 60-uii volume if ,-neth'y• era irv.or-.ca :s cne
separatory funnel and complete che extraction procedure a
second time, combining the extracts in the 250-ml flask.
Perform a third extraction in the .same manner.
Discard the extracted aqueous portion of the sample,
retaining the separatory funnel for washing steps. Pour the
combined extracts through a glass funnel containing anhydrous
sodium sulfate, and collect it in a 500-ml Kuderna-Danish
(K-D) flask equipped with a 10-ml concentrator tube. Rinse
the Erlenmeyer flask and funnel with 20 to 30 ml methylene
chloride to complete the quantitative transfer.
Add 1 or 2 clean boiling chips to the flask and attach a
three-ball Snyder column. Prewet the Snyder column by adding
about 2 ml methylene chloride to the top. Place the K-D
apparatus on a hot water bath (60 to 65°C) so that the concen-
trator 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
temperture as required co complete ;ne concentration in 15 :c 20
III-341
-------
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 1 ml, remove the
K-D apparatus and allow it to drain for at least 10 minutes
while cooling.
Momentarily remove the Snyder column, add 50 ml hexane and a
new boiling chip to the K-D flask, and replace the Snyder
column. Increase the temperature of the water bath to
85 to 90°C. Prewet the Snyder column by adding about 1 ml
hexane to the top. Evaporate the solvent to a volume of
approximately 1 ml, remove the K-D apparatus from the water
bath and allow it to drain and cool for at least 1C) minutes.
Remove the Snyder column and rinse the flask and its lower
joint into the concentrator tube with 1 to 2 ml hexane.
Rinse the 2-liter separatory funnel used in the initial
extraction twice with reagent water and discard the wash.
Pour the hexane extract from the concentrator tube into the
separatory funnel. Rinse the concentrator tube four times
with 10-ml aliquots of hexane. Combine all of the rinses in
the separatory funnel.
Add 50 ml of 10 N sodium nydroxiae solution co cne funnel and
shake for 30 to 60 seconds. Discard the aqueous phase.
Wash the organic layer with 50 ml reagent water. Discard the
aaueous ohase.
•
Wash the hexane layer with ar least two 30-ml aliquots of
concentrated 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.
Wash the hexane layer with two 50-ml aliquots of reagent
water. Discard the aqueous phases.
Prepare a glass funnel packed with sodium sulfate. Pour the
hexane extract through the funnel and collect in the K-D
flask used in the first concentration. Rinse the separatory
funnel with several hexane washes. Pour each wash through
the sodium sulfate column and collect in the K-D flask to
effect a quantitative transfer.
Add 1 or 2 clean boiling chips and concentrate the extract to
approximately 1 ml. Rinse the K-D reservoir with a few ml of
hexane and collect the solvent in the concentrator tube.
Concentrate the extract to 1 ml by blowing down under a stream
of clean nitrogen. Stopper ;rie .concentrator tube vith 2 ^l
III-342
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stopper and store refrigerated and protected from light if
GC/MS analysis or cleanup will not be performed immediately.
If the original sample volume was not measured in a graduated
cylinder, refill the sample bottle to the marked level and
measure the volume taken.
2.1.6 Sample Cleanup
Cleanup procedures may not be required for relatively clean
sample matrices. The cleanup procedures recommended in this
method have been used for the analysis of various clean water
and industrial effluent samples The single-operator precision
and accuracy data presented were obtained using the recommended
cleanup procedures.
The alumina column is frequently used to overcome interferences
and the silica gel column has been used to overcome background
problems. Other cleanup procedures have been described to
overcome special interference problems.3-6
Alumina Column Cleanup
Fill a 300-mm x iO-mm-I.D. chromatograpny column ?ntn actv/ataa
alumina to the 150-mm level, tapping the column gently to
settle the alumina. Add 10 mm anhydrous sodium sulfate on top
of the alumina.
Preeiuie :he column with 30 nl '-.exane at ; -ate :f 1 •nl/min.
Discard the eluate and, just prior to exposure of the sodium
sulfate layer to the air, transfer the 1.0-ml sample extract
onto the column. Use two 2-ml washes of the concentrator
tube with hexane to complete a quantitative transfer.
Just prior to exposure of the sodium sulfate layer to the air
during elution, add 50 ml of 3% methylene chloride/97% hexane
(v/v) and continue the elution of the column. Discard the
eluate.
Elute the column with 50 ml of 20% methylene chloride/80%
hexane (v/v) into a 500-mi K-D flask equipped with a 10-ml
concentrator tube. Concentrate the sample to 1 ml using
standard K-D techniques. Analyze by GC/MS (see paragraph 2.1.7)
Silica Gel Cleanup
Fill a 400-mrn x 11-mm-I.D. chromatography column with silica gel
to the 300-mm level, tapping the column gently to settle the
silica gel.
Add 10 mm anhydrous sodiunTsulfate to the top of the silica
gel.
TII-343
-------
Preelute the column with 50 ml of 20% benzene/80% hexane (v/v)
at a rate of 1 ml/min. Discard the eluate and, just prior to
exposure of the sodium sulfate layer to the air, transfer the
extract to the column. Rinse the extract container with
two 2-ml washes of 20% benzene/80% hexane and transfer the
washes to the column.
Just prior to exposure of the sodium sulfate layer to air
during elution, add 40 ml of 20% benzene/80% (v/v) hexane to the
column. Collect the eluate in a clean 500-ml K-D flask equipped
with a 10-ml concentrator tube.
Concentrate the eluate to 1.0 ml using standard K-D techniques.
Analyze by GC/MS (see paragraph 2.1.7).
2.1.7 GC/MS Analysis
Calibrate the GC/MS system daily as described 1n Subsection H.
The volume of calibration standard must be measured or be
the same as all sample injection volumes. A volume of 2 to 5
ul is suggested.
Analyze standards and samples with the mass spectrometer
operating in the selected ion monitoring (SIM) mode, using a
dwell time to give at least seven points per peak, "or low
resolution-mass spectrometry (LRMS), use ions at m/e 320, 322,
arid 257 for 2,-3,7,8-TCDD and either the ion at m/e 328 for
37Cl4-2,3,7,8-TCDD or m/e 332 for 13C12-2,3,7,8-TCDD. For
high-resolution mass spectrometry (HRPIS), use ions at m/e
319.3965 ana 321.2936 ror :,3,?,3-TC3D and sither the «on
at m/e 327.8847 for -37C!4-2,3,7,8-TCDD or the ion at m/e
331.9367 for 13C12-2,3,7,8-TCDD. Total and Extracted Ion
Current Profiles for TCDD are presented in Figure 2. Electron
capture GC (GC/ECD) may be used to screen samples and to elimi-
nate samples from GC/MS analysis if the results from GC/ECD
analysis are below the GC/MS detection limit. An example of a
GC/ECD chromatogram for two TCDD isomers 1s shown 1n Figure 3.
GC/ECD should not be used for quantisation of samples above
the GC/MS detection limit.
The method performance data reported in Subsection G were gath-
ered using a final extract volume of i.O ml. 'If lower detection
limits are required, the extract may be concentrated further
by carefully evaporating the extract to dryness under a gentle
stream of nitrogen with the concentrator tube in a water bath
at 40°C. Redissolve the extract 1n the final desired volume
of hexane or tetradecane.
The following criteria must be met to make a qualitative
Identification of TCDD:
III-344
-------
Normalized Instrument Response
U'J
rt -.« cort- -n 4* to ro H- -^ _»o> O vl
2" "H ^J=r 0 ... • 10 4k O O W
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rt ii> — i ro 01 3 ro fire i re — •• oi o» ro
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Oin DI 3*3 cu re —••—•• in Q. Q. n> — •
rere TDrt- reo> 03 rt-o *< 3
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-*• Qi O 3 —»_.._•. o rt- -*• O> -•• 3 *" ™
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3r* ire Dirt--*. »«3 3-*- r> "^
reo« o> — • 3 n 3 r*3 i ro
re -•• >-vi re«<3-reo-'. 3-rt-ro cu »*
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co o-f nro 3 o ro rt o-uro o -*
*" ~> C3 C -t» -«•*< -J 3 . ._
en -«cr 001 in -O3 33— ( HI u>"
rJ'O »-i rtrtro oi oico r» 2*
rort ro 3-Oirtrt- 3 .-j CD
o;3 o cos ~~^ 3-0 ro
"••». rt-<» ujroaimre i/aire 3 '
inro 3- -jroxrt xirt-oi rt- -^
— ' rert re -'•.oico rerix* <,,
3 o> - 1, <-•• ro o _
res -i *r -irt -'• 3- — • -t. C
f-Ol -~^ «-»»CLO OOiOl -•. r+
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oa.ro 3- -i rt rt -». re «»-*
< ooco oi 3- -•.-..< m o"
«>co ro3rtoiO3re w
• roTio rrrts o
rore ore i/irtrt-t,
ci cu -a -i o» 3-0
rx — '. A- re 3 rtore -H
roo o»re-^ -hrto
33 -*»TC x> re 3 03- a
wo e ~v. .-* QI re o
t.' -j-t> oi co re 3- — • .
«r o — !ro ro -»• -* oo
C T O CO cr 3
rt rt ro Oi -j rt i
—I o -a ro 3 oi ro
o ro oi rt -»
i
H
SL
o".
O
c
3
**
TJ
"t
C^
—
- in
JZZ5*
/*^
/
*
f
c rn
1 x
V i-»
s 5
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r *^
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S a
\ 6"
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V °
J C
^ ^
> -- CO
> SE
\ « -o
J* i' *"V
-------
Column: 6' x 2 mm I.D. glass, 3% OV-210 on 80/100 Supelcoport.
Carrier: 5% methane/argon
Flow rate: 27 ml/min.
Column temperature: 180°C isothermal
Injector temperature: 235°C
Detector temperature: 300*C
Retention Time - Minutes
Figure 3. Electron capture detector cnromatogram of 45 ng/ml 1,2,3.4-TCDD
i5.26 minutes) and SO ,ig/ml :,3,7,3-TCDD ;3.78 rninuces).-
II1-346
-------
co-eluting impurity may be suspected. In this case, another
set of ions characteristic of the TCDD molecule should be ana-
lyzed. A good second choice of ions is m/e 257 and m/e 259
for TCDD and the related ions for the internal standard.
These ions are in a cluster indicating the loss of one chlorine
and one carbonyl group from TCDD. Suspected impurities such
as DDE, ODD, or PCB residues can be confirmed by checking for
their major fragments, but may require another injection using
different SIM ions or full repetitive mass scans. If the
response for 37ClA-TCDD Is too high, PCB contamination at m/e
328 can be checked by using the PCB ion at m/e 326. These con-
taminants can be removed by alumina column cleanup.
If broad background interference restricts the sensitivity of
the GC/MS analysis, the analyst should employ additional
cleanup procedures and reanalyze by GC/MS. In those circum-
stances where cleanup procedures do not yield a definitive
conclusion, the use of high-resolution mass spectrometry is
suggested.*
111-347
-------
2.2 Analysis of Water Samples for TCDD
Analytical Procedure: evaluated
Sample Preparation: evaluated
2.2.1 Reference
Harless, R. L., E. 0. Oswald, W. K. Wilkinson, A. E. Dupuy,
D. D. McDaniel, and H. Tai, "Sample Preparation and Gas
Chromatography - Mass Spectrometry Determination of
2,3,7,8-Tetrachlorod1benzo-p-dioxin." Anal. Chem. 52:
1239-1245 (1980).* ~
2.2.2 Method Summary
One liter of water is fortified with isotopically-labeled
TCDD and extracted with multiple portions of methylene
chloride. The residue 1s solvent-exchanged into hexane
and washed with KOH, sulfuric acid, and reagent water.
Following neutralization and drying, the extract is concen-
trated and cleaned up on an alumina column. The extract is
passed through a second alumina column, solvent-exchanged
into benzene, and analyzed by GC/MS.
2.2.2 \pplicability
This procedure is applicaole to the determination of TCDD in
aqueous samples. The 1 imte of detection is affected by
sample size, percent recovery during extraction, sample
effects, ?nd electronic noise during analysis.
2.2.4 Precision and Accuracy
The described method produced a mean recovery of 80% for
2.5 to 10 ng 37ClA-TCDD (internal standard) and a mean accuracy
of ±23% for 1 to 1250 pg TCDD in quality assurance samples. The
precision of the GC/high resolution mass spectrometry (GC/HRMS)
technique was determined to be ±20% relative to 2 to 10 pg
TCDD quantification standards during daily operations.
2.2.5 Sample Preparation
Thoroughly mix the original water sample and transfer 1 liter
a 2000-ml separatory funnel. Fortify the sample with 2.5 ng
surrogate standard and mix.
Extract the sample with three 100-ml portions of methyl ene
chloride. Combine the extracts In a 500-ml flask. Add a
Snyder column and evaporate the extract to dryness on a steam
bath. Dissolve the residue in 100 ml hexane and transfer
nuantitativelv into a seoaratorv funnel.
II1-348
-------
2.2.6 Extract Cleanup
Wash the hexane extract with 25 ml IN KOH. Discard the
aqueous phase.
Wash the sample with four 50-ml portions of concentrated
sulfuric acid. Discard the acid wash.
Wash the sample with 25 ml of reagent water. Neutralize the
sample mixture by the addition of powdered N32C03. Separate
the phases and discard the aqueous layer.
Transfer the extract to a drying column consisting of a 1-cm
I.D. x 50 cm glass column containing 10 cm of anhydrous
powdered Na£C03. Collect the hexane in a Kuderna-Danish
evaporative concentrator. Concentrate the sample to a volume
of 3 ml.
Prepare two alumina chromatographic columns by packing 4.5 cm
of neutral alumina into a clean and dry 15-cm x 0.5-cm
disposable Pasteur pipet. Top the alumina with 0.5 cm
anhydrous granular NapSO,;. Wash the column with 4 ml of
methylene chloride and force residual solvent from the column
using a stream of ary n'troaen. 'Store the jolumns 'n an oven,
at 225°C for a minimum of 24 hours. Prior to use, equilibrate
the columns to room temperature by olacement in a aesiccator
containing Drierite.
Prewet one of the columns with 1 ml of hexane- Transfer the
sample concentrate to tne coiumn. tlute tne coiumn *itn
6 ml carbon tetrachionde ana discara tne eiuate. £lute
the column with 4 ml methylene chloride and collect the
eiuate in a 12-ml distillation receiver.
Add a carborundum boiling chip to the receiver and cap it with
a micro-Snyder column. Place the apparatus in a hot water bath
and evaporate the methylene chloride just to dryness.
Add 2 ml hexane to the residue and evaporate the sample just
to dryness. Repeat the process with a second 2 ml portion
of hexane.
Dissolve the residue in 3 ml hexane and transfer the sample
to the second alumina column that has been prewet with
1 ml hexane.
Elute the column with 6 ml carbon tetrachloride and discard
the eiuate. Elute the column with 4 ml methylene chloride
and collect the eiuate in a clean 12-ml distillation receiver.
III-349
-------
Using a hot water bath, evaporate the methylene chloride just
to dryness. Add 2 ml benzene to the receiver and concentrate the
extract to a volume of 100 pi.
Quantitatively transfer the sample to a 2-ml graduated Chroma-
flex sample tube (Kontes Glass Company, K-422560). Utilizing
a slow stream of dry nitrogen, carefully concentrate the
benzene solution to a final volume of 60 yl.
If the analyses are not to be completed immediately, seal the
extract in glass tubing (3 mm x 7 cm) and store at subzero
temperatures.
III-350
-------
3.1- Analysis of Methylene Chloride Extracts of Sediment/Soil Samples for
2,3,7,8-TCOO
Analytical Procedure: evaluated
Sample Preparation: evaluated
3.1.1 Reference
Harless, R. L., E. 0. Oswald, M. K. Wilkinson, A. E. Dupuy,
D. D. McDanlel, and H. Tai , "Sample Preparation and Gas
Chromatography - Mass Spectrometry Determination of
2,3,7,8-Tetrachlorodibenzo-p-dioxin." Anal. Chem. 52:
1239-1245 (1980). 4
3.1.2 Method Summary
Ten to twenty g of well-mixed soil/sediment is fortified with
Isotopically labeled TCDD and placed in a 100-ml boiling
flask. The sample is then refluxed for 2.5 hours with alcoholic
KOH and extracted with multiple portions of methylene chloride.
The residue is solvent-exchanged into hexane and washed with
KOH, sulfuric acid, and reagent water. Following neutraliza-
tion and drying, the extract is concentrated and cleaned up on
a series of alumina columns. The residue is solvent-exchanged
into benzene, concentrated, and analyzed by £C/MS.
3.1.3 Applicability
This procedure is applicable to the determinations of TCDD in
solid-chase samoles such as soil or sediment. The limit of
detection is affected oy sample *e:gnt ana j-;se, percent
recovery during extraction, sample matrix affects, and instru-
ment noise during analysis.
3.1.4 Precision and Accuracy
The described method produced a mean recovery of 80% for
2.5 to 10 ng 37C1^-TCDD (internal standard) and a mean accuracy
of ±23% for 1 to 1250 pg TCDD 1n quality assurance samples.
The precision of the GC-HRMS technique was determined to be ±20%
relative to 2 to 10 pg TCDD quantification standards during daily
operations.
3.1.5 Sample Preparation
Remove extraneous objects from the sample and mix thoroughly.
Weigh out a 10 to 20 g portion of the homogenized sample and
transfer to a 100-ml boiling flask.
Add 5 to 10 ng of ^'Cla-TCDD surrogate standard, 20 ml ethyl
alcohol, and 40 ml of 45% ootassium hydroxide solution to the
sample. Heat the sample and rerlux anile stirring r'or J.5 rsours.
III-351
-------
After cooling, transfer the mixture to a separatory funnel.
Rinse the flask with 20 ml ethyl alcohol and add to the
separatory funnel. Rinse the flask with 20 ml hexane and add
the wash to the sample.
Extract the sample with 25 ml hexane. Transfer the hexane
layer to a clean separatory funnel. Repeat the sample
extraction with three additional 25-ml portions of hexane.
Combine the hexane extracts.
3.1.6 Extract Cleanup
Wash the combined hexane extracts with 25 ml IN KOH. Discard the
aqueous phase.
Wash the sample with four 50-ml portions of concentrated
sulfuric acid. Discard the acid wash.
Wash the sample with 25 ml reagent water. Neutralize the sample
mixture by the addition of powdered N32C03. Separate the phases
and discard the aqueous layer.
Pass the hexane layer through a drying column consisting of
a 1-om-I.D. x 50-cm glass column containing 10 crn anhydrous
powdered Na2C03. Collect the hexane in a Kuderna-Oamsn
avaporative concentrator. Concentrate the sample to a volume
of 3 ml.
-------
Dissolve the residue in 3 ml hexane and transfer the sample
to the second alumina column that has been prewet with 1 ml
hexane. Elute with 6 ml carbon tetrachloride and discard
the eluate. Elute the column with 4 ml methylene chloride
and collect the eluate in a 12-ml distillation receiver.
Using a hot water bath, evaporate the methylene chloride just
to dryness. Add 2 ml benzene to the receiver and concen-
trate the extract to a volume of 100 yl. Quantitatively
transfer the sample to a 2-ml graduated Chromaflex sample tube
(Kontes Glass Company, K-422560). Utilizing a slow stream of
dry nitrogen, carefully concentrate the benzene solution to a
final volume of 60 yl.
If the analyses are not to be completed immediately, seal the
extract in glass tubing (3 mm I.D. x 7 cm) and store at subzero
temperatures.
-------
3.2 Determination of 2,3,7,8-TCDD in Methanol Extracts of Soil and
Sediment
Analytical Procedure: evaluated
Sample Preparation: available
3.2.1 Reference
U.S. Environmental Protection Agency, "Determination of
2,3,7,8-TCDD in Soil and Sediment." U.S. EPA, Region VII
Laboratory, Kansas City, Kansas. 37 p. February 1983.H
3.2.2 Method Summary
A 10-gram soil sample is spiked with an internal standard
of isotopically-labeled 2,3,7,8-TCDD. The wet sample is mixed
with 20 grams anhydrous sodium sulfate prior to extraction
with hexane/methanol using a jar extraction technique. Optional
cleanup procedures to aid in the elimination of interferences
that may be encountered are provided. The extract is concen-
trated to a volume of 0.10 ml. Capillary column GC/MS condi-
tions are described which allow for the separation and measure-
ment of 2,3,7,8-TCDD in the extract.
3.2.3 Apolicabillty
This method is. intended for use In the determination of
2,3,7,8-TCDD in soil and sediment at levels of 1 part per
biTlion and higher. The method is'specific for the 2,3,7,8-
TCDD isomer, since it employs capillary columns which separate
that isomer from :he ;tner 21 "CD ''corners. ~ots1 T.DD :zn
also be estimated by this method. Determination of other
specific TCDD isomers depends on the availability of the
specific Isomer and the separation from other interfering
Isomers. The final measurement process utiMras low resolution
mass spectrometry. Because of the increased possibility for
Interferences at levels below 1 part per billion, the user is
cautioned in extending the method range below that concen-
tration.
This method should be restricted to use only by or under the
supervision of analysts experienced 1n the use of gas
chromatography/mass spectrometry and skil'ed in the inter-
pretation of mass spectra.
3.2.4 Precision and Accuracy
The nominal detection limit for this method is 1.0 part per
billion. However, for certain samples this detection limit
may not be achievable because of interferences. On other
relatively clean samples, the estimated detection limit may be
iower.
II1-354
-------
The following method recovery values for isotopically labeled
2,3,7,8-TCDD from fortified soil-samples have been reported in
three different laboratories (mean recovery ± one standard devia-
tion): Lab A, 70 ± 12%, 50 data points; Lab B, 59 ± 23%, 85 data
points; Lab C, 72 ± 16%, 11 data points.
In Table 2, data are presented indicating the method precision
based on duplicate analyses. The data are presented for the same
lab running the same sample (intralab precision), different labs
running samples taken at the same place at the same time
(interlab precision), and different labs running samples taken
at the same place but on different days (total precision).
3.2.5 Phase Separation
.CAUTION: When using this method to analyze for 2,3,7,8-TCDD,
all of the following operations should be performed in a
containment laboratory with the analyst wearing full protective
covering for all exposed skin surfaces.10
An initial centrifugation step is provided for the phase
separation of very wet soil or sediment samples. If used,
analyze the separated water phase using procedures specified
for water V3ubs2cf;sn J -.) and continue processing the solid
phase. If phase separation is not used, proceed to the sample
extraction step.
Place a 30-g aliquot in a suitable centrifuge bottle. Place
;he sampie*ana i jsuntsr-balanes ** i "rsntr^yae. Centrifuge
the samp!a for 20 ninutss ?t 2000 rom. Remove the sample ana
mark the phase interface on the bottle to estimate the relative
volume of each phase. Using disposable pipettes, transfer the
liquid layer into a clean bottle for analysis as a water sample.
3.2.6 Sample Extraction
Two procedures are provided for extracting dioxins from the
soil or sediment matrix. Option A is a relatively simple jar
extraction with methanol and hexane. Option B is a more
rigorous Soxhlet extraction with toluene.
Option A
Transfer a 10-g aliquot of the solid sample directly into an
extraction jar (250 ml capacity, or larger).
Add 1 ml of a 2.5rng/ml solution of isotopically-labeled 2,3,7,8-
TCDD directly to the soil. The isotopically-labeled TCDD
should be added at several sites over the surface of the soil.
Add 20 q of solvent-extracted anhydrous sodium suifate ana mix
tnorougniy using a -jtainlass :tee! :pcon or icatula. ^llow
III-355
-------
TABLE 2. SUMMARY OF DUPLICATE 2,3,7,8-TCDD RESULTS
(In parts per billion)
Paired Results Mean Result Percent Relative Difference
<1
1.4;
0.9,
1.5,
29.3,
1.8,
0.5,
(Intralab)
<1
0.5
1.2
1.5
20.0
1.1
0.4
—
0.95
1.05
1.5
24.65
1.45
0.45
0
95
29
0
38
' 48
22
(Interlab)
118, 110 114 7
<1, <1 0
104, 65 d4.o 46
175, 170 172 3
1.5, 4.4 2.95 98
0.4, 1.1 0.75 93
9.2, 8.5 8.85 ' 8
24, 27.3 * 25.3 \:
20.i, 15.4 17.75 26
14.5, 13 13.75 11
(Total)
7.0, 9.0 8.0 25
7.0, 3.9 5.45 57
9.0, 3.9 6.45 79
24.7, 1.5 13.1 177
118, 270 194 78
110, 270 190 84
50, 52 51 4
140, 240 190 53
2.6, 0.9 1.75 97
<1, 0.4 - 0
<1, <1 0
<1, <1 • 0
<1, <1 - 0
<1, <1 0
II1-356
-------
the mixture to stand under ambient conditions. Mix again after
2 hours and allow to stand for at least 6 hours. Should the
soil/sodium sulfate mixture form lumps which cannot be easily
broken, it will be necessary to grind the mixture in a glass mortar
with a glass pestle.
Mix the soil/sodium sulfate mixture just before adding solvent.
Add 20 ml methanol, stir, and then add 150 ml hexane.
Seal the sample and place the jar on a wrist-action shaker,
platform shaker, magnetic stirrer, or equivalent device.
Extract the sample vigorously for a minimum of 3 hours.
Prepare a glass funnel with solvent-rinsed filter paper (Whatman
No. 4 or equivalent). Filter the sample extract. Thoroughly
rinse the extraction jar, its contents, and the filter residue
with hexane. Combine the filtered rinses with the sample extract.
Concentrate the hexane/methanol extract to 1 ml using Kuderna-
Danish or rotary evaporator techniques. When using rotary
evaporator concentration techniques, care must be taken to
carefully rinse the apparatus between samples to prevent cross-
contamination of samples. The extract is now ready for cleanup.
Option B
Thoroughly clean the Soxhlet apparatus oy operating ror 2 flours
with toluene prior to use. '
Transfer a 10-g aliquot of sampie airectly . nto j jir.^b: e
glass container sucn as" a Dea«er or flask. Add 1 .nl of a
25-ng/ml solution of isotopically labeled 2,3,7,8-TCDO directly
to the sample. The isotope should be added at several sites
over the surface of the sample.
Add 20 g of solvent-extracted anhydrous sodium sulfate and mix
thoroughly using a stainless steel spoon or spatula. Allow the
mixture to stand under ambient conditions. Mix again after 2 hours
and allow to stand for at least an additional 6 hours. Mix again
just before transferring the sample to an extraction thimble.
Add 10 g of anhydrous sodium sulfate to the extraction thimoie.
Transfer the sample (soil plus sodium sulfate) to the thimble
and cover with a layer of clean glass wool. Rinse the container
with toluene and transfer the washing to the extraction apparatus.
Put 250 ml of pesticide-grade toluene into the extractor.
Operate the apparatus for a minimum of 25 cycles.
Transfer the toluene extract to a 500-ml round-bottom flask.
Rinse the extraction apparatus with iwo £Q-ini /oiumes of
toluene and add the cashes to the extract. Concentrate the
III-357
-------
extract in a rotary evaporator (Biichi/Brinkman, or equivalent)
at 60 to 70*C under a vacuum of 23 to 27 inches of mercury. When
the toluene volume has been reduced to 2 to 3 ml, stop the evapor-
ation. Transfer the extract to an 8-ml glass culture tube for
further concentration. This is accomplished on an N-Evap
Analytical Evaporator (Organomation Associates, Inc..) at 50*C
with a gentle stream of filtered nitrogen.
Rinse the round-bottom flask with at least three 3-rnl volumes
of methylene chloride. Transfer each rinse to the culture
tube for concentration with the extract. When the volume of
the extract has been reduced to 200 to 300 pi, add 1 ml hexane
to the culture tube. Continue concentrating until the volume is
reduced to 200 to 300 ul. Add 1 ml hexane and reduce the extract
volume to 200 to 300 ul. The concentrated hexane extract is now
ready for cleanup.
3.2.7 Sample Extract Cleanup
Cleanup procedures may not be necessary for a relatively clean
sample matrix (i.e., sandy soils). However, most sample types
will require some cleanup. Extract cleanup must be performed
if any of the following conditions are observed:
1. The samole extract cannot be concentrated to 1 ml.
2. Interferences prevent observation or measurement of the
isotopically-labeled 2,3,7,8-TCDO..
3. Interferences are present in the retention time winaow dt
mass 320, 322, or 257.
4. The required detection limit of 1 ppb cannot be achieved.
5. The sample extract is extremely dark colored and viscous.
The following cleanup options are recommended. Other cleanup
procedures may be used if the isotopically-labeled 2,3,7,8-
TCDD recovery is consistently greater than 50%.
Option A
Pack a 1-cm I.D. x 10-cm chromatography column with 1 g
silica gel and 4 g of 40% w/w sulfuric acid/modified silica gel.
Pack a second chromatography column (1 cm I.D. x 30 cm) with
6 g alumina and top with a 1-cm layer of sodium sulfate.
Add hexane to the columns until they are free of channels or
air bubbles. This can be readily achieved using a small
positive pressure (5 psi) of clean nitrogen.
Drain the hexane to just aoove the cop or the silica get ana pi ace
the hexane extract on *OD of +he silica qel. Rinse the extract
III-358
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container with two 0.5-ml volumes of hexane and add to the
column. Elute the extract from the silica gel column directly
onto the alumina column with 45 ml hexane. Discard the
silica gel.
Place 20 ml hexane on the alumina column and elute until the
liquid has dropped below the sodium sulfate layer. Discard
the eluted hexane.
Elute the column with 20 ml of 20% (v/v) methyl ene chloride/
hexane and collect in a 125-ml Erlenmeyer flask.
Reduce the volume of TCDD-containing eluent using a gentle
stream of filtered nitrogen. When the volume has been
reduced to 1 to 2 ml , transfer aliquots, one at a time, to a
2-ml conical mini-vial for further concentration until the
entire fraction has been transferred. Rinse the Erlenmeyer
flask with 1 ml hexane and transfer the rinse to the mini-
vial. Repeat the rinsing procedure. The contents of the
mini-vial must not be allowed to go to dryness during the
extract concentration process. Finally, rinse the walls of
the mini-vial with 500 ul hexane. Store the extract in a
freezer until analysis. Just prior to analysis, reduce the
hexane volume almost to dryness, and add toluene to achieve a
final volume of 100 ul.
Option B
a glass macro-column (2 cm O.D. x 23 cm, and tapered 1
cm). ^acK tne jcuain *rcn i ;;i;g ;f r:l irn'ied ^lass wool.
followed successively oy 1 g silica, 2 g ""h'ca containing
33% (w/w) 1 M NaOH, 1 g silica, 4 g silica containing 44%
(w/w) concentrated HgSO^, and 2 g silica. Add hexane co the
column until it is free of channels or air bubbles. Quan-
titatively transfer the concentrated sample extract to the
column and elute with 45 ml hexane. Collect the entire eluate
and concentrate to a volume of less than 1 ml in a centrifuge
tube.
Construct a chromatography column by packing a 5-ml disposable
pipet (cut off at the 2-ml mark) with a plug of silanized
glass wool and add 1 g activated Woeim oas-ic alumina 'activated
at 600*C for 24 hours) to the tube.
Quantitatively transfer the concentrated extract to the top of
the column using 2 ml hexane.
Elute the column with 5 ml 3% methyl ene chloride in hexane
(v/v) and retain the entire column eluate for analysis.
Elute both columns vmh £0 :ni 30% methylene chloride fv/y)
'r\ s,exane ^nd retain the eluates for analysis.
III-359
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Combine the eluates and concentrate to a volume of less than
1 ml. Quantitatively transfer the extract to a 2-ml conical
mini-vial. Concentrate the extract to near dryness and store
at 5°C.
Prior to GC/MS analysis reconstitute the extract by adding
toluene and adjusting the final volume to 100 yl.
Option C
Certain very dirty samples may require preliminary cleanup
prior to column chromatography. For those situations, the
following procedure is suggested.
Wash the organic extract with 30 ml 20-percent aqueous
potassium hydroxide by shaking for 10 minutes. Remove and
discard the aqueous layer.
Wash the organic extract with 25 ml doubly-distilled water'
by shaking for 2 minutes. Remove and discard the aqueous layer.
CAUTIOUSLY add 50 ml concentrated sulfuric acid to the organic
extract and shake for 10 minutes. Allow the mixture to stand
antil the aaueous and orqanic layers seoarate (approximately
10 minutes). Remove ana discard cne aqueous .ayer. Repeat
cne acid washing procsdure until ,10-color is visible in the
acid layer.
Add 25 ml doubly-distilled water-to the organic extract and
snaice r'or 2. nnnut3£. '.emcve ana alscara *he -iquaous "-.yer.
Add 10 g anhydrous sodium sulfate :o the extract to dry the
organic layer.
Transfer the organic extract to a centrifuge tube. Concentrate
to near dryness by placing the tube in a water bath at 55°C
while passing a gentle stream of filtered, prepurified nitrogen
over the surface of the extract. Reconstitute in hexane
before proceeding with the column chromatography (either
Option A or Option B).
3.2.8 GC/MS Analysis
Table 3 summarizes typical gas chromatographic capillary
columns and operating conditions. Other columns and/or
conditions may be used as long as isomer specificity is
demonstrated by the introduction of a mixture containing all
22 TCDD isomers. -Thereafter, a calibration mixture containing
fewer isomers should be analyzed on a daily basis in order to
verify the performance of the system.
III-360
-------
TABLE 3. RECOMMENDED GC CAPILLARY CONDITIONS
=======================
Column
2,3,7,8-TCDD R.T.
Helium Linear Velocity
Initial Temperature
Initial Time
Split! ess Time
Program Rate
Final Temperature
Final Hold Time
.Split Flow
Septum Purge Flow
Capillary Head. Pressure
A (Silar IOC)
34.5 min
30 cm/sec
100'C
3 min
20°C/min
180°C**
15 min
====================
B (SP2340)
22 min
0.7 ml /min
at 608C
60'C
3 min
1 min
25°C/min
250'C
15 min
30 ml /min
j .til /min
30 psl
====================
C (DB-5)*
13 min
1 ml /min
80°C
1/min
1 min
15'C/min
300°C
15 min
30 ml /min
." -n /mi n
15 psi
not'soecific for the 2,3,7,8-TCDD isomer.
"then 2"/min to 250UC.5
Calibrate the analytical system daily as described in Sub-
section H. The volume of calibration standard injected must
be measured, or be the same for all injections.
Immediately before analysis, adjust the sample extract volume
to 100 ul.
At the option of the analyst, a standard amount of isotopically
labeled 2,3,7,8-TCDD (different from the one used as a surrogate)
may be added to the extract tojponitor variations in instrument
sensitivity. For example, if ""u^-TCDD :s used as the
surrogate, then ^Cjo-TCDD (about 25 ng) can be added to the
extract just before GC/MS analysis. The response from this
standard can be used to calculate percent recovery of the
Isotopically-labeled surrogate. It must not be used to compute
the concentration .of native TCDD.
Analyze standards and samples with the mass spectrometer
loeratino in the selected ion monitoring (SIM) mode using a
dwell time co ^tve at ^aast ieven points per oeak. r">r LRMS.
-jse Ions st ij/e 320, 322, and 257 for 2,3,7,8-TCDD and either
III-361
-------
the 1on at m/e 328 for 37r/]-TCDD or m/e 332 for 13r,-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-TCDD or m/e 331.9367
for 13C-TCDD.
In order to achieve the stated detection limit, the instrumen-
tation must be sensitive enough to provide a signal for all
three ions in at least a 2.5-to 1 signal-to-noise ratio for an
injection of 100 picograms.
If a lower detection limit is required, the extract may be
carefully evaporated to dryness under a gentle stream of
nitrogen with the concentrator tube in a water bath at about
40°C. This must be done immediately before GC/MS analysis.
Redissolve the extract in the desired final volume of solvent.
Inject a l-to-5 til aliquot of the sample extract.
The presence of 2,3,7,8-TCDD is qualitatively confirmed if
the following criteria are met:
1. Isomer specificity is demonstrated and verified.
2. The 320/322 ratio i< within -.he -2nge of 0.57 to 0.87.
3. Ions 320, 322, and 257 must all be present and produce maxi-
mum instrument response simultaneously. The signal to noise
ratio must be 2.5-to-l or better for all 3 ions.
4. The elusion time or native £,., ,7,6-7CDD ,nust equal iwi^nin 3
seconds) the elution time r'or che isotopicaily labeled
2,3,7,8-TCDD.
5. Five percent of the positive samples should be confirmed
by HRMS. Alternately, 5 percent of the positives can be
confirmed by obtaining partial scan spectra from mass 150 to
mass 350.
For quantitation, measure the response of the m/e 32
2,3,7,8-TCDD and the rn^e 332 peak for 13C12-2,3,7,8-
the m/e 328 peak for 37C1d-2,3,7,8-TCDD. Calculate
320 peak for
-TCDD or
the
concentration of native 2,3,7,8-TCDD using the response factor
(RF) and the following equation:
Concentration, ng/g = {As)(Is)/(Ais)(RF)(VO
where:
AS = SIM resoonse for 2,3,7,8-TCDD ion at m/e 320
III-362
-------
A^s = SIM response for the internal standard ion
m/e 328 or 332
Is = Amount of internal standard added to each
sample (ng)
W = Weight of soil in grams
Co-el uting Impurities are suspected if all criteria except the
isotope ratio criteria are achieved. In this case, another
SIM analysis 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, then the presence
of TCDD can be confirmed. Co-eluting ODD, DDE, and PCB residues
can be confirmed, but will require another injection using the
appropriate SIM ions or full repetitive mass scans. If the
response for 37C!-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 37C1 -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.
If broad background interference restricts the sensitivity of
the GC/MS analysis, the analyst should employ additional
cleanup procedures and reanalyze by GC/MS.
In those ffrrjTr.s tineas whe^e these orocedures do not yield a
definitive conclusion, then the use of mgn- resolution ,nass
spectrometry is suggested.
III-363
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4.1 Analysis of Hexane Extracts .of Biological Tissue for TCDD
Analytical Procedure: evaluated
Sample Preparation: evaluated
4.1.1 Reference
Harless, R. L., E. 0. Oswald, M. K. Wilkinson, A. E. Oupuy,
D. D. McDaniel, and H. Tai, "Sample Preparation and
Gas Chromatography/Mass Spectrometry Determination of
2,3,7,8-Tetrachlorodibenzo-p-dioxin." Anal. Chem. 52:
1239-1245 (1980).4 ~
4.1.2 Method Summary
Fish tissue is digested in alcoholic KOH for 2.5 hours. The
sample is then subjected to multiple extractions with hexane.
The combined hexane extracts are washed with KOH, sulfuric
acid, and water. Following neutralization and drying, the
extract is concentrated and cleaned up on an alumina column.
The extract is passed through a second alumina column,
exchanged into benzene, and analyzed by GC/MS.
4.1.3 Applicability
This procedure is applicable to the determination of TCDD in
fish and other lean tissue. The limit of detection is
affected by sample weight and size, percent recovery during
extraction, sample matrix effects, and electronic noise
during analysis.
4.1.4 Precision and Accuracy
The methodology produced a mean recovery of 80% for 2.5 to 10 ng
37Cl4-TCDD (internal standard) and a mean accuracy of ±23% for
1 to 1250 pg TCDD in quality assurance samples. The precision of
the GC-HRMS technique was determined to be ±20% at TCDD levels of
2 to 10 pg during daily operations.
4.1.5 Sample Preparation
Blend the fish tissue in a blender to homogenize the sample.
Weigh out a 10- to 20-g portion of cne blended tissue and
transfer to a 100-ml boiling flask.
Add 5 to 10 ng of 3?C1-TCDD surrogate standard, 20 ml ethyl
alcohol, and 40 ml 45% potassium hydroxide solution. Heat the
sample and reflux, while stirring for 2.5 hours.
After cooling, transfer the mixture to a separatory funnel. Rinse
the flask with 10 ml ethyl alcohol and add the rinse to the sample
III-364
-------
mixture. Rinse the flask with 20 ml hexane and add the wash to
the sample mixture.
Extract the sample with 25 ml hexane. Transfer the hexane layer
to a clean separatory funnel. Repeat the sample extraction with
three additional 25-ml portions of hexane. Combine the hexane
extracts.
4.1.6 Extract Cleanup
Wash the combined hexane extracts with 25 ml IN KOH. Discard the
aqueous phase.
Wash the sample with four 50 ml-portions of concentrated sulfuric
acid. Discard the acid wash.
Wash the sample with 25 ml reagent water. Neutralize the sample
mixture by the addition of powdered N32C03. Separate the phases
and discard the aqueous layer.
Pass the hexane layer through a drying column consisting of a
1-cm-I.D. x 50-cm glass column containing 10 cm anhydrous powdered
Na^COs. Collect the hexane in a Kuderna-Danish evaporative concen-
trator. Concentrate tne sample tc -, 'oiume of 3 7,1.
Prepare two alumina chromatographic columns by packing i,5 cm
neutral alumina into a clean and dry 15-cm x 0.5-cm disposable
• Pasteur pipet. Top the alumina with 0.5 cm anhydrous granular
,-ia2^<\' ^asn ;r.e coi'jrr.n
-------
El lite the column with 6 ml carbon tetrachloride and discard the
eluate. Elute the column with 4 ml methylene chloride and collect
the eluate in a 12-ml distillation receiver.
Using a hot water bath, evaporate the methylene chloride just to
dryness. Add 2 ml benzene to the receiver and concentrate the
extract to a volume of 100 ul• Quantitatively transfer the sample
to a 2 ml-graduated Chromaflex sample tube (Kontes Glass Company,
K-422560). Utilizing a slow stream of dry nitrogen, carefully
concentrate the benzene solution to a final volume of 60 yl.
If the analyses are not to be completed immediately, seal the
extract in glass tubing (3 mm I.D. x 7 cm) and store at subzero
temperatures.
K. CALCULATIONS
Calculate the concentration of TCDD in the sample using the response
factor (RF) determined in Subsection H and Equation 2.
(AS)(IS)
Concentration (pg/1 or ug/g) = • Eq. 2
(Ais)(RF)(V0)
where:
As = SIM response for TCDD ion at m/e 322
A-JS - SIM -esponse *or the 'nternal standard "on at m/e 322
Is = Amount of internal standard added to each extract (»g)
V0 = Sample volume (liters) or mass (grams).
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.
Report results in micrograms per liter or micrograms per gram. When
duplicate and spiked samples are analyzed, report all data obtained with the
sample results.
For samples processed as part of a set for which the spiked sample
recovery falls outside the control limits established in Subsection G, or for
which the internal standard recovery is below 50%, the data for TCDD must be
labeled as suspect.
111-366
-------
L. WIPE TEST PROCEDURE
Use the bottom of a 1-quart paint can to determine the size of the area
to be wipe-tested. The diameter of the paint can corresponds to an area of
about 85 cm2.
Moisten an ashless 12.5-cm diameter piece of filter paper (Whatman 41 or
equivalent) with 1 ml of hexane and wipe the desired area using a circular
motion.
Fold the paper and place in a glass funnel in the manner the paper would
normally be used for filtering. Elute the paper with two 5-ml portions of
hexane, collecting the hexane ^n a 2-oz. bottle.
Concentrate the hexane to approximately 1 ml by blow-down.
Wash the hexane by shaking with 1 ml concentrated sulfuric acid.
Transfer the hexane to a 1-ml autosampler vial for analysis by electron
capture GC. The detection limit of the test is on the order of 1 ug of
2,3,7,8-TCDD per wipe.° Less than 1 ug per sample indicates acceptable clean-
liness'; anything higher warrants further cleanup.8 More than 10 ug on a wipe
sample indicates an acute hazard which requires prompt remedial attention.8
REFERENCES
1. u.j. cnv'ironmeniai Protection Agency. <"!-?seri"at-!on ind Maximum
Time for the Priority °ollutants." U.S. €PA. environmental Monitoring
and Support Laboratory, Cincinnati, Ohio. (In preparation).
2. American Society for Testing Materials. "Standard Practice for Prepara-
tion of Sample Containers and Preservation." ASTM Annual Book of Stand
ards, Part 31, D 3694. ASTM, Philadelphia, Pennsylvania, p. 679
(1980).
3. U.S. Environmental Protection Agency. "Analytically Determined Method
Detection Limits for Priority Pollutants. Methodology as Method
Performance Criteria." U.S. EPA, Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio. ,'In preparation).
4. Harless, R. L., E. 0. Oswald, M. K. Wilkinson, A. E. Dupuy, D. D.
McDaniel, and H. Tai . "Sample Preparation and Gas Chromatography/Mass
Spectrometry Determination of 2,3,7,8-Tetrachlorodibenzo-p-dioxin."
Anal. Chem. 52: 1239-1245 (1980).
5. Lamparski, L. L. and Nestrick, T. J. "Determination of Tetra-, Hepta-,
and
per
,
and Octachlorodibenzo-p-dioxin Isomers in Paniculate Samples at Parts
per Trillion Bevels." Anal. Chem. 52: 2045-2054 '1980).
III-367
-------
6. Longhorst, M. L. and L. A. Shadoff. "Determination of Parts-per-Trillion
Concentrations of Tetra-, Hexa-, and Octachlorodibenzo-p-dioxins in Human
Milk." Anal. Chem. 5£: 2037-2044 (1980).
7. U.S. Environmental Protection Agency. "Handbook of Analytical Quality
Control in Water and Wastewater Laboratories." U.S. EPA, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio. EPA-600/4-079-019
(March, 1979).
8. U.S. Environmental Protection Agency. "2,3,7,8-Tetrachlorodibenzo-p-
dioxin—Method 613", Methods for Organic Chemical Analysis of Municipal
and Industrial Wastewater, Longbottom, J. E., and J. L. Lichtenburg, Eds.,
U.S. EPA, EPA-600/4-82-057, 1982.
9. U.S. Environmental Protection Agency. "Determination of 2,3,7,8-
Tetrachlorodibenzo-p-Dioxin (2,3,7,8-TCDD) in Water," U.S. EPA, National
Enforcement Investigation Center, Denver, Colorado, p. 19 (No date).
10. National Research Council. Committee on Hazardous Substances in the
Laboratory. "Prudent Practices for Handling Hazardous Chemicals in
Laboratories," National Academy Press, Washington, D. C., 1981.
11. U.S. Environmental protection Agency. "Determination of 2,3,7,8-TCDD
1-n Soi* and Sediment." ;j.3. ~?A, Region VII Laboratory, Kansas City
Kansas. 37 p. February 1983.
III-368
-------
SECTION 9
METHODS FOR THE DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS
A. SCOPE
Polycyclic aromatic hydrocarbons (PAHs) are also referred to as poly-
nuclear aromatic hydrocarbons or polycyclic aromatic compounds. To avoid
undertainty, the nomenclature suggested by Bartle et al.* for naming PAH
compounds is used throughout this section. These compounds are usually deter-
mined by a chromatographic technique. Due to the sensitivity and potential
selectivity of the ultraviolet absorbance/fluorescence detection in analysis of
solutions of these compounds2, a high-performance liquid chromatographic (HPLC)
method- is presented here. Several PAH compounds may also be determined by
base/neutral extraction-gas chromatography mass/spectrometry (GC/MS) (Sec-
_tion 3). However, packed-column gas chromatograohy does not adeauately resolve
the following four pairs of compounds: antr.racsne ana phenar.thrsne, :hrysene and
benzo[a]anthracsne, benzcCb^ucranthene and benzoC'O^luoranthene > or 'libenzo-
[ahjantnracene and indenoti ,£,3-cajpyrene. fusee siiica capillary column GC/MS
can separate these four pairs of compounds, but anthracene and phenanthrene,
chrysene and benzo[a]anthracene, and benzo[b]fluoranthene and b'enzo[k]fluor-
used cc analyze unfamiliar samples, compound 1 dent If •'"cations should be sup-
ported by at least one additional qualitative technique. Thin layer chroma-
tography with fluorescence detection is appropriate for this use. Fluorescence
spectra of collected fractions can also be '.still zed ^or confirmation of
identification.^
B. SAMPLE HANDLING AND STORAGE
Due to the variable nature of the sample matrix, both hazardous waste
sampling methods and sample handling, and storage procedures for polycyclic
aromatic hydrocarbon determinations are quite matrix-dependent. Because of the
potential photoreactive nature of the compounds, ail samples should^be pro-
tected from intense light. 5
Grab samples for water analysis must be collected in glass containers. 6
Conventional sampling practices? should be followed, except that the bottles
should not be prerinsed with sample before collection. 6 Composite water
samples should be collected in glass containers and samples should be kept
refrigerated during the compositing period. 6 Automated equipment should be as
free as oossible of potential sources of contamination. Samples should be
refrigerated at -^C r'rom ihe i'rne jf ;oilecfion ':nti"! processing. Csmoles,
extracts. ?nd standards should be stored in amber or foil -wrapped bottles to
III-369
-------
minimize photolytic decomposition. If residual chlorine is suspected of
being present in an aqueous sample, sodium thiosulfate in excess (10%) of
that needed to neutralize the chlorine should be added. If necessary, a
field test kit for measurement of chlorine may be used on a separate sample
aliquot to determine the amount of sodium thiosulfate required. Addition of
250 mg/liter of sodium thiosulfate will neutralize 5 mg/liter chlorine. All
samples should be extracted within 7 days of collection and analysis must be
completed within 40 days of sampling. 6
Samples of soil or sediment should be stored by freezing. 8 Sediment
samples may be acidified with hydrochloric acid to prevent bacterial decom-
position of the PAHS. 9 Mercuric chloride can be used for the same purpose. 10
Samples can be freeze-driedll«12,13 or air-dried at ambient or elevated
temperatures. 9 .14 ,15, 16
Tissue samples (both shellfish and vegetable) should be stored in opaque
or foil-wrapped containers to prevent photolytic decomposition. Shellfish
can be externally cleaned, shucked and drained of excess fluid. A repre-
sentative 4- to 5- gram portion can be dried at 80°C for 48 hours for deter-
mination of moisture content. 14,15, 16 All work with tissue samples should
be performed under subdued yellow tungsten light. 17, 18
Particulate samples to be analyzed for oolycyclic aromatic hydrocarbons
are usually collectad using glass fjbar "v.tars -ind hign-v'olume air samplers. 5
Approximately *Q mg of participates ^hould be ".oil acted. The samples should
be protected from exposure to light.— Samples dra fairly staole for up to 1
year if stored in the dark and refrigerated. 5
C. INTERFERENCES
1. Method Interferences
Method interferences caused by contaminants in solvents, reagents,
glassware, and other hardware used in sample processing may lead to
discrete artifacts and/or elevated baselines in the chromatograms. All
of these materials must be routinely demonstrated to be free from inter-
ferences under the conditions of the analysis by running laboratory
reagent blanks. Glassware must be scrupulously cleaned. 20 clean all
glasswar3, as soon as possible after jse by rinsing *ith 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, except
volumetric glassware 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. Glassware can also be cleaned with chromic
acid cleaning solution, followed by rinsing with distilled water, oven-
•iry'ng it !RO°C, and -o1vent---insinq.'- After "leaning, ilassware ^ho'jld
be sealed and stored in a clean environment to prevent any accumulation
III-370
-------
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in-371
-------
of dust or other contaminants. Store glassware Inverted or capped with
aluminum foil.
2. Matrix Interferences
Matrix Interferences may be caused by contaminants that are coextracted
from the sample. The extent of matrix Interferences will vary consider-
ably from sample to sample. Cleanup procedures can be used to overcome
many of these Interferences, but unique samples may require additional
cleanup approaches to achieve the Method Detection Limit (MOD. The MDL
values for various PAH compounds are given 1n Table 1. For organisms,
or for sediment samples containing small levels of PAH contamination,
the dimethylsulfoxide (DMSO) partitioning sample cleanup (Subsection
J.3.5) should be performed after the column chromatographlc cleanup.
Use of the DMSO cleanup as a first step for such samples will result 1n
large volumes of solvents and formation of emulsions.22
D. SAFETY
The carcinogenic and mutagenlc properties of many polynuclear aromatic
hydrocarbons (PAH) are well established.21 Benzo[a]anthracene, benzo[a]-
pyrene, and d1benzo[ah]anthracene have been tentatively classified as known
or suspected human/mammalian carcinogens.6 Consequently, care must be
taken *o 3vo1d spilling solutions of PAH material on hands or other areas
of the skin. Manipulations Involving neat PAH compounds or concentrated
solutions should be performed In a fume hood and/or primary containment
area.5f23 Personnel performing these procedures must be familiar with
current Occupational Safety and Health Administration regulations regarding
•af<9 handling of £he °«.Hs. VH nersonnel Involved 1n the analysis must have
access to a reference file of material aata handling sneers pertaining co
these chemicals. Additional references dealing with iaboratory .safety
should be consulted to ascertain that the laboratory has an effective safety
program.23,24,25,26
I. APPARATUS
1. Sampling equipment, for discrete or composite water sampling (either of
the two options listed below 1s acceptable).
1.1 Grab sample bottle - Amber glass, 1-Hter or 1-quart volume,
fitted with screw caps lined with Teflon. Foil may be substi-
tuted for Teflon 1f the sample Is not corrosive. If amber bottles
are not available, protect the samples from light. The container
must be washed, rinsed with acetone or methylene chloride, and
dried before use.
1.2 Automatic sampler - 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 oerlstaltic pumo, a minimum length of compressible
sHlcone ruooer tuoing may oe usea. ^erore jse, ;ne ."ompressible
III-372
-------
TABLE 1. METHOD DETECTION LIMITS FOR SOME POLYNUCLEAR AROMATIC HYDROCARBONS
5=BSSS=S3SSS=SS=SSS=S=BSSBS===SB==S====== SS = = = = = = 5SB=S = S = = SSSBSSB = = B = = = = = = = = = = =
Compound MDL
Hazardous Waste Samples*27 (oil)
Benzo[a]pyrene
Naphthalene*
Acenaphthylene*
Acenaphthene*
Fluorene*
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo[a]anthracene
Chrysene
BenzoCbjf1uoranthene
Benzo[k]fluoranthene
Benzo[a]pyrene
Dibenzo[ah]anthracene
Benzo[ghi]pery1ene
Not available
Benzo[a]pyrene
Water Samples16
Soil/Sediment Samples
Tissue Samples*21
A1rSamples28
0.02 ug/g
1.8*
2.3*
1.8*
0.21*
0.64
0.66
0.21
0.27
0.013
0,15
j.JIS
0.017
j.323
0.030
0.076
0.1 ng/g
10 pg/m3
Benzo[a]pyrene
BSBBSSSBSSSSSBBSSBBSSSSSBSSSSSBSSBSSSSSSSSSSSSBSSSSSSSSSBSBS
*UV detection
Isocratic elution for 5 min with acetonitrile + water (4+6), linear grad
ient elution to 100 percent acetonitrile in 25 min; linear velocity = 2
mm/sec.
^Fluorescence detection, unless otherwise Indicated.
+Barley malt matrix
III-373
-------
tubing should be thoroughly rinsed with methanol, followed by
repeated rinsings with distilled water to minimize potential for
contamination of the sample. An integrating flow meter is required
to collect flow proportional composites.
2. High-volume air sampler (Haskin Scientific Ltd., Montreal, Canada, or
equivalent), with glass fiber filters, 20 x 25 cm (Gelman type A, or
equivalent) and recorder paper and ink.
3. Blender.
4. Ultrasonic vibrator (Branson DHA-1000, or equivalent).
5. Clinical centrifuge (International Model CL, or equivalent).
6. Wasserman clinical centrifuge tubes.
7. Rotary evaporator.
8. Chromatographic columns, 10 mm x 200 mm and 14.5 mm x 250 mm.
9. Analytical concentrator.
9.1 (N-Evap, or equivalent) or
9.2 Kuderna-Danish evaporative concentrator (K-D).
9.2.1 Micro-Snyder column for K-D.
10. '-Mgh-Per^crsanc.? L-i^u*c ^hromatogriDh (Perkin-Elmer Series 2/2, or eauiv-
alent), with columns 0.25 x 25 cm and preparative-scale oonaed polar
aminocyano column (for oil samples only) (Perkin-.Elmer ODS-HC SIL-X-1,
or equivalent), ultraviolet absorbance detector (Perkin-Elmer LC-55, or
equivalent), and filter fluorescence detector (Varian Fluorichrom, or
equivalent) or fluorescence spectrophotometer equipped with a flow cell
(Perkin-Elmer, 204A or equivalent), and 25-pl injection syringes (or
autosampler), chart recorders, and accessories.
11. Tri-carb Scintillation Counter Model C-2425, with Automatic External
Standard Option (Packard Institute Company, Downers Grove, Illinois)
or equivalent.
12. Circular metal punch, 46 mm diameter.
13. Analytical balance, with sensitivity of 0.1 mg.
14. Soxhlet extractor, with extraction thimbles.
15. Hotplate/stlrrer, combination.
15. r!ash 3vaoorator 'Siichner, or e-aulvalent).
111-374
-------
17. Separatory funnels, 125 ml, 250 ml, 500 ml, and 2,000 ml, with Teflon
stopcocks.
18. Amber vials, 10 to 15 ml capacity, with Teflon-lined caps or septa.
19. Flasks, pear-shaped, 300 and 500 ml.
20. Buchner funnel, with coarse-porosity fritted disc (Kontes K-955000, or
equivalent).
21. Water bath - Heated, capable of (±2°C) temperature control.
F. REAGENTS
1. Acetonitrile, HPLC grade.
2. Alumina - Aluminum oxide 90; active neutral (activity Stage I); particle
size 0.063-0.200 mm (70-230 mesh ASTM).
3. Benzo[a]pvrene (B[a]P), radio-labeled: 3H-B[a]P ca. 0.1 ng (25,000 dpm) per
sample; l^C-B[a]P (for high levels) ca. 3 ng (1,000 dpm) per sample.
4. Cyclohexane, distilled in .jlass [Burdic!: ind .lackson Snectrograde, or
equivalent).
5. Cyclohexanone, pesticide grade.
o. ueiomzeu water, intsrfar^ncs-'-^e,
7. Dimethyl sulfoxide, spectrophotometric grade.
8. Ethanol.
9. F1or1s1l, 60/100 mesh (Matheson, Colemen, and Bell, or equivalent).
10. Isooctane, distilled in glass.
11. Methanol, pesticide grade.
12. Methylene chloride, pesticide grade.
13. Permafluor V scintillation cocktail (Packard Inst. Co., Downers Grove
Illinois), or equivalent.
14. Potassium hydroxide pellets.
15. Sephadex LH-20.
15. 311ica yet 50 - Particle sfiG ^.363 *o 1.200 urn '70/230 mesh ASTM).
17. Sodium suifate, annyarous.
iii-J/3
-------
18. Sodium thiosulfate, granular (ACS).
19. Stock standard solutions - Stock standards can be prepared from pure
standard materials [phenanthrene (Phn), fluoranthene (F), pyrene (Py),
benzo[a]anthracene (B[a]A), benzo[b]fluoranthene (Bbf), benzo[e]pyrene
(B[e]Py), benz[a]pyrene (B[a]Py), dibenz[ah]anthracene (dB[ah]A),
benzo[b]chrysene (B[b]Ch), indeno[l,2,3-cd]pyrene (I[cd]Py), benzo[ghi]
perylene (B[ghi]P), dibenzo[ai]pyrene (dB[ai]Py), and corenone (Cor),
available from Div. of Chem. and Phys., FDA, Washington, D.C. Some PAH
compounds are also available from Chemical Repository, Illinois Insti-
tute of Technology Research Institute (IITRI; Chicago, Illinois)]. Store
in Teflon-sealed bottles, protected from light, at 4°C. If compound
purity as used is greater than 96 percent, the mass may be used without
correction in calculation of the concentration. Stock standard solutions
should be replaced after 6 months, or sooner if comparisons with check
standards indicate a problem.
G. QUALITY CONTROL
1. Any laboratory using these methods should operate a formal quality control
program. The minimum requirements of such a program consist of an initial
demonstration of laboratory capability and the analysis of spiked samples
as a continuing check on performance. The laboratory should maintain
performance records to define the quality of data that are generated.
Ongoing performance checks should be :onparsd with established performance
criteria to determine *f the results of analyses are within accuracy and
precision limits expected of ihe method-
2. Before performing any analyses, the analyst shoald demonstrate the ability
to janenta -ssultc ;f xccsptnbla ace-racy ind precision. Tlr's ^ay be
accomplished by analyses of four or more samnJes fortified, at a repre-
sentative concentration, with the compound(s) of interest.5
2.1 One or more unfortified samole(s) should be processed, to determine
background levels, and the level of fortification should be twice the
background level.
2.2 The average percent recovery (R) and standard deviation (s) of the
percent recovery should be calculated. These should then be compared
to the values for average recovery (X) and standard deviation (p)
expected for each parameter. If s > 2p or |X - R| > 2p, the analyst
should review potential sources of error in the procedure, and the
test should be repeated. The analysis should not be performed until
these parameters are satisfactory.
2.3 The results of the determination of the average recovery (R) and the
standard deviation (s) of the average recovery should be used to
calculate the Upper and Lower Control Limits:*9
Upper Control Limit (UCL) • R + 3s
Vower Control Limit 'LCD * * - 3s
III-376
-------
2.4 The UCL and LCL can be used-to construct control charts29 to assess
trends in analytical performance. Ten percent of the samples
processed should be collected in duplicate and analyzed as fortified
and unfortified samples in order to calculate-R and s.
2.5 The laboratory should ascertain that all glassware and reagent inter-
ferences are under control, by analyzing a reagent blank each time a
sample or set of samples is analyzed.
2.6 It is recommended that the laboratory adopt additional quality assur-
ance 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 thin-layer chromatography (TLC) or chromatography with a
dissimilar column or detector must be used (see Subsection K). This
may include the use of a mass spectrometer. Whenever possible, the
laboratory should perform analyses of standard reference materials
and participate in relevant performance evaluation studies.
2.7 In the analytical procedures for soil/sediment (Subsection J.3) and
for animal tissue (Subsection J.4.2) samples, use is made of a radio-
iabeied benzo[a]pyrene "ntarnal standard to "indicate method recov-
ery. 22 use of this internal standard in calculation of results
totally corrects for losses of benzoCa^pyrene during purification and
analysis.22 The application of the same recovery figure to other
PAHs in this method corrects for mechanical losses, dilution changes,
or otfier factors ^hich affect the samol"? as a whole, but does not
correct for any differential losses wmcn occur co a varying degree
in the compound(s) of interest.22 if substantial amounts of PAHs
compounds are present in the sample, l^C-labeled benzo[a]pyrene
is used, while for lower levels -^-labeled internal standard is more
appropriate.22
2.8 When using the vegetable tissue method (Subsection J.4.2), the
.recovery from the silica/alumina column chromatographic cleanup is
measured before use. Recoveries for each PAH should be greater than
or equal to 90 percent.21 For extract concentration, it is recom-
mended that extracts from samples containing the more volatile PAHs
(e.g. anthracene and biphenyl) be concentrated by use of a Kuderna-
Danish evaporative concentrator, to prevent losses.20
H. CALIBRATION
1. Liquid chromatograph operating parameters which produce separations as good
as those indicated in Table 1 and Figures 2 and 3 should be established.
Due to rapid advances being made in chromatography, the analyst may modify
operating parameters, as long as the modifications are not detrimental to
R, s (as defined -n Subsection G). or the separation.
III-377
-------
Column: HC-ODS SIL-X
Mobile phase: 40% to 100% Acetonitrile
in water
Detector: Fluorescence
1. Phenanthrene
2. Anthracene
3. Fluoranthene
4. Pyrene
5. Benzo [a] anthracene
6. Chrysene
7. Benzo [b] fluoranthene
8. Benzo [k] floranthene
9. Benzo [a] pyrene
"0. Oibenzo fa.h] anthracene
11. Benzo fg.h.i] peryiene
12. Indeno [1,2.3-cd] pyrene
1 1
4
i
8
i
12
I
16
i
20
i
24
i
28
I
32
•
36
Retention Time, minutes
Figure 2. Chromatogram of PAH standards (from reference 6).
III-378
-------
1. Naphthalene
2. Acenaphthalene
3. Acenaphthene
4. Fluorene
5. Phenanthrene
6. Anthracene
7. Fluoranthene
8. Pyrene
9. Benzo [a] anthracene
10. Chrysene
11. Benzo [b] fluoranthene
12. Benzo [k] fluoranthene
13. Benzo [a] pyrene
14. Dibenzo [a,h] anthracene
15. Benzo [g.h.i] perylene
16. Indeno [1,2.3-cd] pyrene
5 6
10
16
i I i I I I I I
0 4 8 12 16 20 24 28
Retention Time, minutes
32 36
Figure 3. Chromatogram of PAH compounds with JV detection.
' from ""^fersnc2 ^)
III-379
-------
2. Liquid Chromatograph External Standard Calibration
2.1 Prepare calibration standards at three or more concentrations for
each compound of interest by adding volumes of one or more stock
standard solutions to a volumetric flask and diluting to volume with
acetonitrile.
2.2 Inject 2.0 to 5.0 ul of each calibration standard and tabulate peak
height response or area response as a function of mass injected for
each compound for both ultraviolet absorbance and for fluorescence.
2.3 These results can be used to construct calibration curves. If the
response is linear (<10 percent relative standard deviation, RSD),
passage through the origin can be assumed and the average ratio of
peak height or area per mass can be used 1n place of a calibration
curve. The ratio of fluorescence to ultraviolet.absorbance response
factors should not change with time.
3. Liquid Chromatograph Internal Standard Calibration
Calculate the response factor as follows:
Response Factor (RF) = {AS)(C^S)/(A1-S)(CS) Eq. 1
where:
Ais = Response for the internal standard
As - .vasponsa ror ;n3 -amp]a
C^s = Concentration of the internal standard
Cs = Concentration of the sample.
4. The working calibration curve or the response factor should be verified on
each working day by analysis of calibration standards. If the responses
vary from the expected response by more than either ±10 percent (absolute
basis) or more than twice the RSD, the test should be repeated, using a
new calibration standard. Alternatively, a new calibration curve or
calibration factor should be prepared for the compound.
III-380
-------
I. ANALYTICAL PROCEDURES
1.1 Analysis of Hazardous Wastes for Polycyclic Aromatic Hydrocarbons
Analytical Procedure: available
Sample Preparation: available
1.1.1 Reference
Hertz, H. S., J. M. Brown, S. N. Chesler, F. R. Guenther, L. R.
Hilpert, W. £. May, R. M. Parris, and S. A. Wise, "Determination
of Individual Organic Compounds in Shale Oil." Analytical
Chemistry, 52:1650-1657. 1980.
1.1.2 Method Summary
This method is a chromatographic method which uses preparative
liquid chromatography followed by analytical-scale liquid chro-
matography. An oil sample is diluted and fractionated according
to the number of aromatic rings in the molecule. The concentrated
fractions are analyzed by reversed-phase high-performance liquid
chromatography with ultraviolet and fluorescence detection.
1.1.3 Applicability
This method has been demonstrated to De applicable *c ^ale-oil
samoies ana oetroleuro-contaminated marine sediments.26,27
1.1.4 Precision and Accuracy
Precision information /or ;ne prccsuure ': presented *n T2b1e ?,
The method detection limits are 1'stsd -'n Table 1.
TABLE 2. PRECISION FOR HPLC/HPtC ANALYSIS OF SHALE OIL
assssaszssssssssaaasasa333asssssa33=3=33ass3333333333:: 33=333=
Compound Concentration (ppm) Standard Deviation
Acridine 6 1.2
3enzo[a]pyrene 21 1.5
Fluoranthene 53 3
Pyrene 108 8
1.1.5 Sample Preparation
1.1.5.1 Dilution/Extraction
Dilute oil samples to a level of ca. 0.1 g/ml in methyl ene
cnlonae. extract ^.000 9 jr "less oily :ediment xith
111-381
-------
ultrasonic agitation for ca. 2 hours with ether, con-
centrate by evaporation, solvent-exchange the extract into
hexane, and concentrate to approximately 1 ml.
1.1.5.2 HPLC Fractionation
Inject an aliquot containing approximately 15 mg of sample
onto a preparative-scale aminosilane column. Elute the
sample with pentane^? or 2 percent methylene chloride in
hexane^O at a flow of ca. 5 ml/min. These conditions
result in a separation by the number of aromatic rings in
the PAH compound. Table 3 lists some PAH compounds and
their retention characteristics on the aminosilane
column.31 Use previous injections of standards of the
compounds of Interest to determine specific retention
volumes for the PAH compounds of interest. Collect frac-
tions of column effluent 1n centrifuge tubes, add 50 ul
acetonitrile, and reduce the volume to 50 ul. Figure 4
shows a typical fractionation.
1.1.6 High-Performance Liquid Chromatography
Quantify PAH compounds within each fraction (see Table 3) by HPLC
using an octadecy!silane column and a water-acetonitrile gradient
(50 to 100 percent acetonitrile 1n 30 mm at i ml/nun), tnnancea
sensitivity ?nd selectivity can oe ootauied oy use of both detec-
tors in series. See Table 6 (page HI-393) for ultraviolet absorb-
ance and fluorescence characteristics of several PAH compounds.
Figure 5 shows a chromatogram of a samole with both ultraviolet
absorbance and fluoresencs Uex - -i/0 nm; ±*m •= 400 nm) detection.
Figure 6 snows fluorescence spectra ootainea ror individual peaks
in the chromatograms in Figure 5 (Xex = 270 nm).
III-382
-------
TABLE 3. LOGARITHM OF RETENTION INDEX (I)
FOR SOME PAH COMPOUND$31
====================================================
log I
Octadecylsilane Aminosilane Column
Compound _ (sol vent: acetoni tri'1 e/water) (sol vent :hexane)
Two-Ring Aromatics
Naphthalene 2.00 2.00
Biphenyl 2.57 2.25
Acenaphthalene 2.73 2.00
Three-Ring Aromatics
Fluorene 2.78 2.61
Anthracene 3.02 2.95
Phsnanthrene • 3. CO 3.00
four-Ring Aromat-ics
Fluoranthene 3.42 3.39
2enro[a]*l'jorene 3.74 3.46
Benzo[b]f!uorene 3.78 3. S3
Pyrene 3.51 3.68
Naphthacene — 3.93
Benz[a]anthracene 4.00 4.00
Chrysene 3.94 4.03
Five-Ring and Larger Aromatics
Benzo[a]pyrene 4.57 4.30
Perylene 4.43 4.47
Benzo[ghi]pery1ene >5 4.61
Indeno[l,2,3-cd]pyrene >5 4.72
Dibenzo[ac]anthracene 4.84 4.33
D1benzo[ah]anthracene >5 4.93
III-383
-------
.Ill
Tim* ~f
Figure 4. HPLC fractional ion of an oily sediment sample extract with yBonda
pak NH2- Numbers refer to the fractions collected for subsequent
analysis. Conditions: pentane at 3 ml/min, 1.0 absorbance unit
full-scale (aufs), 1.0 ml injected (from Reference 31).
UV JN_^
2S4nm
Fluor*tc*nc«
•x: 270
Mn:4OO
10 20 30
Retention Tim*, minut**
40
Figure 5. Reversed-phase analysis of PAH fraction (4 in Figure 4).
Conditions: 50 to 100 percent acetonitrile in water, linear
gradient in 30 min at 2 ml/min., 0.05 aufs, 200 yl injected
(20 percent of fraction). Upper chromatogram: UV absorption
jetecv.on ut 254 -TH. '.jwer "hromatogram: "uorsscance emission
detection at 400 nm with excitation at 270 nm from Reference 27).
III-384
-------
(Numbers Indicate Wiv*4«ngth* in rim)
Figure 6. Fluorescence emission spectra at 270-nm excitation of peaks
1-7 1n Figure 5 (from Reference 26).
111-385
-------
2.1 Analysis for Polycyclic Aromatic Hydrocarbons In Water Samples
Analytical Procedure: available
Sample Preparation: available
2.1.1 Reference
U.S. Environmental Protection Agency, "Polynuclear Aromatic Hydro-
carbons - Method 610." Methods of Chemical Analysis of Municipal
and Industrial Wastewater, EPA-600/4-82-057, U.S. EPA (1982).6
2.1.2 Method Summary
A measured volume of water (ca. 1 liter) is solvent-extracted into
methylene chloride. The methylene chloride extract is dried and
concentrated to 10 ml or less, the solvent is exchanged to cyclo-
hexane, and cleanup (as necessary) is performed. Following cleanup,
the solvent is exchanged to acetonitrile and analysis is performed
by HPLC, using ultraviolet (UV) and fluorescence detectors. A
silica gel column cleanup procedure is provided to help alleviate
matrix interference problems.
2.1.3 Applicability
This method is a chromatograohic method applicable to the deter-
mination of the compounds listed ,n faDies 4 ana 5 in .vater :nd
wastewater samnles. Table 4 gives the method detection limits.
The actual method detection limits ootainea may vary with sample .
size, the extent of extract concentration, the nature of the inter-
ferences present, and the sample cleanup techniques used.
This method should only be used by analysts experienced in high-
performance liquid chromatographic techniques, or under the super-
vision of such personnel. Each analyst should demonstrate the
ability to generate results of acceptable precision and accuracy,
using the procedure of Subsection G.
2.1.4 Precision and Accuracy
The method detection limit (MOD is the minimum concentration of
analyte which can be measured and reported with 99 percent con-
fidence that the value is above zero. The MDL values listed in
Table 4 were obtained using reagent *ater, but similar results were
obtained using representative wastewater samples.
This procedure was evaluated by a single laboratory, using trip-
licate fortified samples analyzed on two different days.6 The
average recovery and standard deviation values are given in Table 5.
The method has been shown to give linearity of recovery from forti-
fied water samples over the concentration range of 8 times the MDL
to dOO times the MDL, except :hat benzo[nhi"]pery1ene -ecoveries at.
80 times MOL and 800 times MDL were 35 and 45 percent, respectively.0
II1-386
-------
METHOD DETECTION LIMITS FOR WASTEWATER SAMPLES6
=================:===========================================================-=;
Retention Time Capacity Factor Method Detection
Parameter (min) (k1) Limit (ug/1)a
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fl uoranthene
Pyrene
BenzoCalanthracene
Chrysene
Benzo[b]f 1 uoranthene
Benzo[k]fl uoranthene
Benzo[a]pyrene
Dibenzo[ah]anthracene
Benzo[
-------
TABLE 5. SINGLE-OPERATOR ACCURACY AND PRECISION FOR WASTEWATER SAMPLES6
B3SSS3a333333S33S3B3333:SSB3a33S33SS3«aa3a3aSaS33SB3S3S3aBBS3S33S33a33SaBa33SBS3
Parameter
Average
Percent
Recovery
Standard
Deviation
(*)
Spike
Range
(ug/1)
Number of
Analyses
Matrix
Types
Acenaphthene 88
Acenaphthylene 93
Anthracene 93
Benzo[a]anthracene 89
Benzo[a]pyrene 94
Benzo[b]fluoranthene 97
Benzo[ghi]perylene 86
Benzo[k]fl uoranthene 94
Chrysene 88
Dibenzo[ah]anthracene 87
Fluoranthene 116
Fluorene 90
Indeno[l,2,3-cd]pyrene 94
Naphthalene 78
Phenanthrene 98
Pyrene 96
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
11.6-
250-
7.9-
0.64-
0.21-
0.24-
0.42-
0.14-
2.0-
0.4-
0.3-
6.1-
0.96-
20-
3.8-
2.3-
-25
•450
•11.3
•0.66
•0.30
•0.30
•3.4
6.2
•6.8
•1.7
•2.2
•23
•1.4
•70
•5.0
•6.9
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
filtration of the emulsion through glass wool, centrifugation,
or other physical'methods. Collect the methylene chloride
extract m a tSC-rd I.-'ienmeyer .-'asjc.
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.
2.1.5.4 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 1f the requirements of Subsection G.2. are
met.
2.1.5.5 Pour the combined extracts through a drying column containing
10 cm anhydrous sodium sulfate, and collect the extract in the
K-D concentrator. Rinse the Erlenmeyer flask and column with
20 to 30 ml methylene chloride to complete the quantitative
transfer.
2.1.5.6 Add one or two clean boiling chips to the,evaporative flask
and attach a three-ball Snyder column. Prewet the Snyder
column oy adding aoout i mi .netnyiene chloride "o '.he '•.op.
°1ace the K-0 aooaratus on a hot water bath (60 to 65°C) so
III-388
-------
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 appara-
tus 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 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 and protected from light.
2.1.5.7 Determine the original sample volume by refilling the sample
bottle to the mark and transfering the water to a 1,000-ml
graduated cylinder. Record the sample volume to the nearest
5 ml.
2.1.6 Sample Cleanup
Cleanup procedures rcay not be necessary for a relatively clean sam-
ple matrix. The cleanup procedures ."^commended in :his method have
been used for the analysis of various clean waters and industrial
effluents.. If particular circumstances demand the use of an altern-
ative cleanup procadure, ^he analyst, tnould determine the elution
profile and demonstrate that the "«ccvery or eacn compound of
interest is no less than 85 percent.
2.1.6.1 Silica Gel Column Cleanup
Before the silica gel cleanup technique can be utilized, the
extract solvent must be exchanged to cyclohexane. 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 cyclo-
hexane 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-0 apparatus on a boiling (100'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 xlth ^ mini-
mum of cyclohexane. Adjust the extract volume to about 2 mi.
III-389
-------
Prepare a slurry of 10 g activated silica gel in methylene
chloride and place this in a 10-mm-I.D. chromatography column.
Gently tap the column to settle the silica gel and drain the
methylene chloride. Add 1 to 2 cm anhydrous sodium sulfate
to the top of the silica gel.
Preelute the column with 40 ml 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 cyclohexane to complete
the transfer.
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.
Elute the column with 25 ml methylene chloride/pentane (4 +
6, Y+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.
Concentrate the collected fraction to less than 10 ml by K-D
techniques as described in paragraph 2.6.1 using pentane to
'"'rise the walls of the
-------
Column: HC-ODS SIL-X
Mobile pha»0: 40% to 100% Acetonttrite
in water
Detector: Fluorescence
2
•
3
4
6. Bvnzo (•) amhrK»n*
6. ChryMn*
7 Bwuo (b) fluor*nth«n*
8 Bvfizo IK) flonntn^nv
9 Bvnzo [•) pyran*
10 OitMnzo (*.h) «ntt»r»c»n«
11 B*nm (g.h.ij p*rvton«
12. bvteno [1.2.3-cd] pyiww
I
8
I
16
I
20
12 16 20 24 28
Retention Time, minute*
32 36
Figure 7. Liquid chromatogram of polycyclic aromatic hydrocarbons
with Hjorsscsnca detacticn '*—;m r»fer*rc-s 6).
can be compared to spectra of standards recorded under the same con-
ditions, and can be useful for qualitative identification of
peaks.4.31,32 Table 6 lists some polycyclic aromatic hydrocarbons
and their fluorescence and absorbance characteristics in n-heptane
solution.31
Calibrate the system daily as described in Subsection H. 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 tne instrument. Inject 5 to 25 ul 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 yl, and the resulting peak size in height or area units.
Re-equilibrate the liquid chromatographic column at the initial
conditions for at least 10 minutes between injections.
The width of the retention time window used to make identifications
should be based upon measurements of actual retention time variations
of standards over ;ne course QT .; jsy. """hree *imes the standard
III-391
-------
1. Naphthalene
2. Acenaphthalene
3. Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo [a] anthracene
Chryiene
Benzo [b] fluoranthene
Banzo [k] fluoranthene
13. Benzo [a] pyrene
14. Dibenzo [a,h] anthracene
15. Benzo ffl.h.i] perylene
16. Indeno [1.2,3-cd] pyrene
4.
6.
6.
7
8
9
10
11
12
5 6
10
•1 13
15
1
0
i
4
i
8
i
12
t
16
i
20
*
24
28
32
36
Retention Time, minutes
Figure 8. Liquid chromatogram of polycyclic aromatic hydrocarbons with
ultraviolet detection (254 nm)(from reference 6).
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.
If the peak height or area exceeds the linear range of the system,
dilute the extract with acetonitrile and reanalyze. If the peak area
measurement ^s prevented by the oresence of interferences, further
cleanup 1s required.
111-392
-------
TABLE 6. FLUORESCENCE CHARACTERISTICS OF POLYCYCLIC AROMATIC HYDROCARBONS
IN N-HEPTANE AT 298 K
(from reference ?.}
RCRA
Emission Waste
Compound Maxima* (nm) Maxima* (nm) Number
Excitation
Maxima* (nm)
Acenaphthalene**
Acenaphthene
Anthracene
Benz[c]acridine
(3,4-Benzacridine)
Benz[a]anthracene 230,
(1,2-Benzanthracene)
Benz[jk]f1uorene 264,
(1,2-Benzfluorene)
Benz[a]phenanthrene
( 1 ,2-8enzpnenanthrene)
Benz[a]pyrene
(\ 2-Benzoyrene)
Benz[d]pyrene 265,
(3,4-Benzpyrene)
Biphenyl
Carbazole
(Dibenzpyrrole)
Chrysene
Dibenz[ah]anthracene 2B8,
(l,2:5,6-Dibenzanthracene)
Dibenz[ai]pyrene 241,
(1,2:7 ,8-Di benzpyrene)
3,4:9,10-Dibenzpyrene . 241,
Oibenz[ae]pyrene 228,
( 1 ,2 : 4 ,5-Dibenzpyrene)
229,
228,
253.
355,
277,
341,
285,
—
177,
316,
284,
362,
215,
235,
290.
267,
297.
338,
294,
352,
294,
357,
292,
355,
292,
291.
324,
375
287,
358
303,
268,
331
296,
383.
215
248,
318,
306,
319.
373
314,
371,
314,
371,
302,
J7T
300
338,
327
315
288
346
394
254
331
318
332
329
391
329
391
340
320,
321,
377,
446
385,
—
388.
403.
305,
333,
362, 380,
393,
490*
432,
397,
335,
335. 347
399. 422,
407, 432
345, 363
397, 409
427, 454
482
316
347
402, 424
403, 417
447, 459
447, 459,
490
418, 444
—
—
U016
U018
U120
U050
U022
—
—
—
U050
U063
U064
—
—
(continued)
III-393
-------
TABLE 6. (Continued)
RCRA
Excitation Emission Waste
Compound Maxima* (nm) Maxima* (nm) Number
Dibenz[ah]pyrene 270, 297, 310. 396 448. 477. 509
(3,4:8,9-Dibenzpyrene) 418
7,12-Dimethylbenz[a]- 298. 363, 382 407, 424 U094
anthracene
(7,12-Dimethyl-l,2-
benzanthracene)
Fluoranthene 237, 263, 280, 287 461 U021
324, 342, 358
Indeno[l,2,3-cd]pyrene — — U137
Benz[a]anthracene 230, 277, 2S7, 327 385; 407, 432 U018
rtapntnacene 27 1, 278, :93, !94. :?l, 504, 539
4T*. AAO, 469
Naphthalene - 225, 280 323. 335 U165
'eryiene ' ":*• , 336, 1Q8, 425 *38, *65, 449
Phenanthrene 253. 275, 292 346, 364. 385
Pyrene 240, 272, 305, 318, 372, 383, 394
334, 363
aaaasaaaaaaaaaaassaaaaaaaaaaBBasaaaaaBaaBSSBaaaaaaaaasaaaaaaasaaaBaaaaaaaBaaasa
*Most intense wavelengths underlined. Wavelengths determined using a 3-nm
spectral bandpass.
**F1 uorescence attributed to a photodecomposition product or impurity.
111-394
-------
3.1 Analysis of Sediments for'Polycycl'ic Aromatic Hydrocarbons
Analytical Procedure: available
Sample Preparation: evaluated
3.1.1 Reference
Dunn, B. P. and R. J. Armour, "Sample Extraction and Purification for
Determination of Polycyclic Aromatic Hydrocarbons by Reversed-Phase
Chromatography." Analytical Chemistry, 52:13 (1980), pp. 2027-2031.22
3.1.2 Method Summary
Hydrocarbons are extracted from sediment samples by alkaline diges-
tion, and interfering materials are removed by Florisil column clean-
up, dimethyl sulfoxide partitioning, and Sephadex LH-20 column chro-
matography. A radio!abeled internal standard is used to correct for
losses. Twenty polycyclic aromatic hydrocarbons are determined by
reversed-phase high-performance liquid chromatography, using simulta-
neous ultraviolet absorbance and fluorescence detectors.
3.1.3 Applicability
This method is applicable to wet or dry sediment samples.
3.1.4 Precision and Accuracy
The overall recovery of one polycyclic aromatic hydrocarbon, benzc-
[a]pyrene (B[a]P), in sediment' samples 1s 50 to 70 percent.11,12,13
Recoveries of B[a]P from the KOH digestion are generally over 90 per-
cent. --,-'-,-- 3[dj? .-eccver*2s "-en ~* ir*-.':"* column cleanuo are
generally over 90 percent.17 The relative standard deviation of the
method is 6.2 percent for B[a]P.l7
3.1.5 Sample Preparation
3.1.5.1 Sample Extraction
Decant and discard the water layer above the sediment. Weigh
and dry a representative sample for moisture determination.
Carry out all extraction and concentration procedures under
subdued yellow light.
Put 10 to 100 grams wet sediment, 100 ml ethanol, 5 g KOH,
and radioactive B[a]P (either 1,000 dpm 14C-B[a]P, ca. 5 ng,
or 25,000 dpm 3H-B[a]P, ca. 0.1 ng) into a flask. Add a
boiling stone and reflux. Swirl the contents of the flask
and pour Into 100-ml centrifuge tubes. Centrifuge the sus-
pension for 5 min at 500 g, decant the supernatant and wash
the particulate material twice with 50 ml ethanol followed
by stirring and resuspension, centrifugation, and decantation.
Combine the original supernatant, fc.he cashes, and 150 ml
water In a 500-ml seoaratory funnel. Extract three times with
II1-395
-------
200-ml portions of Isooctane. Combine the extracts and wash
four times with 200-nT! portions of warm (60*C) water.
3.1.5.2 Florlsil-Column Cleanup
3.1.5.2.1 Concentration
Reduce the volume of the Isooctane from the extraction to
approximately 10 ml. Use a Kuderna-Danish evaporative
concentrator if losses of the more volatile PAHs are of
concern. If not, a rotary evaporator 1s satisfactory for
this step. After volume reduction, dilute the extract to
100 ml with toluene.
3.1.5.2.1 Column Chromatography
Prepare column of 30 g Florisll (deactivated with 5 percent
water) covered with 60 g anhydrous sodium sulfate in a
glass chromatographic column (40 x 400 mm). Add 100 ml
toluene to the column and elute the toluene until the
liquid level is just at the top of the sodium sulfate layer.
Apply the sample (in toluene) and elute until the liquid
level is at the top of the sodium sulfate layer. Carefully
add 100 ml toluene, elute, add another 100 ml toluene, and
elute again.
<«
3.1.5.3 DMSO Partitioning
Add 5 ml dimethyl sulfoxide (DMSO) to the combined toluene
eiuate r'rom ;ne cc* Jinn and rcncsntrate the solution under
vacuum (at 60") to remove the toluene. Transfer the OMSO
solution to a separatory funnel containing 10 ml Isooctane,
rinse the evaporating flask with 5 ml DMSO, and add it to the
separatory funnel. Shake the mixture and drain the DMSO
layer into a second separatory funnel containing 40 ml water
and 20 ml Isooctane. Add 10 ml DMSO to the first separatory
funnel, shake the mixture, and drain the DMSO into the second
separatory funnel. Shake the second separatory funnel and
transfer the DMSO/water phase into a third separatory funnel
containing 20 ml isooctane. Shake the third funnel, then
discard the DMSO/water layer. Combine the Isooctane layers
from the second and tnird separatory funnels and wash twics
with 40 ml water.
Reduce the volume of the Isooctane solution to approximately
5 ml. Add 10 ml ethanol/toluene (10/90) and evaporate the
mixture to approximately 1 ml. It 1s not necessary to dry the
Isooctane prior to evaporation, as residual water from the
solvent is removed as an ethanol/toluene/water azeotrope
during evaporation. Use a K-D concentrator 1f losses of the
more volatile ?AH compounds are of concern.
II1-396
-------
3.1.5.4 Sephadex LH-20 Chromatography (Optional)
The elution volumes of the Sephadex column should be checked,
using a standard solution, before use and periodically
thereafter. The following procedure is written for elution of
the PAHs between 18 ml and 36 ml. Appropriate changes should
be made if the initial elution volumes differ, or if they
change due to settling of adsorbent.
Transfer the toluene extract from the DMSO partitioning into
a graduated 15-ml tube. Rinse the flask several times with
toluene and add the rinses to the extract. Reduce the volume
.of the extract to approximately 0.5 ml under a stream of dry
nitrogen with gentle wanning. Add an equal volume of ethanol
and apply the sample to a column of Sephadex LH-20 (bed size
10 mm x 200 mm) packed in toluene/ethanol (1/1). Draw the
material onto the column bed using gentle suction and elute
the column with 18 ml toluene/ethanol (1/1). Discard the first
18 ml of eluate. The PAH fraction is recovered from the
column by elution with a second 18 ml of solvent. (The column
can be regenerated for use by washing with 36 ml of solvent.)
Reduce the volume of the PAH fraction to approximately 1 ml.
3.1.5.6 Hign-rerfurmancs u.'cjuid Chrcmatcgr.iiDhy
Transfer tne purified sample "rom t~e ?MSO partitioning step
or the Sephadex column Chromatography step into a 15-mf
concentrator tube. Add 100 ul DMSO and evaporate the
toiuene. Store the purif^d and torcsntrated samples in the
dark in cone vials with Teflon-1^nea screw caos cr Tsrion
septa.
Inject 2- to 10-ul aliquots of the ourified and concentrated
extracts onto the column. Figure 9 shows a chromatogram of
a PAH standard and Figure 10 shows sample chromatograms.
Table 7 gives appropriate operating conditions. Table 6
lists several PAHs and their fluorescence characteristics.
Other chromatographic conditions which have been used can be
found 1n other Subsections under High-Performance Liquid
Chromatography.
Chromatographic peaks can be Identified by retention times,
co-chromatography of reference compounds, and by the response
ratio of ultraviolet absorbance and fluorescence detectors.
If the analytical system is equipped with a stop-flow mecha-
nism, qualitative identification of peaks can be supplemented
by a fluorescence spectrum. Alternatively, fractions of the
HPLC effluent can be collected and analyzed by fluorescence
ana/or ultraviolet ipectroscooy.4 Thin-layer chromatoqraphy
(TLC) (see Subsection K) can also be used for confirmation of
III-397
-------
uv
10
Retention Time, minutes
Peak Identification is as Follows:
1. Phenanthrene
2. Anthracene
3. Fluoranthene
4. Pyrene
5. Triphenyiene
6. Benzo [a] anthracene
7. Chrysene
8. Benzo [e] pyrene
9. Benzo fjl fluoranthene
10. Perytene
11. Benzo [b] fluoranthene
12. Dibenz [a.c] anthracene (shoulder)
13. Benzo [k] fluoranthene
14. Benzo [a] pyrene
15. Dibenz Ja.h] anthracene
16. Benzo [gh,i] perylene
17. Indeno [1.2.3-cd] pyrene
18. Benzo [b] chrysene
19. Coronene
20. Dibenz [ai] pyrene
Figure 3. Chromatogram of polycycllc Aromatic hydrocarbon -?ference comoounds.
Chromatography conditions are described in the text (from reference 22).
III-398
-------
20
UV
I
0
T
10
I
20
Retention Time, minutes
Peak Identification Is as Follows:
1. Phenanthrene
2. Anthracene
3. Fluoranthene
4. Pyrene
5. Triphenylene
6. Benzo [a] anthracene
7. Chrysene
8. Benzo [e] pyrene
9. Benzo [jj fluoranthene
10. Perylene
11. Benzo [b] fluoranthene
12. Dibenz [a.c] anthracene (shoulder)
13. Benzo [k] fluoranthene
14. Senzp (a] pyrene
15. Dibenz [a,h] anthracene
16. Benzo [gh.i] perylene
17. Indeno I1.2,3-cd] pyrene
18. Benzo [b] chrysene
19. Coronene
20. Dibenz [ai] pyrene
c*nure TO. Chromatoqram of sediment extract (from reference 22).
III-399
-------
TABLE 7. HPLC CONDITIONS
Mobile Phase:
A: Water
B: Acetonitrile
Flow rate: 0.5 ml/min
Gradient Program:
1. 40% A 60% B for 6 min
2. 60% B - 99% B in 13 min
3. 99% B
Detectors:
UV Absorbance Detector: 296 nm
Fluorometric Detector:
max . 340 . 38Q nm
PX
(Corning 7-54, 7-60 filters in series)
nm
'Corning 3-73, 4-76 filters in series;
:=================================================================
used for confirmation of identity, as can gas cnromatography/
mass spectrometry (GC/MS) (see Section 3).3,16 The Sephadex
LH-20 column chromatography cleanup should be used only if thr
extract does not give satisfactory riPLC response after the
Florisil and DMSO-partitioning cleanup steps.
3.1.5.7 Determination of B[a]P Recovery
Radio-labeled B[a]P is used to measure method recovery.22
An aliquot of the DMSO solution injected into the HPLC is
analyzed for SH- or l^c-iabeled B[a]P. The percentage
recovery for B[a]P is determined by comparing the recovered
radioactivity to the amount originally added to the sample.
3.1.5.7.1 Counting Procedure
Radioactivity, is determined on a 50 nl aliquot of the
purified sample in DMSO. The DMSO solution is added to
10 ml scintillation fluid, and radioactivity determined
in a scintillation counter, using either 3H or 14C settings
as appropriate.
111-400
-------
Sample counts are corrected for scintillation quenching
through the use of appropriate channel ratio or external
standard techniques.
III-401
-------
4. Analysis of Biological Tissue Samples for Polycycllc Aromatic Hydrocarbons
4.1 Analysis of F1sh and Shellfish Tissue for PAH
Analytical Procedure: available
Sample Preparation: evaluated
4.1.1 Reference
Dunn, B. P. and R. J. Armour, "Sample Extraction and Purification
for Determination of Polycyclic Aromatic Hydrocarbons by Reversed-
Phase Chromatography." Analytical Chemistry, 52:13 (1980)
pp. 2027-2031.22
4.1.2 Method Summary
A 20- to 100-g sample of tissue is digested in alcoholic potassium
hydroxide, the resulting solution is extracted with isooctane, and
the Isooctane extract 1s concentrated by evaporation. The concen-
trated extract is cleaned up by Florisil column Chromatography and
dimethyl sulfoxide partitioning. An optional Sephadex LH-20
column chromatographic cleanup procedure is provided. The purified
and concentrated extract is analyzed by high-performance liquid
Chromatography using both UY absorbance and fluorescence detection.
A radioiaoeied oenzoiajpyrene internal standard it used ;a correct
for Bosses incurred during sample ^reparation.
4.1.3 Applicability
This .iietnoa nas seen demonstrated :o :s uppi "cadi? ;o ;ne*'~. ?*ih
(mussels, clams, oysters) and fish.13
4.1.4 Precision and Accuracy
The overall recovery of B[a]P is 60 to 80 percent, while recovery
from the digestion step and subsequent extraction are essentially
quantitative.17 Recoveries from the Florisll column cleanup are
over 90 percent.17 The precision obtained 1s 6 percent.I7
4.1.5 Sample Preparation
Place 20 to 100 g of tissue in a 300-ml round-oottom rlask and
add 150 ml ethanol, 7 g KOH, two or three boiling chips, and an
aliquot of radioactive benzo[a]pyrene (1,000 dpm 14C B[a]P,
ca. 5 ng; or 25,000 dpm ^H B[a]P, ca. 0.1 ng; or 25,000 dpm of
another radiolabeled PAH of interest). Reflux the mixture
gently for 1.5 hours and add the digest, while still hot, to
approximately 150 ml water in a 1-Hter separatory funnel. The
amount of water used should be adjusted to yield a final mixture
that is 50 to 55% water. Rinse the flask with 50 ml of ethanol
and add :o :he separatory "unne!. "xtract :he nixture three
times with 200 ml isooctane, combine the extracts and wash them
four times witn 200-iTii portions of *»arm (60°C) *aicer.
TH-402
-------
4.1.6 FTorisi!-Column Cleanup
4.1.6.1 Concentration
Reduce the volume of the isooctane from the extraction to
approximately 10 ml. Use a Kuderna-Danish evaporative
concentrator if losses of the more volatile PAHs are of
concern. If not, a rotary evaporator is satisfactory for
this step. After volume reduction, dilute the extract
to 100 ml with toluene.
4.1.6.2 Column Chromatography
Prepare a column of 30 g Florisil (deactivated with 5 percent
water) covered with 60 g anhydrous sodium sulfate in a glass
chromatographic column {40 mm x 400 mm). Add 100 ml toluene
to the column and elute the toluene until the liquid level is
just at the top of the sodium sulfate layer. Apply the sample
(in toluene) and elute the column until the liquid level is at
the top of the sodium sulfate layer. Carefully add 100 ml
toluene, elute the column, add another 100 ml toluene, and
elute the column again.
4,1.7 DMSO Partitioning
Add 5 ml dimethyl sulfoxide (DMSC) to the combined toluene
eluate from the column and concentrate cne solution jnder
vacuum (at 60°) to remove the toluene. Transfer the OMSO
solution to a separatory funnel containing 10 ml isooctane,
rinse tne evaporating r'lasx ,«un 5 .?.! 3M£C, -nd idd it to :he
separatory funnel. Shake the mixture znd drain the DMSO ""ayer
into a second separatory funnel containing 40 ml water and
20 ml isooctane. Add 10 ml DMSO to the first separatory
funnel, shake the .mixture, and drain the DMSO 'nto the second
separatory funnel. Shake the second separatory funnel, and
transfer the DMSO/water phase into a third separatory funnel
containing 20 ml isooctane. Shake the third funnel, then
discard the DMSO/water layer. Combine the isooctane layers
from the second and third separatory funnels and wash twice
with 40 ml water.
Reduce the volume of the isooctane ioiution to approximately
5 ml. Add 10 ml ethanol/toluene (10/90) and evaporate the
mixture to approximately 1 ml. It is not necessary to dry the
isooctane prior to evaporation, as residual water from the
solvent is removed as an ethanol/to!uene/water azeotrope
during evaporation. Use a K-D concentrator if losses of the
more volatile PAH compounds are of concern.
111-403
-------
4.1.8 Sephadex LH-20 Chromatography (Optional)
The elution volumes of the Sephadex column should be checked,
using a standard solution, before use and periodically there-
after. The following procedure is written for elutfon of the
PAHs between 18 ml and 36 ml. Appropriate changes should be
made if the initial elution volumes differ, or if they change
due to settling of adsorbent.
Transfer the toluene extract from the DMSO partitioning into a
graduated 15-ml tube. Rinse the flask several times with
toluene and add the rinses to the extract. Reduce the volume of
the extract to approximately 0.5 ml under a stream of dry nitrogen
with gentle warming. Add an equal volume of ethanol and apply
the sample to a column of Sephadex LH-20 (bed size 10 mm x 200
mm) packed in toluene/ethanol (1/1). Draw the material onto
the column bed using gentle suction and elute the column (by
gentle suction) with 18 ml toluene/ethanol (1/1). Discard
the first 18 ml of eluate. The PAH fraction is recovered from
the column by elution with a second 18 ml of solvent. (The
column can be regenerated for use by washing with 36 ml of
solvent.) Reduce the volume of the PAH fraction to approximately
1 ml.
4.1.9 High-Performance liquid Chromatograpny
Transfer the purified sample from the DMSO partitioning step
or the Senhadex column chromatoqraohv step into a 15-ml
concentrator tube. Aaa 100 yi DMSO ana evaporate the
toluene. Store the purified ana concentrated samples in the
dark in cone vials with Teflon-lined screw caps or Teflon
septa.
Inject 2- to 10-yl aliquots of the purified and concentrated
extracts onto the column. Figure 9, page 111-398, shows a
chromatogram of PAH standards, Figure 11 shows sample chromato-
grams, and Table 7 gives appropriate operating conditions.
Table 6 lists several PAH compounds and their fluorescence
characteristics.
Chromatographic peaks can be identified by retention times, co-
chromatography of reference compounds, and by the ratio of the
responses of ultraviolet absorbance and fluorescence detectors.
If the analytical system is so equipped, qualitative identifi-
cation of peaks can be supplemented by a fluorescence spectrum.
Alternatively, fractions of the HPLC effluent can be collected
and analyzed by fluorescence spectroscopy for confirmation of
identity.4 Thin-layer chromatography (TLC) (see Subsection K)
can also be used for confirmation of identity.17 The Seohadex
LH-20 column cnromatograpny cleanup snouid be used only if the
extract Hoes lot jive satisfactory MPLC "°SDonse if tar '
and DMSO cleanups.
-------
19
20
J
0
1
10
I
20
Retention Time, minutes
Peak Identification is as Follows:
1. Phenanthrene
2. Anthracene
3. Fiuoranthene
4. Pyrene
5. Triphenylene
6. Benzo [a] anthracene
7. Cnrysene
8. Benzo [e] pyrene
9. Benzo fj] fiuoranthene
10. Perylene
11. Benzo [b] fiuoranthene
12. Dibenz [a,c] anthracene (shoulder)
13. Benzo [k] fiuoranthene
14. Benzo {aj pyrene
15. Dibenz [a.h] anthracene
16. Benzo [gh,i] perylene
17. Indeno [1,2,3-cd] pyrene
18. Benzo [b] chrysene
19. Coronene
20. Dibenz [ai] pyrene
figure ii. Chromatograph of
:smoounds ^n nussels (from reference 18).
-------
4.1.10 Determination of B[a]P Recovery
Radio-labeled B[a]P is used to measure method recovery.22
An aliquot of the DMSO solution injected into the HPLC is
analyzed for SH- or l^C-labeled B[a]P. The percentage
recovery for B[a]P is determined by comparing the recovered
radioactivity to the amount originally added to the sample.
4.1.10.1 Counting Procedure
Radioactivity is determined in a 50 yl aliquot of the
purified sample in DMSO. The DMSO solution is added to
10 ml scintillation fluid, and the radioactivity determined
in a scintillation counter, using either SH or i^c settings
as appropriate. Sample counts are corrected for scintillation
quenching through the use of appropriate channel ratio or
external standard techniques.
111-406
-------
4.2 Analysis of Plant Tissues for PAH
Analytical Procedure: available
Sample Preparation: available
4.2.1 Reference
Joe, F. L., Jr., J. Salemme and T. Fazio, "High Performance
Liquid Chromatography with Fluorescence and Ultraviolet
Detection of Polynuclear Aromatic Hydrocarbons in Barley Malt."
J. Assoc. Off. Anal. Chem., 65:1395-1402. 198221.
4.2.2 Method Summary
A 100-gram sample of vegetable tissue is ultrasonically extrac-
ted in cyclohexane, the extract is purified by water-deactivated
silica/alumina Chromatography and dimethyl sulfoxide partition-
ing, and is concentrated and solvent-exchanged into aqueous
acetonitrile/methanol. The resulting solution is analyzed by
reversed-phase high-performance liquid Chromatography.
4.2.3 Applicability
This method is applicable to the determination of PAH com-
pounds in barley malt tissues and :hou!d be amenable to
-------
TABLE 8. FLUORESCENCE RECOVERY (PERCENT) OF PAH
COMPOUNDS FROM BARLEY MALT SAMPLES"
2.5 ppb Spike 5.0 ppb Spike
PAH
Fluorene
Pyrene
Benzo[a]anthracene
Benzo[b]f 1 uoranthene
Benzo[e]pyrene
Benzo[a]pyrene
D^benzo[ah]anthracene
Indeno[l,2,3,cd]pyrene
Benza[ghi]perylene
DibenzoCdi ]pyrene
Coronene
=======================
Recovery3
86
81
78
85
86
83
86
85
91
79
83
===============
RSD&
2.3
1.4
4.4
7.5
8.4
6.6
6.7
10.6
8.0
7.3
9.7
Recovery*
79
82
82
88
97
85
87
87
85
39
87
:==============:
RSDb
7.7
6.5
6.0
6.0
12.0
7.2
7.0
8.1
5.9
2.6
3.5
•2 Average of cri'piicate aetsrminations; cxcvcai:on ^c -3
t> Relative standard deviation, percent
(deactivated with 15 percsnt water), 5 g alumina (deacti-
vated with 10 percent water), and 10 g anhydrous sodium
sulfate, 1n that order, to a 250 mm by 14.5-mm column con-
taining a glass wool plug. Tap gently with each addition.
Fit each column with a 250-ml solvent reservoir.
Wash the column with 50 ml cyclohexane, stopping the
flow when the liquid level just reaches the top of the
sodium sulfate layer. Test the columns as indicated in
paragraph 4.2.6.2 before use.
4.2.6.2 Column Testing
Prepare two columns as described above (one for a blank and
one for recovery measurement). Add 75 ml cyclohexane to one
reservoir and 75 ml cyclohexane solked with 6.25 x 10~2 yg
each of phenanthrene (Phen), fluoranthene (F), pyrene (Py),
benzoiajanthracene i'8[ajA), benzoCbjfluoranthene '3[b]F),
benzo[e]pyrene (B[e]P), benzoCalpyrene (B[a]P), dibenzoCah]-
anthracene iDBLanjA), inaenou,
-------
and coronene (Cor) to the other reservoir. Let the solvent
percolate through the blank and spike columns. Collect the
two eluates in separate 500-ml flasks. Repeat the column
elution with three more 75-ml portions of cyclohexane,
letting each drain just to the top of the sodium sulfate
before adding the next.
Evaporate the combined eluates to approximately 5 ml, using
a flash evaporator with a 40°C water bath. Transfer the
concentrates quantitatively to 15-ml concentrator tubes with
disposable Pasteur pipets and rinse each flask with three 1-
ml portions of cyclohexane. Evaporate the solutions to dry-
ness at 30°C under a gentle stream of purified nitrogen.
Add 0.25 ml of a 0.25 vg/ml benzo[b]chrysene solution and
subject to ultrasonic vibration for approximately 3 minutes.
Analyze by HPLC. Solvents (blank) must be free of interfer-
ences and recovery for each PAH must be 90 percent or better.
4.2.6.3 Sample Chromatography
After the column has been demonstrated to work properly,
transfer the extract to the silica gel/-alumina column, rinse
the Erlenmeyer flask twice with 5-ml portions of cyclohexane,
etna add cr.em ;o ;he column. !"!'.:: 3 :s "i par~qr3ch 4.2.6.2,
collecting the eluate "'n a 500-ml *lask. Evaoorate the com-
bined eiuates to approximately 25 .711, jsing : ->TC water
bath.
4.2.6.4 DMSO Partitioning
Quantitatively transfer the concentrated eluate to a 125-ml
separatory funnel containing 15 ml dimethyl sulfoxide (DMSO)
which has been pre-equilibrated with cyclohexane. Rinse the
flask with 25 ml cyclohexane and add the rinse to the separa-
tory funnel. Shake vigorously for 2 minutes, let the layers
separate, and drain the bottom (DMSO) layer into a 500-ml
separatory funnel containing 90 ml deionized water and 15 ml
cyclohexane. Repeat the extraction of the eluate with two
additional 15-ml portions of DMSO and combine the DMSO
extracts in the 500-ml separatory funnel. Shake vigorously
and let the layers separate. Draw off k,he lower, aqueous
phase into a second 500-ml separatory funnel containing 15
ml cyclohexane. Repeat the extraction, discard the aqueous
phase, and add the organic phase to the organic phase remain-
ing in the first 500-ml separatory funnel. Rinse the second
funnel with two 10-ml portions of cyclohexane and transfer
each to the first funnel. Wash the combined extracts with
100 ml deionized water, shaking for 1 minute. Let the
phases separate and discard the aqueous phase.
111-409
-------
4.2.6.5 Extract Drying and Concentration
Add 50 g anhydrous sodium sulfate to a 60-ml Biichner funnel
with a coarse fritted disc. Rinse the apparatus with 50 ml
cyclohexane, allowing the liquid to drain by gravity.
Discard the rinse. Filter the combined extracts from para-
graph 4.2.6.4 into a 300-ml flask. Rinse the separatory
funnel with two 10 ml portions of cyclohexane and pour each
through the Biichner funnel. Concentrate the dried extracts
and washes to 3 to 5 ml, using a 40°C water bath. Transfer
the concentrate to a 15-ml concentrator tube with a dispos-
able Pasteur pipet. Rinse the flask with three 1-ml por-
tions of cyclohexane and transfer each rinse to the concen-
trator tube. Place the tube in a 30°C water bath and
evaporate to dryness under a gentle stream of purified
nitrogen. Add 0.25 ml of a solution of 0.25 vg/rol benzo-
[b]chrysene (B[b]Ch) in a 80/20 mixture of acetonitrile:
methanol (l:l)/water and subject to ultrasonic vibration for
3 minutes. Filter if necessary, and analyze the resulting
solution by HPLC.
4.2.7 High-Performance Liquid Chromatography
Inject 20 yl extract or standards onto the octadecylsilanc:
"31 u.Tin, 'jsing the ~.CM vent troqram Tiven in Table 9. Figure 12
and Figure 13 show sample chromatograms obtained under these
conditions. Alternative chromatographic conditions can be
found in other Subsections unde" Hiqh-Performance Liauid
Chromatoqraohy. Between inject".ons, fiusn tne sample ioop and
port passages with approximately o mi acetorntrile/ methanol
(1/1) to prevent cross-contamination. Compare retention
volumes of peaks observed with those of standards. Inject
standards after each third sample. Calculate the concentra-
tion of PAH compounds using the internal standard (B[b]Ch)
procedure.
111-410
-------
TABLE 9. HPLC CONDITIONS
333=333=333333333333=3=3333=33=33333=333333333333333333333333===3=33333=3333==3
Mobile Phase:
A: Water
B: Methanol/acentonitrile (1 + 1)
Flow rate: 1 ml/min
Gradient Program:
1. 80% B - 100% B in 20 min
2. 100% B for 20 min
3. 100% B - 80% B in 5 min
4. 80% B for 20 min
Detectors:
UV Absorbance Detector: 289 nm
Fluorometric Detector: 333
xmax : 340 - 380 nm
(Corning 7-54, 7-60 filters in series)
xmax : >400 nm
(Corning 3-73, 4-76 filters in series)
83333333333333333==33S3SS=33333333a333333333=33S3=3==33SS=======a=33======3===3
Ill-All
-------
Fluonsctnci
UV
(f
—I—
.0
1
:o
Tim*, minutvs
—T~
30
PeaK identJTication is as roiiows:
1. Phenanthrene
2. Fiuoranthene
3. Pyrene
4. Benzo [a] anthracene
5. Benzo [b] fluoranthene
6. Benzo [e] pyrene
7. Benzo [a] pyrene
8. Dibenz [ah] anthracene
9. Senzo [bj chrysene
10. Indeno [1,2,3-cd] pyrene
11. Benzo [ghi] perylene
12. Oibenz [ai] pyrene
13. Corenone
Figure 12. HPLC chromatogram of unfortified barley malt sample.
Top: *1uorescence - 333 nm. <-anqe - 0.1 uA.,
Bottom: UV - 289 nm, attenuation 0.02 AUFS.
(from -sfer^ncs 11}
III-412
-------
1. Phenanthene
2. Fluorene
3. Pyrene
4. Benzo [a] anthracene
5. Benzo {e] fluoranthene
6. Benzo [e] pyrene
7. Benzo [a] pyrene
8. Dibenzo [b] anthracene
9. Benzo (bj chrysene
10. Indeno [1.2.3-cd] pyrene
11. Benzo [g.h.i] perylene
12. Dibenzo [a.i] pyrene
13. Coronene
2
i
4
JL
78,9
i
10
20
30
Retention Time, minutes
Figure 13. HPLC chromatogram of 5 ng each of 13 PAH compounds.
Top: fluorescence - 133 *wu
Bottom: UV - 289 nm, attenuation 0.02 AUFS.
reference £1}
III-413
-------
5.1 Analysis of Air for Polycyclic Aromatic Hydrocarbons
Analytical Procedure: available
Sample Preparation: available
5.1.1 Reference
Method 21516, "Methods Manual for Chemical Analysis of Atmospheric
Pollutants." Environment Canada, Alberta Environmental Centre,
Vegreville, Alberta, Canada, 1981.28
5.1.2 Method Summary
Airborne particulate matter is collected on a glass-fiber filter, the
sample is extracted with cyclohexane, the solvent is flash-evaporated,
and the residue is dissolved in methanol and analyzed by HPLC with
a fluorescence detector.
5.1.3 Applicability
This method is applicable to the determination of polycyclic aromatic
hydrocarbons in airborne particulate matter.
5.1.4 Precision and Accuracy
SINGLE LABORATORY PRECISION AND RECOVERY"
('Senzof a jpyrene j
B[a]P Amount (ng) • Coefficient of. Variation (4) Recovery (%)
______.^_^______———_——————————————-— •
5 — 103
10 20.4
112 5.51
142 — 32 -
334 — 100
338 3.16
ssss==ss=z==s=sss=sz=================================================
5.1.5 Sample Preparation
S.I.5.2 Sampling
Condition a glass-fiber filter overnight at room temperature
and humidity or dry in a desiccator for 25 hours. Weigh the
filter and record the weight. Install the filter in a high-
volume sampler equipped with a chart recorder.
Operate the high-volume sampler for 24 hours, with a flow
chart recording the flow. Weigh the filter disc and record
the final weiqht in qrams. Divide the filter disc into 5
equal parts and process eacn according co cne procedure yiven
below.
III-414
-------
5.1.5.2 Extraction
Extract each portion of the filter disc for 6 hours in a
Soxhlet apparatus using 80 to 85 ml pesticide grade cyclo-
hexane.
5.1.5.2 Concentration
Cool the flask and remove the solvent in a flash evaporator
at 40°C. Dissolve the residue in four 1-ml aliquots of
methanol, transfering each into a centrifuge tube. Centrifuge
the methanolic solution and concentrate to 2 ml by blowing
dry nitrogen over the sample. Transfer the clear solution
with a Pasteur pipet into a vial with a Teflon-lined septum
cap and seal tightly.
5.1.6 High-Performance Liquid Chromatography
Analyze samples by HPLC using 10-ul injections and the instrument
conditions given in Table 10. A suggested order of sample pro-
cessing for quality control purposes is as follows:
Blank
i szanaarcis
Blank
o sampies
Blank
2 standards
TABLE 10. HPLC CONDITIONS33
2SSSSSBS5S-SSB=aSB=SBSSSB==BB3=SBaS==SSSS=SBBSSS===SBBBSS=3SSBBSBSSBSSBB=SS==SSB
Mobile Phase;
A: Acetonitrile
B: Water
Flow rate: 1.0 ml/min
Solvent program: Isocratic;
A:B: 75:25 t'Y:V)
Detectors:
UV Absorbance Detector: 289 nm
Fluorometric Detector: 333 nm
1max : 310 nm
ex
' xmax : 390 nm
Aem
BSSSS8SSSSSSB8SSSSSSSSBSBSSBSSSSaSB=SSSBS8SBBSSSBBBSSSBSSSBSSSSSBBSSBSBS=SS==a=
III-415
-------
Sample chromatograms of standards and a sample are shown 1n Figures
14 and 15.
NOTE: Routine quality control must be followed to ensure the pre-
cision and accuracy of the method. With every series of ten extrac-
tions, one sample should be analyzed 1n duplicate with Its mate
being spiked with 100 ng of B[a]P. This will allow the technician
to detect any changes occurring within the total analysis. Also a
blank, 2 standards, and then another blank should be run at a
minimal interval of 5 samples to ensure reproducibility of standard
areas. If these practices are followed, the method should fall
easily within the tolerances quoted for accuracy and precision.
J. QUALITATIVE IDENTIFICATION
1.0 Fluorescence Spectra
If the HPLC system is so equipped, a fluorescence scan can be made of
a given peak. Alternatively, fractions of the HPLC effluent can be
collected and analyzed by fluorescence spectroscopy.4 Table 6 gives
fluorescence data for a large number of PAH compounds in n-heptane
solution. Figure 4 shows emission spectra obtained of several peaks
in an HPLC PAH analysis. If both uv and fluorescence detectors are
used i-n series, the ratio of <:he two resoonses at various wavelengths
can be useful in ascertaining the identity of a pea*, when oenzotdj-
pyrene (B[a]P) and benzol'k jfuorantnene «. 8[k]F) coelute., use has been
made of excitation at 307 nm and measurement of fluorescence inten-
sity at 406 nm, to give the 8[k]F concentration, so that the contri-
bution of B[k"lF to the fluorescence Intensity at 406 nm from excita-
tion at 384 nm is known.;~ I; r.as seen round tnaz cne concentrations
of compounds m air samples wnlcn interfere witn use-of this tech-
nique are typically not high enough to interfere seriously.33
2.0 Gas Chromatography/Mass Spectrometry
Confirmation of the identity of PAHs may be accomplished by the use
of gas chromatography/mass spectrometry (GC/MS).29 See Section 3,
Subsection J.3 of this Chapter for protocol.
3.0 Thin-Layer Chromatography
Confirmation of the identity of individual PAH compounds may be
performed by thin-layer Chromatography (TLC).3^ Samples are prepared
as 1n the HPLC procedures, the extract reduced in volume to approxi-
mately 0.1 ml, and the concentrate applied to a cellulose-acetate
thin-layer plate.
Standard solutions are applied next to the sample spot, and the plate
is developed with ethanol/to!uene/water (17/4/4).34 The PAH bands are
visualized with long-wave ultraviolet light. Caution must be exer-
cised to avoid unnecessary exposure or tne PAH rract'ons co ' ight,
^as been -eoortsd to decomoose them.5
III-416
-------
1. Benzo [e] pyrene
2. Benzo [k] fluoranthene
3. Benzo [a] pyrene
4. Impurity from benzo [e] pyrene
Figure H. Sample chromatogram of PAH standards ^from reference
U 1-41.7
-------
1. Unknown
2. Benzo [k] fluoranthene
3. Benzo [a] pyrene
4. Unknown
figure 15. Sample cnromatcgram jf an air sample I from reference
III-418
-------
The adsorbent at a band of interest can be scraped off and the PAH
desorbed with hot (65°C) methanol (4 x 4 ml). The methanol is added
to 10 ml of 20-percent hexadecane in isooctane, and the methanol and
•fsooctane removed by rotary evaporation.
The PAH concentration in hexadecane is measured fluorimetrically
using the baseline technique.34 Samples and standards are excited at
an appropriate wavelength (see Table 6) and the emission spectrum
recorded over an appropriate wavelength range.^
K. CALCULATIONS
1. Internal Standard Approach
Calculate the response factor (RF) using Equation 1 (Subsection G.2.2),
and calculate the concentration in the sample from:
Concentration = (AS)(I1-S)/(A1-S)(RF)(M0) Eq. 2
where:
\r = Response for the parameter to be measured
ITS = Amount of internal standard added to each extract
A-fS = Response for the internal standard
RF = Response Factor (see CuDsect:'cn 3.2.2;
M0 - Initial sample size (mass or volume).
2. External Standard Approach
Obtain a calibration curve or calibration factor according to Subsection
G.2.2, and calculate the concentration of the sample by interpolation
(using a calibration curve) or by use of Equation 3:
Concentration = (As)(Vf)/(F)(M0) Eq. 3
where:
As = Response of the sample
Vf = Final extract volume
F * Calibration factor; (response)/(concentration)
M0 = Initial sample size (mass or volume).
3. Recovery Corrections 'Subsections J.3, J.4.2)
-------
For analyses where 3H- or l^C-labeled B[a]P are used to indicate losses,
fractions of the HPLC effluent should be collected which correspond to
the peak identified as B[a]P. These fractions should then be assayed for
3H or 14C, as appropriate.
Recovery of B[a]P is calculated by:
Recovery (R) = (CS)/C Eq. 4
where:
C « Radioactivity initially added to sample
Cs = Radioactivity recovered in sample extract
Use of R can then be made to correct results (see above):
Corrected Results = (Concentration)/R Eq. 5
where:
Concentration = Results of 1 or 2 (above)
R = Recovery (Eq. 4)
III-420
-------
REFERENCES
1. Bar-tie, K. D.; M. L. Lee; and S. A. Wise. "Modern Analytical Methods
for Environmental Polycyclic Aromatic Compounds." Chem Soc. Rev.
10:113-158. (1981)
2. Jurgensen A., E. L. Inman, Jr., and J. D. Winefordner. "Comprehensive
Analytical Figures of Merit for Fluorimetry of Polynuclear Aromatic
Hydrocarbons." Anal. Chim. Acta, 1^:187-194. (1981)
3. Sauter, A. D., L. D. Betowski, T. R. Smith; V. A. Strickler; R. G. Beimer;
B. N. Colby, and J. E. Wilkinson. "Fused Silica Capillary Column GC/MS
for the Analysis of Priority Pollutants." HRC CC, 4:366-384. (1981)
4. Fox, M. A., and S. W. Staley. "Determination of Polycyclic Aromatic
Hydrocarbons in Atmospheric Particulate Matter by High Pressure Liquid
Chromatography Coupled with Fluorescence Techniques." Anal. Chem., 48:
992-998. (1976)
5. American Public Health Association. "Methods of Air Sampling and Anal-
ysis." 2nd ed. M. Katz, Ed. Washington, D. C. 984 p'. (1977)
6. U.S. Environmental Protection Agency. "Polynuclear Aromatic Hydrocarbons
- Method 610." Methods of Organic Chemical Analysis of Municipal and
Industrial Wastewater. J. lonabottom and J. licntenburg, eds.
EPA-600/4-82-057, (1982).
7, American Society for Testing and Materials. "Standard Practice for
Sampling Water.'' ASTM Book of Standards, Section il, Vol. 11.01, D337C.
Philadelphia, Pennsylvania. (1983)
8. Dunn, B. P. "Handbook of Polycyclic Aromatic Hydrocarbons in Sediments
and Marine Organisms." Ch. 10, Handbook of Polyaromatic Hydrocarbons.
A Bjrfrsten, ed. Marcel-Dekker, New York, p. 443. (1983)
9. Kawakami and Nishimura, J. Oceanogr. Soc. Japan, 3_2_:175 (1976).
10. Prahl, F. G. and R. Carpenter. "The Role of Zooplankton Fecal Pellets
in the Sedimentation of Polycyclic Aromatic Hydrocarbons 1n Dacob Bay,
Washington." Geochim Cosmochim Acta, £3_: 1959-1972. (1979)
11. Bjorseth, A., J. Knutzen, and J. Skei. "Determination of Polycyclic
Aromatic Hydrocarbons in Sediments and Mussels from Saudafjord, W.
Norway, By Glass Capillary Gas Chromatography." Sci. Total Environ.
1^:71-86. (1979)
12. Bieri, R. H. M. K. Cuenan, C. L. Smith, and C. W. Su. "Polynuclear
Aromatic and Polycyclic Aliphatic Hydrocarbons in Sediments from the
Atlantic Outer Contental Shelf." Int. J. Environ. Anal. Chem. 5:293-310.
(1978)
III-421
-------
13. Gelger, W., and C. Schaffner. "Determination of Polycyclic Aromatic
Hydrocarbons in the environment by Glass Capillary Gas Chromatography."
Anal. Chem., 50:243-249.
14. Brown, R. A., and B. K. Starnes. "Hydrocarbons in the Water and Sediment
of a Wilderness Lake. II." Marine Pollut. Bull., 9:162-165. (1978.
15. Maher, W. A., J. Bagg, and J. David Smith. "Determination of Polycyclic
Aromatic Hydrocarbons in Marine Sediments, Using Solvent Extraction,
Thin-Layer Chromatography and Spectrofluorimetry." Int. J. Environ.
Anal. Chem., 7^:1-11. (1979)
16. John, E. D., M. Cooke, and G. Nickless. "Polycyclic Aromatic Hydrocarbon
in Sediments Taken from the Severn Estuary Drainage System." Bull.
Environ. Contam. Toxicol . 22_:653-659. (1979)
17. Dunn, B. P. "Techniques for Determination of Benzo(a)pyrene in Marine
Organisms and Sediments." Env. Sci. and Tech. 10_: 1080- 1021. (1976)
18. Kellner, R. "Analysis of Airborne Particulates by Physical Methods."
H. Malissa, ed., CRC Press, p. 228. (1978)
19. Inscoe, M. Anal. Chem., 36:2505. (1964)
20. American Society for Testing and Materials. "Standard Practice for
Preparation of Sample Containers and for Preservation," ASTM Book of
Standards, Part 31, D3694. Philadelphia, Pennsylvania. (1980)
21. Joe, c, 1., ]r,; J. Salemne, and T. ^zlo. "High-Performance Liauid.
Chroma tography with Fluorescence and Ultraviolet Detection of
Polynuclear Aromatic Hydrocarbons in Barley Malt." J. Assoc. Off. Anal.
Chem., 65_: 1394-1402. (1982)
22. Dunn, B. P., and R. J. Amour. "Sample Extraction and Purification
for Determination of Polycyclic Aromatic Hydrocarbons by Reversed-
Phase Chromatography." Anal. Chem., 52_: 2027-2031. (1980)
23. U.S. Environmental Protection Agency. "Laboratory Use of Toxic Sub-
stances." Occupational Safety and Health Manual, Chap. 8. U.S. EPA
Washington, D. C. (1979)
24. American Chemical Society. " Safety in Academic Chemistry Laboratories."
3rd ed., American Chemical Society, Committee on Chemical Safety,
Washington, D. C. (1979)
25. Occupational Safety and Helath Administration. "OSHA Safety and Health
Standards, General Industry." 29CFR-1910, OSHA 2206 (Revised
January, 1976). OSHA, Washington, D. C. (1976).
II1-422
-------
26. Center for Disease Control. "Carcinogens-Working with Carcinogens."
Center for Disease Coontrol, Public Health Service, Department
of Health, Education and Welfare, Public Health Service. National
Institute for Occupational Safety and Health, Publication No. 77-206,
August 1977.
27. Tomkins, B. A., R. R. Reagan, J. E. Caton, and W. H. Griest. "Liquid
Chromatographic Determination of Benzo[a]pyrene in Natural, Synthetic,
and Refined Crudes." Anal. Chem, 53:1213-1217. (1981)
28. Alberta Environmental Centre. "Methods Manual for Chemical Analysis of
Atmospheric Pollutants." Method 21516, Benzo[a]p'yrene. Alberta
Environmental Centre, Vagreville, Alberta, Canada. (1981)
29. U.S. Environmental Protection Agency. "Handbook of Analytical Quality
Control in Water and Wastewater Laboratories." U.S. EPA, Washington,
D. C. EPA-600/4-79-019. (1979)
30. Hertz, H. S., J. M. Brown, S. N. Chesler, F. R. Guenther, L. R. Hilpert,
W. E. May, R. M. Parris, S. A. Wise. "Determination of Individual Organic
Compounds in Shale Oil." Anal Chem. 52_: 1650-1657. (1980)
31. Wise, S. A., S. N. Chesler, H. S. Hertz, L. R. Hilpert, W. E. May,
'Chemically-bonded /Vrnnosilane Jtat-.cnary Phase -'sr the -Hqn-oer^omance
Liquid Chromatographic Separation of Polynuclear Aromatic Compounds."
Ana I.. Chem., 49, pp. 2306-2310, 1977.
32. Nielsen, T. "Determination of Polycyclic Aromatic Hydrocarbons in
Automobile Exhaust by Means o^ '-'•'gh-oer^onr-ance Liquid Chromatograohy
with Fluorescence Detection." J. Chromatogr.. 170:147-156. (1979)
33. Alberta Environmental Centre. "Methods Manual for Chemical Analysis of
Atmospheric Pollutants." Method 21515. Benzo[a]pyrene and Benzo[k]-
fluoranthene. Alberta Environmental Centre, Vagreville, Alberta, Canada.
(1981)
34. Kunte, H. "Carcinogenic Substances in Water and Soil. XVIII. Determina-
tion of Polycyclic Aromatic Hydrocarbons by Mixed Thin-layer Chromato-
graphy and Fluorescence." Arch. Hyg. Bakterial., 151:193-201. (1967)
II1-423
-------
IV. PROCEDURES FOR INORGANIC SUBSTANCES
IV-1
-------
SECTION 10
ELEMENTAL ANALYSIS BY ATOMIC ABSORPTION SPECTROMETRY
A. SCOPE
Atomic absorption (AA) spectrometry provides the analytical capability to
quantify more than 40 naturally occurring elements. The analytical procedure
consists of three steps: sample preparation to transform each element into a
tractable chemical form, atomization to reduce ions to neutral atomic species,
and spectrometric measurement to quantify the amount of light of specific
wavelengths absorbed by the reduced atoms.
The procedure can be used to distinguish the elemental partitioning of
elements between environmentally important phases by modifying *he sample
preparation techniques (i.e., filtration to distinguish between 'soluble" and
"total" -.oncsntraticns; extraction or ion exchange to distinquish between
"ionic" ana ''complexea" rorms of the same element).
B. SAMPLE HANDLING AND STORAGE
Due to the variable nature of the sample matrix, hazardous waste sampling,
sample handling, :na ^amp1.a storage procedures for these :2t2rm nations ire
quite matrix dependent. However, storage at reduced temperature '•i°C\ in order
to retard chemical and biological activity, should be practiced.
Grab samples for water analysis should be collected -'n iorosilicate glass,
linear polyethylene, polypropylene, or Teflon containers.1 The containers
should be pre-washed with detergent and tap water, rinsed with 1:1 nitric acid,
distilled water: hydrochloric acid, distilled water, and distilled deionized
water, in that order. Chromic acid may be used to remove organic matter from
glassware, but should not be used with plastics. Glassware must be well rinsed
to remove all traces of chromium after such treatment. A commercial product
containing no chromium, NOCHROMIX, is an available substitute for the cleaning
process. If it can be demonstrated througn use of spiked samples ana Blanks
that certain steps in the above-mentioned cleaning process can be omitted
without influencing analytical results, such steps may be eliminated. Figure 1
summarizes sample handling requirements for water, soil/sediment, tissue and
air samples.
For the determination of dissolved metals in a water sample, the sample
must be filtered through a 0.45-u pore size membrane filter as soon as prac-
tical after collection. The filtrate should then be acidified with 1:1 nitric
dcia to a pri jeiow L. .f j precipitate "orns -ipon acidification, the ^Itrate
should be digested ^s described for a total metals determination.
IV-2
-------
< . Purpose
co 1
Container
Sample Treatment
Preservative
Water Sample
^
iV ^ 4
Soil/Sedimi .it Sample I 1 Tissue
|
* *
Sample
r
| Acidify | | Filter ] | Store Wet | | Dry ] | Freeie | | Sample j
I i
| Sure | | Acidify )
.. i
| Oiyest | | Store | | Oigex
, ' 1 ' V
1 Analyte 1 | Analyie | | Anelyre
T V '
r
I Slo. J 1 Store 1 1 Store 1
,. ~]
J ( Dig,,.. ] [ Oigett J [ Oigett |
1 v J
r
1 1 Anal, <> 1 | Analyie | | Analyre |
T. lal Soluble Tot*l Toi : Total
Water Water Sndinitnt Sedim it Sediment
Concentration Concentration Concentration Concenc<-,.iion Concentration
UP G. P G. i>
None Filter rVo..«
HtlO, HNO, <'1,
(|JK2 pM<2
G. < G. P
Air l> ,' Freeze
No, None
Air Sample 1
' r
| Sample ]
^ f
Store J
1
Oiqell 1
, p
Analyte 1
Total
Paniculate
Concentration
Gl.ti
Fiber Membrane
Filter
None
None
Storage Time
t»0d
9Od
indefinite
Oiyestion Solution Sin, KJ Acid None
Slruruj Acid Sl.orK, -id Strong Acid
Strong Acid
Sa.nph} Amount lOOSOOml toOSOOml
25
25g
200Om>
Figure 1. Handling and sample storag*. informdi ion for samples to be analyzed for metals.
-------
For the determination of suspended metals, a representative volume of
unpreserved sample must be filtered through a 0.45-y pore size filter as
quickly as possible after sample collection. The filter and insoluble mate-
rials are digested with concentrated nitric acid or aqua regia and treated as
other samples.
Aqueous samples to be analyzed for total concentrations of elements should
be acidified to a pH less than 2 for preservation. Nitric, acid is generally
used for this purpose. Samples treated in this manner can be stored for up to
6 months.
Soil/sediment samples may be stored in wet, frozen, or dried state if the
total sample concentration is to be determined. When an operationally defined
procedure such as the EP Toxicity Test is to be performed, the samples should
be stored in a field-moist condition. Plastic or glass sample containers may
be used.2
Tissue samples should be weighed as soon as possible, and may be stored as
received, frozen, or dried. However, consideration of the stability of the
tissue itself must be taken into account to avoid excessive decomposition and
associated handling problems. The problem of storage of tissue samples for
elemental determination is not well studied. However, if tissue samples are
digested as soon as practical, the digest can then be stored as a water sample.
Particulates in air samples to be analyzed for metals are usually col-
lected by filtration. Filter media jsed for this purpose should be glass-fiber
.or membrane filters (0.8 p pore size).3. Glass-fiber filter blanks should be
investigated to establish the extent of zinc contamination and potential matrix
effects. Possible interf^rencas due to Gil^ca from glass-fiber f-'ltar--: can be
removed by centrifugation.3 Untreated filter samples from air samoiina can be
stored indefinitely for most metals.3
C. INTERFERENCES
1. General
Since the absorption of incident radiation is monitored in atomic absorp-
tion (AA) spectrometric analyses, the absorption, scattering, or emission
of radiation of the wavelength of interest by any substance other than the
element being quantified will create a positive or negative interference.
Spectral interferences, or extraneous adsorption of radiation of the
wavelength being monitored, can occur due to the presence of organic
molecules in the flame, and are particularly important at lower wave-
lengths (below 240 nm). Several types of automatic background correction
are available from manufacturers of most AA spectrometers. Narrowing the
slit width of the spectrometer detector can also help eliminate this
interference. Emission by molecular and atomic species can create a
negative Interference. This interference can be corrected by the use of a
pulsed source. Scattering of light by particles in the flame or by an
excessively ~*ch Hame will produce ~ oositive tnectral ''nterferorce.
IV-4
-------
2. Flame Atomization
Since instrumental response in AA spectrometry depends on the free-atom
concentration in the beam of radiation, anything which alters the extent
of free-atom formation between calibration standards and sample solutions
will be an interference. Interferences in flame spectrometry may be
classified into three categories: transport interferences, evaporation
interferences, and gas-phase interferences.
1) Transport interferences arise from a difference between standards
and samples in the rate of delivery of solution to the flame. The
rate of aspiration depends upon the viscosity of the solution being
aspirated and, to avoid these interferences, the viscosity of stand-
ards and samples should be the same. Surface tension differences
between standards and samples can also create transport interferences.
Transport interferences can be minimized by using the method of
standard additions or by matching the viscosities and surface ten-
sions of the standards with the samples being analyzed.
2) Evaporation interferences affect the rate of evaporation of solid
aerosols in the flame. The best-known example of such a phenomenon
is the effect of phosphate on the signal observed for calcium in
fame spectrometr'?c analysis, which 4s due to formation of 2 -efrac-
tory Ca-O-P compound upon desolvation. Evaporation interferences can
• be minimized by three procedures: the addition of a releasing agent
which preferentially combines with the interfering moiety, the use of
* a higher temperature flame such as the nitrous oxide-acetylene flame
- to break up refractory compounds, or measurement of absorbance higher
up in the flame (to allow more time for production of free atomsj.
3) Gas-phase interferences can shift ionization equilibria and alter the
degree of analyte ionization from sample to sample and between
samples and standards. This interference can be compensated for by
adding easily ionized cations such as cesium and potassium to samples
and standards at a concentration of approximately 1000 mg/1 to serve
as an ionization buffer.
3. Flameless Atomization
Flameless atomization usually occurs in an inert atmosphere, which ore-
' eludes any chemical effects such as oxide formation due to the presence of
oxygen. However, spectral interferences can be a problem. Gases gener-
ated by the furnace during atomization may have interfering molecular
absorption bands, and smoke-producing samples can cause scattering of the
incident beam. Also, samples with high levels of organic matter can
produce molecular species with broad uv absorption bands which may include
the wavelength of interest. These samples should be digested prior to
analysis.
Matrix effects are much more probable in furnace atomization than m flame
atcmizatlcn. To help verify the absence of •natr-'x •'nterferencas. 3 *or*i-
fied sample and an unfortified sample should both be diluted by at least
* * t c
A v-5
-------
1:1, the two solutions analyzed, and the results compared to the expected
results. Agreement within 10 percent indicates an absence of substantial
matrix effects. If matrix effects are present, samples should be diluted
and reanalyzed to determine if dilution can eliminate the effects, or
whether chemical matrix modification, use of hydrogen in the purge gas, or
the method of standard additions should be used.
D. SAFETY
When using flame-atomization atomic absorption spectrometry, flashback
can occur as the oxidant is changed from air to nitrous oxide. Special burner
heads, with a modified slot, should be used for nitrous oxide flames. In order
to avoid flashback, the air-acetylene flame should be lit first, the flame
stoichiometry adjusted to be quite fuel rich, and then change the oxidant
from air to nitrous oxide. After using a nitrous oxide flame, always turn the
nitrous oxide off first when shutting down the instrument. In addition, stand-
ard precautions for the use of compressed gases should be foil owed.3.4
Because many of the elements determined by atomic absorption spectrometry
are potentially hazardous, adequate ventilation and exhaust capabilities (fume
hoods and instrument exhaust) are mandatory.
When perchloric acid is used, perchlorate salts can be formed, either i_n
situ or in the ductwork of conventional fume noods. These ccmpouncs are poten-
rTaTiy explosive.4 For this reason, solutions to which perchloric acid has
been added must never be heated to dryness. In addition, neating and evapora-
tion of such solutions should always occur in a fume hood specially designed
for use with perch'loric acid. Such fume hoods have surfaces washed with water
in order to eliminate the potential buildup of sucn compcunas. Standard safety
precautions must be taken with concentrated acids, particular'':/ squa regia, and
normal laboratory safety rules should be enforced for all analysts who may
be exposed to samples or standards of potentially toxic or carcinogenic
metals.3»4
E. APPARATUS
1. Air Sampling Equipment
1.1 High-Volume Sampler (Haskin Scientific Ltd., Montreal, Canada,
or equivalent).
1.2 Flowmeters, chart paper, and ink.
1.3 Glass-fiber filters, 20 cm x 25 cm.
1.4 Membrane filters, 0.8 vsn pore size, (Millipore or equivalent).
2. Soxhlet extraction apparatus, with extraction thimbles.
2.1 Thermometer adaptor, 1 24/40.
2.2 Polypropylene tubes, graduated, £5 mi.
IV-6
-------
3. Analytical balance, 0.1 mg sensitivity.
4. Low-Temperature Asher (Tracer!ab LTA-600, or equivalent).
5. Perchloric acid fume hood.
6. Watch glasses, ribbed, 75 mm diameter.
7. Filter paper, Whatman No. 42, or equivalent.
8. Centrifuge.
9. Atomic Absorption Spectrometer with appropriate hollow cathode lamps.
10. Hotplate.
11. Air compressor, or compressed air cylinder.
I?.. Steam table.
13. Compressed-gas-cyclinder regulators (acetylene, air, nitrous oxide).
r. REAGENTS
,1. Acetylene, commercial' grade (contains acetone).
2. .Air.
3. Nitrous oxide, commercial grade.
4. Aqua regia. Prepare immediately before use by adding three volumes
concentrated HC1 to one volume concentrated HN03.
5. Calibration standards, prepared from stock standards, over the concentra-
tion range of interest. These solutions can be prepared as indicated
below or purchased commercially.
Standard metal solutions: prepare a series of standard metal solutions
containing 5 to 1000 yg/1 by appropriate dilution of the following stock
metal solutions rfith deionized, distilled water containing 1.5 ml concen-
trated HN03/1.
Aluminum: dissolve 1.000 g aluminum metal in 20 ml cone. HC1 by heating
gently and diluting to 1000 ml, or dissolve 17.584 g aluminum potassium
sulfate (also called potassium alum), KA1(S04)2*12H20, in 200 ml deion-
ized, distilled water, add 1.5 ml cone. HN03, and dilute to 1000 ml with
deionized, distilled water; 1.00 ml = 1.00 mg Al.
Calcium: to ?,4972 g calcium carbonate. CaCOo, add 50 ml deionized water
and add dropwise a minimum volume of cone. HC1 (about 10 mi) to erfect
complete solution. Dilute -.3 1000 -nl «it.h ieionized. distilled water-
1.00 ml = 1.00 mg Ca.
-------
Cadmium: dissolve 1.000 g cadmium metal in a minimum volume of 1+1 HC1 .
Dilute to 1000 ml with deionized, distilled water; 1.00 ml = 1.00 mg Cd.
Chromium: dissolve 2.828 g anhydrous potassium dichromate, I^CrgOy, in
about 200 ml deionized, distilled water, add 1.5 ml cone. HN03, and dilute
to 1000 ml with deionized, distilled water; 1.00 ml-= 1.00 mg Cr.
Copper: dissolve 1.000 g iron wire in 50 ml of 1+1 HN03 and dilute to
1000 ml with deionized, distilled water; 1.00 ml = 1.00 mg Cu.
Lanthanum solution: dissolve 58.65 g lanthanum oxide, 1.3203, in 250 ml
concentrated HC1 . Add the acid slowly until the material is dissolved
and dilute to 1000 ml with deionized, distilled water.
Iron: dissolve 1.000 g iron wire in 50 ml of 1+1 HN03 and dilute to
1000 ml with deionized, distilled water; 1.00 ml = 1.00 mg Fe.
Lead: dissolve 1.598 lead nitrate, Pb(N03)2, in about 200 ml of water,
add 1.5 ml cone. HNOs, and dilute to 1000 ml with deionized, distilled
water; 1.00 ml = 1.00 mg Pb.
Magnesium: dissolve 10.0135 g magnesium sulfate heptahydrate,
MgS04-7H70, in 200 ml deionized, distilled water, add 1.5 ml cone. HN03,
and make'up to iOOO ml with aeionized, unfilled *atsr; A.JO .TI'I -
1.00 nsg Mg..
Manganese: dissolve 3.076 g manganous sulfate monohydrate,
in about- 200 ml deionized, distilled water, add 1.5 ml cone. HN03, and
make up :o 1000 ml witn deionized, distilled water; LOG ml = l.CG r.g Mn.
Molybdenum: dissolve 1.840 g ammonium molybdate, (NH^^Moyt^J'A^O,
in deionized water and dilute to 1 liter.
Nickel: dissolve 4.953 g nickelous nitrate hexahydrate,, Ni(N02)2'6H20,
in about 200 ml deionized, distilled water, add 1.5 ml cone. HN03, and make
up to 1000 ml with deionized, distilled water; 1.00 ml = 1.00 mg Ni.
Zinc: dissolve 1.000 g zinc metal in 20 ml 1+1 HC1 and dilute to 1.000 ml
with deionized, distilled water; 1.00 ml = 1.00 mg Zn.
6. Deionized, distilled water.
7. Extraction reagents.
7.1 Reagents for PDCA Extraction
Pyrrol idine dithiocarbamic acid (PDCA): prepare by adding 18 ml of
analytical reagent grade pyrrol idine to 500 ml chloroform in a 1-1
flask. Cool and add 15 ml of carbon disulfide in small portions with
swirl-ing, ana dilute to 1 'itar *ith chloroform, "tcra *n in imber
bottle, refrigerated.
IV-8
-------
Ammonium hydroxide, 2 N: dilute 3 ml cone. NfyOH to 100 ml with
deionized, distilled water.
Bromphenol blue indicator solution: dissolve 0.1 g bromphenol blue
in 100 ml 50-percent ethanol or isopropanol.
HC1, 5 percent (y/v): dilute 2 ml redistilled HC1 to 40 ml with
distilled, deionized water.
7.2 Reagents for APDC/MIBK Extraction
a) Ammonium pyrrolidine dithiocarbamate acid (APDC) solution:
dissolve 1.0 g APDC in deionized, distilled water and dilute to
100 ml. Filter through a 0.45 y pore size membrane filter. Prepare
fresh daily.
b) Buffer: dissolve 272 g sodium acetate in 1500 ml deionized,
distilled water. Add 125 ml glacial acetic acid and dilute to 2
liters. Adjust pH to 3.8 with cone. HN03.
c) Indicator: dissolve 0.02 g methyl red and 0.10 g bromocresol
green In 50 ,nl methanol.
-i] SuK'Jr'c 3c:d, 0.25 N: mix 7,0 cone. 'H-?SOd with deionized.
distilled water and dilute to 1.0 liter.
e) NfyOH (1+1): mix equal volumes cone. NfyOH and deionized,
distilled water.
f) Methyl isobutyl ketone - HPLC grade.
8. Hydrochloric acid, cone. (sp. gr. 1.18; 36.5 to 38.0 percent).
9. Hydrochloric acid, constant-boiling (approximately 19 percent}.
10. Nitric acid, cone. (sp. gr. 1.42; 69.0 to 71.0 percent).
11. Perchloric acid - Nitric acid mixture. Add 10 ml of cone, perchloric acid
to 90 ml of cone, nitric acid.
12. Perchloric acid, 10 percent (v/v): dilute 100 ml cone, acid to 1 "Hter
with water.
13. Nitric acid, 40 percent.
14. LiB02, anhydrous (G. F. Smith No. 304).
15. LiF, powder (Alfa No. 87628).
VS. ^O*.
17. Indicating taSO,*.
IV-9
-------
G. QUALITY CONTROL
1. Any laboratory using these methods should operate a formal Quality
Assurance program.5 The minimum requirements of such a program consist
of an initial demonstration of laboratory capability and continued
monitoring of laboratory performance. Records of laboratory performance
should be maintained and available for reference or inspection. Ongoing
performance checks should be compared with established performance cri-
teria to determine if the results of analyses are within accuracy and
precision limits expected. An unknown performance evaluation sample
should be analyzed at least once a year for all metals of interest as a
check on laboratory performance.5
2. Other quality control steps should be implemented to the extent pos-
sible, including: service contracts for maintenance and calibration of
balances and atomic absorption spectrometer, use of Class S weights for
periodic checks on balances, dating and replacement (as necessary) of
chemicals, analysis of standard reference material(s) at least once a
quarter, analysis of at least 10 percent duplicate samples, monitoring of
standard deviation of all measurements, tabulation of mean and standard
deviation of data, and use of quality control charts.5.
H. CALIBRATION
1. Calibration Standards
Calibration standards are prepared by diluting the stock metal solutions
?,t *:h9 time of analysis. For" best results, calibration standards should
be prepared fresh each time an analysis is to be made and discarded after
use. Prepare a blank and at least four calibration standards in graduated
amounts in the appropriate range. The calibration standards should be
prepared using the same type of acid or combination of acids at the same
concentration(s) as will result in the samples following processing.
Since filtered water samples are preserved with 1:1 redistilled HNOs (3 ml
per liter), calibration standards for these analyses should be similarly
prepared with HN03. Beginning with the blank and working toward the
highest standard, aspirate the solutions and record the readings. Repeat
the operation with both the calibration standards and the samples a
sufficient number of times to secure a reliable average reading for each
solution.
For routine samples, the Calibration Curve Technique can be used to convert
sample absorbances to concentrations (paragraph 2). Where the sample matrix
cannot be accurately matched with standards, the method of standard additions
(paragraph 3) should be used.
2. Calibration Curves
cor Instruments which do not give results in concentration units, a cali-
bration curve is ootainea oy plotting measured aosoroance as a function or
concentration rir a series of standards. The absorbance of the samole is
then compared directly to the curve to determine the concentration of the
TV-i.O
-------
unknown. If sample absorbances fall outside the range covered by the
standards, they should be diluted and reanalyzed.
3. Method of Standard Additions
In the method of standard additions, incremental amounts of the analyte
being quantified in equal volumes are each added to separate but equal
volumes of sample, and the absorbance of each mixture measured by atomic
absorption spectrometry. The amounts of analyte added to the sample
aliquot should vary from 0 to 150 percent of the amount of analyte expec-
ted to be present in the sample aliquot.
The results of these measurements are plotted as a function of added
analyte, and when the resulting curve is extrapolated to zero absorbance,
the intersection with the abscissa gives the.concentration of analyte in
the sample. The method of standard additions is required when matrix
interferences are known or suspected of existing in the sample being
analyzed. Figure 2 presents an example of such a plot.
I. DAILY PERFORMANCE TESTS
A calibration curve consisting of at least one reagent blank and three
standards should be obtained daily and subsequent calibration checks must be
rfenfied with at laast one rsagent biank ana one standard. If 20 or more
samples are analyzed in a day, the calibration curve must be verified by anal-
lysis of a standard (at or near the maximum concentration of the curve) after
each 20 samples. Results must be within 10 percent of the expected value.
IV-11
-------
I
I
(8) (6) (4) (2) 0 2 4
Added Standard (jig/ml)
8
Figure 2. Typical graph for standard addition method. The sample contains
5.6 uq/ml of the element. (From C. T. Kenner and K. W. Busch,
Quantitative Analysis, ftactmilom, rt. i., -979. /&
-------
J. ANALYTICAL PROCEDURES
1.1 Determination of Metals in LMB/LiF Fusion Pellets of Waste Samples
Analytical Procedure: available
Sample Preparation: available
1.1.1 Reference
U.S. Environmental Protection Agency, "LMB/LiF Fusion of HWDS
Solid-Phase and Nonaqueous Samples for Total Metals." U.S.
EPA - National Enforcement Investigation Center, Denver,
Colorado, Method 200.62. 3 p. (no date).7
1.1.2 Method Summary
A 100-mg sample is fused with lithium metaborate (LMB) and
lithium fluoride (LW) for 9 minutes at 975°C in a graphite
crucible in a muffle furnace. After cooling, the fusion pel-
let is transferred to a polyethylene vial, dissolved in 100 ml
of 4-percent HNOs, and filtered through a prewashed 0.45-um
membrane filter to remove any carbon from the graphite cru-
cible that may nave adhered to the pellet. The digest i-s then
analyzed as a liquid sample using atomic absorption spec-
trometry.
1.1.3 Applicability
The method is primarily applicable to hazardous waste samples
and has keen successfully aoplied to high-silica soil, sedi-
ment, clay, sludge, ash, sulfide bearing ore, and oil. Because
of the relatively low temperature used, volatile elements sucn
as arsenic and lead may also be determined in the final solu-
tion.
1.1.4 Precision and Accuracy
When the LMB/LiF fusion sample procedure was used with stand-
ard reference materials and the resulting digests analyzed by
ICAP, recoveries in excess of 90 percent were the general rule
•(Table 2 in ICAP Section). Atomic absorption performance
would be expected to be similar to that reported *or analysis
of sediment samples by atomic absorption.
1.1.5 Sample Preparation
Place the weighing dishes in a vacuum desiccator containing
P205 and CaS04. Obtain and record a constant weight for these
dishes.
Weigh an aliquot of -vet HWDS solid-ohase samole (aoproximately
1 g) into a pre-weighed dish. Place the samples in the desic-
cator ana apply a vacuum.
IV-13
-------
After 48 hours of desiccation, reweigh the samples plus dishes.
(A constant weight is obtained when two weighings separated by
6 hours agree to within 2 percent.) The percent moisture of
the sample is calculated as:
(wet wt.) - (dry wt.)
% moisture = x 100
(wet wt.)
Place 6 to 8 graphite crucibles in a preheated muffle furnace
and heat at 1,000°C for 30 minutes. Remove the crucibles;
allow to cool slightly. Blow off excess graphite and chalky
residue with compressed air.
Heat the crucibles for an additional 30 minutes at 1,000°C.
Repeat the process of blowing off excess graphite and chalky
residue with compressed air.
Store the prepared crucibles in a clean, closed container.
Prepare the lithium metaborate flux by combining 3 parts LiF
and 7 parts LiBO^ (by weight). Mix the mixture thoroughly
and store in a tightly-sealed container as the flux is hygro-
scopic.
Preneat the muffle furnace to 795°C.
Tare the prepared crucible. Weigh 1.0 g 1MB flux into the
•crucible, neigh 0.100 g dry sample into the '"'ux. Carefully
stir the contents of the crucible with a platinum wire to mix
the sample and the flux. Distribute the mixture evenly in
the crucible.
Using tongs, place the crucibles in the furnace. Fuse the
samples for 5 minutes. Check the samples after 5 minutes
(for splattering, etc.) and mix. Return the samples to the
furnace and fuse for an additional 4 minutes (total fusion
time is 9 minutes).
Remove the crucibles and place on a cinder plate to cool.
After the fusion product has cooled, transfer the pellet to an
8-ml polyethylene vial by tapping the bottom of the crucible.
Dissolve the pellet in 100 ml of 4 percent v/v HN03. After
dissolution is complete, filter the sample through a prewashed
0.45 ym membrane filter to remove any carbon from the graphite
crucible that may have adhered to the pellet.
1.1.6 Sample Analysis
Analyze the digests using standard atomic absorption spectrom-
etry proceaures as presented in Subsection J.2.
IV-14
-------
2.1 Analysis of Water Samples for Metals
Analytical Procedure: evaluated
Sample Preparation: evaluated
2.1.1 Reference
U.S. Environmental Protection Agency, "Methods for Chemical Anal-
ysis of Water and Wastes." Environmental Monitoring and Support
Laboratory. U.S. EPA, Cincinnati, Ohio. EPA-600/4-79-020, 1979.l
2.1.2 Method Summary
Water or wastewater samples are digested with acid at elevated
temperature, the digest is diluted to a predetermined volume, and
analyzed by atomic absorption spectrometry.
2.1.3 Applicability
This method is applicable to water samples and municipal and indus-
trial wastewaters for determination of the metals listed in Table 1.
Table 1 also lists the optimum concentration range, the sensi-
tivity, and the detection limit for each metal.
2.1.4 Precision and Accuracy
Table 2 summarizes orecision and accuracy information obtained
with the atomic absorption method for the determination of 32
' r ' ' elements using the flame atomization mode, for 29 elements using
the graphite furnace mode, and for 2 elements using hydride gen-
eration procedures. Accuracy data are expressed as percent b-ias
and precision data are expressed as one standard deviation.
2.1.5 Sample Preparation
General guidance is provided to prepare samples for the determina-
tion of dissolved metals concentrations, total metals concentra-
tions, and particulate metals concentrations. The distinction
between these phases is operationally defined by the filter pore
size used in the filtration process.
2.1.5.1 Dissolved Metals Samples
For the determination of total dissolved metals concentrations,
the sample should be filtered through a 0.45 ym pore size mem-
brane filter at the time of collection, if possible, or as soon
as practical thereafter (the solid material on the filter can be
discarded or retained for suspended solids determination, para-
graph 2.1.5.3). The filtrate should be acidified to a pH less
than 2 with nitric acid. Three milliters of 1:1 acid per liter
1s usually sufficient for this adjustment. If a precipitate
forms upon acicnfication, -digest the filtrate as outlined in
oaraqraoh ?.1.5.2.
IV-15
-------
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=========
TABLE l. (Continued)
================C==================================3
Flame Atomization
Metal
H,
Mn
Mo
Ni
Os
Pd
Pt
K
Re
Rh
Ru
Ag
Na
n
Wave-
length
(nm)
285.2
279.5
313.3
232.0
290.9
247.6
265.9
766.5
346.0
343.5
349.9
328.1
589.6
276.8
Opt i mum
Concentration
Range
(mg/1 iter)
0.02-0.5
0.1-3
1-40
0.3-5
2-100
0.5-15
5-75
0.1-£
5-1000
1-30
1-50
0.1-4
0.03-1
1-20
Sensitivity
(mg/1 iter)
0.007
0.05
0.4
0.15
1
0.25
2
u . u-t
15
0.3
0.5
0.06
0.015
0.5
Detection
Limit
(mg/1 iter)
0.001
0.01
0.1
0.04
0.3
0.1
0.2
J • U 1
5
0.05
0.2
0.01
0.002
0.1
Flame
(type)
Air-acetylene
(oxidizing)
Air-acetylene
(oxidizing)
(fuel rich)
Air-acetylene
(oxidizing)
NgO-acetylene
(fuel rich)
Air-acetylene
(oxiaizing;
Air-acetylene
(oxidizing)
lightly oxidizing)
N20-acetylene
(fuel rich)
Air-acetylene
(oxidizing)
Air-acetylene
(oxidizing)
Ai r-acetylene
(oxidizing)
Air-acetylene
(oxidizing)
Air-acetylene
(oxidizing)
coni'.nuea
IV-17
-------
TABLE 1. (Continued)
: = ss = s:sss3: = = s = = 3S= = = == =
Flame Atomization
=================
Metal
Sn
Ti
V
In
Metal
Al
So
As**
3a
Be
Cd
Cr
Co
Cu
Au
Ir
Fe
Pb
Wave-
length
(nm)
286.3
365.3
318.4
213.9
Waveiengtn
(nm)
309.3
^ 1 -^ -
£i/ .0
193.7
352.5
234.9
228.8
357.9
240.7
324.7
242.8
264.0
248.3
283.3
Optimum
Concentration Detection
Range Sensitivity Limit
(mg/liter) (mg/liter) (mg/liter)
10-300 4 0.8
5-100 2 0.4
2-100 0.8 0.2
0.05-1 0.02 0.005
Furnace Method***
Optimum Concentration
Range Sensitivity
(yg/Hter) (ug/liter)
20-200
:o-:oo
5-100
10-200
1-30
0.5-10
5-100
5-iOO
5-100
5-100
100-1500
5-100
5-100
Flame
(type)
N20-acetylene
(fuel rich)
N20-acetylene
(fuel rich)
Air-acetylene
(oxidizing)
Air-acetylene
(oxidizing)
Detection
.--it
'yg/liter>
3
•*
1
?
0.2
0.1
1
-
1
1
30
1
1
(continued)
IV-18
-------
TABLE 1. (Continued)
========================================
Furnace Method***
Metal
Mn
Mo
Ni
Os
Pd
Pt
Re
Rh
rtU
Se1**
Ag
TT
Sn
Ti
V
Zn
Metal
Wavelength
(nm)
279.5
313.3
232.0
290.9
247.6
265.9
346.0
343.5
349.3
196.0
328.1
275.3
224.6
365.4
318.4
213.9
Wavelength
(nm)
Optimum Concentration
Range Sensitivity
(vg/liter) (ug/liter)
1-30
3-60
5-100
50-500
20-400
100-2000
500-5000
20-400
100-C009
5-100
1-25 . '_
5-100
20-300
50-500
10-200
0.2-4
Gaseous Hydride Methods
Working Range
(ug/liter)
Detection
Limit
(ug/liter)
0.2
1
1
20
5
20
200
5
?0
2
0.2
1
5
10
4
0.05
Detection Limit
dig/liters)
As
193.7
2-20
Se 196.0 2-20 2
»s=r===========================================================================
*Chelation-extfaction method
•'Gaseous hydride method also Available.
***Consult Instrument operators manual for appropriate conditions.
IV-19
-------
OZ-AI
(SBLQ }U30J3d)(^U33Jad) UOL^BLA3Q
nv
£*I
—
L'LS
Z*OI-
S'fr-
--
9£I
z-z-
— —
—
jojja
JOJJ3
jojja
—
9H
£-9
—
L&
86
86
__
66
66
— " "
86
001
£11
• H 3 1
-------
TABLE 2. (Continued)
Flame Atomization
Metal
Feb
Pbb
Mg3
Mnb
Mo a
Mia
Os
Pd
Pt
Ka
Re
Rh
Ru
Concent ration(s)
(mg/liter)
0.840
0.438
0.024
0.010
0.367
0.334
0.101
0.084
0.037
0.025
2.1
8.2
0.^26
0.469
0.084
0.106
0.011
0.017
3.30
1.5
7.5
0.20
1.0
5.0
—
—
—
1.6
6.3
—
—
— —
Standard
Deviation
0.173
0.183
0.069
0.069
0.128
0.111
0.046
0.040
0.025
0.022
0.1
0.2
0.070
3.097
0.026
•0.031
0.027
0.020
0.007
0.02
0.07
0.011
0.02
0.04
—
—
—
0.2
0.5
—
--
• •»
Recovery
(Percent)
„
-_
—
—
— —
--
-_
-_
--
—
100
100
_ —
-_
--
—
—
—
100
96
95
100
97
93
—
—
—
103
102
—
—
__
Accuracy
(Percent Bias)
1.8
-0.7
141
382
2.9
1.8
-0.2
1.1
9.6
25.7
_«.
—
1.5
» - *_
2.1
2.1
• 93
22
— —
--
--
— —
.-
—
—
_.
—
— ^
—
--
—
__
[continued;
IV-21
-------
TABLE 2. (Continued)
===============================
Concentration(s)
Metal (mg/liter)
Age 0.050
Naa 8.2
52
Tia 0.60
3.0
15
Sna 4.0
20
60
Ti
Va 2.0
10
50
Znb 0.310
1.056
0.070 "
0.011
0.007
Concentration(s)
Metal (ug/liter)
Al
Sb
Asa 15
20
50
100
Baa 500
!QOO
Flame Atomi ration
Standard
Deviation
8.8
0.1
0.8
0.018
0.05
0.2
0.25
0.5
0.5
—
0.10
0.1
0.2
0.114
0.028
0.028
0.018
0.026
Furnace Methods
Standard
Deviation
—
—
0.75
0.7
1.1
1.6
2.5
2.2
==========
Recovery
(Percent)
10.6%
102
100
100
98
98
96
101
101
—
100
95
97
— —
--
--
--
— —
Recovery
(Percent)
—
—
90
105
106
101
96
102
Accuracy
(Percent Bias)
rel . error
__
--
__
__
--
— —
--
--
—
~ •
-_
--
-0.7.
11.3
6.6
66.6
206
Accuracy
(Percent Bias)
—
—
«. .
--
--
—
__
--
(continued)
-------
:===============================
TABLE 2. (Continued)
s s s s s = s s s s s s a s s ass 5 = = = = =: = :
Furnace Methods
Metal
Cd
Co
Cu
Au
Ir
Fe
Mn
Mo
Ni
Os
Pd
Pt
Re '
Rh
Ru
Sea
Concentration(s)
(ug/liter)
2.5
5.0
10.0
19
48
77
25
50
100
.5
Standard
Deviation
0.10
0.16
0.33
0.1
0.2
0.8
1.3
1.6
3.7
0.6
Recovery
(Percent)
96
99
98
97
101
102
88
92
97
92
Accuracy
(Percent Bias)
--
10
70
0.4
0.5
9R
100
'csntinuad)
IV-23
-------
TABLE 2. (Continued)
Furnace Methods
Concentration(s) Standard Recovery
Metal (yg/liter) Deviation (Percent)
Aga 25 0.4 94
50 0.7 100
75 0.9 104
Tl
Sn
Ti
v
- Zn
Gaseous 1Jydride Methods
Concentration Standard Deviation
Metal (uq/liter) (ug/liter)
"-,s3 5 . r:
in 0.9
20 1.1
Sea 5 0.6
10 " 1.1
.15 2.9
Accuracy
(Percent Bias)
--
Recovery
(Percent)
Od
93
85
100
100
-101
=================
^Single-laboratory statistics
bInter!aboratory statistics
cRound-robin statistics
Proceed to paragraph 2.1.6 for analysis of the samples. Report
the results as dissolved or filterable metal concentrations.
2.1.5.2 Total Metals Samples
Transfer a 50- to 100-ml, well-mixed sample to a 150-ml or
larger beaker. Add 5 ml concentrated HN03 to the sample and
ayaoorate to near Hr^ness in 3 hot nlats. Caution should be
exercised during this process to ensure that the sample does not
boil.
-------
Cool the sample and add a second 5-ml portion of concentrated
HN03- Cover the beaker with a watch glass and reflux the sample
on a hot plate. Additional acid should be added to the sample
as necessary during the refluxing. The heating should be con-
tinued until the digestion process is complete, as indicated by
the presence of a light-colored residue.
Following digestion, add 1 to 2 ml concentrated HC1 and warm
the beaker slightly. Wash the watch glass and beaker walls with
distilled water. Filter the digestate to remove any remaining
insoluble matter and adjust the volume of the filtrate to a con-
venient volume with distilled water.
Proceed to paragraph 2.1.6 for analyses of the samples. Report
the results as total metal concentrations.
2.1.5.3 Suspended Metal Samples*
The following sample preparation technique is useful for most
elements determined by atomic absorption spectrometry. These
procedures should be equally applicable to air particulate
solids as well as aqueous particulate solids. However, separate
digestion techniques are provided for suspended solids samples to
be :nalyred "or ;ola, ^r-idium, palladium, and olatinum 'oaragraph
2.1.5.4), arsenic and selenium (paragraph 2.1.5.5) or rhenium and
titanium 'paragraph 2.1.5.5).
Filter a well-mixed portion of the unpreserved sample through a
Q.i?, urn pore s-'zs membrane filter. Record the volume of samole
•filtered. (The exact volume of sample necessary will vary
inversely with the concentration and the composition or tne
suspended solids.)
Transfer the membrane filter to a 250-ml beaker, add 3 mi concen-
trated HN03, cover with a watch glass, and heat gently. Increase
the temperature of the hot plate after the membrane dissolves.
When the acid has nearly evaporated, allow the beaker and watch
glass to cool, and add 3 ml concentrated HN03. Continue heating
the sample until digestion is completed as indicated by the pres-
ence of light-colored residue or the complete absence of partic-
ulate matter.
Remove the watch glass and evaporate the sample to near dryness.
Add 5 ml 1:1 hydrochloric acid and warm the beaker to dissolve
the residue. (If the sample is to be analyzed for silver or
1f the graphite furnace method is to be used, 1 ml of 1:1 nitric
acid should be used in place of the hydrochloric acid.)
Wash down the watch glass and the walls of the beaker with de-
*oni?.ed. -11 stilled water. Filter the samole to remove any silicates
or other remaining insoluble materials. Adjust the volume or cne
uigest co a convenient -volume with distilled watsr.
IV-25
-------
Proceed to paragraph 2.1.6 to analyze the digest. Report the
results as the participate concentration.
2.1.5.4 Samples to be analyzed for gold, iridium, palladium, and platinum
are acidified with 3 ml concentrated HN03 and heated to near
dryness. Add 5 ml aqua regia, heat on a steam bath for 30 min-
utes. Remove the watch glass and allow the sample to evaporate
to near dryness. Transfer the digest to a convenient-size vol-
umetric flask and dilute to volume with distilled water. Pro-
ceed to paragraph 2.1.6 for analysis and report the results as
the particulate concentration.
2.1.5.5 For samples to be analyzed for arsenic or selenium, add 2 ml
30 percent hydrogen peroxide, 1 ml concentrated HN03, and heat for
1 hour. Transfer the digest to a 100-ml volumetric flask, add
10 ml 1-percent nickel nitrate, and dilute to volume with distilled
water.
Proceed to paragraph 2.1.6 to analyze the digest. Report the
results as the particulate concentration.
2.1.5.6 For samples to be analyzed for rhenium, acidify with 1 ml con-
centrated HN03. Warm the sample on 3 steam bath for 15 minutes.
Allow the sample to cool, filter to remove any resiauai soiias,
and dilute the digest to a convenient volume *n'th distilled
water.
When titanium analyses are to be run, the digestion procedure is
modified siigntly. Acidify che sample with i mi concentrated
riNC3 and 1 ml concentrated H2S04. Heat the .sample j/itil the
evolution of $03 fumes. Allow the sample to cool, filter to
remove any residual solids, and dilute the digest to a convenient
volume with distilled water.
Proceed to paragraph 2.1.6 to analyze the digest. Report the
results as the particulate concentration.
2.1.6 Sample Analysis
Samoles to be analyzed by atomic* absorption spectrometry can be
processed using three separate techniques. These tecnniques are
Direct Flame Atomization (paragraph 2.1.6.1), Graphite Furnace
(paragraph 2.1.6.2), or Cold Vapor Analysis (mercury). Samples
can also be analyzed using chelation-extraction but specific
guidance is not provided here.
2.1.6.1 Direct Flame Atomization.
Prepare a series of working metal standards by diluting the
appropriate stocx ioiutions *ith aeionizea, uisffilea water
containing 1.5 ml concentrated HNO-^/l. These solutions should
be prepared fresh on the day of use.
IV-26
-------
Install the appropriate hollow cathode lamp in the instrument.
Align the lamp and set the source current according, to the
manufacturer's instructions. Turn on the instrument and allow
both the instrument and lamp to warm up. This process usually
requires 10 to 20 minutes.
Select the slit width and wavelength as appropriate for the
analyte being determined. However, the recommended wavelength
should only be used as a guide. Because of calibration differences,
the actual wavelength should be based on maximum sensitivity
after the instrument has completely warmed up.
Turn on appropriate gases, ignite flame, and adjust the flow of
fuel and oxidant to give maximum sensitivity for the metal being
measured. When using a nitrous oxide flame, a T-junction valve
or alternate switching valve should be employed for rapidly
changing from nitrous oxide to air to prevent flashbacks when the
flame is turned on or off.
Atomize deionized distilled water acidified with 1.5 ml concentrated
HN03/1 and check the aspiration rate for 1 minute. If necessary,
adjust the aspiration rate to 3 to 5 ml/min. Zero the instrument.
Atomize a standard and adjust the burner alignment (up, down,
siaeways) until a maximum signal /asponse is obtained.
Aspirate a series of jietal standards chat bracket the expected
.range of sample concentrations and record the absorbance of each
standard. Rinse the atomizer with deionized distilled water
containing 1.5 ,r,: ^cncsntratad HN03/"1 ';stween ?ach standard.
Atomize the digested water samples and record their absorbances.
Rinse the atomizer with dilute nitric acid between each sample.
Samples scheduled for iron and manganese or calcium and magnesium
analysis should be premixed with calcium chloride or lanthanum
chloride solution, respectively. This is accomplished by mixing
four volumes of digested sample with one volume of the appropriate
salt solution.
When determining metal concentrations by atomic absorption, the
following sequence of sample processing is recommended:
a. Analyze a set of standards.
b. Analyze five samples.
c. Analyze a duplicate of the fifth sample.
d. Analyze five.additional samples.
e. Analyze a duplicate of the fifth sample.
f. Analyze a fifth sample that has been spiked.
.g. Analyze a standard.
,1. Repeat Jteps a
i. Reanalyze standards.
IV-27
-------
This approach will Incorporate certain aspects of the quality
control program into the analytical procedure. Specifically,
the suggested sequence allows for evaluation of instrument
stability, replicate analysis, and spike recovery.
Prepare a standard curve by plotting the absorbance of each
standard versus concentration for each metal. Use the standard
curve to convert sample absorbance to metal concentration.
Calculate sample concentrations as indicated in Subsection L.
2.1.6.2 Graphite Furnace Procedure
The use of graphite furnaces or carbon rod atomizers is considered
to be an approved test method because it is essentially an atomic
absorption technique.8 However, the method is not meant for use
with all samples. The method should only be considered as an
alternative to conventional flame atomic absorption spectrophotom-
etry when one or more of the following conditions exist;!
a. Greater analytical sensitivity is required.
b. Sample size is limited.
c. Samples have a high dissolved solids content and cannot be
aspirated into a flame.
These conditions will usually result in the use of a graphite
furnace with water samples oecause of che higher metal concentra-
tions in sediments and the relative ease of increasing sediment
sample size during the digestion step.
Once d decision has been made to utilize a graphite ^urnace,
install the attachment according to manufacturer's instructions.
Align the atomizer as required and warm up the instrument.
Optimum instrument conditions for the graphite furnace will
generally be identical to those used for flame atomization.
Check individual operation manuals for differences with specific
metals (usually arsenic and selenium). Warm up the background
corrector, which should always be used with the graphite furnace.
Prior to the analysis of a new series of samples or the use of a
new sample cup, it is recommended that the atomizer be decontam-
inated. This can oe accomplished by operating the instrument in
the maximum temperature mode for approximately 2 seconds.
The analysis is conducted by transferring a known volume of stand-
ard or sample, 5 to 20 yl, to the sample cup. The recommended
method of sample transfer would be through the use of an automatic
sampler attachment. Other methods such as the use of oxford or
Eppendorf pipettors can be used; but an increase in analytical
variability 1s to be expected as a result. The sample is then
aUDjectea to a drying cycle :o ramove solvent, in Ashing :yc!e to
destroy organic matter, and an atomizing cycle (Table 3). The
metai concentration is quantified during che atomization cycla.
IV-28
-------
TABLE 3. GRAPHITE FURNACE OPERATING CONDITIONS FOR SELECTED METALS
==============================================================================
Conditions AT As Cd Cr Cu Fe
Drying Time,
sec.
Drying Temp.,
°C
Ashing Time,
sec.
Ashing Temp. ,
°C
Atomizing Time,
sec.
Atomizing Temp. ,
°C
Purge Gas
Wavel ennth,
nm
Conditions
Drying Time,
sec.
Dryina Temo. ,
°C
Ashing Time,
sec.
Ashing Temp.,
°C
Atomizing Time,
sec. .
Atomizing TemD. ,
°C
Purge Gas
Wavelength,
nm
30
125
30
1300
10
2700
Ar
309.3
Pb
30
125
30
500
10
2700
Ar
283.3
30
125
30
1100
10
2700
Ar
193.7
Mn
30
125
30
1000
10
2700
Ar
279.5
30
125
30
500
10
1900
Ar
228.8
Mo
30
125
30
1400
15
2800
Ar
313.3
30
125
30
1000
10
2700
Ar
357.5
Ni
30
125
30
900
10
2700
Ar
232.0
30
125
30
900
10
2700
Ar
324.7
Se
30
125
30
1200
10
2700
Ar
196.0
30
125
30
1000
10
2700
Ar
£48. -j
Zn
30
1 "C
30
400
10
2500
Ar
213.9
:============================================
IV-29
-------
V. SCREENING AND GENERAL SAMPLE CHARACTERIZATION PROCEDURES
-------
SECTION 19
METHODS FOR THE DETERMINATION OF OXIDANTS
A. SCOPE
These methods are applicable .to the measurement of the oxidant content
In air^ and agueous samp1es2»3 as well as to aqueous extracts of both hazardous
waste samples^ and solid phase samples such as soil or sediment.
B. SAMPLE HANDLING AND STORAGE
It is not possible to recommend preservation techniques or storage times
for samples that contain reactive chemical soecies. Therefore, these proce-
dures should be completed as rapidly as possible.
C. INTERFERENCES
1. General
Since the tests are not specific'for one particular chemical species,
:ny :uDSuanc3 ±at -^ac'is -jith ;atu£3Vuni ;ccnde *o '•
-------
Peroxyacetyl nitrate gives a response equivalent to approximately 50
percent of that of an equlmolar concentration of ozone. Concentrations
In the atmosphere may range up to 0.1 ppm.
Hydrogen sulflde and reducing dusts or droplets can act as negative
Interferences.
D. APPARATUS
1. Standard laboratory glassware.
2. Microburet, 2 ml, 10 ml.
3. Absorber: all-glass midget 1mp1ngers, graduated 1n 5-ml Increments.
(Other bubblers with nozzle or open-end Inlet tubes may be used. Fritted
bubblers tend to give relatively low results.) Implngers must be kept
scrupulously clean and dust free. Traces of grease can be removed by
alcoholic potassium hydroxide, followed by washing with laboratory
detergent and rinses with tap and distilled water.
4. A1r-meter1ng device: a glass rotameter capable of measuring a flow of
0.5 to 3 liters per minute with a test meter to ensure an accuracy of ±2
percant *z racommended.
5. Air pumo: an appropriate suction dump capable jf drawing the required
sample flow for intervals of up to 30 minutes 1s suitable. It is
desirable'to have a needle valve or critical orifice for flow control.6
A trao should be Installed upstream of the pump to protect the needle
vaive and pump against accidental flooding <»;tn aosoroing reagent -no
consequent corrosion.
6. Spectrophotometer: any laboratory instrument suitable for measuring the •
aosorbance of the iriiodide Ion at 352 nm *itfi stoppered tubes or rjvettss
(suitable for near ultraviolet use) 1s recommended.
E. REAGENTS
All reagents are made from analytical-grade chemicals. They are stable
for several months in well-stoppered bottles.
1. Glacial acetic acid.
2. Potassium Iodide: crystals.
3. Phenylarslne oxide, 0.00564 N: dissolve approximately 0.8 g phenylarsine
oxide powder 1n 150 ml 0.3 N NaOH solution. After settling, decant 110 ml
Into 800 ml distilled water and mix thoroughly. Adjust the pH into the
range 6 to 7 with 6 N HC1 and dilute to 950 ml with distilled water.
Standardize against a standardized Iodine solution prior to use. Com-
mercial ly-preparea pnenyiarslne oxide solutions are dvaiiaoie ,Wallace ana
T1erman. or
-------
4. Double-distilled water, used for all reagents: redistill distilled water
1n an all -glass still after adding a crystal each of potassium permanga-
nate and barium hydroxide.
5. Absorbing solution (1 percent KI in 0.1 M phosphate buffer): dissolve
13.6 g potassium dlhydrogen phosphate (KH2P04), 14.2 g dlsodlum hydrogen
phosphate (Na^HPO^), or 35.8 g of the dodecahydrate salt (^HPO^^hUO) ,
and 10.0 g potassium Iodide 1n sequence and dilute the mixture 1:1 with
double distilled water. Keep at room temperature for at least 1 day
before use. Measure pH and adjust to 6.8 ± 0.2 with NaOH or KHoPO^ This
solution can be stored for several months in a glass-stoppered brown
bottle at room temperature without deterioration. It should not be
exposed to direct sunlight.
6. Stock solution 0.025M 12 (0.05N): dissolve 16 g potassium iodide and
3.173 g resublimed iodine successively and dilute the mixture to exactly
500 ml with double-distilled water. Keep at room temperature at least
1 day before use. Standardize shortly before use against 0.025 M Na2S?03.
The sodium thlosulfate is standardized against primary standard potassium
biiodate (KH( 103)2) or potassium bichromate (I
6.1 0.001M !2 solution: plpet sxactly 4.00 ml of the 0.025-M stock
solution into a 100-ml low-actinic volumetric flask and dilute to
*.*i9 iiars '-ntn iosormng solution. Dorset "rom strong light.
Discard after use.
6.2 Calibrating iodine solution: 4.09 ml of the 0.001-M I? solution
(or equivalent volume for other molarlty) is diluted with absorbing
solution just before use to 100 ml (final volume) to make the final
concantration equivalent to 1 ui of u3/;nl >3ee Calculations; .
Discard this solution after use.
Sulfur dioxide absorber: flashfired glass fiber paper is impregnated with
chromium tnoxide, as follows:^ Drop 15 mi of aqueous solution containing
2.5 g chromium trioxide and 0.7 ml cone, sulfuric acid uniformly over
400 cm2 of paper, dry in an oven at 80 to 90°C for 1 hour and store 1n a
tightly capped jar. Half of this paper suffices to pack one absorber.
Cut the paper in 6- x 12-mm strips, each folded once into a V-shape, pack
into an 85-ml U-tube or drying tube and condition by drawing air that has
been dried over silica gel through the tube overnight. The absorber is
active for it ""east 1 tjonth, When H becomes '-Msibly vet from samolfng
humid air, it must be dried with dry air before further use.
V-4
-------
F. ANALYTICAL PROCEDURES
1.1 Ox'idant Capacity of Hazardous Waste Samples. Reserved.
-------
2.1 Oxidant Capacity of Aqueous Samples
Analytical Procedure: evaluated
Sample Preparation: available
2.1.1 Reference
U.S. Environmental Protection Agency, "Oxldant Determination for
HWDS Samples." U.S. EPA, National Enforcement Investigations
Center, Denver, Colorado. Method 330.65, 2 p. No date.
2.1.2 Method Summary
Potassium Iodide is added to an acidified (pH 4 or less) sample
aliquot (aqueous phase or aqueous extract of a solid phase). The
sample is titrated with phenylarsine oxide, using starch as an
indicator, to quantify the amount of liberated iodine. The oxidant
strength of the sample is expressed as an equivalent concentration
of chlorine.
2.1.3 Apolicability
The method is suitable for both liquid and solid-phase samples.
Mowever, *~~r solia-onase •samoles, *he ^rocadur? only measures the
extractable or exchangeable oxidant capacity as defined Dy the
conditions ':sed to orepare the «oHd-onase extract.'' The range of
the test vrill vary with the size of the sample aliquot titrated.
Data based on the determination or' totai resiauai chionne using
the starch-iodine titration method is presented In Table 1. While
the ourpose was to determine residual chlorine rather than oxidant
capacity, the analytical technique is tne same and the data pro-
vides an estimate of expected procedure performance.
TABLE 1. PRECISION OF OXIDANT DETERMINATION FOR AQUEOUS SAMPLES2
3333BB333333B33333a33::BB3BS3333333333:S33333333333533B33B333BB33=3333B33333=33:s3
Sample
Matrix
Distilled water
Distilled water
Drinking water
River water
Domestic sewage
Raw sewage
Replicates
3
3
7
7
7
7
Chlorine
Concentration
mg/1
0.41
3.51
0.84
0.84
0.87
0.55
Std.
Qev.
mg/1
0.05
0.12
0.04
0.02
0.07
0.09
Rel.
Std. Oev.
%
12.2
3.3
4.3
2.7
7.6
16.0
"-5
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2.1.5 Sample Preparation
Obtain an aqueous aliquot of the sample to be analyzed as
Indicated below:
a. Shake 2 g of solid-phase hazardous waste with 200 ml
distilled water for 1 hour. Decant the liquid phase and use
100 ml for the oxldant capacity determination.
b. P1pet 100 ml of aqueous-phase hazardous waste sample or water
sample Into a disposable beaker.
c. Prepare an aqueous extract of soil /sediment samples as
explained In paragraph (a).
Standardize the phenylarslne oxide tltrant against a fresh Iodine
solution. Place 10 ml 0.0282 N Iodine solution 1n a flask and add
1 g potassium Iodide. Titrate with phenylarslne oxide to the
starch endpoint. Adjust to 0.00564 N. 1.00 ml = 200 »g chlorine.
To the 100 ml aqueous extract or water sample, add 5 ml acetic
add. While stirring on a magnetic stirrer, add 1 g KI. Add 1 ml
starch indicator solution.
After the blue color is homogeneously distributed, titrate the
sample with 0.00564 N phenylarsine oxide (PAO) until a colorless
condition persists 1n the sample for at least 15 seconds. This is
*he sndooint
-------
3.1 Determination of Oxidant Capacity in Soil Samples. Reserved.
V-8
-------
4.1 Determination of Oxidant Capacity in Biological Tissue Samples.
Reserved.
V-9
-------
5.1 Determination of Oxldants 1n A1r
Analytical Procedure: available
Sample Preparation: available
5.1.1 Reference
American Public Health Association, "Methods of A1r Sampling and
Analysis." 2nd ed; APHA, Washington, D.C. Method 411, pp. 556-559.
(1977).
5.1.2 Method Summary
This is a method for manual determination of oxidants (Including
ozone) in air over the range of approximately 0.01 to 10 ppm (as
ozone). The method is based on sampling with a midget impinger and
determination of the iodine liberated from a 1-percent potassium
iodide solution at pH 6.8 ± 2.1*7 The Iodine is determined
spectrophotometrically by measuring the absorption of triIodide
ion at 352 nm. The stoich-'ometry is approximated by the following
equation;!
03 * 3
-------
upstream from the impinger. Butt-to-butt glass connections with
lightly greased Tygon tubing may also be used for connections with-
out losses 1f lengths are kept minimal. Pipet exactly 10 ml of the
absorbing solution Into the midget Impinger and sample at a flow
rate of 0.5 to 3 liters per minute. Note the total volume of the
air sample. If the sample air temperature and pressure deviate
greatly from 25*C and 760 mm Hg, measure and record these values.
Sufficient air should be sampled so that the equivalent of 1 to
10 ul of ozone 1s absorbed. Approximately 1 ul of ozone can be
obtained in the absorbing solution at an atmospheric concentration
of 0.01 ppm by sampling for 30 min at 3 1/min.l Do not expose the
absorbing reagent to direct sunlight. If appreciable evaporation
has occurred, add distilled water to restore the volume to the
10-ml graduation mark.
5.1.6 Sample Analysis
Assemble a train consisting of a rotameter, U-tube with chromium
trioxide paper (optional), midget Impinger, needle valve or criti-
cal orifice^ and pump. Connections upstream from the Impinger
should be ground glass or inert tubing outt lointed witn polyvinyl
tubing. FluorosiUcon or fluorocarbon grease should be used
sparingly, s-'oet exactly 10 ml of the absorbing loluticn Into the
midget Impinger. Sample at a rate of 0.5 to 3 I/mm ror up to 30
min, The T]QW rate and tne time of samolinq snouia oe aajustsd to
obtain a sufficiently large concentration of oxidant m the
absorbing solution. Approximately 1 ill of ozone can be obtained
in the absorbing solution at an atmosoherlc concentration of 0.01
opm oy sampling ror 30 min 3t 3 I/mm. Measure cne air temperature
and pressure, and calculate the totai volume of cf-.e air sampie. Do
not expose the absorbing reagent to direct sunlight.
The sample 1s absoroed and read in A0 fli of absoroing reagent.
Calibrating solutions are made up to 10 ml to facilitate the
calculations. For greater precision these can be made up to
25 ml or more.
It is more convenient to standardize the instrument using iodine
solutions than accurately-known gas samples. See paragraph 5.1.2
for postulated stoichiometr-'c relationshios. To obtain a ranqe of
concentration values 1n standardization, add several amounts of
calibrating solution from 0 to 10 ml to a series of !0-ml volu-
metric flasks. Dilute to volume with absorbing reagent.
Read the absorbance of each standard at 352 nm.
Plot the absorbances of the standards versus the ul of 03 in 10 ml
absorbing reagent. The plot should ^ollow Beer's law.
If appreciaole evaporation of the dosoroing solution nas occurrsa
•lur-'ng sampling, 5dd double-distilled water to bring the liauid
volume to 10 ml.
-------
Within 30 to 60 minutes alter sample collection, read the absorb-
ance of the sample solution 1n a cuvette or tube at 352 run against
a reference cuvette or a tube containing double-distilled water.
Measure the absorbance of the unexposed reagent at 352 nm and
subtract the value from the absorbance of the sample.
G. CALCULATIONS
1. Aqueous Samples
Adjust the sample tltratlon volume based on the blank tltratlon results
(subtract the blank PAO tltratlon results or add the net equivalent of the
back-titratlon results).
Calculate the oxidant content (OC) of the solid-phase sample as:
35,450 mg E
OC (ug Cl2/g) a - x - x - x 1,000
S eq G
where:
OC = oxidant concentration
Vi » corrected volume of PAO used, ml
Hi » normality of "AO
S * volume of sample titrated, ml
E = volume of water extract prepared, ml
5 - weiqht of «olid ohase extracted, q.
The oxidant concentration of an aqueous sample is calculated as:
(YiHNii 35,450 mg
OC (mgCl2/D - - x -
S eq
where the terms are defined as above with 35, 450 mg/eq being the
equivalent weight of chlorine.
Air Samples
Standard conditions are taken as 760 Torr and 25*C, at which the molar gas
volume 1s 24.47 liters.
Record the volume of the sample collected in liters. Generally the
correction of the sample volume to standard conditions Is slight and may
be omitted. However, for greater accuracy, corrections may be calculated
•w Tieans of the ideal oas law.
V-12
-------
The calibrating iodine solutions are calculated on the basis, of equiva-
lence of. 63 and 12, as indicated in Subsection E.6.2. Hence, a 100-ml
portion of final solution equivalent to 1 yl Os/ml contains the
following amount of iodine:
100 x 10-6
U = = 4.087 x 10'° moles
12
-6
24.47
This is equivalent to 4.09 ml of 0.001 M (or 0.002N) \2 solution.
The total yl of 03/10 ml of reagent are read from the calibration curve.
The concentration of 03 in the gas phase in yl/1 or ppm is given by:
total yl ozone per 10 ml
03 ppm
volume of air sample in liters
The cone, of 03 in terms of yg/1 at 750 Torr ana 25°C is ootainea *nen
desired from the value of ul/1 by:
ppm-x 48.00
ug 03/ liter = - - 1,362
.17
V-13
-------
REFERENCES
1. American Public Health Association. "Methods of A1r Sampling and Anal-
ysis." 2nd ed., APHA, Washington D.C. pp. 556-559. (1977).
2. U.S. Environmental Protection Agency. "Methods for Chemical Analysis of
Water and Wastes." U.S. EPA, Environmental Monitoring and Support Labo-
ratory, Cincinnati, Ohio. EPA-600/4-79-020, (1979).
3. American Public Health Association. "Standard Methods for the Examination
of Water and Wastewater." 15 Ed. Washington, D. C., (1980).
4. U.S. Environmental Protection Agency. "Oxidant Determination for HWDS
Samples." U.S. EPA, National Enforcement Investigations Center, Denver,
Colorado. Method 330.65, 2 p. No date.
5. Saltzmann, B. E., and A. F. Wartburg, Jr. "Absorption Tube for Removal
of Interfering Sulfur Dioxide in Analysis of Atmospheric: Oxidants."
Anal. Chem. 37:779, (1965).
5. Lodge, J. P., Jr., j. 3. Pate, 3. c. Ammons, and A. F. Swansons,
"The Use of Hypodermic Needles as Critical Orifices in Mir Sampling.'1
Air Poll. Cont. Assoc. J., 16:197-200, 11966).
7. Byers, D. H., and B. E. Saltzmann. "Determination of Ozone in Air by
Neutral and Alkaline Iodide Procedures." J, Am. Indust. Hyg. Assoc.,
19:251-7, (19585.
-------
SECTION 20
METHOD FOR THE DETERMINATION OF REDUCTANT CAPACITY
A. SCOPE
This method is applicable to the measurement of sample reductant capacity
in aqueous samples and aqueous extracts of hazardous wastes, soil and sediment
samples. The reductant capacity of each sample is expressed as an equivalent
concentration of iodine.
B. SAMPLE HANDLING AND STORAGE
The concentration of total reductants in the sample Is expressed as an
equivalent concentration of iodine. However, there is no recommended
preservative or molding time '"or 'odine.1- As ^ rule, itany suostances that
contribute to the reductant capacity of a sample can react with oxygen.
Therefore, it ts recommended that sample container: be completely filled xnth
sample, when possible, to exclude oxygen. Also, the samples should be stored
at 4*C and the storage period kept to a minimum.
~,he rsductant capacity of 'he samoie 's
-------
E. REAGENTS
1. Potassium Iodide, 1 percent w/v: dissolve 1 g KI 1n 100 ml of demin-
erallzed water.
2. Standard Iodine solution, 0.0282 N: dissolve 25 g KI 1n a small volume of
distilled water 1n a 1-Hter volumetric flask. Add the appropriate amount
of 0.1 M stock Iodine solution (approximately 282 ml) and dilute to
1 liter with distilled water. For accurate work, this solution should be
standardized daily against an arsenite or thiosulfate solution. Commer-
cially-prepared iodine solutions are available from Fisher Scientific Co.,
Pittsburgh, Pennsylvania.
3. Stock iodine solution, 0.1 N: dissolve 40 g KI in 25 ml distilled water.
Add 13 g resublimed iodine and stir until dissolved. Transfer to a
1-liter flask and dilute to volume. Standardize against a 0.1-N arsenite
solution using a starch solution as an endpoint indicator. To improve
accuracy, ensure that the solution is saturated with COj at the end of the
tltration by bubbling C02 through the solution or adding HCl to shift the
carbonate equilibrium and liberate C02- Alternately, this solution may be
standardized against the phenylarsine oxide solution used in the oxidarn;
capacity determination.
4. Starch solution: add 1 g starcn to 1 liter of coiling, aistillea water.
Stir and. let settle overnignt. Use tne clear suoernatant. This solution
may be preserved with 1.25 g salicylic acid, 4 g zinc chloride, or a
combination of 4 g sodium propionate and 2 g sodium azide/1.
V-16
-------
F. ANALYTICAL PROCEDURES
Analytical Procedure: available
Sample Preparation: available
NOTE: Measurement of this parameter 1s based on the analysis of an aqueous
phase or aqueous extract. Therefore, all matrices have been combined into
a single procedure. The reductant capacity for solid-phase samples is
operationally defined by the extraction procedure and may not measure the
total reductant capacity of the sample.
1.1 Reference
U.S. Environmental Protection Agency, "Reductant Determination for
HWDS Samples." U.S. EPA, National Enforcement Investigation Center,
Denver, Colorado. Method 331.60. 2 p. No date.2
1.2 Method Summary
An aliquot of sample is added to a trliodide solution. The
decrease in Intensity of the yellow color, due to the presence of
reducing agents, 1s measured spectrophotometrically at 352 nm. The
:otai reducing capacity jf ;he sample is expressed as an equivalent
iodine concentration.
1.3 Applicability
"he .nethod :s ;ui"abla "or both "-'quid- ird ral'd-ohase samoles.
However, for solid-phase samples, *he -srocsdure only Treasures tne
extractable or exchangeable reductant concentration as defined by
the conditions used to prepare the solid-phase extract. A sample
containing 10 mg/1 salflde lan be visually detected with the
procedure and a spectrophotometer can detect 0.5 mg/1 sulflde.
1.4 Precision and Accuracy
Precision and accuracy Information is not available at this time.
1.5 Procedure
P1pet 5 ml of 1% KI solution into a series of test tubes. Add
0.025 ml of 0.0282 N Iodine solution to all but one of the test
tubes.
P1pet 0.010 ml of sample Into one of the test tubes containing the
Iodine solution. The rapid bleaching of the yellow color Indicates
the presence of a reducing agent. If the yellow color 1s completely
dissipated, dilute the sample and test again until some color
remains.
Set the spectropnotometer wavelength at CS2 ,im. Zero ^he s
photometer with the 11 KI solution 1n quartz cuvettes. Measure and
record the absorbance of each sample and a standard iodine solution.
V-17
-------
G. CALCULATIONS
The reductant content of each sample 1s calculated based on the ratio of
the sample absorbance to the absorbance of the standard Iodine solution.
(X)(VI)(N)(D) x 1000
Reductant cone, (meq/1) a
(S)(V2)
where:
X = absorbance reading for sample
S * absorbance r |