PB85-154367
Master Analytical Scheme for
Organic Compounds in Water
Part 1. Protocols
Research Triangle Inst.
Research Triangle Park, NC
Prepared for
Environmental Research Lab., Athens, GA
Jan 85
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
NIKS
-------
PE85-15U367
EPA/600/4-84/010a
January 1985
MASTER ANALYTICAL SCHEME FOR
ORGANIC COMPOUNDS IN WATER:
PART 1. PROTOCOLS
by
E. D. Pellizzari, L. S. Sheldon, J. T. Bursey,
L. C. Michael and R. A. Zweidinger
Analytical Sciences Division
Chemistry and Life Sciences Group
Research Triangle Institute
P.O. Box 12194
Research Triangle Park, NC 27709
Contract No. 68-03-2704
Project Officer
A. W. Garrison
Analytical Chemistry Branch
Environmental Research Laboratory
U.S. Environmental Protection Agency
Athens, GA 30613
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
ATHENS, GA 30613
-------
TECHNICAL REPORT DATA
(Please read !RwuctWns on the reverse before corn pleting)
1 REPORT NO 2
EPA/600/4-84/O lOa
3 RECJ F S AçcgspIot.p o
I 4 oi Ji S
4 TITLE AND SUBTITLE
Master Analytical Scheme for Organic Compounds
in Water; Part 1. Protocols
5 REPORT DATE
January 1985
6. PERFORMING ORGANIZATION CODE
7 AUTHORISI
E.D. Pellizzari, L.S. Sheldon, J.T. Bursey,
L.C. Michael and R.A. Zweidinger
8 PERFORMING ORGANIZATION REPORT NO.
9 PERFORMING ORGANIZATION NAME AND ADDRESS
Research Triangle Institute
P.O. Box 12194
Research Triangle Park NC 27709
10 PROGRAM ELEMENT NO
ABWD1A
11 CONTRACT/GRANT NO
68—03—2704
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency——Athens GA
Office of Research and Development
Environmental Research Laboratory
Athens GA 30613
13 TYPE Of RE!9 I 4. Jp P RIOO COVERED
Finai, ,iio—ii8i
14.SPONSORINGAGENCYCODE
EPA/600/Ol
15 SUPPLEMENTARY NOTES
Part 2: Appendices to Protocols, EPA/600/4-84/OlOb
16. ABSTRACT
A Master Analytical Scheme (MAS) has been developed for the analysis of volatile
(gas chromatographable) organic compounds in water. In developing the MAS, it was
necessary to evaluate and modify existing analysis procedures and develop new tech-
niques to produce protocols that provide for the comprehensive qua] itative—quantitativ
analysis of almost all volatile organics in many types of water. The MAS provides for
analysis of purgeable and extractable, as well as neutral and ionic water soluble,
organics in surface and drinking waters and in leachates and various effluents. Nomi-
nal lower quantifiable limits range from 0.1 pg/L to 100 ig/L, depending on chemical/
physical class of the analyte and complexity of the aqueous matrix. Recoveries are
reported for about 280 model compounds of a wide variety of chemical classes and
physical properties dosed into representative samples of several major types of water.
a DESCRIPTORS
b IDENTIFIERS/OPEN ENDED TERMS
C. COSATI Field/Group
17 KEY WORDS AND DOCUMENT ANALYSIS
.- .. JTION STATEMENT
RELEASE TO PUBLIC
19 SECURITY CLASS (ThuReport)
UNCLASSIFIED
21 NO OF PAGES
35
20 SECURITY CLASS (Thu page)
UNCLASSIFIED
22 PRICE
EPA Form 2220-1 (9-73)
1
-------
DISCLAIMER
The information in this document has been funded wholly or in part
by the United States Environmental Protection Agency under Contract No.
68—03—2704 to Research Triangle Institute. It has been subject to the
Agency’s peer and administrative review, and it has been approved for
publication as an EPA document. 4ention of trade names or commercial
products does not constitute endorsement or recoinniendation for use by
the U.S. Environmental Protection Agency.
11
-------
FOREWORD
Nearly every phase of environmental protection depends on a capability
to identify and measure specific contaminants in the environment. As part of
this Laboratory’s research on the occurrence, movement, transformation, impact
and control of environmental contaminants, the Analytical Chemistry Branch
characterizes chemical constituents of water and soil.
Chemists working in governmental, industrial, and academic laboratories
have long recognized a need for a comprehensive analytical methodology for
organic compounds in water. To this end, research was begun in 1978 to
develop a qualitative—quantitative scheme for the analysis of organics of
all volatility classes (amenable to gas chromatography), of almost all
functional groups, in almost any water sample. The result is the Master
Analytical Scheme (MAS) for Organic Compounds in Water, a compilation of
protocols that empioy new techniques and modifications of existing analysis
procedures. Users may apply the MAS as a set of protocols for a comprehensive
analysis or use individual protocols separately for analysis of organic
fractions of particular interest. Among its many applications are epidemio—
logical studies, wasteload allocations, permit application evaluations, trends
analyses, watershed management studies, exposure assessments, landfill hazard
evaluations, and aqueous source characterizations.
Rosemarie C. Russo, Ph.D.
Director
Environmental Research Laboratory
Athens, Georgia
iii
-------
PREFACE
The Master Analytical Scheme (MAS) represents the first effort to
develop a comprehensive qualitative—quantitative scheme for the analysis of
organic compounds in water. The MAS is a set of analytical protocols that
includes a broad scope of organics with a wide variety of functional groups
and physical properties. These protocols provide for the GC/MS/computer
analysis of the usual purgeable and extractable compounds, with the addition
of various neutral and ionic water soluble compounds; in fact, the I4AS is
applicable to any compound that can pass unchanged through a gas chromato—
graph, or can be derivatized to do so. Recoveries have been determined
from distilled and drinking water, industrial and municipal effluents, and,
in some cases, surface water and energy effluents, so the protocols are
expected to be applicable to most water types. One unique feature of the
MAS is its comprehensiveness. Another is its qualitative—quantitative
aspect: an extensive data base of mass spectrometer detector response and
recovery factors allows computer estimation of concentration without re-
course to standards for each analyte.
In developing the MAS, existing analytical techniques were evaluated
and modified and new techniques were developed to produce the comprehensive
protocols. Development was in two stages. An interim set of protocols was
developed by October 1980; analysis of environmental samples by these
protocols revealed several important deficiencies that were subsequently
corrected by additional experimental work. The final result is this first
edition of MAS protocols.
Two companion reports resulted from MAS development: Experimental
Development of the MAS for Organic Compounds in Water, and Literature Review
for Development of the MAS for Organic Compounds in Water. The user can
refer to the experimental development report for information on techniques
considered for MAS incorporation and experiments dealing with technique
optimization and recovery studies. The literature review, which covers
iv
-------
material through June 1982 on techniques for analysis of organics in water,
was the starting point for experimental development, and will also be of
interest to many users. Neither companion report is essential to MAS use,
however; this report (Part I: Protocols and Part II: Appendices to Protocols)
stands alone as the handbook for implementation. Part II includes: Appendix
A — specific instructions on fabrication of the purge and trap apparatus and
ancillary devices for purgeable organics, Appendix B — hard copy of computerized
relative molar response and recovery data for standards and analytes, and
Appendix C — documentation of MASQUANT computer program for quantification
of MAS data.
The prospective MAS user should thoroughly familiarize himself with
Chapters 1 and 13 of this report (the Introduction and GC—MS—COMP Analysis
Procedures) for an overview and a guide to use of the protocols.
pp 1 icat ions
The MAS was developed for two general types of application:
(1) The analysis of carefully selected samples to answer the question,
“What compounds are present above detection limits and approxi-
mately how much of each is present?” It has been demonstrated
repeatedly that one cannot predict reliably what chemicals will
be found in surface waters, drinking water, or even industrial
effluents for established manufacturing processes (Donaldson,
W. T., Env. Sci. and Technol., 11, 348—351, 1977). Therefore, a
strong need exists for broad spectrum identification and measure-
ment methods for organic chemicals in such water. Applying the
MAS should be cost effective in areas such as:
(a) Drinking Water — in epidemiological studies and as early
wai:ning for toxic pollutants below acutely toxic levels.
(b) Industrial Wastewaters — in wasteload allocations, permit
application evaluation and long—range projections for the
state of the environment.
(c) Surface Waters — in trends analysis, assessments of abate-
ment program effectiveness, watershed management (including
exposure assessment), and incident investigation.
V
-------
(d) Landfill Leachates - in exposure assessment, evaluation of
landfill performance, and diagnosis of problems.
(e) 1 Envjronmenta] . Processes — In chemical characterization of
aqueous sources as related to fate and transport of organic
compounds.
The complete set of MAS protocols may be applied for a survey
analysis, or each protocol may be used as a separate entity for
analysis of an organic fraction (e.g., purgeable volatiles) of
particular interest.
(2) The second application addresses the need for repetitive analysis
of selected pollutants for which methodology has not been devel-
oped. In the qualitative analysis of most samples that contain
chemicals at high enough levels to merit further consideration,
several pollutants will be identified for which methods have not
been suggested (the vast majority of organic compounds, in fact,
falls into this category). By applying only the part(s) of the
HAS that relate to the pollutant(s) of interest, repetitive
measurements for these pollutants can be made at a modest cost
prior to the development of optimized new methods. In such
applications a simpler selective detector can usually be substi-
tuted for the mass spectrometer.
vi
-------
ABSTRACT
A Master Analytical Scheme (HAS) has been developed for the analysis
of volatile (gas chromatographable) organic compounds in water. In develop-
ing the MAS, it was necessary to evaluate and modify existing analysis pro-
cedures and develop new techniques to produce protocols that provide for
the comprehensive qualitative-quantitative analysis of almost all volatile
organics in many types of water. The HAS provides for analysis of purgeable
and extractable, as well as neutral and ionic water soluble, organics in
surface and drinking waters and in leachates and various effluents. Nominal
lower quantifiable limits range from 0.1 ig/L to 100 pg/L, depending upon
chemical/physical class of the analyte and complexity of the aqueous matrix.
Recoveries are reported for about 280 model compounds of a wide variety of
chemical classes and physical properties dosed into representative samples
of several major types of water.
This report was submitted in fulfillment of Contract No. 68—03—2704
by Research Triangle Institute under the sponsorship of the U.S. Environmental
Protection Agency. This report covers a period from September 1978 to July
1983 and work was completed as of July 1983.
vii
-------
CONTERTS
Disclaimer .
Preface iv
Abstract . vii
Acknowledgement xii
1. Introduction i
1.1 Organization of MAS Protocols 2
1.2 HAS Overview 4
1.3 Time Requirements for the HAS 29
1.4 Summary of Recovery and Precision for HAS Protocols . 29
2. Sampling and Sample Handling for Volatile Organics (VO). . 36
2.1 Introduction 36
2.2 Materials and Reagents 38
2.3 Preparation of Standard Solutions 39
2.4 Clean-up Procedures 41
2.5 Preparation of Control and Blank Samples 42
2.6 Field Collection 42
3. Sampling and Sample Handling for Neutral Water Soluble
Organics (NEWS) 46
3.1 Introduction 46
3.2 Materials and Reagents 48
3.3 Preparation of Standard Solutions 50
3.4 Glass Cleaning Procedures 51
3.5 Preparation of Control and Blank Samples 52
3.6 Field Collection 52
4. Sampling and Sample Handling for Extractable Organics
(ESSA, WARN)
4.1 Introduction 55
4.2 Materials and Reagents 57
vii i
-------
CONTENTS (coat’d.)
4.3 Preparation of Standard Solutions 59
4.4 Glass Cleaning Procedures 66
4.5 Preparation of Control and Blank Samples 66
4.6 Field Collection 68
5. Sampling and Sample Handling for Other Ionic Compounds
(VOSA, NOVA, SAIl) 71
5.1 Introduction 71
5.2 Materials and Reagents 73
5.3 Preparation of Standard Solutions 76
5.4 Glass Cleaning Procedures 79
5.5 Preparation of Control and Blank Samples 79
5.6 Field Collection. 79
6. Purge, Trap and Analysis of Volatile Organics (VO) . . . . 84
6.1 Introduction 84
6.2 Apparatus and Reagents 85
6.3 Preparation for Analysis 92
6.4 GC/MS/CONP Analysis 97
7. Elevated Temperature Purge, Trap, and Analysis of
Neutral Water Soluble Organics (NEWS) 108
7.1 Introduction 108
7.2 Apparatus and Reagents 109
7.3 Preparation for Analysis 115
7.4 GC/MS/COMP Analysis 117
8. Batch Liquid-Liquid Extraction and Analysis of
Semivolatile Strong Acids (ESSA) 128
8.1 Introduction 128
8.2 Apparatus and Reagents 129
8.3 Preparation for Analysis 133
8.4 Sample Extraction and Derivatization 140
8.5 GC/MS/CONP Analysis 146
9. Extraction and Analysis of Weak Acids, Bases and
Neutrals (WARN) 153
ix
-------
CONTENTS (cont’d.)
A. Sorbent Accumulator Column (WARN-SC)
9.1
9.2
9.3
9.4
9.5
9.6
9.7
9.8
9.9
9.10
9.11
9.12
9.13
9.14
9.15
9.16
Introduction.
Apparatus and Reagents
Preparation for Analysis
Sample Extraction
C. Continuous Liquid-Liquid
With Flow-Under Extractor
Introduction
Apparatus and Reagents.
Preparation for Analysis.
Sample Extraction
170
171
174
178
183
184
189
194
199
• 199
205
205
209
209
• 209
217
217
218
224
233
236
Introduction
Apparatus and Reagents
Preparation for Analysis
Sample Extraction
B. Batch Liquid-Liquid Extraction (WABN-BL)
154
155
159
166
Extraction
(WABN-FU)
D. Cleanup/Fractionation of WARN Fractions
Introduction
Apparatus and Reagents
Preparation for Fractionation
Sample Fractionation
E. GC/MS/COMP Analysis of WARN-SC, WABN-BL,
or WABN-FU Sample Extracts
9. 17 Introduction
9.18 Preparation for Analysis.
9.19 Analysis
10. Anion-Exchange, Distillation and Analysis of
Volatile Strong Acids (VOSA) . . .
10.1 Introduction
10.2 Apparatus and Reagents. . . .
10.3 Preparation for Analysis
10.4 Ion-Exchange Separation and Derivatization.
10.5 GC/MS/COMP Analysis . . .
x
-------
CONTENTS (cont’d.)
11. Anion-Exchange and Analysis of Nonvolatile Acids (NOVA). 244
11.1 Introduction 244
11.2 Apparatus and Reagents 245
11.3 Preparation for Analysis 251
11.4 Ion—Exchange Separation and Derivatization. . . . . 261
11.5 GC/MS/CONP Analysis 265
12. Cation—Exchange and Analysis of Strong Arnines (SAM). . . . 274
12.1 Introduction 274
12.2 Apparatus and Reagents. . . . . . 275
12.3 Preparation for Analysis 281
12.4 Ion-Exchange Separation and Derivatization 293
12.5 GC/MS/COMP Analysis 296
13. GC/MS/CONP Analysis Procedures - General Instructions
for All Protocols 310
13.1 Introduction 310
13.2 Apparatus and Materials . . 310
13.3 Operational Parameters . . 312
13.4 Analysis of System Performance Solutions (SPS)
and Samples 318
APPENDICES - PART II
A Fabrication of Purge and Trap Apparatus and Ancillary
Devices 1
B RMR and Recovery Data File for Internal and External
Standards and Analytes in MASQUANT 24
C Description of Computer Program MASQUANT for Processing
Data Collected Using MAS 172
x i
-------
ACKNOWLEDGEMENTS
Development of the Master Analytical Scheme would not have been
possible without the efforts and contributions of B. Bickford, D. Dodd, P.
Dodd, B. Hargrove, J. Harry, P. Hyldburg, M. Jones, R. Porch, D. Rosenthal,
C. Sparacino, J. Storm, K. Tomer, R. Wiseman, and S. Yung of Research
Triangle Institute; A. Alford—Stevens, C. H. Anderson, J. J. Ellington, A.
F. Haeberer, J. M. MeGuire, J. D. Pope, and W. M. Shackelford of the Analyti-
cal Chemistry Branch, Athens Environmental Research Laboratory; and W. T.
Donaldson of the Athens Environmental Research Laboratory. J. E. Gebhart,
D. Perry, L. Rando, and J. F. Ryan of Gulf South Research Institute made
significant contributions in the early stages of MAS development. H. S.
Hertz and E. White, V, of the National Bureau of Standards developed the
MAS internal standards.
xii
-------
CHAPTER 1
INTRODUCTION
This set of analytical protocols for the Master Analytical Scheme
(HAS) is predicated on gas chromatography/mass spectrometry/computer
(GC/MS/ COMP) as the final instrumental technique. As such, the HAS is
designed for a comprehensive qualitative/quantitative analysis of purgeable,
extractable, and water soluble organics in drinking and surface waters,
municipal, industrial and energy-related effluents, and leachates. Although
designed to span the complexity encountered in these water types, procedures
are included that define the water quality and allow for optimal detection
limits for that water sample. If the nominal detection limit for qualita-
tive GC/HS analysis is assumed to be 10 ng for an organic compound, then
the limits for the HAS range from 0.1 ppb (e.g., volatile organics in
drinking water) to 100 ppb (e.g., nonvolatile strong acids in energy
effluents) depending upon the chemical/physical class of the analyte and
complexity of the matrix. In general, the quantitative limits of detection
for target compounds with known retention times may be 5-10 times lower.
More information on limits of detection are given in the introduction of
each HAS operational protocol.
The prospective user has the latitude of applying all the protocols or
just those that cover organic group types of interest. Thus, each protocol
stands alone, containing the elements for determining water quality, col-
lecting the sample, adding internal standards and processing the sample
with subsequent analysis according to prescribed GC/MS/COMP conditions.
The purpose of this introduction is to provide an overview of the
current HAS analytical operations and techniques, with recovery data for
about 280 model compounds used during HAS development. This section also
serves as a guideline for determining which protocols apply to specific
classes or groups of compounds of interest.
Chap. 1 - 1
-------
1.1 ORGANIZATION OF HAS PROTOCOLS
Table 1.1 shows how these HAS protocols are organized and related to
material in this introduction, and explains some HAS terminology. An
understanding of the physical/chemical classifications of organics and how
these relate to the analytical operational protocols and to the chapters in
this volume is necessary for application of the HAS to a water sample.
Symbols are for convenience in identifying each major HAS fraction, corres-
ponding to a chemical/physical property group. Operational protocols in
Table 1.1 are brief descriptions of the techniques for separation of
organics from the water sample. Column 4 of Table 1.1 lists the tables,
included in this introductory chapter, that provide recovery data for the
applicable chemical classes and individual analytes for each protocol.
These are the approximately 280 analytes for which recovery data were
generated during development of the HAS. Finally, Table 1.1 lists the HAS
chapters that are pertinent to analysis of each major HAS fraction.
Generally, protocols for each fraction are divided into two chapters: one
for sampling and sample handling and one for separation of the organics
from water and analysis. Chapter 13 includes general GC/MS/COMP procedures
common to all fractions.
The HAS user should study this introduction for an overview of the
entire analytical scheme. If he is interested in analyzing for compounds
contained in one or only a few protocols, he should refer to Table 1.1,
which will lead him to the appropriate recovery tables (of 1.3-1.9).
Examination of the chemical classes and individual analytes in these
tables will indicate the appropriate protocols to apply. The tables give
recoveries for all analytes tested. In several cases, recoveries are
unacceptably low (<40%). Inclusion of these analytes serves to bracket the
limits of compound types amenable to each protocol. Compounds whose
recovery is indicated as unacceptable by one protocol are usually listed in
another table at greater than 40%, indicating acceptable recovery by the
other protocol. Recovery data, as well as relative molar response (RMR)
factors, are also given in Appendix B (and in the computer data bank of
HASQUANT). Use of Appendix B values and HASQUANT for quantitation are
described in Chapter 13.
Chap. 1 - 2
-------
C)
Table 1.1. ORGANIZATION OF MAS PROTOCOLS
rigs
Symbol
Chemical/Physical
Property Group
Operational Protocol
Example Classes
and Analytes a
and Recoveries
Pertinent
Chapters and Subjects
VO (volatile
organics)
volatile, neutral, low
water solubility
purge and trap
Table 1 3
2 SamplinR and Simple Handline for VO
6 Purge, Trap and Analysis of VO
MEWS (neutral
water soluble)
volatile, neutral, high
water solubility
elevated temperature
purge and trap
Table 1 4
3 Sampling and Sample Handling for
NEWS
7. Elevated Temperature Purge. Trap,
and Analysis of NEWS
WARN (weak
acids and
bases, and
neutrals)
pH 8 extractable weak
acids, weak bases, and
neutrals
sorbent (accumulator)
column partitioning
(pH 8 0) (WARN-SC)
Table 1.5
4. Sampling and Sample Handling for
Extractable Organic.
9. Extraction and Analysis of WARN
I. Sorbent Accumulator Column
V. GC/IIS/COflP
batch liquid-liquid
partitioning (p11 8 0)
(WARN-B c .)
Table I S
4. Sampling and SampLe Handling for
Extractable Organica
9. ExtractIon and Analysis of WARN
11. Batch Liquid-Liquid
IV Clean-up
V. GC!MS/COtIP
flow-under liquid-
liquid partitioning
(p11 8 0) (WABN-FU)
Table 1.5
6 Sampling and Sample Handling for
Extractable Organic.
9. Extraction and Analysts of WARN
Ill. Flow-Under
V. GC/HS/COIIP
ESSA (extract—
able aemivola—
tile strong
acids)
pH 1 extractable, semi—
volatile strong acids
batch liquid—liquid
partitioning (pH 1 0)
Table 1.6
4. Sampling and Sample Handling for
Estractable Organic.
8 Batch Liquid-Liquid Extraction and
Analysis of ESSA
VOSA (volatile
strong acids)
volatile carboxylic acids
.
anion exchange/distills-
Lion
Table 1.7
5. Sampling and Sample Handling for
Other Ionic Compounds
10. Anton-Exchange, Distillation, and
Analysis of VOSA
NOVA (nonvola-
tile acids)
very strong, nonextract-
able, nonvolatile acids
anion exchange
Table 1 8
5. Sampling and Sample Handling for
Other IonLc Compounds
11.
SAIl (strong
aminea)
primary and tertiary
strong amines (SM-PT)
cation exchange
Table 1 9
Anion-Exchange and Analysis of NOVA
5 Sampling and Sample Handling for
Other Ionic Compounda
12 Cation-Exchange and Analysis of
SAIl
secondary strong amLnes
(SAN-S)
all groups
13 GC/NS/COMP Analysis Procedures -
General Instructions for All
aTb I in Chapter 1
-------
If specific compounds of interest are not found in the recovery
tables, the user must look for similar compounds in the tables to decide
which protocols to apply (see Section 1.4 — Summary of Recovery and Preci-
sion for HAS Protocols). He will then need to use chemical judgment in
selection of recovery and RNR factors of structurally similar compounds
from corresponding parts of Appendix B to apply towards estimates of
analyte concentrations. More guidance in this procedure is given in
Chapter 13.
1.2 HAS OVERVIEW
Figure 1.1 depicts a flow diagram of the procedures for implementation
of the HAS. Each step is summarized below.
1.2.1 Sample Handling
Seven sub-samples are required for a comprehensive sample analysis:
one for each protocol class. Procedures are prescribed in the protocols
for sample collection, storage and preservation. Volatile organic (VO
fraction) samples are collected in septum—capped bottles with no headspace.
Methylene chloride is added to all extractable and ionic compound samples
as a bacteriocide, while hexane is used as “keeper” solvent layer for
extractable compounds. Chlorine determination indicates the level of
sodium thiosulfate necessary to stoichiometrically reduce any residual
chlorine left from water treatment. All samples are stored at —4°C in the
dark.
Various water quaii’tç scouting measurements help in the selection of
appropriate analytical procedures, which are optimized according to water
quality rather than sample “type” (e.g., drinking water or municipal
effluent). Headspace gas analysis by GC of a separate small sample is
employed to determine the dilution necessary for VO purge and trap analy-
sis. A trial shake-out with inethylene chloride of a small aliquot of the
extractable (WABN) sample shows whether emulsion formation is a problem,
and thus whether the flow-under extractor must be used. Conductivity
measurements indicate maximum sample volume allowable for isolation of
ionic compounds by ion-exchange resin without exceeding resin capacity.
Chap. 1 - 4
-------
pH 8.0
(3 alterna-
tive tech-
tuques)
• Derivatization of 5 fractions
o ESSA——————————methyl esters/ethers
o VOSA——————————benzyl esters
o NOVA ——methyl esters/ethers
o SA11—PT——————Schiff bases
o SM—S ——pentafluorobenzyl amines
• Clean—up of pH 8 extractables (Silica column)
o WABN—BL 3 subfractions (WABN—BL1,
WASN-BL2,
WABN—BLJ)
• Evaporation/concentration of 8 fractions
• Addition of external standards
I ________
CC-MS—C0111’ • VUSA——-— -
Analysis _______ SNOVA—————————
(10 maximum S SAN-PT-—-----—--
fractions) S SAfl—S———————————————————————————————
S W&BN——————-—————
o WAIN-EL 1WABN-BL 1
(Silica I WABN—BL2———————
subfractions) WAZN-BL3—-——
Qua itative r. Computer Searches
Analysis L’ Manual interpretation
Qua ’titativc r• Manual Calculations
Analysis L’ Operator Interactive Computer Program (NASQUANT)
Figure 1.1. Master Analytical Scheme Flow Diagram.
Purgeables (2)
• Collection (7 Sub—samples)- -— —lo Extractables (2)
Sample ] • Storage/preservation Other Ionic Compounds (3)
Handling . Water quality scouting measurements (conductivity, headapace
I L gas analysis, ulsion index, pH, and chlorine determination)
Addition of • Volatiles E Purge and Trap on Tenax CC (VO)
Internal
Standards S Neutral, Water Soluble, Low..___.... [ 0 Heated Purge and Trap (NEWS)
Molecular Weight Compounds
ro pH 1.0 [ Semlvolacile Strong acids (ESSA)
I r* tch Liquid—Liquid (WABN—BL)
I I (aeparatory funnel)
Isolation from_______ I
Aqueous Matrix • Extractables—4 o
5 Contjnuous Flow—under (WABN—flJ)
(emulsion prone samples)
*Sorbent Accumulator (WABN—SC)
(drinking water only)
Volatile Strong Acids (VOSA)
• Other Ionic Compounds Jo Nonvolatile Strong Acids (NOVA)
(4 fractions from 1° Primary and Tertiary Anines (SM—PT)
— ion—exchange resins) Secondary Amines (SAN—S)
V
Extract ____________
Processing
S VO——————————————thermal desorption into CCC
• NEWS————— ———thermal desorpcion into CCC
• ESSA—— ___ ———————— ——— — —————— CCC
CCC
CGC
CCC
CCC
CCC
CCC
CCC
CCC
Chap. 1 - S
-------
1.2.2 Internal Standards
Prescribed deuterated internal standards (Table 1.2) are added to each
sub-sample before processing or storage, preferably in the field. Selection
and packaging assures that from one to nine standards of the total of 20
will appear in each extract for GC/MS analysis; retention times are such
that the standards span the chromatographic window in most cases. These
standards are used for monitoring recovery during analysis, for quantifying
sample components, and for relative retention time calculations.
The initial sets of MAS standards were prepared by the National
Bureau of Standards (NES). After assuring chemical and isotopic purity of
each standard compound, NBS determined its stability and compatability for
packaging in solution with other internal standards. Methanol solutions
(water solutions for highly water—soluble compounds) were prepared and
packaged for sample dosing in the combinations shown in Table 1.2.
Purgeable NBS standards are packaged in glass capillary ampoules that
are placed in the sample bottle and crushed with a magnetic stirbar after
the water sample has been collected. For other sample aliquots, internal
standards are packaged in vials such that emptying the entire content of
the vial into the prescribed sample volume produces the Concentrations of
standards shown in Table 1.2. Low concentration standards sets are gener-
ally used for drinking and surface waters. Higher concentration sets are
used for effluents.
1.2.3 Isolation of Organics
After addition of internal standards, the seven subsamples are proc-
essed as follows. (Protocol symbols are in parenthesis.)
1.2.3.1 Volatile Organics (VO)--
Highly volatile (purgeable) organics (Table 1.3) are analyzed by a
modification of the Bellar-Lichtenberg (EPA’s Method 624) method, using a
custom built purge and trap system (Appendix A) designed especially for
capillary GC columns. Sodium sulfate is used to “salt outtt the organics in
a 200 niL sample, which is purged at 30°C. More concentrated samples are
first diluted to 200 niL in accordance with the total concentration of
purgeable organics as indicated by GC scouting of the headspace. Dilution
prevents saturation of the GC/MS/CONP and decreases foaming potential.
Chap. 1 - 6
-------
Table 1. 2. INTERNAL AND EXTERNAL STANDARDS EMPLOYED IN THE MASTER ANALYTICAL SCHEME
Internal Standards
bro oethane-d 0 56 6 6
an isolr-2 ,4 ,6 d 3 4 2 25 6
chlorobenzene-d 7 2 5 4
naphthalene-d 8 5 8 48 7
t-butanol-d 112 9 554 0
nitrobenzen -d 5 20 1 103 5
o-xy lene—d 2 8 104.7
iiaphtha1en d 20.2 500 7
nitrobenzene- 94 2 538 8
l-phenyL-d -etkanoi -- 520 9
acetopheno e-d -- 104 7
propiophenone- 5 -- 98 1
pery lene-d 12 1 1 98 1
acridine-d 9 19 8 100 3
phenol-d 5 112 7 400 4
heptanoic acid-d 13 13.8 111 0
benzoic acid-d 5 1.08 100.3
butyric scid-d 7 15 7 535 9
2-naphthalenesulfonic 99 4 497.5
acid-d 7 1120
n-buty lasine-d 118 3 554 3
2-phenylethyl_? ,l,2,2_ 121 2 530.3
d 4 -asine
N -ethy l—2-f luorobenzyl_ 107.2 522 1
amine
aConcentration after dosing into the appropriate volume of sample These concentrations are based upon the “400 series ”
standard solutions prepared by NBS.
NAS
Symbol
Operational
Protocol
a
pg/ L
Low High Esternal
Standard
VO (volatile organics)
Purge and Trap
NEWS (neutral Water
soluble)
WABN (weak acids, bases
and neutrals)
Elevated Temperature
Purge and Trap
Sorbent Cblumn,
8atch L-L, or
Flow-under
Partitioning (pH 8 0)
ESSA (extractable semi-
volatile strong scics)
VOSA (volatile strong
acids)
NOVA (nonvolatile acids)
SAIl (strong amities)
Hatch L-L
Partitioning (p11 1 0)
Anion-exchange/
Distills tion
Anion-exchange
Cation-exchange
perflu rotoluene
4-fluoro—7-iodotoluene
4 -fluoro-2-jodotolueoe
2-fluorobiphenyl
2-fluorobiphenyl
4-f luoro-2- iodotoluene
2-fluorobiphenyl
4 -fluoro-2-iodotoluene
2-fluorobiphenyl
4 -fluoro-2-aodotoluene
production run of internal
-------
Table 1.3. EXA {PLE CLASSES AND ANALYTES RECOVERED BY
THE PURGE AND TRAP ANALYTICAL PROTOCOL (VO)
r• Volatility range
I —13°C to 220°C BP
Slightly soluble to
I nonsoluble in water
1.. Relatively nonpolar
Applicable Chemical
Classes Example
Drinking Water
Recoveries
Analytes Mean ± S.D. (C.V.)
Overall
Mean ±
a
Recoveries
S.D. (C.V.)
Aromatic benzene 78 ± 6 (7) 76 ±
Hydrocarbons toluene 125 ± 6 (5) 125 ± 6 (5)
p-xylene 108 ± 13 (12) 98 ± 13 (14)
ethylbenzene 96 ± 5 (5) 96 ± 7 (7)
1,3,5-trimethylbeazene 65 ± 3 (5) 77 ± 18 (23)
1,2,4—trimethylbenzene 97 ± 2 (2) 94 ± 4 5 b
t—butylbenzene 113 ±d 3 113 ± 3 (3)
sec-butylbenzene - 113 ± 3 (3)
o-diethylbenzene 98 ± 6 -
-diethylbenzene 61 ± 4 (6) 81 ± 28 (3k)
4-methylisopropylbenzene 100 ± 5 (5) 100 ± 5 (5)
naphthalene - 74 ± 2 (3)
diphenylmetharie 59 ± 5 (9)
Halogenated f luorobenzene 116 ± 5 (5) 116 ± 5 ( 5 )b
Aromatics chlorobenzene 98 ± 10 (10) 91 ± 15 05 b
bromobenzene 123 ± 12 (10) 123 ± 12 (10)
-bromotoluene 104 ± 4 (3) 104 ± 4 (3)
—iodotoluene 96 ± 8 (8) 106 ± 15 (14
l,2-dichlorobenzene 116 ± 10 (9) 116 ± 10 (9)
l,3-dichlorobenzene 90 ± 4 (5) 103 ± 18 (l )
1,4-dichlorobenzene 83 ± 4 (4) 83 ± 4 (4)
2-bromochlorobenzene 73 ± 3 (4) 92 ± 27 (30)
(continued)
-------
Table 1.3 (cont’d.)
Applicable Chemical
Classes Example
Drinking Water
Recoveries
Analytes Mean ± S.D. (C.V.)
Overall
Mean ±
a
Recoveries
S.D. (C.V.)
4-bromochlorobenzene - 106 ± 2 (2)
l,2,4-trichlorobenzene 88 ± 3 (3) 101 ± 21
a,a,ct-trichlorotoluene 71 ± 11 (16) 70 ± 11 (16)
Misc. Aromatics anisole 62 ± 1 (2) 68 ± 8 (12)
phenyl ether - 47
Aliphatic and cyclopentane 95 ± 4 (5) 113 ± 25 (22)
Alicyclic cyclohexane 79 ± 8 (10) 85 ± 8 (10)
Hydrocarbons n-hexane 90 ± 17 (19) 105 ± 21 (20)
n-heptane - 120 ± 4 (3)
n—octane 103 ± 11 (11) 103 ± 1 (1)
n-nonane 80 ± 32 (40) 79 ± 32 (40)
n-decane 70 ± 10 (14) 89 ± 9 (10)
n-undecane - 50 ± 4 (8)
n-dodecane 64 61 ± 5 (7)
n-tridecane 52 ± 18 (34) 44 ± 11 (26)
n-tetradecane 58 ± 22 (38) 49 ± 13 26 b
dipentene 96 ± 15 (16) 96 ± 15 (16)
Halogenated chloroform 74 ± 4 (6) 78 ± 6 (7)
Aliphatic bromochioromethane 48 ± 3 (7) 95 ± 61 (71)
Hydrocarbons trans-1,2-dich loroethylene 82 ± 6 (7) 87 ± 8 b
1,2—dichloroethane 102 ± 10 (10) 102 ± 10 (10)
1,2-dibromoethane 68 ± 5 (8) 77 ± 13 (16)
trichioroethylene 118 ± 9 (8) 118 ± 9 (8)
allyl chloride - 64 ± 12 (19)
tetrachloroethylene 82 ± 7 (8) 77 ± 6 (8)
1,2—dichioropropane - 128 ± 12 (lg)b
2-bromo-1-chloropropane 99 ± 4 (4) 99 ± 4 (4)
1,2-dibromopropane 90 ± 4 (4) 90 ± 4 (4)
(continued)
-------
Table 1.3 (cont’d.)
Applicable Chemical
Classes
Example Analytes
Drinking Water
Recoveries
Mean ± S.D. (C.V.)
a
Overall Recoveries
Mean ± S.D. (C.V.)
Halogenated
Aliphatic
Hydrocarbons
(continued)
2-bromobutane
1,4-dibromobutane
l-chlorohexane
1-bromohexane
117 ± 3 (3)
94 ± 7 (7)
128 ± 11 (9)
129 ± 7 (5)
117 ± 3 ( 3 )b
103 ± 13 (l3
128 ± 11 (9
129 ± 7 (5)
Misc. O,S Compounds
propylene oxide
diethyl ether
2—methyl furan
allyl ether
bexyl ether
carbon disulfide
thiophene
67 ± 15 (22)
—
65 ± 3 (5)
87 ± 9 (10)
68 ± 16 (23)
-
120 ± 7 (6)
67 ± 15 ( 22 )b
115 ± 5 (4)
80 ± 21 (26)
98 ± 15 (16)
70 ± 3 (4)
100 ±
120 ± 7 (6)
Deuterated Standards
d 5 -bromoethane
2,4,6-d 3 -aniso le
d 5 -chlorobenzene
d 8 —naphthalene
91 ± 20 (23)
77 ± 11 (15)
109 ± 6 (6)
—
85 ± 16 (19)
57 ± 15 (25)
120 ± 20 (16)
99 ± 10 ( 10 )C
aThe recovery is a mean value for triplicate determinations in drinking water, spiked at
0.2 to 1.8 ppb (nominally 1 ppb), arid triplicate determinations in a 60/40 industrial/muni-
cipal wastewater, spiked at 30 to 87 ppb (nominally 50 ppb). S.D. = standard deviation,
C.V. = coefficient of variation.
bRecovery is for triplicate determination in drinking water only.
CRecovery is for triplicate determination in industrial/municipal wastewater only.
r .J
dNot determined.
-------
Purged organic vapors are collected on a Tenax GC sorbent trap, from which
they are thermally desorbed into a liquid nitrogen cold trap. Before
desorption of the purged sample components into the cold trap, an “external”
standard, perfluorotoluene, is added to the cold trap through an injection
port system. The injection port is installed between the sorbent trap and
the cold trap. The total condensate is then flash evaporated into a fused
silica capillary for analysis by GC/MS/COMP. Comparison of MS signals for
the external standard with those for the internal standards purged from the
sample allows calculation of recoveries of the internal standards, thus
monitoring performance of the entire analytical operation.
1.2.3.2 Neutral Water Soluble Compounds (NEWS)-—
Low molecular weight, water soluble, non-extractable compounds
(Table 1.4) are purged from a 10 niL water sample containing 20% sodium
chloride at 80°C and trapped on Tenax, using the same equipment as for the
VO fraction. To achieve lower limits of detection for drinking water, a
200 m l sample is concentrated by azeotropic distillation in a Peters’
(Peters, T. I., Anal. Chem., 52, 211, 1980) apparatus to produce a 3 niL
aqueous condensate enriched in neutral organics. This condensate is then
purged as above. -
1.2.3.3 Organics Extracted at pH 8 (WABN)--
Compounds of intermediate volatility, most of which are water insoluble
(Table 1.5), are analyzed by batch liquid—liquid extraction of 1 L of water
sample with methylene chloride in a separatory funnel. Adjustment of
sample pH to 8.0 allows reproducible extraction of the weak acids, (e.g.,
most phenols) and weak bases, (e.g., most anilines) as well as neutral
compounds.
For some samples, however, batch liquid-liquid extraction is not
suitable. An initial trial solvent extraction in a stoppered graduated
cylinder indicates whether emulsion formation is likely to be a problem.
For emulsion-prone samples, continuous liquid-liquid extraction with
methylene chloride in a flow-under extractor should be used. For samples
in which extractable organic concentration is expected to be low, such as
drinking water and some surface waters, XAD-4 resin sorbent accumulator
columns are used for sorption/concentration from 10—15 L of water. The
organics are desorbed using methanol followed by methylene chloride.
Chap. 1 - 11
-------
Table 1.4. EXAMPLE CLASSES AND ANALYTES RECOVERED BY THE
ELEVATED TEMPERATURE PURGE AN]) TRAP PROTOCOL (NEWS)
r Very soluble to
I slightly water
soluble
I Volatility range
I --35°C to 225°C BP
L Relatively Polar
Drinking Water
Applicable Chemical Recoveries
Classes Example Analytes Mean ± S.D. (C.V.)
Overall
Mean ±
a
Recoveries
S.D. (C.V.)
Alcohols 1-propanol 104 ± 4 (4) 108 ± 19
1-butanol 120 ± 12 (10) 120 ± 12 (10)
1-pentanol 129 ± 14 (10) 113 ± 22 (20)
1-hexanol 117 ± 13 (11) 131 ± 32 (25)
1-heptanol 94 ± 10 (11) 106 ± 25
1-octanol 100 ± 11 (11) 100 ± 11 (l1
cyclohexanol 122 ± 10 (8) 122 ± 10 (8)
furfuryl alcohol - 64 ± 10 (16)
Aldehydes propionaldehyde 99 ± 41 (41) 99 ± 41 ( 41 )b
n-butyra ldehyde 53 ± 17 (32) 72 ± 23 (32)
n-valeraldehyde 93 ± 27 (29) 81 ± 23 (29)
crotonaldehyde 81 ± 20 (25) 83 ± 21 (25
furfural - 36 ± 6 (16)
Esters methyl formate 25 ± 13 (54) 45 ± 4 (g)C
methyl acetate — 93 ± 5 ( 6 )C
ethyl acetate 35 ± 7 (19) 89 ± 5 ( 6 )C
n-propyl acetate 40 ± 8 (20) 79 ± 4 ( 5 )C
allyl acetate 51 ± 9 (18) 79 ± 13
n-butyl acetate 34 ± 11 (33) 72 ± 4 (5)
ethyl butyrate 27 ± 9 (35) 63 ± 2 ( 4 )C
(continued)
-------
Table 1.4 (cont’d.)
Applicable Chemical
Classes
Example Analytes
Drinking Water
Recoveries
Mean ± S.D. (C.V.)
a
Overall Recoveries
Mean ± S.D. (C.V.)
Ethers
tetrahydrofuran
dioxane
62 ± 14 (23)
23 ± 0.5 (2)
79
50
± 25 (3l
± 7 (14)
Ketones
methyl ethyl ketone
cyclopentanone
cyclohexanone
53 ± 20 (34)
52 ± 6 (11)
65 ± 3 (4)
69
52
65
± 22 (32
± 6 (11)
± 1 (2)
Nitriles
acrylonitrile
propionitrile
isobutyronitrile
benzonitrile
30 ± 9 (31)
95 ± 6 (7)
62 ± 7 (12)
90 ± 8 (9)
89
95
57
91
± 5 (6)
± 6 (7)
± 7 (14)
± 8 (9)
Nitro Compounds
nitromethane
1-nitropropane
nitrobenzene
67 ± 3 (4)
84 ± 8 (9)
92 ± 9 (10)
70
93
96
± 5 (7)
± 13 (14)
± 6 (7)
Deuterated Standards
d 9 -t-butanol
d 5 -nitrobenzene
93 ± 21 (23)
87 ± 12 (14)
98
93
± 7 (7)
± 9 (10)
aRecove is a mean value for triplicate determinations from drinking water, spiked at
0.8 to 1.2 ppb (nominally 1 ppb), and for triplicate determinations from a 60/40 industrial/
municipal wastewater, spiked at 40 to 63 ppb (nominally 50 ppb). S.D. standard devia-
tion, C.V. = coefficient of variation.
bHean recovery for triplicate determinations from drinking water only.
Cflean recovery for triplicate determinations from vastewater only.
‘-a
( )
d
No data.
-------
Table 1.5. EXAMPLE CLASSES AND ANALYTES RECOVERED BY ACCUMULATOR COLUMN AND BATCH L-L
PARTITION PROTOCOLS AT pH 8.0 (WABN)
Weak Acids
Applicable Chemical
Classes
Overall
Accumulator
Physical/Chemical Column b
Properties Encompassed Example Analytes (WARN-SC)
Recovery (Mean
± S 0 (C V
Batch
L-l.
(WABN_BL)C
No Clean-up
d
With Clean-up
C)
83
Weak Bases
A lkaneg
pX < 7
8
B P —115 up to
limit of chroma-
tography
phenol
p—creso l
2 ,3-dimethy lphenol
o-isopropylphenol
nitr opheno1
-chloro-2-methylpheno1
55
63
95
88
± 6 (II)
± IS (23)
± 5 (5)
± 9 (10)
-
9O
12 ± 32
-
74 ± 7
98 ± 43
(44)
(9)
(44)
61 + ii (l8) +
62+27 (44)* +
58 14 (24) *
57 1
49 i 10 (22) +
118 I I
l-naphthol
(I0)**
p—t-hutylpheno l
2— itro—p—cresol
2 ,4-dich lorophenol
2,4,6-trichlorophenol
di -t-butyl .4-methy lpheno l
4-ciu loro-3-methylphenol
86
32
76
.
± 5 (6)
± 17 (53)
78
66
73
± 7 (9)
-
80 ± 13
76 ± 16
-
-
-
(16)
(21)
89 + 18 (20)
77 6 (8)**
61 + 9 (15)
81 + 26 (32)
30 + 6 (l2) +
69 1 14 (20)**
66 + 9 (l4) +
.
pK > 3 5
a D iico
up tO
limit of chroma-
tography
pyridine
0-picoline
aniline
lutidine
indole
2 ,3,6—trimethy lpyridine
2,6—dimethylani line
-chIoroanj1ine
qulnoline
2—nitroanillne
2 , 4 —dimethy lquinoltne
nicotine
carbazole
2-aminobiphenyl
diphenylamine
tributylamine
caffeine
dibenzy lamine
N.N-dinethy ldodecy lamine
atrazine
36
34
95
95
84
81
95
91
77
90
62
82
82
85
82
86
53
71
-
± 4 (11)
± 11 (32)
± 6 (6)
±6 (6)
± 1 (1)
± 10 (11)
± 5 (5)
± 13 (14)
± 4 (5)
± I (1)
± 10 (16)
± 2 (2)
± 8 (8)
± 1 (I)
± 0 (0)
± 5 (6)
± 8 (15)
69
± 7 (10)
58 ± 24
66 ± I?
84 ± 34
57 ± 31
86 ± 36
82 ± 25
12 ± 6
88 ± 25
77 ± 24
.
80 ± 19
84 ± 17
79 ± B
91 ± 15
76 ± 20
70 ± Il
92 ± 16
62 ± 18
68 ± 14
90 ± S
(42)
(26)
(40)
(54)
(42)
(31)
(8)
(28)
(31)
(24)
(20)
(10)
(17)
(27)
(25)
(Il)
(29)
(20)
(6)
49 + 13 (26) +
63 + 25 (40) +
—
66 + 13 (30)**
60 + 21 (35)**
86 + 18 (21)**
80 + S (6)**
53 + 16 (31)**+
.
55 + 20 (36)**
-
13 + 16 (22)
55 + 13 (24)*$
87 + II (13)*
40 1 10 (25)**,
68 12 (18)*k+
43 • 12 (29) +
74 I 13 (18)**
78 4 12 (16) +
.
B P 150°C up to
limit of chroma—
tography
Water/ClI C1 ,
partitioB c8effi-
cient
-------
Table 1.5 (cont’d.)
Applicable Checiical
Classes
Overall
Accumulator
Physical/Chemical Column b
Properties Encompassed Example Analytes (WABN-SC)
Recovery IMean
± S
0 (C.V )I
a
Batch
L-L
(VABN_BL)C
No Clean-up
— d
With Clean-up
C)
Oi
(‘I
Alaphatic Ketones,
2-heptanone
66
-
55 • S
(l0)
Alcohols, and
butoxyethanol
-
65 t 16
(24)
70 + 10
(l5) +
Esters
2-octanone
butyl propionate
52
77
t 10
(20)
70 ± 10
61 1 13
(14)
(21)
55 + 15
31 + 6
(27)4*4
(2l) +
usophorone
85
14
(5)
78116
(21)
71 + 25
(35)4*
tenchone
0-terpineol
n-decanol
dihydroluran
64
92
94
89
± 14
± 1
± 1
± 2
(22)
(1)
(1)
(2)
74 1 23
75 ± 24
95 1 39
.
(31)
(32)
(41)
63 + 19
Ill + 27
11 + 20
(31)**+
(24)4*
(28)
dimethyl adipate
methyl stearete
49
91
± 8
(16)
62 ± 23
77 ± 14
(43)
(18)
49 + 21
91 + 14
(43) *+
(15) *
Misc AlIphstic
Compounds
1,4—dicyanobutane
butyl carbamate
du—L—butyldisulfide
bis(2—chloroe thoxy)ethane
trubutylphosphate
a ldrun
70
40
58
92
86
83
1 29
± 4
± 10
± 11
1 2
± 23
(36)
(10)
(17)
(12)
(2)
(28)
135 1 38
60 1 16
-
78 1 26
93 1 10
-
(28)
(26)
(33)
(11)
104 + 37
57 + 6
65 4 17
75 + 7
96 + 3
(36)**
(11)
(26)**
(9)
(3)4+
Aromatic Hydrocarbona
2—methylnaphtha lenr
acenaphthene
biphenyl
1,8-dimethy lnaphtha lene
Iluorene
2.3,5-trimethylnaphthalene
anthracene
pyrene
9,10-dimethylanthracene
chrysene
perylene
65
76
79
78
87
84
78
73
60
62
1 lB
± 17
1 15
1 16
± 15
± 22
± 1
1 2
± 4
43
1 13
(27)
(22)
(19)
(20)
(17)
(26)
(1)
(3)
(7)
(20)
71 1 14
78 1 21
80 1 17
71 1 13
85118
76 1 11
—
80 1 1
45 1 21
78 1 27
54 ± 13
(19)
(27)
(21)
(18)
(21)
(15)
(9)
(47)
(35)
(24)
48 + 9
50 + 13
—
48 + 9
84 + 12
48 • 6
66 16
78 + 19
63 + 26
95 + IS
118140
(L9)+
(77)*+
(l8)*,
(14)4*
(13)**
(24)**
(24) *
(41)4*
(16)**
(36)**
Halogen.ted Aromatic
Compound.
benzyl chloride
4-chlorobenzonitrjje
3-ch lorobeozaldehyde
o— ch loroiniso le
3.4-dichlorobenzaldehyde
1,2,4—trich lorobenzene
2-bromoch lorobensene
4 -t luoro-2-iodo(oltuene
1,2—dichloronaphtha lene
l, 2 , 4 ,S-tetracl ulorohenzene
p—dibromobenzene
4-browophenyl phenyl ether
heaachlorobenzenu
35
80
57
100
71
56
62
87
65
91
64
± 2
± 9
-
± 6
± 10
± 5
± 19
± 26
± 1
77
± 12
± 14
± 6
(6)
(Ii)
(10)
(10)
(7)
(34)
(42)
(1)
(18)
(15)
(9)
63 1 28
79 ± 29
-
lB 129
78 ± 28
73 ± 13
-
82 ± 14
77 ± 25
69 ± 17
80 ± 2
80 ± 2
-
(65)
(37)
(37)
(37)
(18)
(17)
(33)
(25)
(3)
(3)
B2 4 14
47 12
80 + 11
50 + 9
55 + 9
107 + 14
— -
-
68 + 22
53 13
43 + 7
80 18
95 6
(17)
(26)*
(14)**
(18)
(17) ’
(13) *
(33)4*
(25)*+
(17)
(22)**
(16)*+
(continued)
-------
Table 1.5 (cont’d.)
Applicable Che.tcal
Class(es)
Physical/Chemical
Propertiex Encompassed
Example Analytes
Overall
Recovery IPlean I
S 0 (C V )Ja
Accumulator
Column b
(WABN-SC)
Ratch L-L
(WABN_BL)C
No
Clean-up
d
With Clean-up
Aromatic Aldehydes
and Ketonea
“
benzaldehyde
o-totua ldehyde
acetophenone
salicylaldehyde
anisaldehyde
-
88 ± 15 (17)
92 ± 2 (2)
—
96216 (17)
89
66
73
72
2 31 (35)
± 24 (36)
-
2 46 (60)
± 10(14)
43 + 6 (l5)**
105 + 19 (l8) *
—
67 + 17 (25)
62 + 9 (l4) ’+
Aromatic Esters and
Sulfonates
“
phenyl acetate
benzyl acetate
methylbenzene sulfonate
methyl-p-toluene sulfonate
diethylphthalate
ethyl-p-toluene sulfonate
dimethylphthalate
dibutylphthalate
butylbenzylphtha late
diethylhexylphthalate
74
79 ± 10 (13)
36 ± 6 (17)
46 2 8 (17)
8716 (7)
53 ± 4 (7)
7418(11)
-
64
74 ± 12 (16)
74
81
67
108
91
107
96
97
102
•
2 12 (16)
± 30 (37)
± 47 (70)
±7 (6)
± 36 (40)
239 (37)
± 10 (10)
± 27 (28)
32 + 6 (18)**
55 + 8 (l4)**
-
—
89 + 6 (7)a.*
70 1 18 (26)**
60 + 7 (12)**
138 + 3 (2)
74 + 13 (18)**
104 19 (l8) .
Niac Aromatic Compounds
‘
2,3 -dihydroben ofuran
nitrobenrene
benzothiazole
phenylcarbamate
2, 4 -dinstrotoluene
beozylsulfide
diphenylsulfone
triphenylphosphate
diphenylmercury
tetraphenyltin
—
83
85212(14)
—
89 2 10 (11)
67 ± 7 (10)
69 ± 4 (6)
84 2 21 (24)
-
47 ± 5 (11)
67
86
71
84
86
93
112
94
2 13 (20)
± 19 (22)
28(11)
-
± 6 (7)
± 1 (1)
± 29 (31)
2 12 (11)
± 19 (20)
48 + 9 (18)fr*
58 13 (22)**+
58 5 (8)**
48 + 9 ( 18)a*
105 11 (1l)**
71 j 12 (17)**
67 8 (12)**
71 7 (l0)**
81 16 (20)**
72 24
Deuterated Internal
Standards
d 10 -xylerie
d 5 —phenol
d 5 -ace tophenone
d 5 -phenylethanol
d 5 -nitrobenzene
d 5 -propiophenone
d 8 -naphthaleoe
d 9 -acridine
d 12 -perylene
76 26 (8)
55 ± 6 (11)
87 ± 10 (11)
7828(10)
82210(12)
93 ± 4 (4)
79 ± 3 (4)
87 ± 14 (10)
62 ± 13 (21)
58
85
75
80
79
73
71
84
80
2 13 (23)
± 34 (40)
± 10 (14)
222 (27)
± 13(17)
± 11 (16)
± 9 (12)
± 13 (IS)
46 + 12 (26)*
66 1 26 (40)**+
—
62 + 20 (32)**
55116 (29)
42 1 8 (l8)
40 1 4 (lI) *
77 1 ii (15)A++
29 (36)
78 28 (36) .
1 The flow-under extraction protocol for emulsion-prone samples (WABN-FIJ) requires the determination of recoveries in the sample
matrix under investigation, aince it has been determined that large variations occur from one sample matrix to another Therefore,
no recoveries are given here MASQIJANT requires the user to input recovery factors as they are determined on these samples
bRecoveries are for triplicate determinations !rom drinking water, spiked at 0 5 to 5 ppb (nominally I ppb)
are For triplicate determinations from a 60/40 industria 1/municipal wastewater, spiked at IS to SO ppb (nominally 25 ppb)
dvith clean-up step included, * subiraction I, ** = aubfraction 243, (recoveries of compounds in subfraction 3 were not deter-
mined separately) *+ r aubfraction 2+3, and * subfraction I are From reagent water, interIerence in wastewater prevented
recovery determination
e not determined
t Single determination
-4
0 ’
-------
1.2.3.4 Organics Extracted at pH 1 (ESSA)-—
Extraction at pH 8 does not efficiently recover strong acids or
bases. Strong bases are extracted on ion-exchange resins, but a new
procedure has been developed for semivolatile strong acids (Table 1.6).
This involves batch liquid-liquid extraction of a 1 L sample with methylene
chloride at pH 10 to remove most neutrals and bases (discarded), after
which the sample is made to pH 1.0 with HC1 and the semivolatile strong
acids are extracted with methyl-t-butyl ether. This procedure includes
most carboxylic acids and strongly acidic phenols. The lower molecular
weight carboxylic acids, however, are included in a separate volatile acids
protocol (VOSA); they are too volatile to be efficiently recovered during
liquid-liquid extraction and subsequent extract processing. In addition,
some acids, e.&., sulfonic acids, are too ionic to be extracted under these
conditions and are included in the nonvolatile strong acid analytical
protocol (NOVA).
This is followed by derivatization with diazomethane to form the
corresponding methyl esters or ethers before GC/MS analysis.
1.2.3.5 Other Ionic Compounds (VOSA, NOVA, SAN)--
Compounds that are easily dissociated in water have not previously
been included in analytical schemes because of difficulties with extraction
and chromatography. New techniques, however, were developed to allow
inclusion of most of these compounds in the MAS. Ion-exchange resins are
used to separate four classes of ionic compounds from the sample matrix
using three separate aliquots of the sample.
“ Volatile” strong acids (VOSA) , such as acrylic acid, octanoic acid,
and other volatile carboxylic acids (Table 1.7) are separated from the
water on Biorad AG 1-X8 anion exchange resin, then eluted with sodium
bisulfate in an acetone:water solution. The volatile acids are distilled
from the eluate, converted to nonvolatile salts, then derivatized with
benzylbromide to form benzyl esters.
“ Nonvolatile” strong acids (NOVA) , e.g., naphthalene sulfonic acids,
(Table 1.8) are also separated from water on Biorad AG l-X8 resin. They
are eluted with HC1 in methanol, the solvent is evaporated, and the acids
are methylated with diazomethane.
Chap. 1 - 17
-------
Applicable Chemical
Classes
Example Analytes
Drinking Water
Recoveries
Mean + S.D. (C.V.)a
Carboxylic Acids
valeric
benzoic
dichloroacetjc
£-t oluic
n-octanoic
o-methoxybenzoic
o—chlorobenzoic
n-nonanoic
£-nitrobenzoic
2-naphthoic
n—decanoic
lauric
2, 4 -dichlorophenoxyacetic
palmitic
oleic
-t-buty1benzoic
myristic
82 ± 3 (4)
92 ± 5 (6)
73 ± 7 (10)
91 ± 6 (6)
82 ± 8 (9)
90 ± 6 (7)
91 ± 2 (2)
85 ± 7 (9)
88 ± 8 (9)
94 ± 8 (9)
87 ± 5 (6)
106 ± 12 (11)
110 ± 10 (9)
89 ± 10 (11)
63 ± 11 (17)
96 ± 4 (5)
90 ± 18 (20)
Phenols
2-nitro-2-creSol
2,4—dichiorophenol
2,3,6-trichiorophenol
peatachloropheno l
2,4,5-trichiorophenol
88 ± 8 (9)
88 ± 4 (5)
93 ± 5 (5)
100 ± 19 (19)
99 ± 4 (4)
Deuterated Internal
Standards
d 13 —heptanoic acid
d 5 -benzoic acid
65 ± 8 (12)
92 ± 5 (6)
aRecovery is a mean value for triplicate determinations in
spiked at 50—100 ppb (nominally 55 ppb). S.D. = standard
coefficient of variation. Recoveries were not determined
waters.
Table 1.6 EXAMPLE CLASSES AND ANALYTES RECOVERED BY
BATCH L-L PARTITION PROTOCOL AT pH 1.0 (ESSA)
r B.P. —200°C up to
limit of chromatog—
I raph (methyl esters)
L PKa < 8.0
drinking water,
deviation, C.V. =
in more complex
Chap. 1 - 18
-------
Table 1.7. EXAMPLE CLASSES AND ANALYTES RECOVERED BY
ANION-EXCHANGE/OtSTILLATION ANALYTICAL PROTOCOL (VOSA)
Applicable Chemical
Classen
Example Analytes
Drinking Water
Recoveries
sean ± SD (C V
)
a
Oversit Recoveries
Plean ± S 0 (C V )
Relative
Retention (C
2-Fluorobiphenyl
d 7 -Butyic Acid,
Beozyl Eater
Volatile Carboxylic
Acids
acetic
acrytic
n-propanoic
iaobutyric
methacrylic
n-butyric
-methytbutyric
isovaleric
cro tonic
n—valeri e
2— methyl-2-buteootc
2-methylcyclopropane-
carboxylic
3,3—di methylacry l lc
2-ethylbutyric
3-hexenoic
2—hexenoic
n-hexanoic
cyciopentylacetic
n-heptanoic
cyclohexane carbozylic
n-octano ic
cyc loheay lacettc
n-nonanotc
Tiglic acid
-
119 • 8 (1)
101 ± 5 (5)
-
8016(7)
—
93 t 9 (10)
105 ± 11 (10)
102 ± 10 (8)
88 ± 10 (11)
-
92 ± 6 (6)
91 ± 9 (10)
67 ± 11 (16)
70 ± 7 (10)
62 ± 10 (16)
70 ± 7 (10)
54 8 1 (13)
62 ± 10 (16)
32 ± 2 (6)
54 ± 7 (13)
39 ± 6 (15)
-
77 ± 4 (5)
90
42
57
51
76
88
77
15
74
72
57
61
61
60
41
48
32
46
37
77
c
± 25
—
±29
±19
± 8
± Il
± 15
8 22
± 12
—
8 11
± 17
± 9
8 9
± 3
8 9
± 6
± 13
8 2
8 7
± 3
-
± 4
(28)
(69)
(34)
(14)
(23)
(17)
(28)
(16)
(24)
(23)
(15)
(14)
(4)
(16)
(13)
(27)
(6)
(15)
(9)
( 5 )d
0 623 (0 23)
0 186 (0 1.1)
0 806 (0 09)
0886 (0.05)
0941 (004)
0970 (0 05)
1 031 (0 10)
1 040 (0 14)
1 069 (0 05)
1.146 (0 04)
1 161 (0.23)
1.122 (0 22)
1.151 (0.24)
1 184 (0 06)
I 220 (0 27)
1 285 (0 33)
1 317 (1.0)
1 379 (0.34)
1.473 (0 10)
7. 55? (0 09)
1 626 (0 09)
1.690 (0 10)
1 713 (0 12)
0 648 (0.02)
0 817 (0 09)
0 639 (0 10)
0 ia (008)
0986(007)
1 010 (0.07)
1 066 (0.10)
1.075 (0.14)
1.113 (0.06)’
1 192 (0.07)
1 120 (0.26)
1.160 (0.32)
1 190 (0.28)
1.231 (0.05)
3.261 (0.30)
1 328 (0.34)
1 370 (1.0)
1.421 (0 55)
1.533 (0 09)
1 619 (0.09)
1.692 (0.10)
1 757 (0.10)
1 845 (0.10)
Deuterated Internal
Standard
8 7 -butyric acid
93 8 3 (3)
85
± 12
(14)
0 961 (0.03)
1 00
5 Rrcove , is mean value (or triplicate determinations from drinking water, spiked at 0 3 ppb, and for triplicate determinations from a 60/40
industrial/municipal wastewater, apiked at 120 ppb S 0 standard deviatton, C V coefficient of variation
bRelative retention, as their bensyl Cater derivatives, to 2-fluorobipheny] and d 7 -butyric acid, benzyl ester DB-l fused silica capillary
column, 30 II a 0 34 s I 0 a film thickness
determined
drinking water only
(•‘ Boiling point range
I —l00°C-2 ’ 0°C
I (free acids)
L PKa 6 0
-------
Table 1.8. EXAMPLE CLASSES AND ANALYTES RECOVERED BY
ANION-EXCHANGE ANALYTICAL PROTOCOL (NOVA)
r Boiling point range
I —250°C to limit of
I chromatography
I (methyl esters)
L PKa < 6.0
Applicable Chemical
Classes Example
Deionized Water
Recoveries
Analytes Average ± Range
Overall
Mean ±
Recoveriesa
S.D. (C.V.)
c
Carboxylic Acids malonic 78 78 d
succinic 106±0 87 ± 40 (45)
azelaic 73 0 64 ± 19 ( 30 )e
rn-nitrobenzoic - 68 ± 5 (7)8
terephthalic 55 ± (2)
3,4-dichlorobenzoic 42 ± 4 (9)
2 ,4,5-trichlorophenoxyacetic 67 ± 8 (13)
oleic 9 ± 2 (22)
Sulfonic Acids methanesulfonic 115 ± 6 (5) 115 ± 6 ( 5 )C
benzenesulfonjc 130 84 ± 38 (45)
p-toluenesulfonic 74 98 ± 23 (24)
4-chlorobenzenesul.fonjc 53 92 ± 6 (7)8
2-naphthalenesulfonic 110 ± 15 ( 14 )g
Miscellaneous benzenephosphoric 104 ± 50 (50)’
benzenephosphonic 144 140 ± 20 (l4
pentachlorophenol 62 ± 7 (11)6
(continued)
-------
Table 1.8 (cont’d.)
Applicable Chemical
Classes
Example Analytes
Deionized Water
Recoveries
Average ± Range
Overall Recoveries
Mean ± S.D. (C.V.)
Deuterated Internal
2—naphthalenesulfonic
110 ± 15
( 14 )g
Standard
acid-d 7 1120
aAll recoveries at nominal 50 ppb; S.D. = standard deviation, C.V. = coefficient of
variation.
bsingie determination.
CRecovery from deionized water only.
dRecovery from deionized water and one municipal and three industrial effluents.
eRecovery from deionized water and one municipal and two industrial effluents.
NOt determined.
Recovery from one industrial effluent.
hlRecovery from one municipal and two industrial effluents.
‘Recovery from two industrial effluents.
-------
Strong amines (SAN) , such as hexylamine, dibutylamine, and 2,6-dimethyl-
piperidine (Table 1.9) are isolated from the water sample on Biorad AG
SOW-X8 cation exchange resin, then eluted with sodium hydroxide in acetoni-
true: water solution. The eluate is acidified (HCL), the solution is
evaporated to dryness, and the amine hydrochloride salts are dissolved in
base and extracted with methyl-t-butyl ether. The extract is split, half
is derivatized with pentafluorobenzy]. bromide to make the pentylfluorobenzyl
tertiary amines from the secondary amines (SAN-S), and half is derivatized
with pentafluorobenzaldehyde to make Schiff bases of the primary amines
(SAN-PT).
Tertiary amines are also separated by this protocol arid quantified
(underivatized) in the primary amine fraction. Certain weak bases (e.g.,
anilines) may be detected in these fractions, but are measured in the pH 8
extractable fraction (WABN), where they are extracted more efficiently.
(Quaternary amines are not addressed by the MAS.)
1.2.4 Extract Processing
Extractable and ionic fractions require further processing before
GC/MS. The necessary derivatization steps, for example, are described
above arid summarized in Figure 1.1.
The pH 8 extractable fraction (WABN—BL) of many industrial effluents
will require clean-up and sub-fractionation before effective separation can
be achieved, even on capillary column. First, however, a scouting procedure
is implemented to determine whether clean-up is necessary. The crude
extract is analyzed by GC using a packed column and flame ionization
detection; baseline rise relative to a separately run standard is the
evaluation criterion. Clean-up, if necessary, is on a silica gel column
from which three fractions (WABN-BL1, -BL2, and -BL3) are eluted using
pentane, methylene chloride, methanol, and their mixtures.
Concentration of extracts for GC/MS analysis is by Kuderna-Danish
evaporation down to 4 mL, followed by nitrogen blowdown to 0.5 mL or
1.0 mL using a modified Snyder column. ‘External” standards (Table 1.2)
are added to each final extract just before GC/MS analysis to monitor
recovery of the deuterated internal standards that were added to the
original water samples. The external standard for the purgeable fractions
Chap. 1 — 22
-------
Table 1.9. EXAMPLE CLASSES AND ANALYTES RECOVERED BY
CATION-EXCHANGE ANALYTICAL PROTOCOL (SAN)
r Boiling point range
I -30 to 300°C
I (free amines)
L 1 a > 6
Primary and Tertiary
Amines (SAN-PT)
Secondary Amines
(SAN-S)
allylamine
n-buty lamine
i sobutylamine
sec-butylarnine
t—butylamine
n-hexylamine
cyclohexylamine
2 -ethylhexylamine
benzylamine
1 ,2-ethane diamine
tri-n-butylamine
diallylamine
di-n-propy lamine
piperidine
morpholine
2—methylpiperidine
di-n-butylamine
2 , 6 -dimethylpiperidine
dicyclohexylamine
101 ± 7 (7)
45 ± 9 (20)
70 ± 1 (1)
60 ± 0 (0)
86 ± 26 ( 31 )b
75 ± 11 ( 15 )C
76 ± 18 ( 24 )C
77 ± 19 (25)
58 ± 24 (41)”
58 ± 16 (27)
76 ± 10 (13)
75 ± 10 ( 13 C
66 ± 8 (12)
73 ± 20 ( 27 )C
75 ± 25 ( 33 )C
Drinking Water
Applicable Chemical Recoveries a
Classes Example Analytes Mean ± S.D. (C.V.)
Overall Recoveries
Mean ± S.D. (C.V.)
r)
0 )
F..,
80 ± 8 (10)
136 ± 34 (25)
108 ± 45 (42)
21 ± 9 (43)
98 ± 19
70 ± 18
22 ± 12
( 20 )b
(26)
(55)
144 ± 12 (9)
118 ± 113 (11)
112 ± 9 (8)
75 ± 12 (16)
46 ± 15 (23)
32 ± 49
56 ± 30
56 ± 23
57 ± 25
40 ± 12
(155
53 b
42 b
(31)
(continued)
-------
Table 1.9 (cont’d.)
Drinking
Water
Applicable Chemical
Recoveries
Overall Recoveries
Classes
Example
Analytes
Mean ± S.D.
(C.V.)
Mean ±
S.C.
(C.V.)
Internal Standards
75 ± 27
(36)
75 ± 20
( 27 )b
d 9 -butylamine
d 4 -phenylethylaznine
N-ethyl-2-f luorobenzylamine
aRecove for triplicate determination; S.D. = standard deviation, C.V. = coefficient of
variation.
bRecovery from drinking water, spiked at 35 ppb, and two municipal and three industrial
effluents spiked at 110 ppb.
CRecovery from two municipal and three industrial effluents spiked at 110 ppb.
-------
(VO and NEWS) is perfluorotoluene. External standards for all the other
fractions are 2-fluorobiphenyl and/or 4-fluoro-2-iodotoluene.
1.2.5 Gas Chromatography
As shown in Figure 1.1, as many as 10 extracts or fractions may be
obtained from one sample if the entire HAS protocol is applied- (this may
be reduced to 7 if cleanup of the pH 8 (WABN) extract is not necessary, and
if the primary and secondary amine fractions can be mixed for a single
GC/MS analysis). Glass or fused silica capillaries are prescribed. Bonded
phase (e.g., Durabond DB-1 or DB-5), wide-bore, thick film (1.0 p), 30 or
60 N fused silica columns are recommended for inertness, stability, and
sample capacity. In all cases performance standards (see Quality Assurance)
rather than specific columns are specified (see Quality Assurance) in the
analytical protocol. No more than four different GC columns should be
necessary for the entire HAS. The analytical protocol for each fraction
prescribes optimum GC conditions.
1.2.6 Qualitative Analysis
Sample components are identified by established GC/MS/COMP techniques.
No research was conducted on the HAS identification procedures. CC/MS data
are stored on tape or disk. Internal standards in each extract are used as
reference points for retention time measurements as well as for quantifica-
tion. Compounds are identified by computer searching of mass spectra data
banks or by manual interpretation.
1.2.7 Quantitative Analysis
Extensive recovery studies were conducted during development of the
HAS (Tables 1.3-1.9). Approximately 280 model compounds from a wide
variety of chemical classes and physical property groups were dosed into
representative samples of several major types of water (distilled and
drinking water, and municipal and/or industrial effluents). Recoveries
were determined and average recovery factors are stored in a computer data
bank. Relative molar response (RNR) factors (relative to the deuterated
internal standards), based on MS selected ion peak areas, were also deter-
mined and stored in the data bank. (Appendix B is a hard copy of these
data.) The HAS user can use these data banks and a computer program devel-
oped for the HAS (HASQUANT, Appendix C) to calculate the concentration in
Chap. 1 - 25
-------
the original water sample of these model compounds as they are identified.
For compounds that are not in the data bank, concentrations can be estimated
by using RIIR and recovery factors for structurally similar compounds in the
data bank. (See summary tables in Section 1.4.)
During development of the MAS, it was determined that GC/NS data
should be collected in the full scan mode rather than by selective ion
monitoring. It was also shown, however, that quantification should be
based on peak areas (or height) of selected ions of compounds rather than
on the total ion current of that compound. Thus, RMR factors involve
ratios of ion peak areas and moles of the model compound to ion areas and
moles of the internal standard selected for quantification (usually the
closest eluting standard). These ion areas depend strongly on the GC/MS
instrumentation and MS tuning. Thus, it is necessary for each MAS user to
either develop his own RMR values or alternatively measure a few compound
RMRs at the MS tune specified and use a linear regression plot of these
against tabulated RI ffis of the same compounds to develop a correction
factor (slope) for the tabulated RIIRs. Tabulated R 1R values are those in
the computer data bank and are provided as part of the MAS protocols (Appen-
dix B).
R1 fR values developed by the linear regression correction procedure
will not be as accurate as those measured directly by the user. The user
can measure his own RMR factors using one or more solutions of the model
compounds within each protocol class (e.g., VO, NEWS, ESSA, etc.) contain-
ing all of the appropriate internal standards for that class. For example,
development of RJIRs for the MAS computer data bank for MASQUANT required
analysis of about 15 solutions containing up to 35 compounds in each.
These solutions were analyzed in replicate (usually six runs) on a Finnigan
4021 GC-MS-COMP system.
Even if the user develops his own RMR data bank, it is necessary for
him to track the tune of his mass spectrometer daily by chromatographing a
system performance mixture prescribed in each analytical protocol. If the
tune is significantly different from that used when his RKR values were
measured, the same linear regression procedure can be used to correct the
tabulated RMRs for the current tune. The linear regression and correction
can be done by MASQUANT.
Chap. 1 - 26
-------
To use MASQUANT for quantification, the user must provide sample
component identities, some information as to the specific HAS analytical
steps used, selected ion peak areas for each sample component ‘and the
appropriate (nearest) internal standard, and the concentration of the
internal standard in the original water sample. If the identified sample
component is one of the model compounds, the computation program will
select appropriate RMR and recovery factors for that component and calculate
its concentration in the original sample. Complete instructions for HAS
quantification are given in Chapter 13 and Appendices B and C.
As mentioned above, if the identified sample component is not a model
compound, RMR and recovery factor of a structurally similar compound in the
data bank may be used for an estimate of concentration (see summary tables
in Section 1.4). In addition to this obvious source of error, an additional
error is involved in using any recovery factors from the data bank. Since
sample matrices used for recovery studies were only representative of the
various water types, and since all recovery values for all matrices studied
were averaged to give the factor in the data bank, errors will occur in
applying these factors to other samples and the error is dependent on the
matrix differences between the sample being analyzed and the representative
recovery samp]es.
Footnotes on Tables 1.3-1.9 give more information on sample matrices
used for recovery studies. Separate recovery values are given for drinking
water for all HAS fractions except nonvolatile acids. These are more
accurate than HASQUANT data for drinking water analysis since the MASQUANT
(Appendix B) data bank values are averages over all water types studied.
On the other hand, recoveries using the WABN-FU protocols (for industrial
effluents) are so matrix dependent that there is no representative sample
matrix for recovery studies (see Footnote a, Table 1.5). The user must
generate his own recoveries for his own sample matrix.
1.2.8 Quality Assurance/Quality Control
As discussed above, extensive recovery studies with model compounds
were performed during development of each HAS procedure. Statistical
analysis techniques were applied to replicate recovery determinations to
arrive at the precision associated with each compound’s recovery. The same
Chap. 1 - 27
-------
statistical approach was used in measuring compound RMRs. The sum of the
tabulated recovery precision and RMR precision values for an analyte gives
the best precision to be expected with use of the appropriate HAS protocol.
This should be considered when expressing the quality of data generated by
application of the HAS. Irthe user generates his own R 1R and/or recovery
data, the precision and accuracy will be different from the tabulated
values since they will include terms for user operational errors, different
levels of GC/MS/COMP instrument precision, and new matrix effects.
Of most importance are the quality control steps prescribed for the
user in each protocol. Some of these steps are outlined below.
1.2.8.1 System Performance Solution--
One important quality assurance step is theuse of standard performance
solutions to check performance of the GC/MS/COMP each day. The HAS pres-
cribes a performance standard solution and corresponding criteria of
acceptance for each analytical protocol. Solutions include compounds to
measure GC peak asymmetry, separation number, resolution, polarity, and
column acidity and basicity; capacity of the capillary column; inertness of
the GC to MS transfer line; limits of detection of the HS/COHP system; and
tune of the MS. These solutions also contain deuterated internal standards
and the external standard appropriate to each protocol for RFIR verification
and periodic determination of the RMR correction factor by linear regression,
if necessary.
1.2.8.2 Internal and External Standards--
Comparison of the recovered quantity of deuterated internal standards
to the quantity of external standard added to the extract just before CC/MS
analysis reveals recovery deficiencies, thus serving as a check to indicate
malfunctions of the HAS analytical procedure. The primary use of deuterated
internal standards is for quantification; reference to an internal standard
is generally accepted as the most accurate quantitation technique available
for the GC/MS analysis of organics in water. Internal standards may also
be used as retention time indices for an aid in compound identification.
1.2.8.3 Sample Scouting--
As described earlier, several sample scouting measurements are pres-
cribed to characterize water quality and in turn allow selection of the
Chap. 1 - 28
-------
appropriate and optimal analytical techniques for a particular water
sample. Scouting measurements also help in determining optimum sample size
and dilution factor for certain protocols.
1.2.8.4 Blanks, Controls, Duplicates, and Surrogate Samples--
Requirements and procedures for field and laboratory blanks, spiked
field and laboratory controls, and duplicate and surrogate samples are
specified in each sampling protocol. Procedures for cleaning glassware and
apparatus and other steps to assure quality of measurement are also speci-
fied throughout the HAS.
1.2.8.5 HAS Test Samples--
For each HAS protocol, instructions are given for preparing controls
(for quality assurance) by dosing known amouhts of analytes into reagent
water. Test samples for practicing and checking HAS procedures may be
prepared in the same way.
1.3 TIME REQUIREMENTS FOR THE HAS
A very preliminary estimate of time per comprehensive HAS sample, or a
corresponding quality assurance sample, is 80 hours. This is for a labora-
tory analyzing only a few, say 10 to 50, samples per year, using personnel
who are experienced in trace organic analysis of water and set up with the
equipment and techniques used for the HAS.
It should be remembered that the HAS protocols were developed and
designed as separate entities so that a laboratory could analyze only the
fractions appropriate to its mission. The cost for analyzing pH 8 extract-
ables, for example, might be only 10% of that for a comprehensive HAS
analysis.
1.4 SUMMARY OF RECOVERY AND PRECISION FOR HAS PROTOCOLS
Tables 1.10—1.12 summarize the recovery data reported in Tables 1.3-1.9
for the chemical classes corresponding to each HAS protocol. Footnotes to
Table 1.10 give information on sample matrices and spiking levels used for
recovery studies. Several observations can be made regarding these data
(Table 1.11): 1) recoveries for volatile (purgeable) organics are highest
(these purgeables data are for a wide variety of compound classes from
several types of water); 2) neutral water soluble organics, a new class of
organic analytes, are recovered adequately with adequate precision; and
Chap. 1 - 29
-------
Table 1.10. SUMMARY OF ALL MAS RECOVERY DATA
(INCLUDING DEUTERATED INTERNAL STANDARDS)
PROTOCOL CLASS
Recovery
Mean
CV
Mean
Chemical Class
Compounds
Range
Recovery,
Range,
CV,
(Examples)
Studied
t
%
Footnotes
VOLATILE (PURGEABLE) ORGANICS (VO)
Aromatic Hydrocarbons 9 59-113 85 335 11 a
(benzese, napbthsleoe)
Halogeaated Aromatics 7 91-106 100 2-30 15 a
(cblorobenzese, 1,2,3—tn-
chlorobenzene)
Misc Aromatic Compounds I — 68 — 12 a
(anisole)
Aliphatic and Alicyclic Hydro-
carbons 11 44—120 82 1-40 16 a
(cyclohexase, e—tridecane)
Halogenated Alip atic Hydrocar-
bons 7 77-118 90 4-16 9 a
(chloroform, I ,4—dibromobutace)
Miscellaneous Oxygen & Sulfur
Compounds 5 70-115 93 3-26 11 a
(diethyl ether, hexyl ether)
Deutenated Standards 4 57—120 90 10—25 18 a
Cd —bromoethane, 2,4,6—d -
a isole, d —chlorobenzene,
d 8 -naphLha ene)
NEUTRAL WATER SOLUBLE ORGANICS
(NEWS)
Alcohols 4 106—131 115 18—25 22 b
(1-propanol, 1-heptanol)
Aldebydes 3 72-83 79 25—32 29 b
(n-butyraldehyde, crotonalde—
hyde)
Esters 7 45-93 74 4-17 7 c
(methyl formate, ethyl butyrate)
Ethers 2 50—79 65 14—31 23 b
(tetrahydrofuraii, dioxane)
Xetones 2 65-69 67 2-32 17 b
(methyl ethyl ketone, cyclo—
hexanone)
Nitniles 4 57-95 83 6-14 9 b
(acrylonitnile, benzonitnile)
Nitro Compounds 3 70-96 86 5-13 9 b
(nitrometbane, ostrobenzene)
Deuterated Standards 2 93-98 96 7-10 9 b
(d 9 -t-butanol d 5 - nitrobenzene)
WEAK ACIDS, BASES, AND NEI.TFRALS
(WARN-SC and WABN-BL)
Weak Acids
(phenol, 2,4-dicblorophenol)
accumulator column 6 55-95 71 5-23 11 d
batch liquid—liquid 12 49-118 71 5-27 13 e
Weak Bases
(aniline, carbazole)
accumulator co1 ,jmn 16 53—95 82 0-16 7 d
batch liquid-liquid 15 40-86 64 6-40 24 e
Alkanes
(n—decane, n-tridecane)
accumulator column 8 42-66 52 6-20 15 d
batch liquid—liquid 11 45-82 62 13—31 21 f
Aliphacic Ketones, Alcohols,
and Esters
(feochone, methyl stearste)
accumulator column 7 49—94 75 1—22 10 d
bitch liquid—liquid 9 49111 71 10—43 25 e
(continued)
Chap. :1 - 30
-------
Table 1.10 (cont’d.)
Misc Aliphatic Compounds
(di-t—butyldisulfide, tributyl-
phosphate)
accumulator Column
batch liquid-liquid
Aromatic Hydrocarbons
(2-methylnaphthsleoe pyrene)
accumulator column
batch liquid-liquid
halogecated Aromatics
(o-cbloreaoisole, hexacbloro-
beozene)
accumulator column
batch liquid-liquid
Aromatic Aldehydes and Ketonea
(o-tolualdebyde, acetophenone)
accumulator column
batch liquid-liquid
Aromatic Esters and Sulfonates
(benzylacetate, ethyl—i- toluene—
sulfonate)
accumulator column
batch liquid—liquid
Misc. Aromatic Compounds
(nitrobeozene, tetraphenyltin)
accumulator column
batch liquid-liquid
Deuterated Standards
(d 1 -rylene, d -phenol,
d — cetophenon , d -phenyl—
e hanol, d -oitrob nzene, d -
propiophen ne, d 8 -naphthale e,
d 9 -acridine, d -perylene)
accumulator lumn
batch liquid-liquid
EXTRACTABLE SEMIVOLATILE STRONG
ACIDS (ESSA)
Carboxylic Acids
(beezoic acid; palmitic acid)
Phenols
(2—nitro—i—cresol, penta—
chiorophenol)
Deuterated Standards
(d -heptinoic acid, d 5 -benzoic
acfl)
VOLATILE STRONG ACIDS (VOSA)
Volatile Carboxylic Acids
(acrylic acid, n—octaooic acid)
Deuterated Standards
(d 7 —butyric acid)
NONVOLATILE ACIDS (NOVA)
Carborylic Acids
(succinic acid; 2, 4 1 5—tri-
chiorophenoxyacetic acid)
Sulfonic Acids
(benzeoesulfo ic acid; 2—
napbtbaleaesulfonic acid)
Misc Nonvolatile Acids
(benzenephospboric acid,
pentachloropbenol)
Deuterated Standards
(2—naphtbale esulfonic acid-
d 7 1120)
6 40-92
4 57—104
10 60-87
7 48-118
10 56—100
9 43—107
3 88-96
4 43-105
6 46—87
7 55—138
6 47-89
10 48-105
9 55-93
8 40-78
17 63—110
5 88-100
2 65-92
(continued)
PROTOCOL CLASS
Recovery
Mean
CV
Mean
Chemical Class
Compound,
Range
Recovery,
Range,
CV,
(Examples)
Studied
%
%
I
I Footnotes
72 2-36 18 d
75 9—36 21 e
74 1—27 16 d
79 13-41 24 e
73 1—42 16 d
68 13-33 20 e
92 2-17 12 d
69 6-19 13 e
69 7—17 12 d
84 3—19 11 e
74 6-24 13 d
68 8-33 17 e
78 4—21 10 d
58 11-40 26 e
89 2-20 8
94 4—19 8 g
79 6—12 9 g
16 46-90 65 4—34 19 h
I - 85 — 14 b
6 42-87 64 2-45 18
4 84—110 96 7—45 23 i
3 62—140 102 11—50 25 i
1 — 110 - 14
Chap. 1 - 31
-------
Table 1.10 (cont’d.)
Compounds
Recovery
Range
Mean
Recovery,
CV
Range,
Mean
CV,
Studied
%
t
% Footnotes
R0TOC0L CLASS
Chemical Class
(Examples)
STRONG AMINES (SAM-PT and SAS-S)
Primary and Tertiary Am ,oes
(m—butylamine, tri—n-butyl-
amine)
Secondary Amines
(diallylamine, 2-methyl-
piperidine)
Deuterated Staodards
(d 0 -butylamine, d -pbeoylethyl-
amine, N-etbyl-2-fluorobenzyl-
amine)
aMean recoveries are for triplicate determinations from drinking water, spiked at 0 2 to 8 ppb
(nominally 1 ppb), plus triplicate determinations from a 60/60 industrial/municipal wastel,ater,
spiked at 30 to 87 ppb (nominally SO ppb)
bMean.recoveries are for triplicate determinations from drinking water, spiked at 0 8 to 1 2 ppb
(nominally I ppb), plus triplicate determinations from a 60/40 industrial/municipal vastewater,
spiked at 40 to 63 ppb (nominally 50 ppb)
CHean recoveries are for triplicate determinations from 60/40 industrial/ciunicipal wastewater
only, spiked at 60 to 63 ppb (nominally 50 ppb)
dMeao recoveries are for triplicate determinations from drinking water, spiked at 0 5 to 5 ppb
(nominally I ppb), using XAD—4 resin sorbent columns
eMean recoveries are for triplicate determinations from a 60/40 industrial/municipal wastewater,
or, for about 1/4 of the total compounds, from reagent water spiked at 15 to SO ppb (nominally
25 ppb), using batch liquid-liquid extraction, with clean—up included
1 Mean recoveries are for triplicate determinations from reagent water only, with clean—up steps
included (Interferences prevented recovery determinations from wastewater)
tIean recoveries are Iron triplicate determinations from drinking water only, Spiked at 50-100 ppb
(nominally 55 ppb) Recoveries were not determined from more complex waters
bMeafl recoveries are for triplicate determinations from drinking water, spiked at 0 3 ppb, plus
triplicate determinations from a 60/40 industrial/nunicipal wastewater, spiked at 120 ppb
‘Mean recoveries are for triplicate determinations from several industrial and municipal
effluents
P1ean recoveries are for triplicate determinations from three industrial and two municipal effluents
spiked at 110 ppb, and including, in some cases, triplicate determinations from drinking water
spiked at 35 ppb
kReCoveriei determined for only one (d 9 -butylamine) of the three internal standards
11
58-86
72
12—41
26
j
6
40-98
63
20-53
36
j
1
—
75
-
27
Chap. I - 32
-------
Table 1.11. SUMMARY OF ALL HAS RECOVERY DATA BY PROTOCOL CLASS
Protocol Class
No.
Compounds
Mean
Recovery, %
Mean
CV, %
Volatile (purgeables) Organics (VO)
44
89
13
Neutral Water Soluble Organocs (NEWS)
27
84
14
Weak Acids, Bases, and Neutrals (WABN)
accumulator column (WABN-SC)
batch liquid-liquid (WABN-BL)
a b
87 ‘
96 a
b
74
69
b
12
20
Extractable Semivolatile Strong Acids
(ESSA)
24 b
89 b
9 b
Volatile Strong Acids (VOSA)
17
66
19
Nonvolatile Acids (NOVA)
14 C
85 C
20 C
Strong Amines (SAN-PT and SAM-S)
18
327 a
69
76 d
28
16 d
aSixty...nine compounds were used for both accumulator column and batch liquid-liquid recovery studies;
the total number of different compounds in this table is therefore 258.
bESSA and WABN—SC recovery data are for drinking water only.
CNOVA recovery data are for industrial and municipal effluents only.
dHean recoveries and mean CVs were calculated from values for the 327 individual compounds.
Pu
-------
Table 1.12. SUMMARY OF HAS RECOVERY DATA FOR ORGANICS IN DRINKING WATER BY PROTOCOL CLASSa
aFor triplicate determinations from
in drinking water.
bLevel spiked into water sample
Protocol Class
No.
Compounds
Spiking
Range
(ppb)
Nominal
Spiking
Leveib
(ppb)
Mean
Recovery
%
Mean
CV
%
Volatile (Purgeable) Organics
(VO)
52
0.2-1.8
1
90
10
Neutral Water Soluble Organics
(NEWS)
25
0.8—1.2
1
84
16
Weak Acids, Bases, and Neutrals
(WABN, accumulator column)
87
0.5-5
1
74
12
Extractable Semivolatile Strong
Acids (ESSA)
24
50-100
55
89
9
Volatile Strong Acids (VOSA)
18
—
0.3
82
10
Strong Amines (SAN-PT and SAN-S)
11
217
-
35
81
82 c
16
12 c
drinking water.
Nonvolatile Acids (NOVA) were not determined
cHean recoveries and mean CVs were calculated from values for the 217 individual compounds.
-------
3) two classes of ionic compounds, volatile strong acids and strong amines,
are characterized by relatively low recoveries and poor precision. It is
also seen from Table 1.11 that recovery of organics using accumulator
columns is better than with batch liquid-liquid extraction in a separatory
funnel, and that precision is also better. Matrix effects may be more
important than the extraction technique, however; only drinking water was
extracted by accumulator column, whereas more complex matrices were extract-
ed by the batch technique.
Table 1.12 shows summarized recovery data for organics in drinking
water only, by MAS protocol. (These data were integrated into the total
recovery data of Tables 1.10 and 1.11.) Spiking ranges and nominal spiking
levels are significantly lower than those used for recoveries from industri-
al and municipal effluents (see footnotes to Table 1.10. Recoveries for VO
and NEWS compounds are practically the same as those given in Table 1.11;
i.e., matrix effects or spiking levels did not make a significant difference
in the summarized data. Extractable semivolatile strong acids were recov-
ered well from drinking water, with good precision, but the spiking level
was relatively high. The other ionic classes of organics (VOSA and SAN)
were recovered at significantly higher levels and with better precision
from drinking water than from other matrices (cf. Table 1.11).
Mean recoveries over all protocols for water types studied (Table 1.11)
for 327 determinations (258 different compounds) was 76% with a mean rela-
tive standard deviation (for 3 or more measurements) of 16%. For drinking
water (Table 1.12), the mean recovery for 217 spiked compounds was 82%,
with a mean RSD of 12%.
Chap. I - 35
-------
CHAPTER 2
SAMPLING AND SAMPLE HANDLING FOR VOLATI1E ORGANICS (VO)
2.1 INTRODUCTION
This section describes the collection and handling of drinking water,
surface water, and municipal and industrial wastewater effluent samples
for the analysis of volatile (purgeable) organics (VO). For all waters, a
12 rnL and a 250 mL water sample is collected for scouting measurements and
analysis, respectively. Immediately after collection and scouting, resid-
ual chlorine in drinking water and treated municipal water will be quenched
using sodium thiosulfate. Samples are collected in containers with no
headspace, capped with crimp-top, Teflon-lined septum caps, and the bottles
inverted for shipment and storage. Upon arrival at the laboratory, inter-
nal standards are released into the samples, which are then stored at 4°C
until analysis.
Table 2.1 illustrates the sampling regime with quality control and
assurance samples for up to 20 different field samples. Duplicates,
surrogates, field and laboratory controls, and field and laboratory blanks
are prescribed. The exact number is dependent upon the number of field
samples to be collected. For example, if 11 field samples are to be
collected, then two duplicates (a duplicate of each of two field samples),
one surrogate, and one each of field and laboratory controls and blanks
are incorporated as part of the QC/QA. Thus, a total of 18 analyses would
be performed for 11 field samples (Table 2.1).
Control samples will be prepared by adding known quantities of repre-
sentative target compounds to reagent (free of VO compounds) water.
Blanks are unspiked reagent water. Laboratory controls and blanks, after
preparation, are stored at 4°C in the laboratory until analysis. Field
controls and blanks are shipped to the sampling site and handled in the
same manner as field samples in terms of cooling, shipping and storing.
Chap. 2 - 36
-------
No. of Sarz3ples
to be Collected
Duplicates
Surrogates
Controls
Blanks
Total No.
of Analyses
Field
Laboratory
Field
Laboratory
1
2 1 — — 1 1 5
3 1 — 1 1 6
4 1 1 1 1 8
5 1 — 1 1 1 9
6 1 — 1 1 1 10
7 1 1 1 1 11
8 1 1 1 1 12
9 1 1 1 1 13
10 1 — 1 — 1 1 14
11 2 1 1 1 1 1 18
12 2 1 1 1 1 1 19
13 2 1 1 1 1 1 20
14 2 1 1 1 1 1 21
• 15 2 2 1 1 1 1 23
16 2 2 1 1 1 1 24
17 2 2 1 1 1 1 25
18 2 2 1 1 1 1 26
19 2 2 1 2 2 2 30
20 2 2 2 2 2 2 32
-------
Upon the return to the laboratory, representative VO target compounds
are spiked into replicate field samples (the number of samples treated in
this manner is specified in Table 2.1) for use as surrogate samples.
Comparison of the analytical results for controls, blanks and duplicates
to results for field samples will provide a measure of uncontrolled contam-
ination and/or analyte losses during sample collection, shipment, and
storage, the homogeneity of the sample matrix and the accuracy and preci-
sion associated with sample preparation and analysis. Recovery of internal
standards and spiked compounds from the surrogate samples will provide
information on matrix effects on method accuracy and precision (i.e., if
there are two or more surrogates).
If more than 20 samples are to be collected, the additional field
samples are matched with the prescribed number of duplicates, surrogates,
etc. For example, if 33 samples are to be collected, then lines for 20
and 13 samples, respectively, are consulted and the number of duplicates,
surrogates, etc., are a sum of these two.
2.2 MATERIALS AND REAGENTS
The following materials are required for collecting a set of 20 water
samples plus two duplicate and two surrogate samples and two each of field
and laboratory controls, and two each of field and laboratory blanks for
the analysis of purgeable volatile organics (VO).
(1) Thirty-two, narrow mouth, 243 mL amber glass bottles with Teflon
lined septum screw cap or (crimpable) caps. One 3 dram (12 mL),
septum-capped vial. Thirty-two Teflon coated magnetic stirring
bars; one for each bottle.
(2) One Bausch and Lomb Mini Spec 20 portable spectrophotometer.
(3) One 25 ml mixing cylinder (Hach Chlorine Determination kit).
(4) Materials (e.g., “Blue Ice® ) for chilling the sample during
shipment and storage; insulated shipping containers for samples.
(5) 25 ml volumetric flasks for preparing standard solutions; 2 ml
glass ampules.
(6) 10 IJL GC-type microliter syringe, Teflon tipped plunger.
(7) Reagents:
Chap. 2 - 38
-------
(a) reagent water - reagent water is defined as water inwhich
no interferences are observed at the detection limit for
the parameters of interest. Two liters of water from a
purification system (Millipore Super_Q® or equivalent),
purged at 80°C for 30 mm with cryogenically (LN 2 ) cleaned
He gas at 15 mL/min may be used.
(b) Thirty-two microcapsules containing internal standards (NBS
prepared) as shown in Table 2.2.
(c) 25 mL standard solution (Table 2.3). Purgeables standard
solutions are prepared from pure, neat reagent materials.
(d) 250 mL Na 2 S 2 O 3 solution - 0.05 N in reagent water.
Ce) DPD Total Chlorine Reagent Powder Pillow (Hach Chemical
Co.).
2.3 PREPARATION OF STANDARD SOLUTIONS
2.3.1 Internal Standards
These microcapsules are used as received from EPA (or NBS, Table 2.2).
2.3.2 Standard Solution of Purgeable Compounds
(1) Prepare a standard solution by accurately measuring 3 pL of neat
liquid standard (Table 2.3) using a 10 .iL syringe into the
expanded area of a 25 mL volumetric flask containing about 25 mL
Table 2.2. NES MICROCAPSULES CONTAINING PURGEABLE
INTERNAL STANDARDS IN METhANOLa
Internal Standard
b
pg/L
Set
No.
1
Set No.
3
d 5 -bromoethane
0.56
6.6
2,4,6-d 3 -anisole
4.2
25.6
d 5 -ch lorobenzene
7.2
5.4
d 8 -naphtha lene
5.8
48.7
aConcentrations are based upon the production run made by NBS.
This production run is the “400” series.
b .
Concentration after dosing in 250 mL of water. Set No. 2 was
an experimental set and is not prescribed in the HAS and, thus,
it is not listed here.
Chap. 2 - 39
-------
Table 2.3. PURGEABLE STANDARD SOLUTION
Chemical
Density
(@ 20°C)
niL
diethyl ether
0.714
86
cyclopentane
0.746
90
n-dodecane
0.749
90
n-tetradecane
0.763
92
dichioromethane
1.327
160
chloroform
1.483
178
1,2-dichioroethane
1.235
148
2-bromohexane
1.174
141
1-bromo—4-chlorobenzene
1.576
190
1,2,4-trichlorobenzene
1.454
174
2-iodotoluene
1.678
201
benzene
0.878
105
ch lorobenzene
1.105
133
tetrach loroethy lene
1.623
195
1,3,5-trimethy lbenzene
0.894
107
trichioroethylene
1.464
176
of methanol. After adding all
times to mix. Store at 4°C for
volumetric flask. Aliquots may
and sealed (the transfer should
stored for up to 4 weeks.
(2) Standard Solution With Internal Standards (Optional) . Prepare
the standard solution containing internal standards by accurately
delivering 3 pL with a microliter syringe of the neat deuterated
substances (>98% chemically and isotopically pure, Table 2.2)
into the 25 niL volumetric flask containing purgeable standards
(above, 1). Densities are given in Table 2.4. This is done if
NBS microcapsules are not available. Store at 4°C for no longer
than 3 days in the volumetric flask (see above for transferring
to ampules).
compounds invert flask several
no longer than 3 days in the
be transferred to 2 niL ampules
be done at 4°C). Ampules may be
Chap. 2 - 40
-------
Table 2.4. INTERNAL STANDARD SOLUTION (OPTIONAL)
Chemical
Density
(@ 20°C)
Level 1
Level 2
, La
b
pg/L
La
b
pg/L
d 5 -brbmoethane
d 8 -naphthalene
1.460
1.025
3
1.4
1.9
30
14.0
9.6
d 5 -chlorobenzene
1.106
3
1.1
30
11
2,4,6-d 3 -anisole
0.996
3
0.96
30
9.6
a
pL of neat material added to 25 mL methanol.
bpg/L is final concentration in water sample if 2.0 iL of this
solution is added to 250 mL.
C 60 mg is weighed into 25 mL volumetric flask before adding
methanol.
d 30 mg is weighed into 25 mL volumetric flask before adding
methanol.
2.3.3 Internal Standard Solution (If NBS prepared standards are not
available )
Prepare deuterated internal standard solution (Table 2.4) by accurate-
ly pipetting 3 .iL of the deuterated compounds into a 25 mL volumetric
flask Containing 25 mL of methanol. After all internal standards have
been added, invert the flask several times to mix. Store at 4°C for no
longer than 3 days in the volumetric flask (see above 2.3.2(1) for long
term storage).
2.4 CLEAN-UP PROCEDURES
(1) All glassware to be used, including sample bottles, should be
washed with Amway SA-8 laundry detergent, rinsed several times
with deionized water, and baked for a minimum of 4 h at 500 to
550°C. All cleaned glassware is immediately capped or covered
with precleaned foil to prevent contamination.
(2) The exteriors of the NBS microcapsules containing deuterated
internal standards are rinsed with toluene and pentane, air
dried at room temperature and stored in a clean screw-capped
vial.
Chap. 2 - 41
-------
(3) Teflon lined septa are sonicated for 10 mm in pesticide grade
methanol followed by 10 mm in pesticide grade pentane and dried
in a vacuum oven at 60°C for 1 hr and stored in a screw-capped
vial.
2.5 PREPARATION OF CONTROL AND BLANK SAMPLES
(1) Place a clean magnetic stirbar into each of eight 250 raL sample
bottles. Place one NBS microcapsule containing the internal
standards into each 250 mL smuple bottle. If NBS standards are
not available, see section 2.6.0(8) for optional internal stand-
ard procedures. For drinking water use set No. 1 (Table 2.2).
For dirtier waters, set No. 3 may be employed. Fill each bottle
with reagent water, leaving no headspace, and seal with Teflon
lIned septum caps.
(2) Inject through the septum cap of 4 of the bottles with a micro—
liter syringe 2 pL of purgeable standard solution (Table 2.3) to
produce the control samples. Inject as far below the septum as
possible.
(3) Label and number bottles 1 to 8, 4 as blanks and 4 as controls.
(4) Invert all eight bottles and store at 4°C.
(5) Field controls (2) and field blanks (2) are shipped to the
collection site and are handled, stored and shipped in the same
manner as field samples (at 4°C). The remaining laboratory
blanks and controls are stored at 4°C in the laboratory.
2.6 FIELD COLLECTION
(1) Carry to the field:
(a) Chlorine measurement kit (Hach); 25 mL mixing chamber; Nini
Spec 20.
(b) Na 2 S 2 O 3 quenching solution.
(c) Tweezers, pipettes.
(d) Twenty—four NBS microcapsules containing internal standards
(Sets 1 or 3, Table 2.2) [ see 2.6.0(8): optional].
(e) 2 field control and 2 field blank samples (kept at 4°C).
(f) Twenty—four clean, empty 250 mL sample bottles (which have
been capped with septum seals to keep clean) and 24 Teflon
Chap. 2 - 42
-------
lined septa, stored in a clean sample vial. Crimping
device, if crimp caps are jised. Twenty-four magnetic
stirbars (Teflon-coated).
(g) One 3 dram septum capped vial.
(2) A preliminary water sample (- 25 mL) is collected at the field
site to determine the total chlorine content (only for drinking
water, final treated municipal effluent, or water suspected of
containing chlorine). This measurement is made with a Bausch
and Lomb Mini Spec 20 portable spectrophotometer. The water
sample is added to a 25 mL mixing cylinder along with the con-
tents of a DPD Total Chlorine Reagent Powder Pillow. The mixture
is shaken and the color is allowed to develop 3 to 6 minutes.
Percent transmittance, measured at 530 nm, is converted to mg/L
total chlorine using Table 2.5.
(3) Rinse each sample bottle three times with the water sample to be
collected and drain, then place a clean magnetic stirbar (handle
with clean tweezers) and a NBS microcapsule (use tweezers)
containing internal standards in each bottle. Use Set No. 1
(Table 2.2) for drinking water and Set No. 3 for dirtier waters.
Table 2.5. TOTAL CHLORINE (mg/L as Cl) VS. S TRANSMITTANCE
ST
5 T
Units
Tens
0
1
2
3
4
5
6
7
8
9
10
4.00
3.84
3.68
3.54
3.42
3.30
3.18
3.08
2.98
2.88
20
2.80
2.71
2.63
2.55
2.48
2.41
2.34
2.28
2.21
2.15
30
2.09
2.04
1.98
1.93
1.88
1.82
1.78
1.73
1.68
1.64
40
1.59
1.55
1.51
1.47
1.43
1.39
1.35
1.31
1.28
1.24
50
1.20
1.17
1.14
1.10
1.07
1.04
1.01
0.98
0.95
0.92
60
0.89
0.86
0.83
0.80
0.78
0.75
0.72
0.70
0.67
0.64
70
0.62
0.60
0.57
0.56
0.52
0.50
0.48
0.46
0.43
0.41
80
0.39
0.37
0.34
0.32
0.30
0.28
0.26
0.24
0.22
0.20
90
0.18
0.16
0.14
0.13
0.11
0.09
0.07
0.05
0.04
0.02
Reprinted from: Water and Wastewater Analysis Procedures Manual,
Hach Chemical Co., 1975, p. 2—29.
Chap. 2 - 43
-------
(4) A stoichiometric quantity (plus 10% excess) of Na 2 S 2 O 3 solution
(Figure 2.1) is added to each bottle based upon the total chlo-
rine determination for that sampling point.
(5) Sample bottles are then filled with the water sample, leaving no
headspace, and sealed with Teflon lined septa. Remember, 4 du-
plicates (a duplicate of 4 of the samples) are collected; 2 will
serve as surrogate water samples. A 3 dram (12 mL) vial is also
filled with sample water and sealed (no internal standards are
added). This 12 mL sample will be used for scouting measurements
at the laboratory.
(6) All sample bottles are inverted and shipped on ice (e.g., “Blue
Ice®ht) directly to the laboratory by an appropriate air carrier
(e.g., Federal Express) in well insulated cartons.
(7) As soon as the samples arrive at the laboratory, spike two of
the duplicate water samples with 2 .iL (drinking water), or 50 pL
(for dirtier waters) of the purgeable standard solution by
injecting through the septum cap with a microliter syringe.
Inject as far below the septum as possible. These are the
surrogate samples.
(8) Break NBS internal standard capsules by placing bottles (all
field samples and quality control and assurance samples) on a
magnetic stirrer (slightly off center) and using violent stirring
conditions completely crush the capsule.
Optional . If NBS microcapsules containing deuterated internal
standards are not employed, the internal standards are added
(immediately after headspace scouting has been completed, see
Analysis Protocol, Chapter 6) by injecting 2 iL of the internal
standard solution (Table 2.4) in methanol. Use Level 1 solution
for drinking water, Level 2 for dirtier waters. Inject as far
below the septum as possible. To insure quantitative transfer
of the standards from the syringe to the water, a 10 pL GC-type
syringe with a Teflon- tipped plunger is used. The injection
technique involves first drawing 2 pL of air, followed by 2.0 iJL
Chap. 2 - 44
-------
of standard, followed by a second 2 pL of air. In this way the
actual volume of standard to be injected into the sample can be
observed in the syringe. After injection of the standard into
the sample, an aliquot (—5 giL) of the sample is drawn up into
the syringe and expelled back into the sample to insure quantita-
tive introduction of the standards. Mix by placing on a magnetic
stirbar plate.
(9) All samples are stored at 4°C until analysis (bottles inverted).
Analysis should be performed within 10 days of sample collection.
Figure 2.1. Total chlorine vs volume of 0.O5MNa 2 S 2 O 3 added to a
1 L sample. (If a 250 inL sample is collected then
add 1/4 of the volume indicated).
7
6.
I .,
m
U
U
S.
0
3.
S
Slope • 3.38
0:2 o: o6 o:a i:o 1:2 i: 1:6 i:s 2:0 2:2 2. 2 6 28
Vo1 e 0 05 25203 (at)
Chap. 2 - 45
-------
CHAPTER 3
SAMPLING AND SAMPLE HANDLING FOR NEUTRAL WATER SOLUBLE ORGANICS (NEWS)
3.1 INTRODUCTION
This section describes the collection and handling of drinking water,
surface water, and treated municipal and industrial wastewater effluent
samples for the analysis of low molecular weight, volatile water soluble
compounds. For drinking water, a 200 mL sample will be collected. A
20 mL sample will be collected for all other water types. Immediately
after collection, residual chlorine will be quenched using sodium thiosul-
fate, the sample pH will be adjusted using H 2 S0 4 or NaOH, and internal
standard solutions will be pipetted into each sample. Samples will be
capped, then stored at 4°C until analysis.
Table 3.1 illustrates the sampling regime, including quality control
and assurance samples for up to 20 different field samples. Duplicates,
surrogates, field and laboratory controls, and field and laboratory blanks
are prescribed. The exact number is dependent upon the number of field
samples to be collected. For example, if 11 field samples are to be
collected, then two duplicates (one duplicate of each of two field samples),
one surrogate, and one each of field and laboratory controls and blanks
are incorporated as part of the QC/QA. Thus, a total of 18 analyses would
be performed for 11 field samples (Table 3.1).
Control samples will be prepared by adding known quantities of repre-
sentative target compounds to reagent (free of NEWS compounds) water.
Blanks are unspiked reagent water. Laboratory controls and blanks, after
preparation, are stored at 4°C in the laboratory until analysis. Field
controls and blanks are shipped to the sampling site and handled in the
same manner as field samples in terms of cooling, shipping and storing.
Upon the return to the laboratory, representative NEWS target compounds
are spiked into replicate field samples (the number of samples treated in
this manner is specified in Table 3.1) for use as surrogate samples.
Chap. 3 - 46
-------
No. of Sawples
to be Collected
Duplicates
Surrogates
Controls
Blanks
Total No.
of Analyses
Field
Laboratory
Field
Laboratory
1
1
—
2 1 - 1 1 S
3 1 - 1 1 6
4 1 1 1 1 8
5 1 1 1 1 9
6 1 1 1 1 10
7 1 1 1 1 11
8 1 1 1 1 12
9 1 1 1 1 13
10 1 — 1 — 1 1 14
11 2 1 1 1 1 1 18
C)
12 2 1 1 1 1 1 19
13 2 1 1 1 1 1 20
14 2 1 1 1 1 1 21
15 2 2 1 1 1 1 23
16 2 2 1 1 1 1 24
17 2 2 1 1 1 1 25
18 2 2 1 1 1 1 26
19 2 2 1 2 2 2 30
20 2 2 2 2 2 2 32
-------
Comparison of the analytical results for controls, blanks and dupli-
cates to results for field samples will provide a measure of uncontrolled
contamination and/or analyte losses during sample collection, shipment,
and storage, the homogeneity of the sample matrix, and the accuracy and
precision associated with sample preparation and analysis. Recovery of
internal standards and spiked compounds from the surrogate samples will
provide information on matrix effects on method accuracy and precision (if
more than two surrogates are included).
If more than 20 samples are to be collected, the additional field
samples are matched with the prescribed number of duplicates, surrogates,
etc. For example, if 33 samples are to be collected, then lines for 20
and 13 samples, respectively, are consulted and the number of duplicates,
surrogates, etc., are a sum of these two values.
3.2 MATERIALS AND REAGENTS
The following materials are required for collecting a set of 20 water
samples plus two duplicate and two surrogate samples and two each of field
and laboratory controls and blanks for the analysis of neutral water
soluble organics (NEWS).
(1) Thirty-two narrow mouth, amber glass, sample bottles with Teflon
lined screw caps. For drinking water, 250 mL bottles calibrated
to 200 mL are used, for all other water types 25 mL bottles
calibrated to 20 mL are used. Bottles are calibrated by adding
an accurately measured volume of water, then marking the level
of the water miniscus by scratching the outside surface. Bottles
should be calibrated before cleaning.
(2) Graduated pipettes (1.0 niL) for preparing and aliquoting standard
solutions.
(3) Pasteur pipettes plus pipette bulbs.
(4) One Bausch and Lomb Mini Spec 20 portable spectrometer.
(5) One 25 mL mixing cylinder (Hach Chlorine Determination Kit).
Materials (e.g., Blue Ice ) for chilling the sample during
shipping and storage; insulated shipping containers for samples.
(7) Volumetric flasks (10 niL and 100 mL) for preparing standard
solutions.
Chap. 3 - 48
-------
(8) Syringes for preparing standard solutions — 100 and 250 liL.
(9) Teflon lined screw cap bottles for storing standard solutions.
(10) Narrow range pit paper.
(11) Reagents
(a) reagent water - reagent water is defined as water in which
no interferences are observed at the detection limit for
the parameters of interest. A water purification system
(llillipore Super_Q® for equivalent) may be used to generate
reagent water.
(b) NIBS ampoules containing deuterated NEWS internal standards
in water (Table 3.2). If not available prepare as described
under Section 3.3.1.
Cc) 10 mL NEWS standards (Table 3.3). Control standards are
prepared from pure stock solutions.
(d) sodium hydroxide - 0.1N in reagent water (must be demon-
strated as contaminant free; Chapter 4).
(e) sulfuric acid solution — 0.1N in reagent water (must be
demonstrated as contaminant free; Chapter 4).
(f) 250 mL Na 2 S 2 0 3 solutioti - 0.05 N in reagent water (must be
demonstrated as contaminant free; Chapter 4).
(g) DPD Total Chlorine Reagent Powder Pillow (Hach Chemical
Co.).
Table 3.2. NBS ANPOULE CONTAINING NEWS
INTERNAL STANDARDS IN WATER
a
Concentration ( .ig/Ampoule)
Spiked
Concentration
(ppb)
200 b
20 mLC
Compound
Set No.
1
Sample
Sample
t-Butanol-d 9
112.9
75.3
376
Nitrobenzene-d 5
20.1
13.4
67
aConcentration is for the “400” series production run; Set No. 1 contains
7.5 ml. water.
b 10 niL from Set No. 1 is added.
C 05 mL from Set No. 1 is added.
Chap. 3 - 49
-------
Table 3.3. NEWS STANDARD SOLUTION
Compound
Density
(@ 2°C)
Am
Sol
(i.JL t
ount
ution
o 10
Stock
Added
mL water)
Standard
(pg/
reagent
Solution
rnL in
water)
propionitrile
0.782
60
4.6
n-butyral4ehyde
0.817
15
1.2
dioxane
1.034
30
3.1
cyclopentanone
0.949
20
1.9
hexanol
0.827
40
3.2
3.3 PREPARATION OF STANDARD SOLUTIONS
3.3.1 Internal Standard Solution, Table 3.4 (If NBS standards not
available)
(1) With a 50 t.iL syringe, draw up and accurately weigh 25 mg of t-
butanol-d 9 and 5 mg of nitrobenzene-d 5 into a 10 rnL volumetric
flask containing 9 mL of reagent water. Bring to final volume
with reagent water and mix well. This is the stock solution.
(2) Transfer this stock solution into a Teflon sealed screw cap
bottle. Store at 4°C and protect from light.
(3) With a 250 l.JL syringe, accurately measure 100 IlL of this stock
solution and inject into a 20 mL volumetric flask containing
19 mL of reagent water. Make to volume with reagent water and
well. This is the standard solution.
(4) With a 1 mL graduated pipette, transfer 1.0 mL portions of this
solution to 0.5 dram vials. Seal with Teflon lined, septum seal
screw caps. Store at 4°C and protect from light.
(5) Fresh standards must be prepared every six months. If signs of
degradation or evaporation occur, they should be replaced more
frequently.
(6) When compound purity is 96% or greater, the weight can be used
without correction to calculate concentration. Compounds which
are less than 96% pure cannot be used for standards.
3.3.2 Standard Solutions of NEWS Compounds (Table 3.3 )
(1) Prepare a standard stock solution. With a 100 pL syringe, accu-
rately measure the specified volume of each liquid standard
Chap. 3 - 50
-------
Table 3.4. DEUTERATED INTERNAL STANDARD SOLUTION (OPTIONAL)
Spiked
Standard Concentration
Concentration
(ppb)
200 mLa
20
Compound
(pg/mL)
Sample
Sample
t-Butanol-d 9
12.5
62
620
Nitrobenzene-d 5
2.5
12
120
aConcentration when 1 mL of solution is spiked into a 200 mL water
sample.
bConcentration when 1 mL of solution is spiked into a 20 mL water
sample.
(Table 3.3) into a 10 mL volumetric flask containing 9 mL water.
flake to volume with reagent water and mix well. Transfer into a
Teflon sealed screw cap bottle. Store 4°C and protect from
light.
(2) Prepare the standard solution. With a 250 i.iL syringe, accurately
measure 100 pL of the control stock solution into a 100 mL
volumetric flask. Dilute to volume with reagent water and mix
well. Transfer into a Teflon sealed screw cap bottle. Store at
4°C and protect from light.
(3) Fresh standards must be prepared every six months. If signs of
degradation or evaporation occur, they should be replaced more
frequently.
(4) When compound purity is 96% or greater, the weight can be used
without correction to calculate concentration. Compounds which
are less than 96% pure cannot be used for standards.
3.4 GLASS CLEANING PROCEDURES
(1) All glassware to be used including sample bottles should be
washed with Amway SA-8 laundry detergent rinsed several times
with deionized water and baked for a minimum of 4 hours at 500
to 550°C. All cleaned glassware is immediately capped or covered
with foil (precleaned with hexane) to prevent contamination.
Chap. 3 - 51
-------
(2) Teflon liners and Teflon lined septa are sonicated for 10 minutes
in pesticide grade methanol followed by 10 minutes in pesticide
grade pentane.
3.5 PREPARATION OF CONTROL AND BLANK SAMPLES
(1) Aliquot reagent water into eight sample bottles. For drinking
water 200 mL samples will be placed in 250 mL bottles, for all
other water types, 20 mL will be aliquoted into 25 mL bottles.
Number the bottles 1 to 4.
(2) Measure total chlorine in a separate water sample and, if neces-
sary, quench using sodium thiosulfate (Section 6, step 2 and 3).
(3) Spike all samples with 1 mL (0.5 mL for 20 mL sample) of internal
standard solution.
(4) Spike and label the sample bottles as described in Table 3.5.
(5) Adjust the sample pH to 6-7 in all bottles using sulfuric acid
or soditim hydroxide solution. Use narrow range pH paper to
measure pH.
(6) Field controls and field blanks are shipped to the collection
site and are handled, stored and shipped in the same manner as
field samples. Laboratory blanks and controls are stored at 4°C
in the laboratory.
3.6 FIELD COLLECTION
(1) Water samples (20 field samples plus two duplicate and two
surrogates) are collected by rinsing sample bottles three times
Table 3.5. CONTROL AND BLANK SAMPLES
Bottles
Amount Spikeda
Disposition
Sample Type
1,2
1 niL
send
to
field
field control
3,4
1 inL
store
in
lab
laboratory control
5,6
none
send
to
field
field blank
7,8
none
store
in
lab
laboratory blank
aspiked with standard NEWS compounds (Table 3.3).
Chap. 3 — 52
-------
with sample water then filling to the calibration mark. Drinking
water samples are collected (200 mL) in 250 mL bottles. For all
other water types 20 mL are collected in 25 mL bottles.
(a) Addition of NBS Standards--The deuterated intern l standards
are pipetted (use 1.0 mL pipet) into each water sample
using volumes given in Table 3.2.
(b) Addition of Deuterated Internal Standards (Optional if NBS
ampoules not available)--Internal standard solutions
(1.0 mL) in 0.5 dram vials (Table 3.4) are added to each
water sample. Rinse the vials three times with sample
water and add to sample bottle.
(2) A preliminary water sample ( 25 mL) is collected for the purpose
of determining total chlorine content. This measurement is made
at the sample collection site and utilizes the Bausch and Lomb
Mini Spec 20 portable spectrophotometer. The water sample is
added to a 25 mL mixing cylinder along with the contents of a
DPD Total Chlorine Reagent Powder Pillow (Hach Chemical Co.).
The mixture is shaken and the color is allowed to develop for 3
to 6 minutes. Percent transmittance, measured at 530 nni, is
converted to mg/L total chlorine using Table 3.6.
(3) Residual chlorine is quenched by adding a stoichiometric quantity
of Na 2 S 2 O 3 (Figure 3.1) to the bottles containing the water
sample by delivering the appropriate volume of the 0.05M solution.
(4) Adjust sample pH to 6 to 7 with sodium hydroxide or sulfuric
acid. Use narrow range pH paper to measure pH.
(5) Samples should be shipped on ice (e.g., “Blue Ice® ) directly to
the laboratory by an appropriate air carrier (e.g., Federal
Express) in well insulated cartons.
(6) As soon as the samples arrive at the laboratory spike the two
surrogate samples with 1.0 mL of the NEWS standard solution
(Table 3.3) using a 1 mL graduated pipette.
(7) All samples should be stored at 4°C until analysis. All samples
should be processed within 21 days of collection.
Chap. 3 - 53
-------
Table 3.6
TOTAL CHLORINE (mg/L as Cl) VS. % TRANSMITTANCE
%T
% T Units
Tens
0
1
2
3
4
5
6
7
8
9
10
4.00
3.84
3.68
3.54
3.42
3.30
3.18
3.08
2.98
2.88
20
2.80
2.71
2.63
2.55
2.48
2.41
2.34
2.28
2.21
2.15
30
2.09
2.04
1.98
1.93
1.88
1.82
1.78
1.73
1.68
1.64
40
1.59
1.55
1.51
1.47
1.43
1.39
1.35
1.31
1.28
1.24
50
1.20
1.17
1.14
1.10
1.07
1.04
1.01
0.98
0.95
0.92
60
0.89
0.86
0.83
0.80
0.78
0.75
0.72
0.70
0.67
0.64
70
0.62
0.60
0.57
0.56
0.52
0.50
0.48
0.46
0.43
0.41
80
0.39
0.37
0.34
0.32
0.30
0.28
0.26
0.24
0.22
0.20
90
0.18
0.16
0.14
0.13
0.11
0.09
0.07
0.05
0.04
0.02
Reprinted from: Water and Wastewater Analysis
Chemical Co., 1975, P. 2-29.
Procedures Manual, Rach
Figure 3.1. Total chlorine vs volume of 0.OSMNa 2 S 2 O3 added to a 1 L
sample. (If a 200 mL sample is collected then add 1/5 of
the volume indicated).
I
1
Slop. • 3 31
2
l•o l•2 I 11 IS 20 2
I 0 02 11 1135303 (.1.)
Chap. 3 - 54
-------
CHAPTER 4
SAMPLING AND SAMPLE HANDLING FOR EXTRACTABLE ORGANICS
(ESSA, WABN)
4.1 INTRODUCTION
This section describes the collection and handling of drinking water,
surface water, and treated municipal and industrial wastewater effluent
samples for the analyses of solvent-extractable compounds. Separate
samples will be collected for the analysis of acid extractable compounds
(ESSA) and pH 8 extractable compounds (WABN). For drinking water, a
11.35 L sample will be collected for pH 8 extractable compounds only. For
all other analyses a 1 L sample will be collected. Immediately after
collection, internal standard solutions will be added to each sample. At
the same time, residual chlorine will be quenched using sodium thiosulfate,
the sample pH will be adjusted using 11 2 S0 4 or NaOH, and the volumes of
each recorded. ?lethylene chloride (2%, v/v) will be added as a preserva-
tive for all ESSA samples and WABN drinking water samples. A “keeper”
solvent (60 mL of 35% methylene chloride, 65% hexane) will be added to the
other WABN samples. Samples will be capped, then stored at 4°C until
analysis.
Table 4.1 illustrates the sampling regime with quality control samples
for 20 different field samples. Duplicates, surrogates, field and labora-
tory controls, field and laboratory blanks, and procedural blanks are
prescribed. This regime is for WABN or ESSA samples; quality control
samples are repeated for each sample type. The exact number is dependent
upon the number of field samples to be collected. For example, if 11 field
samples are to be collected, then two duplicates (one duplicate of each of
two field samples), one surrogate, one each of field and laboratory con-
trols and blanks, and three procedural blanks are incorporated as part of
the QC/QA. Thus, a total of 21 analyses would be performed for 11 field
samples (Table 4.1). If both WABN and ESSA fractions are analyzed, a
Chap. 4 - 55
-------
Table
4.1
NUMBER
OF QUALITY
CONTROL SAMPLES
ASSOCIATED
WITH
FIELD SAMPLES
No. of
Samples
to be
Collected -
Duplicates
Surrogates
Controls
Blanks
Total No.
Analyses
Field
Laboratory
Procedural
Field
Laboratory
r)
U,
0 ’
1
1.
1
1
1
5
2
1
—
—
—
1
1
1
6
3
1
—
I
—
1
1
1
8
4
1
—
1
-
1
1
1
9
5
1
—
I
—
1
1
2
11
6
1
—
1
-
1
1
2
12
7
1
-
1
-
1
1
2
13
8
1
—
1
—
1
1
2
14
9
1
—
1
—
1
1
2
15
10
1
1
1
1
1
1
2
18
11
2
1
1
1
1
1
3
21
12
2
1
1
1
1
1
3
22
13
2
1
1
1
1
1
3
23
14
2
1
1
1
1
1
3
24
15
2
2
1
1
1
1
3
26
16
2
2
1
1
1
1
3
27
11
2
2
1
1
1
1
4
29
18
2
2
1
1
1
1
4
30
19
2
2
1
2
2
2
4
34
20
2
2
2
2
2
2
4
36
-------
total of 42 analyses would be required. An exception to this schedule is
prescribed for wastewater samples where two 1 L field samples are collected
for each WABN sample in addition to the QC samples in Table 4.1. Only one
of these is analyzed unless the sample is found to be emulsion prone
(Chapter 9 Part II, section 9.4).
Control samples will be prepared by adding known quantities of repre-
sentative target compounds to reagent water (free of ESSA and WABN com-
pounds). Blanks are unspiked reagent water. Laboratory controls and
blanks, after preparation, are stored at 4°C in the laboratory until
analysis. Field controls and blanks are shipped to the sampling site and
handled in the same manner as field samples in terms of cooling, shipping,
and storing. Upon return to the laboratory, representative ESSA or WARN
target compounds are spiked into replicate field samples (the number of
samples treated in this manner is specified in Table 4.1) for use as
surrogate samples.
Comparison of the analytical results for controls, blanks and dupli-
cates will provide a measure of uncontrolled contamination and/or analyte
losses during sample collection, shipment, and storage, the homogeneity of
the sample matrix, and the accuracy and precision associated with sample
preparation and analysis. Recovery of internal standards and spiked
compounds from the surrogate samples will provide information on matrix
effects on method accuracy and precision (if two or more surrogates are
included).
If more than 20 samples are to be collected, the additional field
samples are matched with the prescribed number of duplicates, surrogates,
etc. For example, if 33 samples are to be collected, then lines for 20
and 13 samples, respectively, are consulted and the number of duplicates,
surrogates, etc., are a sum of these two.
4.2 MATERIALS AND REAGENTS
The following materials are required for collecting a set of 20 water
samples plus two duplicate samples and two surrogate samples and two each
of field and laboratory controls, and two each of field and laboratory
blanks for the analysis of both ESSA and WARN compounds.
Chap. 4 - 57
-------
(1) Fifty-six (eighty-four, if all samples are wastewaters) narrow
mouth glass sample bottles with Teflon lined screw-caps. For
drinking water, three one gallon bottles are used to collect each
WABN sample, for all other water types, one quart bottles are
used (up to eighty-four one gallon bottles and twenty-eight one
quart bottles may be required). Each sample bottle is calibrated
by filling with 3.78 L of reagent water for a 1 gal container and
940 mL of reagent water for a 1 quart container. The water level
is marked on the bottle and then the sampling containers are
emptied.
(2) Twenty 20 mL polyethylene or polypropylene vials for scouting
samples.
(3) 10 mL graduated pipettes.
(4) Pasteur pipettes plus pipette bulbs.
(5) One Bausch and Lomb Mini Spec 20 portable spectrometer.
(6) One 25 niL mixing cylinder (Hach Chlorine Determination Kit).
(7) Materials for chilling the sample during shipping and storage -
insulated shipping containers and “Blue Ice®t .
(8) Volumetric flasks (1000, 500, 100, and 10 mL) for preparing
standard solution
(9) Teflon-lined screw-cap bottles for storing standard solutions
(10) Graduated cylinders - 1000 niL, 50 niL.
(11) Syringes for preparing standard solutions - 100, 250, and 1000 pL.
(12) One hundred 3 dram vials with 15 mm screw caps (Supelco 3-3112)
and Teflon lined rubber septa (Supelco 3-3115).
(13) Teflon lined rubber septa (Supelco 3-3115).
(14) Reagents
(a) reagent water — reagent water is defined as water in which
no interferences are observed at the detection limit for the
parameters of interest. A water purification system (Milli-
pore Super-Q or equivalent) may be used to generate reagent
water.
(b) 5.0 mL of each internal standard solution (for use if
NIBS ampoules not available; Tables 4.2 and 4.3). Set 1
Chap. 4 - 58
-------
Table 4.2. INTERNAL STANDARD SOLUTSIONS - ESSA
Compound
Conc
(pg/S mL
ent
in
ration a
methanol)
Spiked
Concentration (ppb)
Set 1
Set 2
Set 1
Set
2
benzoic acid-d 5
1
100
1
100
heptanoic acid-d 13
15
100
15
100
aA1SO pg spiked into 1 L of sample.
standards are used for spiking drinking water and other
relatively clean samples. Set 2 standards are used for
spiking municipal and industrial wastewaters. Internal
standard solutions can be prepared from pure certified
standards or certified solutions of standards.
(c) 10 mL each standard solution (Table 4.4 and 4.5) for ESSA
and WABN. Standards are prepared from pure stock solutions.
Cd) sodium hydroxide solution (iON) - dissolve 40 g NaOH in
reagent water and dilute to 100 mL.
(e) sulfuric acid solution (1+1) - slowly add 50 mL H 2 S0 4 (sp.
gr. 1.84) to 50 mL of reagent water.
(f) 250 niL Na 2 S 2 0 3 solution - 0.05 N in reagent water.
(g) DPD Total Chlorine Reagent Powder Pillow (Hach Chemical
Co.).
(h) methylene chloride - distilled in glass (Burdick and
Jackson).
(i) acetone - distilled in glass (Burdick and Jackson).
(j) methanol — distilled in glass (Burdick and Jackson).
(k) 35% inethylene chloride in hexane (v/v) - distilled in glass
(Burdick and Jackson).
(1) NBS ampoules containing deuterated ESSA and WABN internal
standards in methanol (Tables 4.6 and 4.7); optional.
4.3 PREPARATION OF STANDARD SOLUTIONS
4.3.1 Set 1 ESSA Internal Standard Solutions - Table 4.2 (For use if NBS
ampoules are not available; for drinking and surface waters)
Chap. 4 - 59
-------
Table 4.3. INTERNAL STANDARD SOLUTIONS - WABN
Set No. 1
Set No. 2
Spiked
Compound
Density
(@ 20°C)
Volume
or Weight
Added to
(pL)
(mg)
500 cnL
pg/oiL
in
Methanol
Concentration (ppb)
Volume
or Weight
Added to
(pL)
(ing)
500 oiL
pg/S oiL
in
Methanol
Spiked
Concentration
(ppb)
Drinking Surfacg
Watera Water
o-Xy lene-d 10
0.98
3
5.9
1.6 2.6
10
.
98
104
Nitrobenzene-d 5
1.25
100
250
66 110
40
500
530
Naphthalene-d 8
1.06
20
42
11 19
50
530
560
Acridine-d 9
S
20
40
11 18
10
100
106
Phenol-d 6
S
100
200
53 88
40
400
420
Propiophenone-d 5
I OS
-
-
- -
10
105
110
l-Phenyl-d 5 -ethanol
1.06
-
-
- -
50
530
560
Perylene-d 12
S
1
2
0.53 0.88
10
100
110
Acetophenone-d 5
1.07
-
—
— -
10
107
110
(3 gal/sample total).
81.0 oiL is added to each 1 gal aliquot of sample
b
1.0 oiL is added to 940 oiL of sample.
C ount added to 940 oiL of municipal, industrial or energy effluent sample.
C-,
0
-------
Table 4.4. STANDARD SOLUTION FOR ESSA
Compound
De
((P
nsities
20°C)
Concentrationa
pg/mL
2,3,6-trichlorophenol
sb
500
pentachlorophenol
S
500
n-nonanoic acid
0.906
450
n-decanoic acid
S
500
dichioroacetic acid
1.563
780
trichioroacetic acid
S
500
2,4,5-trichiorophenoxy—
S
500
acetic acid
benzoic acid
S
500
-toluic acid
S
500
2-nitrobenzoic acid
S
500
3,4-dichlorobenzoic
S
500
acid
o-methoxybenzoic acid
S
500
2,5-dinitrophenol
S
500
o—mercaptobenzoic acid
S
500
a .
Concentration in the secondary dilution.
bSl.d
(1) Accurately weigh 10.0 mg of benzoic acid - d 5 and 150 mg of
heptanoic acid - d 13 into a 500 niL volumetric flask. Dissolve
these materials in methanol and dilute to the mark.
(2) Transfer this stock solution into a Teflon sealed screw cap
bottle. Store at 4°C and protect from light.
(3) Accurately measure 5.0 ml of this stock solution and transfer to
a 500 niL volumetric flask. Dilute to volume with methanol and
mix well.
(4) Transfer 5.0 niL portion of this solution to 3 dram vials. Seal
with Teflon lined, septum seal, screw caps. Store at 4°C and
protect from light.
Chap. 4 - 61
-------
Table 4.5. STANDARD SOLUTION FOR WABN
Densities
Compound (@ 20°C) Concentration pg/mLa
pyridine 0.978 490
a-picoline 0.943 670
2-(n-butoxy)ethano l 0.903 450
benzaldehyde 1.044 520
aniline 1.022 510
2,3,5-tri methylpyridine 0.917 460
acetophenone 1.030 520
2,3-dihydrobenzofuran 1.065 530
o-cresol 500
isophorone 0.923 460
l,2,4-trichlorobenzene 1.454 720
-chloroani1i.ne 1.206 600
n-terpioeol 0.933 470
tributylamine 0.778 390
2-nitrocresol S 500
n—decanol 0.829 410
4-chlorocreso l S 500
-t-buty1pheno1 S 500
n-tridecane 0.756 380
nicotine 1.010 500
2, 4 -dimethy lquino line S 500
dimethylphthalate 1.190 600
n-pentadecane 0.769 380
1,2-dich loronaphtha lene S 500
diphenylamine S 500
tributyiphosphate 0.979 490
n—heptadecane 0.777 390
anthracene S 500
n-octadecane 0.777 390
dibuty lphtha late 1.043 520
n-eicosane S 500
pyrene S 500
perylene S 500
a 0 t in secondary dilution.
bSld
C’ ap. 4 - 62
-------
Table 4.6. NBS AMPOIJLES CONTAINING ESSA INTERNAL STANDARDS IN METHANOL
Compound
Concentration
(pg/Arnpoule)
a
Conce
Spiked
ntration (ppb)
Set No.
1 b Set No.
2
Set No.
1
Set No.
2
Benzoic-d 5 acid
2.15
200.6
2.15
200.6
Heptanoic-d 13 acid
25.1
202.9
25.1
202.9
a . . I, tt
Concentration in the 400 series production run; each ampoule contains
5.0 mL methanol.
bFor drinking and surface waters.
cFor municipal, industrial and energy effluents.
Table 4.7. NES AIIPOULES CONTAINING WARN INTERNAL STANDARDS IN METHANOL
Compound
Concentration
(pg/ampoule)a
Spiked
Concentration
(ppb)
Set
No. 1
Set
d
No. 2
Drinking
Water
Surface
WaterC
Set No. 1 Set No.
2
o-Xylene-d 10
2.8 104.7
0.74
3.0
111
Nitrobenzene—d 5
94.2 538.8
24.9
100
573
Naphthalene—d 8
20.2 500.7
5.3
21.5
533
Acridine-d 9
19.8 100.3
5.2
21.1
107
Phenol-d 6
Propiophenone-d 5
112.7 400.4
98.1
29.8
---
119.9
---
426
104
1-Phenyl-d 5 -ethanol
520.9
---
554
Pery lene—d 12
1.1 98.1
0.29
1.2
104
Acetophenone-d 5
--- 104.7
-—-
---
111
in the “400” series production run; each ampoule Contains
5.0 mL methanol.
bior drinking water, 1 ampoule is added to each 1 gal of sample (3 gal or
11.35 L total per sample).
or surface water, 1 ampoule is added per 940 mL of sample.
or municipal, industrial, and energy effluent samples, 1 ampoule is
added per 940 inL of sample.
eNO present.
Chap. 4 - 63
-------
(5) Fresh standards must be prepared every six months. If signs of
degradation or evaporation occur they should be replaced more
frequently.
(6) When compound purity is 96% or greater, the weight (or volume)
can be used without correction to calculate concentration.
Compounds that are less than 96% pure cannot be used for stand-
ards.
4.3.2 Set 2 ESSA Internal Standard Solutions - Table 4.2 (For use if NES
ampoules are not available; for industrial and energy wastewater
effluents)
(1) Accurately weigh 100 mg of benzoic acid - d 5 and 100 mg of
heptanoic acid — d 13 ; dissolve in methanol in a 500 niL volumetric
flask. Dilute the sample to volume with methanol and mix well.
(2) Accurately measure 50 niL of this stock solution and transfer to
a 500 mL volumetric flask. Dilute to the mark with methanol and
mix well.
(3) Transfer 5.0 mL portions of the dilute solution into 3 dram
vials. Seal with Teflon lined, septum seal screw caps. Store
at 4°C and protect from light.
(4) Prepare fresh standards every six months. If signs of degrada-
tion or evaporation occur they should be replaced more frequently.
4.3.3 Set 1. WABN Internal Standard Solutions - Table 4.3 (For use if NBS
ampoules are not available; for drinking and surface waters)
(1) Accurately weigh the amount of solid compounds indicated in
Table 4.3 for Set 1 and dissolve in methanol in a 500 niL volumet-
ric flask. With a 100 IJL syringe accurately measure the volumes
of the liquid standards into the same flask. Dilute the stock
solution to the mark with methanol and mix well.
(2) Transfer this stock solution into a Teflon sealed screw cap
bottle. Store at 4°C and protect from light.
(3) Accurately measure 50 niL of this stock and transfer to a 500 mL
volumetric flask. Dilute to volume with methanol and mix well.
(4) Transfer 5.0 niL portion of this solution to 3 dram vials. Seal
with Teflon lined, septum seal, screw caps. Store at 4°C and
protect from light.
Chap. 4 - 64
-------
(5) Fresh standards must be prepared every six months. If signs of
degradation or evaporation occur they should be replaced more
frequently.
(6) When compound purity is 96% or greater, the weight can be used
without correction to calculate concentration. Compounds which
are less than 96% pure cannot be used for standards.
4.3.4 Set 2 WABN Internal Standard Solutions - Table 4.3 (For use if NIBS
ampoules are not available; for industrial and energy wastewater
effluents)
(1) Accurately weigh the amount of solid compounds indicated in
Table 4.3 for Set 2 and dissolve in methanol in a 500 mL volumet-
ric flask. With a 500 pL syringe accurately measure the volumes
of the liquid standards into the same flasks. Dilute the stock
solution to the mark with methanol and mix well.
(2) Accurately measure 50 mL of this stock solution and transfer to
a 500 mL volumetric flask. Dilute to the mark with methanol and
mix well.
(3) Transfer 5.0 mL portions solution into 3 dram vials. Seal with
Teflon lined, septum seal screw caps. Store at 4°C and protect
from light.
(4) Prepare fresh standards every six months. If signs of degrada-
tion or evaporation occur they should be replaced more frequently.
4.3.5 Standard Solutions (Tables 4.4 and 4.5)
(1) Prepare individual stock solutions (50 mg/mL) for each standard
compound.
- Solids - accurately weigh about 0.500 grams of pure material.
Dissolve the material in acetone, dilute to volume in a
10 niL volumetric flask.
— Liquids - with a 1000 pL syringe accurately add 500 I.iL of
pure material to methanol (Tables 4.4 and 4.5) in a 10 niL
volumetric flask. Dilute to volume.
When compound purity is 96% or greater, the weight can be used
without correction to calculate the concentration of stock
standards. Compounds which are less than 96% pure cannot be
used for standards.
Chap. 4 - 65
-------
(2) Transfer stock solutions into a Teflon sealed screw cap bottle.
Store at 4°C and protect from light. Prepare fresh solutions
every six months. If signs of degradation or evaporation occur,
they should be replaced more frequently.
(3) Prepare standard solutions individually for. ESSA and WABN com-
pounds as shown in Tables 4.4 and 4.5. With a 250 pL syringe,
accurately add 100 i.JL of each stock standard to a 10 mL volumet-
nc flask and dilute to volume with methanol. Transfer to a
Teflon sealed screw cap bottle. Store at 4°C and protect from
light.
4.4 GLASS CLEANING PROCEDURES
(1) All glassware to be used, including sample bottles should be
washed with Amway S-A-8 laundry detergent, rinsed several times
with deionized water and baked for a minimum of 4 hours at 500
to 550°C. All cleaned glassware is immediately capped or covered
with precleaned foil to prevent contamination.
(2) Teflon liners and Teflon lined septa are sonicated for 10 minutes
in methanol followed by 10 minutes in pesticide grade pentane.
Store in a clean vial.
4.5 PREPARATION OF CONTROL AND BLANK SAMPLES
(1) Dispense aliquots of reagent water into sixteen sample bottles.
For drinking water, 3.50 L samples will be placed in 1 gallon
bottles. Number the bottles 1 to 16.
(2) Measure total chlorine in the water sample and, if necessary,
quench using sodium thiosulfate (Section 4.6, step 3).
(3) Spike samples 1-8 with WABN internal standard solutions
(Table 4.3) and 9—16 with ESSA internal standards (Table 4.2).
If drinking or surface water samples are to be collected spike
with 5.0 mL of set 1 standard (1 vial of set 1 internal standard).
If municipal, industrial or energy effluents are to be collected
spiked with 5.0 mL of set 2 standards (1 vial of set 2). If NBS
standards are available, see 4.6(1)(a) below.
(4) Spike with standard solutions (Tables 4.4 and 4.5) and label
sample bottles as described in Table 4.8.
Chap. 4 - 66
-------
Table 4.8. CONTROL AND BLANK SAMPLES
Sample
Bottle Spike Handle pH Sample Type
1 & 2 1.0 mL WABNa standard send to field 6-8 field control WABN
solution
3 & 4 1.0 mL WABN standard store in lab 6—8 laboratory control
solution WABN
5 & 6 none send to field 6-8 field blank WABN
7 & 8 none store in lab 6-8 laboratory blank
WAEN
9 & 10 1.0 mL ESSAb standard send to field 4-5 field control ESSA
solution
11 & 12 1.0 mL ESSA standard store in lab 4-5 laboratory control
solution ESSA
13 & 14 none send to field 4-5 field blank ESSA
15 & 16 none store in lab 4—5 laboratory blank
ESSA
aPH 8 extractable organics
bacid extractable organics
-------
(5) Adjust the sample pH in all bottles as- specified in Table 4.8
using sulfuric acid or sodium hydroxide solution. Use narrow
range pH paper to measure pH.
(6) Add 60 niL of 35% (v/v) unethylene chloride in hexane to all WABN
samples except drinking water, where 2% (v/v) methylene chloride
is added. Add 2% (v/v) methylene chloride to all ESSA samples.
Cap bottles and mix well.
(7) Field controls and field blanks are shipped to the collection
site and are handled, stored, and shipped in the same manner as
field samples. Laboratory blanks and controls are stored at 4°C
in the laboratory.
4.6 FIELD COLLECTION,
(1) Water samples are collected by rinsing sample bottles three
times with sample water, then filling to the calibration mark.
(a) Addition of NBS Standards--For drinking and surface waters,
5.0 niL (1 ampoule) of Set No. 1 standard is added to the
appropriate volume of water (see Tables 4.6 and 4.7).
Municipal, industrial, and energy wastewater effluent
samples are spiked with 5.0 niL of Set No. 2 (Table 4.7).
In either case rinse the vials containing the internal
standard solutions three times with sample water and add to
the sample bottle.
(b) Addition of Deuterated Internal Standards (Optional if NBS
ampoules are not available)--Proceed as in (a) above, using
internal standard solutions prepared in Sections 4.3.1,
4.3.2, 4.3.3, or 4.3.4 (see Tables 4.2 and 4.3 for amounts).
(2) A preliminary water sample ( 25 mL) is collected for the purpose
of determining total chlorine content. This measurement is made
at the sample collection site and utilizes the Bausch and Lomb
Mini Spec 20 portable spectrophotometer. The water sample is
added to a 25 mL mixing cylinder along with the contents of a
DPD Total Chlorine Reagent Powder Pillow (Hach Chemical Co.).
The mixture is shaken and the color is allowed to develop for 3
to 6 minutes. Percent transmittance, measured at 530 nm, is
converted to mg/L total chlorine using Table 4.9.
Chap. 4 - 68
-------
Table 4.9. TOTAL CHLORINE (mg/L as Gi) VS. % TRANSMITTANCE
%T
%TUnits
Tens
0
1
2
3
4
5
6
7
8
9
10
4.00
3.84
3.68
3.54
3.42
3.30
3.18
3.08
2.98
2.88
20
2.80
2.71
2.63
2.55
2.48
2.41
2.34
2.28
2.21
2.15
30
2.09
2.04
1.98
1.93
1.88
1.82
1.78
1.73
1.68
1.64
40
1.59
1.55
1.51
1.47
1.43
1.39
1.35
1.31
1.28
1.24
50
1.20
1.17
1.14
1.10
1.07
1.04
1.01
0.98
0.95
0.92
60
0.89
0.86
0.83
0.80
0.78
0.75
0.72
0.70
0.67
0.64
70
0.62
0.60
0.57
0.56
0.52
0.50
0.48
0.46
0.43
0.41
80
0.39
0.37
0.34
0.32
0.30
0.28
0.26
0.24
0.22
0.20
90
0.18
0.16
0.14
0.13
0.11
0.09
0.07
0.05
0.04
0.02
Reprinted from: Water and Wastewater Analysis Procedures Manual, Hach
Chemical Co., 1975, p. 2—29.
(3) Residual chlorine is quenched by adding a stoichiometric quanti-
ty of 0.05 H Na 2 S 2 0 3 (Figure 4.1, a 10% excess is used and is
already incorporated here) to the bottle containing the aqueous
sample.
(4) Samples collected for the analysis of WABN compounds are adjusted
to pH 6 to 8. Samples collected for the analysis of ESSA com-
pounds are adjusted pH 4 to 5. Use narrow range pH paper to
measure pH. Use sodium hydroxide or sulfuric acid for pH adjust-
ment. (Note: verify contamination free as described in
Chapter 9.)
(5) Add 60 mL of 35% (v/v) rnethylene chloride in hexane to all WABN
samples except drinking water as a preservative, cap the samples,
and shake well. Add 2% (v/v) methylene chloride to WABN drinking
water samples and ESSA samples. The samples must be maintained
at 4°C from the time of collection until extraction.
(6) Samples should be shipped on ice (e.g., “Blue Ice® ) directly to
the laboratory by an appropriate air carrier (e.g., Federal
Express) in well insulated cartons.
Chap. 4 - 69
-------
(7) As soon as the samples arrive at the laboratory, the surrogate
samples must be spiked with target compounds. Samples for the
analyses of extractable strong acids (ESSA) and weak acids,
bases and neutrals (WARN) are spiked with the corresponding
standard spiking solutions (Tables 4.4 aiiTd 4.5). Drinking water
and surface water samples are spiked with 100 pL of the appropri-
ate ESSA standard solution into a 940 mL of sample using a
250 pL syringe. Industrial, municipal and energy wastewaters
are spiked with 1 mL of the appropriate ESSA standard solution
into 940 mL of sample using a 1 mL graduated pipette.
For WABN compounds, 100 pL of the WARN standard solution is
spiked into each 1 gal of drinking water and 940 mL of surface
water. The municipal, industrial, and industrial effluent
samples are spiked with 1.0 mL of the WARN standard solution
(940 mL of sample).
Figure 4.1.
2 04 06 06 10 12 24 16 ii 20 23 34 36 36
V.1 0 06 U S303 C m l.)
Total chlorine vs volume of 0.OSM Na 2 S 2 0 3 added to a
1 L sample. (If a 11.35 L sample is collected, 11.35x
the amount of Na 2 S 2 0 3 must be added.)
7
‘I
U
1’
I
Slop. • 3 II
2
Chap. 4 - 70
-------
CHAPTER 5
SAMPLING AND SAMPLE HANDLING FOR OTHER IONIC COMPOUNDS
(VOSA, NOVA, SAM)
5.1 INTRODUCTION
This section describes the collection and handling of drinking
water, surface waters, and treated municipal and industrial wastewater
effluent samples for the analysis of ionic “intractable” compounds.
Separate samples will be collected for the analysis of low molecular
weight carboxylic acids (VOSA), nonvolatile organic acids (NOVA), and
amines (SAM). For drinking water, and surface water, a 3.5 L sample will
be collected. For all other water types, a 1 L sample will be collected.
Immediately after collection, internal standard solutions will be pipetted
into each sample. At the same time, residual chlorine will be quenched
using sodium thiosulfate, the sample pH will be adjusted using H 2 S0 4 or
NaOH, and 2% (v/v) methylene chloride will be added as a preservative.
Samples will be capped, then stored at 4°C until analysis.
Table 5.1 illustrates the sampling regime with quality control samples
for up to 20 different field samples. Duplicates, surrogates, field and
laboratory controls, and field, laboratory, and procedural blanks are
prescribed. This regime is for VOSA, NOVA, or SAM samples; quality control
samples are repeated for each sample type. The exact number of controls
is dependent upon the number of field samples to be collected. For example,
if 11 field samples are to be collected, then two duplicates (one duplicate
of each of two field samples), one surrogate, and one each of field and
laboratory controls and blanks are incorporated as part of QC. During
sample processing three procedural blanks would also be run. Thus, a
total of 21 analyses would be performed for 11 field samples (Table 5.1).
If VOSA, NOVA, and SAM fractions are all analyzed, a total of 63 analyses
would be required.
Chap. 5 - 71
-------
c. J
U i
No. of
Samples
to be
Collected
Duplicates
Surrogates
Controls
Blanks
Total No.
Analyses
Field
Laboratory
Field
Laboratory
Procedural
1
1
—
—
—
1
1
1
5
2
1
-
-
—
1
1
1
6
3
1
—
1
—
1
1
1
7
4
1
—
I
—
1
1
1
9
5
1
—
I
—
1
1
2
11
6
1
—
1
—
1
1
2
12
7
1
—
1
—
1
1
2
13
8
1
—
1
—
1
1
2
14
9
1
—
I
—
1
1
2
15
10
1
1
1
1
1
1
2
18
11
2
1
1
1
1
1
3
21
12
2
1
1
1
1
1
3
22
13
2
1
1
1
1
1
3
23
14
2
1
1
1
1
1
3
24
15
2
2
1
1
1
1
3
25
16
2
2
1
1
1
1
3
27
17
2
2
1
1
1
1
4
29
18
2
2
1
1
1
1
4
30
19
2
2
1
2
2
2
4
34
20
2
2
2
2
2
2
4
36
-------
Control samples will be prepared by adding known quantities of repre-
sentative target compounds to reagent (free of VOSA, NOVA, and SAN com-
pounds) water. Blanks are unspiked reagent water. Laboratory controls
and blanks, after preparation, are stored at 4°C in the laboratory until
analysis. Field controls and blanks are shipped to the sampling site and
handled in the same manner as field samples in terms of cooling, shipping
and storing. Upon return to the laboratory, representative VOSA, NOVA, or
SAN target compounds are spiked into replicate field samples (the number
of samples treated in this manner is specified in Table 5.1) for use as
surrogate samples.
Comparison of the analytical results for controls, blanks and dupli-
cates will provide a measure of uncontrolled contamination and/or analyte
losses during sample collection, shipment, and storage, the homogeneity of
the sample matrix, and the accuracy and precision associated with sample
preparation and analysis. Recovery of internal standards and spiked
compounds from the surrogate samples will provide information on matrix
effects on method accuracy and precision (if two or more surrogates are
included).
If more than 20 samples are to be collected, the additional field
samples are matched with the prescribed number of duplicates, surrogates,
etc. For example, if 33 samples are to be collected, then lines for 20
and 13 samples, respectively, are consulted and the number of duplicates,
surrogates, etc., are a sum of these two.
5.2 MATERIALS AND REAGENTS
The following materials are required for collecting a set of 20 water
samples plus two duplicate and two surrogate samples and two each of field
and laboratory controls, and two each of field and laboratory blanks for
the analysis of organics (VOSA, NOVA, and SAM).
(1) Ninety—six narrow mouth, amber g1as , sample bottles with Teflon
lined screw caps. For drinking and surface water, one gallon
bottles calibrated to 3.5 L are used, for all other water types,
one quart bottles calibrated to 920 mL are used. Bottles are
calibrated by adding an accurately measured volume of water,
then marking the level of the water miniscus by scratching the
outside surface. Bottles should be calibrated before cleaning.
Chap. 5 - 73
-------
(2) 10 mL graduated pipettes for adding internal standard solutions.
(3) Pasteur pipettes plus pipette bulbs.
(4) One Bausch and Lomb Nin [ Spec 20 portable spectrometer.
(5) One 25 mL mixing cylinder (Hach Chlorine Determination Kit).
(6) M terials (e.g., “Blue Ice®l$) for chilling the sample during
shipping and storage; insulated shipping containers for samples.
(7) Volumetric flasks (1000, 500, and 10 mL) for preparing standard
solutions.
(8) Graduated cylinders - 1000 caL, 50 caL.
(9) Syringes for preparing standard solutions — 100 and 250 pL.
(10) Graduated pipettes for preparing standard solutions - 1 caL.
(11) Teflon lined screw cap bottles for storing standard solutions.
(12) One hundred 3 dram vials with 15 mm screw caps (Supelco 3—3112)
and Teflon lined rubber septa (Supelco 3-3115).
(13) Teflon lined rubber septa (Supelco 3-3115).
(14) Reagents
(a) reagent water — reagent water is defined as water in which
no interferences are observed at the detection limit for
the parameters of interest. A water purification system
(Millipore Super-Q or equivalent) may be used to generate
reagent water.
(b) 1000 caL internal standard solution (For use if NBS ampoules
are not available; Table 5.2). Set 1 standards are used
for spiking drinking and surface water samples. Set 2
standards are used for spiking municipal and industrial
wastewater effluents and energy effluents. The same inter-
nal standard solution is used for VOSA, NOVA, and SAN
analyses. Internal standard solutions can be prepared from
pure certified standards or certified solutions.
Cc) 10 caL standard solutions (Table 5.3) for VOSA, NOVA, and
SAIl analyses. Standards are prepared from pure stock
solutions.
(d) sodium hydroxide solution - 0.lN in reagent water (must be
demonstrated as contaminant free; Chapter 10, 10.3.6.3,
Chapter 11, 11.3.6.3, and Chapter 12, 12.3.6.3).
Chap. 5 - 74
-------
Table 5.2. INTERNAL STANDARD SOLUTIONSa
Amount
Seti b
Compound (pg/7.5 mL)
Set2
(iig/l0 )C
n-Butylamine-d 9 74
370
n-Butyric acid—d 7 14
480
2-Naphthalene sulfonic acid-d 7 H 2 O 100
N-Ethyl-2- fluorobenzylamine 100 d
500
500 d
2-Phenylethyl-l,l ,2,2—d 4 -amine 97
480
aprepared in reagent water.
bAlso pg spiked into 3.5 L of sample.
CA1S 0 pg spiked into 920 mL of sample.
dDensity unknown - calculated assuming d = 1.
Table 5.3. STANDARD SOLUTIONS
Concentration
Solutions (pg/mL)
Solvent
Low Molecular Weight Volatile Acids (VOSA)
Acrylic acid 530
acetone
Methylbutyric acid 470
acetone
2-Methylcyclopropane carboxylic acid 510
acetone
2-Hexenoic acid 450
acetone
Octanoic acid
Nonvolatile Acids (NOVA)
Benzene sulfonic acid 500
acetone
Nitrobenzojc acid 500
acetone
Pentachioroptienol 500
acetone
Chlorobenzene sulfonic acid 500
acetone
2 , 4 ,5-Trichlorophenoxyacetic acid 500
acetone
Aniines (SAN)
Allylamine 380
?IBEa
Diallylamine 390
MBE
Hexylamine 380
MBE
Benzylamine 490
MBE
Dibenzy lamine 510
MBE
2-Methylpiperidine 420
MBE
a
Methyl-t-butyl ether.
Chap. 5 - 75
-------
(e) sulfuric acid solution - 0.1N in reagent water (must be
demonstrated as contaminant free; Chapter 10, 10.3.6.3,
Chapter 11, 11.3.6.3, and Chapter 12, 12.3.6.3).
(f) 250 mL Na 2 S 2 O 3 solution - 0.05 N in reagent water (must be
demonstrated as contaminant free; Chapter 10, 10.3.6.3,
Chapter 11, 11.3.6.3, and Chapter 12, 12.3.6.3).
(g) DPD Total Chlorine Reagent Powder Pillow (Hach Chemical
Co.).
(h) methylene chloride - distilled in glass.
(i) acetone - distilled in glass.
(j) methyl t-butyl ether - pesticide quality or equivalent.
(k) NBS ampoules containing VOSA, NOVA and SAN deuterated
internal standards in water (Table 5.4); optional.
5.3 PREPARATION OF STANDARD SOLUTIONS
5.3.1 Set 1 Internal Standard Solution - Table 5.2 (For use if NBS ampoules
are not available; for drinking and surface waters)
(1) Accurately weight 66.6 mg of 2-naphthalene sulfonic acid-d 7 .H 2 0,
then dissolve in reagent water in a 500 mL volumetric flask.
With a 100 pL syringe accurately measure 65 iL of the liquid
standards into the same flask (for d 7 —butyric acid use 18 iiL).
To determine the quantity of N-ethyl-2-fluorobenzylamine added
to the solution, weigh the dry syringe then weigh the syringe
containing neat standard. The difference between these two
weights is the weight of material delivered to the solution.
The syringe must be rinsed with solvent to assure that all of
the neat material is delivered. Dilute the sample to volume
with reagent water and mix well.
(2) Transfer this stock solution into a Teflon sealed screw cap
bottle. Store at 4°C and protect from light.
(3) Accurately measure 100 mL of this stock and transfer to a 1000
niL volumetric flask. Dilute to volume with reagent water and
mix well.
Chap. 5 - 76
-------
Table 5.4. NBS ANPOULES CONTAINING VOSA, NOVA, AND
SAN INTERNAL STANDARDS IN WATER
Compound
Concentration
(pg/Ampoule)a
Set No.
1 b
Set No.
2
n—Butyl-d 9 —amine
n-.Butyric—d 7 acid
2—Naphthalene—d 7 —sulfonic acid H O
N-ethyl-2-fluorobenzylamine
2-Phenylethyl-1,l,2,2-d 4 -amine
118.3
15.7
99.4
107.2
121.2
554.3
535.9
497.4
522.1
530.3
aConcentration in the “400” series production run; Set No. 1 and
Set No. 2 contains 7.5 and 10.0 mL water, respectively.
A1so, pg spiked into 3.5 L of sample.
Also, pg spiked into 920 inL of sample.
(4) Transfer 7.5 niL portions of this solution to 3 dram vials. Seal
with Teflon lined, septum seal, screw caps. Store at 4°C and
protect from light.
(5) Fresh standards must be prepared every six months. If signs of
degradation or evaporation occur they should be replaced more
frequently.
(6) When compound purity is 96% or greater, the weight (or volume)
can be used without correction to calculate concentration.
Compounds that are less than 96% pure cannot be used for stand-
ards.
5.3.2 Set 2 Internal Standard Solutions - Table 5.2 (For use if NBS
ampoules are not available; for industrial and energy wastewater
effluents)
(1) Accurately weigh 50 mg of 2-naphthalene sulfonic acid - d 7 H 2 O,
then dissolve in reagent water in a 1000 niL volumetric flask.
With a 100 pL syringe, accurately measure 50 pL of the liquid
standards into the same flask. Dilute the sample to volume with
reagent water and mix well. To determine the quantity of N-
ethyl-2-fluorobenzylamine added to the solution, weigh the dry
syringe then weigh the syringe containing the neat compound.
Chap. 5 - 77
-------
The difference between these two weights is the weight of material
delivered to the solution. The syringe must be rinsed well with
solvent to assure that all of the neat material is delivered.
(2) Transfer 10 mL portions solution into 3 dram vials. Seal with
Teflon lined, septum seal screw caps. Store at 4°C and protect
from light. -
(3) Prepare fresh standards every six months. If signs of degrada-
tion or evaporation occur they should be replaced more frequently.
5.3.3 Standard Solutions (Table 5.3)
(1) Prepare individual stock solutions (50 mg/mL) for each standard
compound.
- Solids - accurately weight about 0.500 gram of pure material.
Dissolve the material in distilled in glass quality acetone,
dilute to volume in a 10 mL volumetric flask.
- Liquids - with a 1 mL graduated pipette. Accurately add
500 iJL of standard compound to the specified solvent
(Table 5.3) in a 10 mL volumetric flask. Dilute to volume.
(2) Transfer stock solutions into a Teflon sealed screw cap bottle.
Store at 4°C and protect from light. Prepare fresh solutions
every six months. If signs of degradation or evaporation occur,
they should be replaced more frequently.
(3) Prepare three standard solutions for: 1) the low molecular
weight carboxylic acids; 2) the nonvolatile organic acids; and
3) the amines as shown in Table 5.3. With a 250 iL syringe,
accurately add 100 ilL of each stock standard to the specified
solvent in a 10 mL volumetric flask and dilute to volume. This
gives the concentration in Table 5.3. Transfer to a Teflon
sealed screw cap bottle. Store at 4°C and protect from light.
Prepare fresh standards every six months. If signs of degrada-
tion or evaporation occur, they should be replaced more frequent-
ly.
(4) When compound purity is 96% or greater, the weight (or volume)
can be used without correction to calculate the concentration of
stock standards. Compounds which are less than 96% pure cannot
be used for standards.
Chap. 5 - 78
-------
5.4 GLASS CLEANING PROCEDURES
l) All glassware to be used, iz cluding sample bottles, should be
washed with Amway S-A-8 laundry detergent, rinsed several times
with deionized water and baked for a minimum of 4 hours at 500
to 550°C. All cleaned glassware is immediately capped or covered
with foil (precleaned with hexane) to prevent contamination.
(2) Teflon liners and Teflon lined septa are sonicated for 10 minutes
in pesticide grade methanol followed by 10 minutes in pesticide
grade pentane. Store in a clean vial.
5.5 PREPARATION OF CONTROL AND BLANK SA iPLES
(1) Dispense aliquots of reagent water into 24 sample bottles. For
drinking water, 3.5 L samples will be placed in 1 gallon bottles.
For all other water types, 920 mL will be aliquoted into 1 quart.
bottles. Number the bottles 1 to 24.
(2) Measure total chlorine in the water sample and, if necessary,
quench using sodium thiosulfate (Section 6, step 2 and 3).
(3) Spike all samples with internal standard solutions (Table 5.2 or
5.4). If drinking or surface water samples are to be collected
spike with 7.5 mL (1 vial) of set 1 standard. If municipal,
industrial or energy effluents are to be collected spiked with
10 mL (1 vial) of set 2 standards.
(4) Spike with standard solutsion (Table 5.3) and label sample
bottles as described in Table 5.5.
(5) Adjust the sample pH in all bottles as specified in Table 5.5
using sulfuric acid or sodium hydroxide solution. Use narrow
range pH paper to measure pH.
(6) Add 2% (v/v) methylene chloride to all samples. Cap bottles and
mix well.
(7) Field controls and field blanks are shipped to the collection
site and are handled, stored, and shipped in the same manner as
field samples. Laboratory blanks and controls are stored at 4°C
in the laboratory.
5.6 FIELD COLLECTION
(1) Water samples are collected by rinsing sample bottles three
times with sample water, then filling to the calibration mark.
Chap. 5 - 79
-------
Table 5.5. CONTROL AND BLANK SAMPLES
U i
0
Bottle Spike
Handle
Sample
pH
Sample Type
1,2 1.0 mL VOSA standard solution
send to field
7-8
field control VOSA
3,4 1.0 mL VOSA standard solution
store in lab
7-8
laboratory control VOSA
5,6 none
send to field
7-8
field blank VOSA
7,8 none
store in lab
7-8
laboratory blank VOSA
9,10 1.0 mL NOVA standard solution
send to field
7-8
field control NOVAb
11,12 1.0 mL NOVA standard solution
store in lab
7-8
laboratory control NOVA
13,14 none
send to field
7-8
field blank NOVA
15,16 none
store in lab
7-8
laboratory blank NOVA
17,18 1.0 mL SAM standard solution
send to field
4-5
field control SAN
19,20 1.0 mL SAM standard solution
store in lab
4-5
laboratory control SAM
21,22 none
send to field
4-5
field blank SAM
23,24 none
store in lab
4-5
laboratory blank SAN
aLOW molecular weight carboxylic acids.
bNlil acids.
-------
(Remember that separate water samples for each of the VOSA, NOVA
and SAN protocols must be taken and spiked with internal stand-
ards.) Internal standards are added to each water sample. NBS
ampoules (Table 5.4) or laboratory prepared standards (Table 5.2)
are used. For drinking water and surface waters, 7.5 mL (1 vial)
of the Set No. 1 is used. Municipal, industrial, and energy
wastewater effluents are spiked with 10 niL (1 vial) of Set
No. 2. Rinse the vials containing the internal standard solutions
three times with sample water and add to the sample bottle.
(2) A preliminary water sample ( “ .25 ml) is collected for the purpose
of determining total chlorine content. This measurement is made
at the sample collection site and utilizes the Bausch and Lomb
Mini Spec 20 portable spectrophotometer. The water sample is
added to a 25 niL mixing cylinder along with the contents of a
DPD Total Chlorine Reagent Powder Pillow (Hach Chemical Co.).
The mixture is shaken and the color is allowed to develop for 3
to 6 minutes. Percent transmittance, measured at 530 nm, is
converted to mg/L total chlorine using Table 5.6.
Table 5.6. TOTAL CHLORINE (mg/L as Cl) VS. % TRANSMITTANCE
%T
% T Units
Tens
0
1
2
3
4
5
6
7
8
9
10
4.00
3.84
3.68
3.54
3.42
3.30
3.18
3.08
2.98
2.88
20
2.80
2.71
2.63
2.55
2.48
2.41
2.34
2.28
2.21
2.15
30
2.09
2.04
1.98
1.93
1.88
1.82
1.78
1.73
1.68
1.64
40
1.59
1.55
1.51
1.47
1.43
1.39
1.35
1.31
1.28
1.24
50
1.20
1.17
1.14
1.10
1.07
1.04
1.01
0.98
0.95
0.92
60
0.89
0.86
0.83
0.80
0.78
0.75
0.72
0.70
0.67
0.64
70
0.62
0.60
0.57
0.56
0.52
0.50
0.48
0.46
0.43
0.41
80
0.39
0.37
0.34
0.32
0.30
0.28
0.26
0.24
0.22
0.20
90
0.18
0.16
0.14
0.13
0.11
0.09
0.07
0.05
0.04
0.02
Reprinted from: Water and Wastewater Analysis Procedures Manual, Hach
Chemical Co., 1975, p. 2-29.
Chap. 5 - 81
-------
(3) Residual chlorine is quenched by adding a stoichiometric quantity
of 0.05 N Na 2 S 2 O 3 (Figure 5.1, a 10% excess is used and is
included in calculation) to the bottle containing the aqueous
sample.
(4) Samples collected for the analysis of VOSA and NOVA fractions
are adjusted to pH 7 to 8. Samples collected for the analysis
of SAN are adjusted to pH 4 to 5. Use narrow range pH paper to
measure pH. Use sodium hydroxide or sulfuric acid for pH adjust-
ment.
(5) Add 2% (v/v) methylene chloride to all samples as a preservative,
cap the samples, and shake well. The samples must be maintained
at 4°C from the time of collection until extraction.
(6) Samples should be shipped on ice (e.g., “Blue Ice “) directly to
the laboratory by an appropriate air carrier (e.g., Federal
Express) in well insulated cartons.
(7) As soon as the samples arrive at the laboratory, the surrogate
samples must be spiked with target compounds. Samples for the
analyses of VOSA, NOVA, and SAN compounds are spiked with the
corresponding standard spiking solution (Table 5.3). Drinking
water and surface water samples are spiked with 1.0 mL of the
appropriate solution using a graduated pippette. Industrial,
municipal and energy wastewaters are spiked with 1 mL of the
appropriate solution using a 1 mL graduated pipette.
Chap. 5 - 82
-------
Volume O.05M Na 2 S 2 O 3 (mL)
Figure 5.1. Total chlorine vs volume of O.O5MNa S 2 0 3 added to a
1 L sample (if a 3.5 L sample is col ected, 3.5x the
amount of Na 2 S 2 O 3 must be added).
6
5.
4.0
E
.—
1.4
0
3.
4 J
Slope = 3.38 mg-L -mL
2.
1.
O 6 O 8 LO 1.2 1.4 L6 1.8 2.0 2.2 2:4 2.6 2.8
Chap. 5 - 83
-------
CHAPTER 6
PURGE, TRAP AND ANALYSIS OF VOLATILE ORGANICS (VO)
6.1 INTRODUCTION
6.1.1 Principle of the Nethod
This analytical protocol is for the determination of volatile organic
(VO) compounds in drinking water, surface waters, and municipal, industrial
and energy wastewater effluents. The procedure utilizes helium gas purging
through buffered water samples at high ionic strength to effectively
partition semisoluble and insoluble volatile organic compounds between the
gaseous and aqueous phases, followed by adsorption from the gaseous phase
onto a Tenax GC sorbent trap. A custom built purge and trap system is
employed in combination with capillary gas chromatography-mass spectrometry.
(Fabrication of the system is described in Appendix A; it is also required
for the NEWS compounds protocol, see Chapter 7). After trapping on Tenax,
the organics are thermally desorbed and sample components are cryofocussed
in a liquid nitrogen trap. An external standard (perfluorotoluene) is
added, and the VO compounds are flash evaporated onto a capillary column.
6.1.2 Detection Limits and Sample Size
Since the same initial sample size is not used for all water types,
detection limits will depend upon the final volume of water sample purged
which is determined from a headspace scouting method. The maximum water
volume purged is 200 mL; however, dilutions of 1 to 100 may be necessary
for some industrial effluents (the purged volume is always 200 mL). If
the detection limit for qualitative GC/NS analysis of each VO compound is
10 ng, then the nominal detection limits may range from 0.1 ppb (for
200 mL of drinking water) to 10 ppb, depending upon the dilution factor
required. Usually, the limits of detection for quantitative analysis of
target compounds are a factor of five lower.
Chap. 6 - 84
-------
6.1.3 Interferences
Background contamination from impure purging and carrier gases may be
experienced if prescribed precautions are not instituted. A hydrocarbon
background may occur from the laboratory atmosphere; e.g., from the solvent
employed with electrostatic printer! plotters used with GC/MS systems.
The solvent may adsorb on the purging glassware that is stored nearby
before use.
Some phenols and amines, because of their polarity, may tail severely
during gas chromatographic analyses and mask compounds of interest. If
the pH of the sample is adjusted to seven, as directed, the phenolics and
amines are mostly in an ionic state and will not purge.
6.1.4 Recoveries and Scope
Table 1.3 (Chapter 1) presents the mean percent recovery and standard
deviation for VO compounds in drinking water and an industrial/municipal
effluent. Compound classes most amenable to this technique include ethers,
aliphatic hydrocarbons, halogenated aliphatic hydrocarbons, aromatic
hydrocarbons and halogenated aromatic hydrocarbons. Esters are recovered
only moderately and aldehydes and ketones are recovered poorly; volatile
compounds in these classes are best handled by the NEWS protocol (Chap-
ter 7). Generally, compounds exhibiting good recoveries (>60%) are those
with boiling points below 200°C (vapor pressure —l nun Hg @ 25°C), and in
several instances below 250°C.
6.2 APPARATUS AND REAGENTS (To Process Six Samples)
(1) A custom built purge and trap system (Fig. 6.1, fabrication des-
cribed in Appendix A).
(2) Six purge flasks, 200 mL capacity (Fig. 6.2), calibrated (Sec-
tion 6.3.4). Sample transfer system (Fig. 6.3).
(3) Reagents
(a) Sodium sulfate, anhydrous powder (ACS grade and free of VO
compounds, Section 6.3.3), 350 g.
(b) pH 7.0 Phosphate buffer; 0.95 g K 2 HPO 4 + 2.96 g NaH 2 PO 4
7 H 2 0 (6 weighed aliquots).
(c) 1.5 L reagent water (high purity water, purged for 30 mm @
90°C, >50 mL/min with Ultra—pure Helium gas).
Chap. 6 - 85
-------
Figure 6.1. Purge and trap system with injection system (front view).
0
p.
p p
a’
a’
OC lnj.ction Syit.m
-------
0.25 in O.D.
MicroFlex
Valve (K-749100)
Threaded Adaptor
(K. 423800)
Medium Porosity
Frit
Figure 6.2.
Purge vessel (200 mL capacity).
Valve (K-423600)
(K .423600) —
200 mL
Capacity
Threaded Adaptor
(K-423800)
6.0 mm o.d. X
1.0 mm i.d.
35cm
Chap. 6 - 87
-------
Twoay Valve (G)
Three-Way
Sample Valve (B)
Gasket (A)
SS Reducing,
Union (C)
SS Hypodermic
Tubing (E)
in
Co
Sample Needle (D)
SS Hypodermic
Needle ( I)
Magnetic
Stirring Bar
Figure 6.3. Sample transfer schematic.
-------
(d) Ultra-pure Helium gas.
Ce) NBS deuterated VO internal standards in glass capsules (see
Table 2.2, Chapter 2).
(f) Internal standard solution (optional if NBS capsules are
not available; see Table 2.4, Chapter 2).
(g) External standard, perfluorotoluene (50 pL). Acceptable
chemical purity is >98%.
(h) Headspace analysis calibration mixture (Table 6.1).
(i) System performance solution (Table 6.2).
(4) Thermostated water bath. To accommodate two 250 mL bottles at
30-32°C.
(5) Two cryogenic traps (Fig. 6.4): one is for purifying helium to
prepare reagent grade water, the second for providing liquid
nitrogen to the cryofocussing trap of the injection system. Two
1 L Dewar flasks, copper tubing, SS tubing, pressure relief
valve, and flow control valve V (Fig. 6.4).
(6) Syringes:
(a) 1 j.iL, 5 iL, 10 pL (Hamilton Gas_Tight® or equivalent).
(b) 5 and 10 mL gas sampling syringes with shut-off valve.
Table 6.1. } [ EADSPACE CALIBRATION HIXTURE
Chemical
b.p. (°C)
Density
(@ 20°C)
Volumea
(pL)
diethyl ether
35
0.714
14
cyclopentane
49
0.746
13
chloroform
61
1.483
7
thiophene
84
1.065
9
n-heptane
98
0.684
15
toluene
111
0.867
12
ethyl butyrate
120
0.878
11
phenyl ether
170
1.075
9
1,4-dibromobutane
197
1.789
6
l,2,4,5—tetrachlorobenzene
244
1.858
5
aVi of neat substance added to a 100 mL volumetric flask con-
taining methanol.
Chap. 6 - 89
-------
Table 6.2. SYSTEM PERFORMANCE SOLUTION FOR VO COMPOU1 1DSa
Density
Chemical (@ 20°C)
b
Concentration
(ng/ iL)
AmoUnt
Injected
(rig)
l-bromo-4-fluorobenzene 1.495 1,490 300
perfluorotoluene 1.770 1,770 350
d 5 -bromoethane 1.460 1,460 290
d 5 -chlorobenzene 1.106 1,100 220
2,4,6-d 3 -axiisole 0.996 990 200
d 8 -naphthalene l. 02 Sc 1,020 200
n-octane 0.702 700 140
n—decane 0.730 730 150
acetophenone 1.028 1,030 210
1-octanol 0.827 820 160
5-nonanone 0.822 820 160
2,6-dimethyiphenol 0.968 970 190
2,6-dimethylaniline 0.972 970 190
ethy lbenzene 0.867 860 170
—xylene 0.861 860 170
n-nonane 0.718 1,000
1,3,5-trimethy lbenzene 0.865 52 e 10
aSee Table 6.6 for the function of each test component in the system per-
formance evaluation.
bSoivent is methylene chloride; 25 pL of neat chemical is delivered to a
25 mL volumetric flask containing 25 mL of solvent.
CThe mass of d 8 -naphthalene added is determined by weighing the solid
material.
d
175 liL is added.
e 15 pL is added.
Chap. 6 — 90
-------
Figure 6.4. Cryogenic system for generating liquid nitrogen
from a gas reservoir.
1/4” o.d. X 24”
Copper Tubing
1/4” to 1/16”
Foam
Control Valve
Pressure
Cap
Tubing
To Two-Stage
Regulator
X 18’
SS Tubing
Cryogenic
Trap
1 L Liquid Nitrogen Dewar
-------
(7) 60 m x 0.32 mm i.d., DB—l (1.0 p film thickness) fused silica
capillary (or equivalent).
(8) One gas chromatograph (packed column) with flame ionization
detector.
(9) 1.8 m X 0.2 cm i.d. glass column packed with 3% OV-17 on 80/100
Gas Chrom Q.
(10) 1 liter glass gas bulb (Fig. 6.5), heating mantle, Teflon®
coated magnetic stirbar, and magnetic stirrer.
6.3 PREPARATION FOR ANALYSIS
6.3.1 He Purification
Helium, which is used for preparing reagent grade water, must be
passed through a liquid nitrogen-cooled trap to remove volatile components.
This trap is constructed of 0.25 in o.d. X 24 in copper tubing, coiled and
immersed in a liquid nitrogen Devar bath (see right side of Fig. 6.4).
The copper tubing must be cleaned by rinsing with pentane and air drying
under a heat gun prior to use to remove any potential contaminants.
6.3.2 Distilled/Deionized Water Purification
Interfering volatile substances in distilled water and/or deionized
water are removed by purging at 90°C with purified helium at >50 mLfmin
for at least 30 mm. Water purified in this manner should be used the
same day of its preparation.
6.3.3 Sodium Sulfate Purification
Sodium sulfate purity must be verified prior to use. This is accom-
plished by purging 60 g of anhydrous Na 2 SO 4 , contained in the purge and
trap vessel, with 500 niL purified helium, and analyzing by GC/MS (Sec-
tion 4.4). If a significant increase in the number or magnitude of peaks
is observed relative to a dry, empty vessel purge, a new lot of Na 2 SO 4
must be examined or the existing material must be purified. Sodium sulfate
can be purified by heating to 400°C in an oven for 2 hr in a 2 cm diam. X
20 cm quartz tube with a nominal flow of pre-purified nitrogen gas at
20 niL/mm. The purified salt should be cooled to approximately 100°C in
the quartz tube under N 2 flow, after which it may be transferred to a
nitrogen-purged, clean, glass bottle with a Teflon—lined screw cap. Once
a satisfactory lot of Na 2 SO 4 has been identified, sufficient quantity
Chap. 6 - 92
-------
should be obtained to complete the entire sample batch. Sodium sulfate
(anhydrous powder) obtained from J. T. Baker Chemical Co., Phillipsburg,
NJ is generally satisfactory for use here, but purity must be verified.
6.3.4 Calibration of Purge Flasks and Gas Bulb
Each purge flask (Fig. 6.2) must be calibrated at a volume equivalent
to the total volume of the sample plus the buffer salt and sodium sulfate.
To determine this volume, 54 g of anhydrous sodium sulfate powder, 0.95 g
K 2 }1P0 4 and 2.96 g HaH 2 PO 4 • 7H O are dissolved in 200 mL of water at 30°C.
The total volume (—212 niL) is measured with a graduated cylinder. An
equivalent volume of water is transferred to each purge flask and a perma-
nent mark is made at the appropriate height on the outside of each purge
flask.
The gas bulb in Figure 6.5 is calibrated by filling with reagent
water and then measuring the volume of water it held with a graduate
cylinder. The gas bulb volume is used to calculate the volume of external
standard, perfluorotoluene, to be added to each VO fraction analyzed.
6.3.5 Determination of Dilution Factor Scale
The degree of sample dilution required (if any) to provide successful
GC/NS/CONP analysis of the actual samples must be determined relative to
the operable dynamic range of the mass spectrometer being employed. This
is accomplished by purging reagent water spiked with the components given
in Table 6.1. Two standard solutions (0.1 and 1.0 ppm/component) are each
prepared in triplicate by injecting 12 .iL and 120 i L into each of two
12 niL vials filled (no headspace) with reagent water. An aliquot of each
standard solution (4 mL, 2 mL, 1 mL corresponding to l:50 1:100, 1:200
dilutions) is transferred to the purge flask containing 200 niL of reagent
water and the appropriate salts and analyzed by GC/NS as described under
Section 6.4.4.
The GC/MS area counts for principal quantitation ions (as given in
Appendix B) are determined (Table 6.3). The dilution factor of each
aqueous standard that gives results that are approximately midpoint of
the dynamic range of the GC/MS system (e.g., approximately 400 ng of in-
jected analyte) is then indicated as shown, for example, in Figure 6.6 for
0.1 and 1 ppm. For the other VO levels in samples the remainding dilution
factors are extrapolated.
Chap. 6 - 93
-------
Heating Mantle
Figure 6.5.
Gas bulb and heating mantle for producing
gaseous external standard, perfluorotoluene.
Magnetic
Stirrer
Chap. 6 - 94
-------
100 1,200,000
4.’
0
10 - 100,000 u
I 4 . ’
I C)
I I x
I I
I I
I I I
I 41
I I
I I
I I
I 0
0.1 I
I I 4)
I I $1
I I
0 ’ I
0.01 - 100
U’
4)
I x
I I
0.001 I ________ ________ ________ ______
l- 10 1- ’ 150 1- -500 1-’-5,000 1- 50,0O0
Dilution Factor (As determined by GC/MS)
I I I I
20 ml 4 xmL 400 iiL 40 pL 4 pL
Sample Volume Added to Purge Vessel
Figure 6.6. Determination of dilution factor for
sample analysis from headspace scouting.
-------
Table 6.3. EXANPLE OF DYNAIIIC RANGE RESULTS
Analyte Level
Analyzed by GC/MS
Final Dilution
Factor
Nean Area Counts
For 10 Components
100
l 200
60,000
200
1+100
125,000
400 a
l 50
245,000
1000
l 2O0
620,000
2000
l- l00
1,250,000
4000
l- 50
-
aApproximate midpoint of dynamic range of GC/NS system (defined as
capillary capacity, but could also be analog/digital interface
saturation on some systems).
b
AID saturation.
6.3.6 Calibration of GC/FID System for Sample Scouting
The GC/FID system is calibrated for response by analyzing the head-
space of four standard solutions of VO compounds (Table 6.1) prepared at
0.01, 0.1, 1 and 10 ppm/component. For example, to prepare a 1 ppm/compo-
nent mixture, 100 pL of the standard solution (Table 6.1) is injected into
a 3 dram (12 mL capacity), septum-capped vial containing 10 mL of 30%
aqueous (reagent water) Na 2 SO 4 solution. After equilibration at 50°C for
1 hr in a water bath, 10 pL of headspace gas is withdrawn and analyzed by
GC/FID on a 1.8 m X 0.2 cm i.d., 3% OV-17 on 80/100 Gas Chrom Q column,
temperature programmed from 30° to 200°C at 20°C/win after a 5 win hold at
30°C, with carrier gas flow of 30 niL/win.
The mean of the GC area (or peak height) counts for all peaks are
plotted on a graph similar to that shown in Figure 6.6 for each standard
solution. Thus, the GC/FID response is equated to the GC/MS response and
dilution factor required for successful GC/NS analysis.
6.3.7 Sample Scouting
A 10 niL aliquot of the scouting sample (collected in a 3 dram vials)
is transferred to a 3 dram (12 niL), septum-capped vial containing 3 g of
pure anhydrous Na 2 SO 4 . After equilibration at 50°C for 1 hr in a water
Chap. 6 - 96
-------
bath, 100 pL of headspace gas is withdrawn and analyzed by GC/FID on a
1.8 in x 0.2 cm i.d., 3% OV-17 on 80/100 Gas Chrom Q column, temperature
programmed from 300 to 200°C at 20°C/mu after a 5 mm hold at 30°C, with
carrier gas flow of 30 mL/min.
The degree of dilution required for the corresponding water samples
is then determined from Figure 6.6.
6.4 GC/MS/COJIP ANALYSIS
Prior to beginning GC/MS/COHP analysis of VO compounds, the analyst
should consult the general procedures described in Chapter 13, entitled
“Capillary GC/NS/CONP Analysis Procedure - General Instructions for All
Protocols.”
6.4.1 Preparation of Gaseous External Standard
A 1 L gas bulb (Fig. 6.5) with a septum port and containing a Teflon
coated magnetic stirring bar is flushed for 3 miii ( ‘-3 L/inin) with He,
while the gas bulb is in a heating mantle (35°C) positioned on an operating
magnetic stirrer. Approximately 0.5-0.7 pL (the exact volume is recorded)
of perfluorotoluene (PFT) is injected into the gas bulb with both stopcocks
closed and stirred for 30 mm to attain complete volatilization and mixing.
6.4.2 GC/MS/COMP Analysis
6.4.2.1 GC/MS/COMP Operating Parameters--
The recommended GC/NS operating parameters are given in Table 6.4 for
the analysis of VO compounds. Other capillary columns which meet the
system performance criteria may be substituted for the recommended fused
silica (See Section 13.4).
6.4.2.2 tIS Calibration--
The mass spectrometer is calibrated (Chapter 13) using the manufactur-
er’s recommended approach. The acceptability of the calibration results
is checked upon analysis of the system performance solution (SPS).
6.4.2.3 Analysis of SPS (Quality Control)-—
Prior to analysis of the SPS or any samples, the sorbent trap (Tenax
GC) on the purge and trap system is conditioned at 220°C for 30 mm each
day when the purge and trap system is used. The 6-port valve (E, Fig. 6.1)
should be in position A during conditioning. The temperatures on the
system are set as indicated in Table 6.5.
Chap. 6 - 97
-------
Table 6.4. GC/NS OPERATING CONDITIONS FOR VO COMPOUND ANALYSIS
GC Column
GC Carrier GaB
Carrier Gas Flow
Carrier Gas Sweep Rate
Temperature Program
Injector Temperature
MS Transfer Line Temperature
Injection Mode
Injection Volume (PFT vapor
in helium)
Ionizing Energy
Ion Source Temperature
Scan Range
Scan Speed
60 m DB-l wide-bore fused
silica capillary column
(1.0 p film thickness)
Helium
1.8 mL/min
28 cm/sec
35°C for 5 mm; 45°C for
5 mm; prograed to 240°C
at 4°C/mm
200°C
260°C
Spl itless
1.0 mL
70 eV
250°C
35-400
Scan 0.95 sec, hold 0.05 sec
Table 6.5. TEMPERATURE SET-POINTS ON
PuRGE AND TRAP SYSTEM (FIG. 6.1)
Parameter
Temperature (°C)
.
6-port valve, E (8)
160
•
transfer line (20) from purge
flask exit to 6-port valve
160
transfer lines (21 and 22) from
6—port valve to sorbent trap
160
•
transfer line (5) from 6-port
to injection system
160
.
injection port and valve, G and
II (1)
200
.
cryofocussing trap (2)
— bakeout
- operational modes
240
* cryogenic
* sample injection (flash evaporation)
-195
220
Tenax GC trap (4)
•
- bakeout
- sample adsorption
- sample desorption
220
ambient (—23)
200
a
( ) denotes the item numbered in Fig. A-i and Table A-2 or A-3
of Appendix A.
Chap. 6 - 98
-------
The system performance mixture (Table 6.2) is analyzed each day
before any samples are run. A 0.2 pL injection is made directly into the
injection port (G, Fig. 6.1; valve in position “B”) with the cryogenic
trap cooled to liquid nitrogen temperatures. After 4 mm the liquid
nitrogen cooling of the cryofocussing trap is discontinued, the trap
temperature is raised to 220°C and data acquisition is started.
The SPS raw data are extracted from the run, test parameters calculat-
ed and plotted or tabulated (Tables 6.6 and 6.7). Up to 14 days of SPS
analytical results can be historically recorded in Table 6.6. This allows
the analyst to follow subtle trends which may develop, and anticipate GC
or MS maintenance required. Calculation of test parameters and discussion
of their use is given in Chapter 13.
A check of RMRs on a daily basis is also calculated from SPS raw data
and tabulated (Table 6.7) and compared to that set developed for the
historical data bank. The usage of this data is also discussed in
Chapter 13.
6.4.3 Determination of RJIRs
RMRs for the User’s data bank (as opposed to the daily RHR checks
discussed in the above paragraph) are determined by injecting either a
gaseous mixture (of deuterated internal standards, PET, and analytes
eluting prior to anisole) or a methylene chloride solution (of deuterated
internal standards, PFT, and analytes eluting after chlorobenzene) into
the injection port of the purge and trap system (G, Fig. 6.1).
6.4.3.1 Gaseous Mixtures of Analytes and Deuterated Internal
Standards--
A gaseous mixture is prepared for VO compounds in a similar manner to
the gaseous PET procedure (Section 6.4.1). The amount of compound/mL of
gas is calculated from the neat volume injected (converted to mass based
on its density) and the volume of the bulb.
6.4.3.2 Solution of Analytes and Deuterated Internal Standards--
This solution is prepared in the same manner as the SPS (Table 6.2).
6.4.3.3 GC/MS/COMP Analysis for RMR Determination——
The standard mixtures of analytes and internal and external standards
are injected into the injection port and trapped in the cryofocussing trap
Chap. 6 - 99
-------
Dates:
Run Id Code
GC Column
Notebook R
and
efer
Program:
ences:
MS SYSTEII CHECXS
High/Low Mass Balance
Perfluorotoluene:
m/z 69 74 79 93 98 108 111 148 167 186 217 218 236 237
::::: ::::i::::: ::::i:::: ::::: :::::
Bromofluorobenzene.
Abundance Observed
Day
Table 6.6. GC-MS SYSTEM PERFORMM4CE TEST FOR VOLATILE ORGANICS (VO)
Ion Relative Abundance Criteria
50 15 to 40% of m/z 95
75 30 to 60% of eli 95
95 100%
96 5 to 9% of m/z 95
173 <2% of eli 174
174 >50% of e/z 95
175 5 to 9% of m/z 174
176 >95%, <101% of e/z 174
177 5 to 9% of eli 176
These abundances are initially established by the MAS user and subsequently become his
guideline for acceptability of calibration
(continued)
Chap. 6 - 100
-------
Table 6.6 (cont’d.)
Test Components Criteria
Linit of
Detection Trimetby lbenzene S:N (m/z 51) > 4:1
10
Z
8
(#
6
4,
‘ I I I I I I I I I I
Day
GC SYSTEM CHECKS
1. Acetophenone (ui/z 120 or 105) • 7, PAF < 300
Peak 2. 1-Octanol ( /z 70 or 84) £ 7, PAl < 250
Asy netry 3. 5-Nonanone (n/z 57) U Z PAl < 160
300 No. 1
No2
° 200
No.3
100
I I I I I I I I I —
Day
Acidity/
Basicity
1. Acetophenone (TIC)
2. 2,6-Dimethylphenol (TIC)
3. 2,6—Di. ethy1ani1ine (TIC)
Ratio2:1 O7toJ.3 •
Ratio 3:1 = 0.7 to 1 3 U
1.4
1.2
0
. 1.0
C I
0.8
0.6
I I I I I —
Day
(continued)
Chap. 6 - 101
-------
Table 6.6 (cont’d.)
Test Co!rponents Criteria
Separation n-Octane ( /z 43) SN > 40
Number n-Decane ( /z 43)
48
46
z 44
U)
42
40
• I U U I U I I I I I I
Day
Ethylbenzene (rn/i 91) R > 1.0
Resolution -Xylene (rn/i 91)
1.6
1.4
12
1.0
0.8
0.6
U I I U I U I I U I
Day
Capillary
Capacity n-Nonane (rn/z 43) PAP > 70
100
u
<
n.
d 80
70
I I I I I I I I I I I I
Day
COMNEWTS:
Chap. 6 - 102
-------
Table 6.7. GC-MS SYSTEM PERFORMANCE TEST-MAS:
R1IR CHECK FOR VOLATILE ORGANICS (VO)
Date: Run ID Code:
DATA
Amount Ion Area Area
Standard MW (ng) u N (m/z) (Ran 1) (Run 2)
Perfluorotoluene 236 186
(PET) 236
deBromoethane 114 113
J 115
d -Chlorobenzene 118 82
J 117
119
d -A niso1e 111 82
-) 111
d 8 —Naphtha lene 136 136
MATRIX OF STANDARD ION RMRs
Standard Ion 186 236 113 115 82 117 119 82 111 136
PET 186
236
-
—
d 5 -Bromoethane 113
115
-
-
d 5 -Chlorobenzene 82
117
119
-
—
—
d 3 -Aniso] .e 82
111
-
d 8 -Naphthalene 136
A MW
= ng A = ion area or height
x/y A MW ng ng ng injected
y y x xanalyte
Con wrS: y = standard
Chap. 6 - 103
-------
(Section 6.4.4). A 1 inL volume of the gaseous mixture is used. A 0.2 pL
sample of the liquid solution is injected in an identical manner to the
introduction of the SPS (Section 6.4.2.3)
The GC/NS/COMP operating parameters were given in Table 6.4.
Table 13.8 may be used to tabulate RNR raw data.
6.4.4 Analysis of Blanks, Controls, Samples and Surrogates
A water sample (243 mL bottle) to be analyzed by this purge and trap
method is equilibrated at 30—32°C in a thermostated water bath for 20 mm.
A clean 200 niL calibrated purge flask is filled with 54 g of Na 2 SO 4
and prepurged with purified helium at 25 niL/mm for 5 mm with valves A
and B closed, C disconnected, and D connected and open (Fig. 6.1). Then,
valves B andC are opened and C connected via the Teflon line to the purge
flask; valves A and D are meanwhile closed during the transfer step
(Fig. 6.1). For drinking water, 200 niL is delivered to the purge and trap
vessel (to the calibration mark, Section 6.3.4) by pressurizing the sample
vessel (Fig. 6.3) using a pressure which transfers the sample in —4-5 mm.
The long and short stainless steel needles are inserted respectively
through the Teflon faced septum (H) just prior to transfer of the samples.
The purge vessel is vigorously shaken (in place) as the sample is intro-
duced and until the Na 2 SO 4 is completely dissolved. (Valve C is open so
that compounds that volatilize into the purge vessel’s headspace pass to
the Tenax GC trap which is at ambient temperature during the purging
step). Close valve B and the two-way pressurizing valve (G, Fig. 6.3).
The water sample is then purged with helium at 25 niL/mm for 20 mm with
valves C and D open (Fig. 6.1). The 6-port (E) and the purge gas valves
(F) are in the “A” positions during purging. All valves on the purge
vessel are then closed for a “dry” purge step. A 10 mm “dry” purge
(25 mL/min) to remove excess water from the Tenax GC trap is performed
with the 6-port valve (E) in the “A” position and the purge gas valve (F)
in the “B” position.
A 1.0 niL gas sample of PFT is injected into the injection port (G,
with valve H in “B” position, Fig. 6.1) to introduce the external standard
into the cryofocussing trap (which has been cooled to liquid nitrogen
temperature), 6 mm after “dry” purging has commenced.
Chap. 6 - 104
-------
Several points need to be emphasized for obtaining reproducibility of
PFT measurements by GC/MS;
(1) The needle on the gas sampling syringe (10 niL) should have a
rounded solid tip with a side port for gas entrance and exit.
(The large gauge of a regular tapered needle point cuts slices
out of septa.)
(2) The septum on the injection port must be tight to remain leak-
free. A decrease in column head-pressure can be observed on the
gauge during injection if the septum is not very tight, even
when the side-port needle is used.
(3) Any septum-sweep flow in the injection port must be closed off.
As much as 30% of the 1 niL injected can be lost out the septum
purge vent.
(4) The Teflon plunger and/or surfaces in the syringe can retain
about 10% of the previous PFT analyte. That is, if 1 mL PFT
vapor gives a GC area of 100,000 counts, a repeat 1 niL injection
of air will yield about 10,000 counts of PFT. If the syringe
plunger is pulled up to 2 niL and depressed to 0 niL 5-6 times
after PFT injection the area count on a subsequent 1 niL injection
of air is less than 2,000 counts.
(5) The syringe should have a closure such that the 1 niL PFT vapor
sample can be locked in the syringe and the sample pressurized
before injection into the GC; i.e., the plunger at 1 niL gradua-
tion is depressed to 0.7 niL before the needle is inserted into
the injection port to equalize pressure in the syringe and
injection port. (Follow syringe manufacturer’s instruction.)
By observing these precautions, the precision of PFT GC/MS response can be
maintained at less than 10% relative standard deviation throughout an 8 hr
period. However, the PFT gas should be produced fresh each day.
At the end of the “dry” purge step the 6-port valve (E) is rotated to
the “B” position (desorb mode), and the injection port valve (H) to the
“A” position. The Tenax GC trap is desorbed by heating to 200°C with the
He flow at 15 niL/mm. After 8 ruin, the liquid nitrogen cooling of the
cryofocussing trap is terminated, the injection port valve (H) is rotated
Chap. 6 - 105
-------
to the “B” position, the temperature on the cryofocussing trap is raised
to 220°C, data acquisition started and the GC program conditions are
initiated (Table 6.4).
The purge vessel is removed and thoroughly rinsed with deionized
water, using suction to pull water through the frit in order to remove
residual salt before caking ensues.
For water samples that require dilution prior to purging (as indicated
by headspace scouting, Section 6.3.7), the purge vessel is charged later
with Na 2 SO 4 and buffer salts. Reagent water is added so that the addition
of the water sample will brings the final volume to 212 mL (calibration
mark). The vessel is shaken to dissolve the salts, with the valves closed.
The appropriate water sample volume is withdrawn from the sample bottle
into a glass syringe (10 i.iL, 100 pL, 1 rnL or 10 mL, depending on dilution
desired) and then injected through valve A of the purge vessel. Valves B
and D are closed during addition of the sample aliquot, valve C is open.
Purging of the water sample is identical as described above.
6.4.5 Sample Data Analysis
Qualitative and quantitative procedures are described in Chapter 13.
Table 6.8 may be used to tabulate sample analyte raw data, prior to using
the HASQUAJ T software program or manual quantitation.
Chap. 6 - 106
-------
Table 6.8. RAW ANALYTE DATA FOR VO FRACTION
A •MW n
nfl — x _____
6 x RMR A •MW
x/y y y
A •MW
y y
this information is also
automatically.
A = ion area or height
ng = ng recovered from volume
of water processed
x = analyte
y = standard
Date:
Run I.D. Code:
Notebook Reference:
Vol. Water Processed
Sample Identification:
Vol. Water CL):
(to which i were added)
(mL):
STANDARDS
Spec-
I.D. trum
No. No.
Perfluorotoluene
d 5 -Bromoethane
2,4,6—d 3 —Anisole
d 5 -Chlorobenzene
d 8 -Naphthalene
Weight Ions
(ng ) (m/z) Area
MW;
Ka
186,236:
113,115: ,______
81,111: ,______
82,117:
136: ,______
,
,
,
,
,
ANALYTES
Class No.
I.D.
No.
.
Spectrum Ions
No. (mfz) Area
MWa
,
,
,
,
,
,
,
,
,
,
,
,
,
,
—
a 1 f calculations are performed manually then
needed, whereas MASQUANT performs functions
Chap 6. - 107
-------
CHAPTER 7
ELEVATED TEMPERATURE PURGE, TRAP, AND ANALYSIS
OF NEUTRAL WATER SOLUBLE ORGANICS (NEWS)
7.1 INTRODUCTION
7.1.1 Principle of the Method
This analytical protocol is for the determination of volatile low
molecular weight, water soluble compounds in drinking water, surface
waters, and municipal, industrial and energy wastewater effluents. The
procedure utilizes a prepurge of a 10 mL sample with helium at room
temperature (to remove VO compounds) and then gas purging while heating
(80°C) the water sample (containing 20% NaCl to yield high ionic strength)
with adsorption of the NEWS compounds on a Tenax GC sorbent trap. A
custom built purge and trap system is employed in combination with capil-
lary gas chromatography/mass spectrometry. (Fabrication of the system is
described in Appendix A; it is the same system used for analysis of VO
compounds, see Chapter 6.) This approach is used for all water types
except drinking water. For drinking water, lower limits of detection are
achieved by concentrating a 200 mL sample to 3 mL using azeotropic distil-
lation in a Peter’s (Peters, T. I., Anal. Chem., 52, 211, 1980) apparatus.
This condensate (5 mL after rinsing the apparatus) is transferred to the
purge vessel, made to 20% in NaC1, and then purged at 80°C. In either
case, the organics are thermally desorbed from the Tenax, the sample
components are cryofocussed, an external standard (perfluorotoluene) is
added, and then the NEWS compounds are flash evaporated onto a capillary
column.
7.1.2 Detection Limits and Sample Size
If the detection limit for qualitative GC/MS analysis of each NEWS
compound is 10 ng, then the nominal detection limits for drinking water
and all other water types will be 0.05 and 1 ppb, respectively. Usually,
the quantitative limits of detection for target quantitative analysis of
compounds are lower.
Chap. 7 - 108
-------
7. 1.3 Interferences
Background contamination from impure purging and carrier gases may be
experienced if prescribed precautions are not instituted.
7.1.4 Recoveries and Scope
Table 1.4 (Chapter 1) presents the mean percent recovery and standard
deviation for NEWS compounds in drinking water and an effluent. Compound
classes most amenable to this technique include aidehydes, ketones, ni-
trues, alcohols, ethers and nitro containing substances. Esters are not
recovered wei] from the azeotropic distillation step and thus cannot be
quantitatively analyzed in drinking water.
7.2 APPARATUS AND REAGENTS (To Process Six Samples)
(1) A custom built purge and trap system (Fig. A—i, fabrication
described in Appendix A).
(2) Six purge flasks, 10 mL capacity (Fig. 7.2).
(3) Peter’s Apparatus (Fig. 7.3), Vigreau column, 500 mL round
bottom flask and heating mantle. The Peter’s device is not
commercially available and must be fabricated by a giassblower.
(4) Reagents
(a) NaCl (ACS grade and free of NEWS compounds), 15 g.
(b) 1.5 L reagent water (high purity water, e.g., Milli-Super Q®
water, purged for 1 hr @ 90°C, >50 mL/min).
(c) Ultra-pure Helium gas.
(d) NES deuterated NEWS internal standards in water (see
Table 3.4, Chapter 3).
(e) Internal standard solution (optional if NES standards are
not available; see Table 3.4, Chapter 3).
(f) External standard, perfluorotoluene (50 pL). The chemical
purity should be >98%.
(g) System performance solution (Table 7.1).
(h) RMR solution.
(i) 0.006N phosphate buffer (pH 7.0).
(5) Two cryogenic traps: one is used to purify helium for prepara-
tion of reagent grade water, the second for providing liquid
nitrogen to the cryofocussing module of the injection system
Chap. 7 - 109
-------
Figure 7.1.
Purge and trap system (front view).
10 p ç i
@I 1@.
GC Snj.c*,on Syzwn
-------
Valve (K.423600)
MicroFlex
Valve (K-749100)
Threaded Adaptor
(K-423800)
Medium Porosity
Frit
Figure 7.2.
Purge vessel (10 mL capacity).
lOmL
Capacity
Valve
(K-423600)
Threaded Adaptor
(K. 423800)
6.0 mm o.d. X
1.0 mm i.d.
1 6 cm
Char.. 7 — 111
-------
Vigreau
Column
Figure 7.3.
Cooling
Water
In
Peter’s concentration-collection apparatus.
Cooling
Water
Out
Distillate
Overflow Tube
Distillation
Pot
Distillate
Chamber
(3mL)
24/40 Joint
Heating
Mantle
Chap. 7 - 112
-------
Table 7.1. SYSTEM PERFORMANCE SOLUTION FOR NEWS COMpOUNDSa
Density
Chemical (@ 20°C)
b
Concentration
(ng/pL)
Amount
Injected
(ng/pL)
1-bromo-4-fluorobenzene 1.495 1,490 300
perf luoroto luene 1.770 1,770 350
d 9 -t-butano l - 300
d 5 -nitrobenzene 300
n-octane 0.702 700 140
n-decane 0.730 730 150
acetophenone 1.028 1,030 210
1-octanol 0.827 820 160
5-nonanone 0.822 820 160
2,6-dimethyiphenol 0.968 970 190
2,6—dimethylaniline 0.972 970 190
ethy lbenzene 0.867 860 170
p-xylene 0.861 860 170
n-nonane 0.718 5 , 020 d 1,000
1,3,5-trimethylbenzene 0.865 52 e 10
aSee Table 7.4 for the function of each test component in the system per-
forinance solution.
bSolvent is methylene chloride; 25 pL of neat chemical is delivered to a
25 mL volUmetric flask containing 25 niL of solvent.
CThe mass of external and internal standards added is determined by
weighing the 10 IlL syringe.
d
175 pL is added.
e 15 pL is added.
(Fig. 7.4). Two 1 L Dewar flasks, 0.25 in O.D. copper tubing,
0.125 in I.D. SS tubing.
(6) Syringes:
Chap. 7 - 113
-------
1/4” o.d. >( 24”
Figure 7.4. Cryogenic system for generating liquid nitrogen
from a gas reservoir.
1/4” to 1/16”
Foam
Control Valve
Pressure
C)
1/16” o.d.,
0.020” i.d. x i8’
SSTubing
Tubing
To Two-Stage
Regulator
Cap
Cryogenic
Trap
Nitrogen Dewar
-------
(a) 1 pL, 10 liL, 50 .iL, 250 l.iL (Hamilton Gas_Tight® or equiva-
lent).
(b) Two 10 mL and one 5 mL gas sampling syringes with shut-off
valve.
(7) 60 m X 0.32 mm i.d., DB-5 fused silica capillary (or equivalent).
1.0 j film thickness.
(8) 1 liter glass gas bulb (Fig. 7.5), heating mantle, Teflon®
coated magnetic stirbar, and magnetic stirrer.
(9) Five 5 mL glass ampoules, one Teflon® lined screw cap vial.
7.3 PREPARATION FOR ANALYSIS
7.3.1 He Purification
See Section 6.3.1, Chapter 6.
7.3.2 Distilled/Deionized Water Purification
Interfering volatile substances in distilled water and/or deionized
water are removed by purging at 95°C with purified helium at >50 mL/min
for 1 hr.
7.3.3 Preparation of System Performance Standard (Table 7.1)
With a 50 iL syringe, accurately measure 25 pL of pure liquids into a
25 mL volumetric flask containing about 24 niL of methylene chloride (inject
beneath the liquid surface). For the deuterated standards (liquids),
weigh the syringe after filling and after delivering the external standard
to the volumetric flask. Determine the weight added by difference. For
13,5,-trimethylbenzene, add 1.5 pL. With a 250 i.iL syringe add 175 pL of
n-nonane. Make the volume to 25 niL with methylene chloride. Transfer
into a series of 2 niL Teflon® sealed screw cap vials. Store at 4°C.
If compound purity is 96% or greater, the weight can be used without
correction to calculate the concentration of stock standards. If the com-
pound is less than 96% pure, then it cannot be used as a standard.
7.3.4 Preparation of RMR Solulion
Accurately measure 5 IiL of neat NEWS compound (those of interest to
you) using a 10 IJL syringe and inject into a 25 niL volumetric flask con-
taining 24 niL of reagent water. Also, weigh an equivalent amount of
d 9 -t-butanol and d 5 -nitrobenzene in a syringe and transfer to the flask.
Make the volume to 25 ml. Purity criteria are the same as for the SPS
(Section 7.3.3 above).
Chap. 7 - 115
-------
GC Plug Septum
Heating Mantle
Magnetic
Stirrer
Figure 7.5. Gas bulb and heating mantle for producing
gaseous external standard, perfluorotoluene.
Chap. 7 - 116
-------
Transfer aliquots of the RHR solution into 5 niL glass ampoules, seal
and store at 4°C. (May be stored in Teflon® lined screw cap vial when in
use.)
7.3.5 Azeotropic Distillation of Drinking Water
Assemble the Peter’s apparatus as shown in Figure 7.3. Make the
200 mL water sample 20% (w/v) in NaC1 and buffer to pH 7.0 with 0.006N of
phosphate ions. Heat the sample to refluxing temperature and reflux for
30 mm. Transfer the 3 niL condensate from the distillate chamber to a
clean glass vial (10 mL) sealable with a Teflon® lined screw cap. Rinse
the still’s distillate chamber with 2 niL of reagent water and also transfer
this to the vial. Use a 5 mL glass syringe for transfers.
7.3.6 Calibration of Gas Bulb
The gas bulb in Figure 7.5 is calibrated by filling with reagent
water and then measuring the volume of water it held with a graduate
cylinder. The gas bulb volume is used to calculate the volume of external
standard, perfluorotoluene, to be added to each NEWS fraction analyzed.
7.4 GC/MS/COMP ANALYSIS
Prior to beginning GC/MS/CONP analysis of NEWS compounds the analyst
should consult the general procedures in Chapter 13 entitled “GC/MS/CONP
Analysis Procedure — General Instructions for All Protocols.”
7.4.1 Preparation of Gaseous External Standard
A 1 L gas bulb (Fig. 7.5) with a septum port and containing a Teflon
coated magnetic stirring bar is flushed for 3 mm (—3 L/min) with He,
while the gas bulb is in a heating mantle (35°C) and positioned on a
magnetic stirrer. Approximately 0.5-0.7 pL (the exact volume is recorded)
of perfluorotoluene (PFT) is injected into the gas bulb and stirred for
30 mm to attain complete volatilization and mixing.
7.4.2 Preparation for Analysis
7.4.2.1 GC/MS/COMP Operating Parameters--
The recommended GC/MS operating parameters are given in Table 7.2 for
the analysis of NEWS compounds. Other gas chromatographic capillaries
which meet the system performance criteria may be substituted for the
recommended fused silica (See Section 13.4).
Chap. 7 - 117
-------
Table 7.2. GC/MS OPERATING CONDITIONS FOR NEWS COMPOUND ANALYSIS
GC Column 60 m DB-5 wide-bore fused silica
capillary column (1.0 p film
thickness)
GC Carrier Gas Helium
Carrier Gas Flow Rate 1.8 mL/min
Carrier Gas Sweep Velocity 28 cm/sec
Temperature Program 35°C for 5 mm; 45°C for 5 mm;
programmed to 200°C at 4°C/mm
Injector Temperature 200°C
MS Transfer Line Temperature 260°C
Injection Mode Splitless
Injection Volume (PIT vapor in 1.0 mL
helium)
Ionizing Energy 70 eV
Ion Source Temperature 250°C
Scan Range 35—400
Scan Speed Scan 0.95 sec, hold 0.05 sec
7.4.2.2 MS Calibration—-
The mass spectrometer is calibrated using the manufacturer’s recom-
mended approach (Chapter 13). The acceptability of the calibration results
is verified upon analysis of the system performance standard (SPS).
7.4.2.3 Analysis of SPS (Quality Control)--
Prior to analysis of the SPS or any samples, the sorbent trap (Tenax
GC) on the purge and trap system is conditioned at 220°C for 30 mm ini-
tially each day when the purge and trap system is to be used. The valve
CE, Fig. 7.1) is in position “A.” The temperatures on the system are set
as indicated in Table 7.3.
The system performance mixture (Table 7.1) is analyzed initially each
day before any samples are run. A 0.2 pL injection is made directly into
Chap. 7 - 118
-------
Table 7.3. TEMPERATURE SET-POINTS ON
PURGE AND TRAP SYSTEM (FIG. 7.1)
Parameter Temperature (°C)
a
• 6—port valve (8) 160
• transfer line (20) from purge flask 160
to 6-port valve
• transfer lines from 6-port valve to 160
sorbent trap (21 and 22)
• transfer line from 6-port valve to 160
injector block (5)
• GC injection port and valve (1) 200
• purge vessel temperature (outside) 95
• hyofocussing trap (2)
- bakeout 240
- operational modes
* cryogenicb -195
* sample injection 220
• Tenax GC trap (4)
- bakeout 220
- sample adsorption ambient (—23)
- sample desorbtion 200
a( ) denotes the item numbered in Fig. A-7 and Tables A-2 and A-3
of Appendix A.
the injection port (H, valve in position “B,” Fig. 7.1) with the cryofocus-
sing trap cooled to liquid nitrogen temperature. After 4 mm the liquid
nitrogen cooling of the cryofocussing trap is discontinued, the trap
temperature is raised to 220°C and data acquisition is started.
The SPS raw data are extracted from the run, and test parameters
calculated and plotted or tabulated (Tables 7.4 and 7.5). Up to 14 days
of SPS analytical results can be historically recorded in Table 7.4. This
allows the analyst to follow subtle trends which may develop, and antici-
Chap. 7 - 119
-------
Table 7.4. GC-MS SYSTEM PERFORMANCE TEST FOR NEWS COMPOUNDS
Dates: __________ __________ Run Id Code __________ __________
GC Column end Program: ______________________________________________________________________
Notebook References:
MS SYSTEM CHECKS
High/Low Mass Balance
Perfluorotoluene:
m/z 69 74 79 93 98 108 111 148 167 186 217 218 236 237
Abundance*()(,)()()()()()()()( )() (.)()( )
(.3
Bromofluorobenzene:
Abundance Observed
Day
Ion Relative Abundance Criteria
50 15 to 40% of w/z 95
75 30 to 60% of mlz 95
95 100%
96 5 to 9% of m/z 95
173 (2% of m/z 174
174 >50% of m/z 95
175 5 to 9% of w/z 176
176 >95%, <101% of m/z 174
177 5 to 9% of m/z 176
These abundances are initially established by the HAS user and subsequently become his
guideline for acceptability of calibration.
(continued)
Chap. 7 — 120
-------
Acidity/
Basicity
1. Acetophenone (TIC)
2. 2,6-Dirnethyiphenol (TIC)
3. 2,6—Dirnethylaniline (TIC)
Ratio 2:1 = 0.7 to 1.$ I
Ratio 3:1 = 0.7 to 1.3 U
, . . I
Day
(continued)
Table 7.4 (cont’d.)
Test Components Criteria
Lirnit of
Detection Trimetby lbenzene S:N (rn/a 51) > 4:1
10
u
6
4.
I I I I I I I I I I I
Day
CC SYSTEM CHECKS
1. Acetophenone (rn/a 120 or 105) • % PAF < 300
Peak 2. 1-Octanol (rn/z 70 or 84) A % PAF ( 250
Asyetry 3. 5-Nonanone (rn/a 57) a S PAF < 160
300 No.1
No.2
<
° 200
dP
No.3
100
I I I I I I I I I j
Day
1.4
1.2
0
- 4
4.4
(5
0.8
0.6
Chap. 7 — 121
-------
Table 7.4 (cont’d.)
Test Components Criteria
Separation n—Octane (rn/z 43) SN > 40
Number n-Decane (rn/z 43)
48
46
z 44
42
40
I I I I I I I I I —
Day
Ethylbenzene (rn/i 91) R > 1.0
Resolution 2-Xylene (rn/z 91)
1.6•
1.4
1.2
1.0 .
0.8
0.6
I I I I I I I I I I I I
Day
Capillary
Capacity n-Nonane (rn/i 43) % PAl’ > 70
100
I i .
<
0.
d 80
70
I I 1 I I I I j , i I J
Day
CO 1ENT5:
Chap. 7 - 122
-------
Table 7.5. GC-NS SYSTEM PERFORMANCE TEST-tIAS:
RMR CHECK FOR NEUTRAL WATER SOLUBLE ORGANICS (NEWS)
Date:
Run
ID Code:
DATA
Standard
NW
Amount
(ng)
pM
Ion Area
(m/z) (Run
Area
1) (Run
2)
Perf luoroto luene
(PFT)
236
186
236
d 9 -t-butanol
83
64
d -nitrobenzene
128
82
128
MATRIX OF STANDARD
Standard
ION RIIRs
Ion
186
236
64 82
128
PFT
186
-
236
-
d 9 -t-butanol
64
d 5 -nitrobenzene
82
-
128
-
A NW ng
x/y = AX . MWX . ng
y y x
A
n
x
y
= ion area or
= ng injected
analyte
= standard
height
COMMENTS:
pate any GC or MS maintenance required. Calculation of test parameters
and discussion of their use is given in Chapter 13.
A check of RHRs on a daily basis is also calculated from SPS raw data
and tabulated (Table 7.5) and compared to that set developed for the
historical data bank. The usage of this data is also discussed in
Chapter 13.
7.4.3 Determination of RNRs
RMRs for the user’s data bank (as opposed to the daily RMR check
Chap. 7 - 123
-------
discussed in the above paragraph) are determined by injecting 1 pL of the
standard solution containing NEWS compounds and deuterated standards (see
Section 7.3.4) into a 10 mL empty purge vessel (Fig. 7.2) and performing a
“dry” purge.
A clean 10 mL purge flask is assembled in place on the purge and trap
stand (Fig. 7.1). The line at valve C is disconnected. Valves A and B
are closed, and C and D are open. Purge the vessel for 5 mm with He at
20 mL/min. Stop purge and connect line to valve C. Inject 1 lJL of the
standard solution through valve A ini:o the purge vessel’s air space.
Close valve A. Purge vessel for 15 mm at 20 mL/min. After 7 mm from
conunencement of purging, introduce the external standard into the cryofo-
cussing trap as described in Section 7.4.4.3 below and continue from that
point with remainder of the purging, traping and analysis steps. The
GC/MS/CONP operating parameters were given in Table 7.2. Table 13.8 may
be used to tabulate RNR raw data.
7.4.4 Analysis of Blanks, Controls, Samples, and Surrogates
7.4.4.1 Drinking Water--
Add 1 g NaCl (free of NEWS compounds) to a clean 10 ml purge vessel
(Fig. 7.2) and assemble on the purge and trap stand (Fig. 7.1). With
line at C disconnected and valve B closed, prepurge with purified helium
for 2 miii at 20 mL/min and then reconnect. Valves B and D should then be
closed, valves A and C open. Transfer the 5 mL condensate obtained from
the Peter’s still (Section 7.3.5) to the purge vessel by using a 10 ml
glass syringe and injecting through valve A on the purge vessel. Close
valves A and C and shake the purge vessel until the NaCl has dissolved.
Proceed to Section 7.4.4.3 for elevated temperature purge, trap, and
GC/ZIS/COMP analysis.
7.4.4.2 Other Water Types--
Add 2 g NaCl (free of NEWS compounds) to a clean 10 mL purge vessel
(Fig. 7.2) and assemble on the purge and trap stand (Fig. 7.1). With
line at C disconnected and valve B closed, prepurge the salt for 2 miii at
20 mL/uiin. Do not reconnect line at C. Transfer a 10 mL water sample to
the purge vessel by injecting through valve A. Close valves A, C, and D
and shake the purge vessel in place until the NaCl has dissolved. Open
valve C and D and prepurge the water sample with helium for 3 mm at
Chap. 7 - 124
-------
20 mL/min to remove VO compounds. Reconnect line at C. Proceed to
Section 7.4.4.3.
7.4.4.3 Elevated Temperature Purge, Trap, and GC/MS/COMP Analysis--
Wrap the tubular heater (I, Fig. 7.1) with the thermocouple around
the purge vessel and heat the water to 95°C (the actual water temperature
is 80°C, since the thermocouple is next to the heater) for 5 mm. [ Be
sure that the Tenax GC trap is at ambient temperature and the 6-port CE)
and the purge gas (F) valves are in position “A.”) The water sample is
then purged with helium at 30 mL/min for 20 mm with valves C and D open
and A and B closed. Shut off the purge vessel temperature controller.
When purging is complete, an 8 mm “dry” purge (30 mL/min) of the Tenax GC
trap is performed with the 6-port valve (E) in position “A” and the purge
gas valve (F) in position “B.” All valves except D on the purge vessel
are closed during the “dry” purge step.
A 1.0 mL gas sample of PFT is injected into the injection port (G,
injection valve in “B” position, Fig. 7.1) to introduce the external
standard into the cryofocussing trap (which has been cooled to liquid
nitrogen temperature, a process which may take 5 mm) beginning 5 mm
after “dry” purging has commenced.
Several points need to be emphasized for obtaining reproducibility of
PFT GC/MS response:
(1) The needle on the gas sampling syringe (10 mL) should have a
rounded solid tip with a side port for gas entrance and exit.
(The large gauge of a regular tapered needle point cuts slices
out of septa.)
(2) The septum on the GC must be tight to remain leak-free. A
decrease in column head-pressure can be observed on the gauge
during injection if the septum is not very tight, even when the
side-port needle is used.
(3) Any septum-sweep flow in the injection port must be closed off.
As much as 30% of the 1 mL injected can be lost out the septum
purge vent.
(4) The Teflon plunger and/or surfaces in the syringe can retain
about 10% of the previous PFT analyte. This is, if 1 mL PFT
vapor gives an area of 100,000 counts, a repeat 1 mL injection.
Chap. 7 - 125
-------
of air will yield about 10,000 counts of PFT. If the syringe
plunger is pulled up to 2 mL and depressed to 0 mL 5-6 times the
area count on a subsequent 1 mL injection of air is less than
2,000 counts.
(5) The syringe should have a closure such that the 1 mL PFT vapor
sample can be locked in the syringe and the sample pressurized
before injection into the GC; i.e., the plunger at 1 mL gradua-
tion is depressed to 0.7 mL before the needle is injected into
the injection port to equalize pressure in the syringe (follow
manufacturer’s instructions).
By observing these precautions, the precision of PFT GC/MS response can be
maintained at less than 10% relative standard deviation throughout an 8 hr
period. However, the PFT gas should be produced fresh each day.
At the end of the “dry” purge step the 6-port valve (E) is rotated to
the “B” position (desorb mode), and the injection port (G) valve to the
“A” position (Fig. 7.1). The Tenax CC trap is desorbed by heating to
200°C with the He flow at 15 mL/min. After 8 miii, the liquid nitrogen
cooling of the cryofocussing trap is terminated, the injection port valve
is rotated to the “B” position, the temperature on the cryofocussing trap
is raised to 220°C, and data acquisition started with the GC program
conditions given in Table 7.2.
The purge vessel is removed and thoroughly rinsed with deionized
water, using suction, to pull water through the frit, to remove residual
salt before caking ensues.
7.4.4 Sample Data Analysis
Qualitative and quantitative procedures are described in Chapter 13.
Table 7.6 may be used to tabulate sample analyte raw data prior to using
the MASQUANT software program or manual quantitation.
Chap. 7 - 126
-------
Table 7.5. RAW ANALYTE DATA FOR NEWS FRACTION
Date:
Run I.D. Code:
Notebook Reference:
Vol. Water Processed (mL):
Sample Identification:
Vol. Water CL):
(to which i were added)
STA1 DARDS
Spec-
I.D. trum Weight* Ions
No. No. (ng ) (m/z) Area ?1W
Perfluorotoluene 186,236: —
d 9 -t-Butanol 64: ,______ —
d 5 -Nitrobenzene 82,128: —
*Amount of internal standard in volume of water processed
Ka
,
,
,
ANALYTES
Class No.
I.D.
No.
Spectrum Ions
No. Cm/z) Areas
MWa
,
,
,
,
,
,
,
, : ,
,
, :
,
,
,
,
a 1 f calculations are performed manually then this information is also
needed, whereas ?IASQUANT performs functions automatically.
A • MW n A = ion area or height
x x ‘ -7
ng = A X • ng = ng recovered from volume of
y x/y y water processed
x = analyte
ngy y = standard
K=R MW
x/y y
Chap. 7 — 127
-------
CHAPTER 8
BATCH LIQUID-LIQUID EXTRACTION AND ANALYSIS OF
SE}IIVOLATILE STRONG ACIDS (ESSA)
8.1 INTRODUCTION
8.1.1 Principle of the Method
Batch liquid-liquid extraction (BLLE) is used to extract and concen-
trate semivolatile carboxylic acids and some of the more acidic phenolic
compounds from aqueous samples. The sample is collected in a 1 L bottle
with 20 mL of methylene chloride. The extraction is performed by adjusting
the pH to 10 and extracting in a separatory funnel with 100 mL of methylene
chloride. This extract Contains neutral and basic compounds and is dis-
carded. The pH of the sample is then adjusted to <4 with HC1 and the
sample is made 0.1 N in HC1. Sodium chloride is added to give a final
concentration of 20% w/v. The sample is then extracted two times with 300
niL of methyl-t-butyl ether (flEE). The ether extracts are combined, dried
by passing through a sodium sulfate column, and evaporated to —4 mL using
a macro Kuderna-Danish evaporator. The sample is further concentrated to
1 niL by nitrogen blowdown using a modified micro-Snyder column. A 0.3 mL
aliquot of gaseous HC1 in methanol (1 H) is added to facilitate derivatiza-
tion of halogenated acids. The acids are converted to their methyl esters
or ethers using diazomethane reagent in diethyl ether. After completion
of the reaction the solvent volume is reduced to 1.0 mL for all waters
except surface and drinking water which are evaporated to 0.5 niL. This
extract is submitted for GC/HS analysis.
8.1.2 Detection Limit and Sample Size
Since all water types have the same initial ESSA sample size, detec-
tion limits will depend upon the final volume of sample concentrate. If
the detection limit for GC/MS analysis of each extractable organic is
estimated at 10 ng and samples are concentrated from an original volume of
1 L, then using a 1 i.iL injection size, nominal detection limits for specific
water types are as follows:
Chap. 8 - 128
-------
5 ppb in surface and drinking waters (0.5 ml final volume)
10 ppb in industrial and municipal wastewaters and energy effluents
(1.0 ml final volume)
If the sample cannot be concentrated to the specified volume due to high
levels of contaminants, nominal detection limits are proportionately
higher. Usually the limits of detection for quantitative analysis of
target compounds are a factor of five lower.
8.1.3 Interferences
The pre-extraction of the sample with methylene chloride at pH 10
reduces major interferences from neutral and basic organic compounds.
8.1.4 Recoveries and Scope
Table 1.6 in Chapter 1 presents average percent recovery and standard
deviations for a variety of ESSA compounds.
8.2 APPARATUS AND REAGENTS
The following materials are required for processing a set of nine
samples plus two procedural blanks. Nine is the maximum number of field
samples which should be processed at a time. The samples which should be
run during these analyses are listed in Table 4.1. The three procedural
blanks should be run before any samples are collected or processed. The
extraction! concentration procedure will require two working days for a
set of eight samples and one procedural blank.
(1) Nine separatory funnels (2000 ml) with Teflon stopcocks.
(2) Nine 1 L glass bottles with Teflon lined screw caps for collecting
the extracting solvent prior to solvent evaporation.
(3) One 1 L graduated cylinder for measuring sample volumes.
(4) Nine chromatographic columns for sodium sulfate solvent drying.
300 mm X 10 mm i.d. with a glass wool plug at the bottom.
Approximate dimensions are shown in Figure 8.1.
(5) One Soxhlet extractor (50 ml capacity minimum) for extracting
glass wool.
(6) Eighteen glass funnels to fit chromatography columns and separa-
tory funnels.
(7) Pre—extracted glass wool ( 50 mL) for drying columns. Glass
wool should be precleaned by extracting in a Soxhiet overnight
with methylene chloride.
Chap. 8 - 129
-------
30cm
a a
glass wool
“lcrnLd.
— wool
sodium sulfate
(13 cm)
“..85g
Teflon
Figure 8.1. Sodium sulfate drying column.
(8) Boiling chips — Hengar granules are ground with a mortar and
pestle and sieved to obtain a 60/80 mesh. These granules are
cleaned by extracting 5 g of chips three times with l50 mL
methylene chloride.
(9) Water bath - Heated, with concentric ring cover, capable of
temperature control (±2°C). The bath should be used in a hood.
(10) Balance - analytical, capable of accurately weighing to the
nearest 0.001 g.
(11) One manifold with a temperature controlled water bath for nitrogen
blowdown. A manifold with nine spaces is preferred but not
essential.
(12) Pasteur pipettes.
(13) One 5 mL pipette for spiking water samples with internal standard
solution.
Chap. 8 - 130
-------
(14) Eight glass vials (1 dram) with 13 mm screw caps (Supelco 2-3213)
and Teflon lined rubber septa (Supelco 2—3216).
(15) Diazomethane generation/distillation apparatus (Aldrich Macro
Diazoid set Zl0,851-0, Aidrichimica Acta 14 No. 3 1981) all
clear glass seals (Caution the presence of ground glass or
scratched glass may catalyze explosive reaction of diazomethane).
(16) Four ring stands or ring supports suitable for supporting a 2000
mL separatory funnel.
(17) Nine 10-15 mL centrifuge tubes with screw caps.
(18) One gas chromatographic column - 30 m x 0.32 mm I.D. DB-5
(1.0 p film thickness) fused silica capillary column.
(19) Ten mL volumetric flasks for preparing standard solutions.
(20) Syringes for preparing standard solutions -
(21) Graduated pipettes for preparing standard solutions.
(22) Thirty 15 niL glass vials with Teflon lined screw caps for storing
standard solutions.
(23) One gas chromatograph suitable for capillary column chromatography
with flame ionization detection and all required accessories
including syringes, gases, and a strip chart recorder.
(24) Nine Kuderna-Danish (K-D) apparatus. The apparatus consists of:
(a) Three-ball macro-Snyder columns (Kontes No. K503000) -
300 mm length with 24/40 joints.
(b) Evaporation flask (Kontes No. K570002) - 500 niL with 24/40
top joint and 19/22 lover joint.
Cc) Concentrator tube (Kontes No. 1 (570000) - 4 niL graduated,
with 19/22 joint. Attach to evaporator flask with springs
(Kontes No. 1(503000-0232). Ground glass stopper (size
19/22 joint) is used to prevent evaporation of extracts.
(25) Materials and Reagents
(a) 1.5 L methylene chloride (Burdick and Jackson, distilled in
glass).
(b) 80 niL concentrated HC1 (high purity).
(c) 100 niL 6N NaOH. Weigh 24 g of NaOH into a 100 niL volumetric
flask and dilute to the mark with reagent water. (High
purity).
Chap. 8 - 131
-------
(d) 80 g anhydrous sodium sulfate.
Ce) 5 niL internal standard solution (Table 4.2, Chapter 4 or if
available, NES Ampoules as given in Table 4.6, Chapter 4).
(f) 1 inL external standard solution: 4-fluoro-2-iodotoluene
(30 mg) and 2-fluorobiphenyl (30 mg) in 10 niL CH 2 C1 2 .
(g) 1 mL surrogate standard solution (Table 4.4, Chapter 4).
(h) 1 niL system performance solution (Table 8.1).
(i) 5 L methyl t-butyl ether (MBE) (Burdick and Jackson, dis-
tilled in glass).
(j) sodium chloride, 1.6 kg.
Table 8.1. GC/MS SYSTEM PERFORMANCE SOLUTION FOR ESSAa
Density
Compound (@ 20°C)
Concentration
pg/mL in Methanol
2,6-dimethyiphenol sb 300
acetophenone 1.030 310
1-tetradecano]. 0.823 250
1-octadecene 0.789 240
n-octadecane 0.777 230
DFTPP S 500
n-eicosane S 300
n-heneicosane S 300
methyl stearate S 10
d -heptanoic acid 0.948 280
(methyl ester)
d -benzoic acid 1.13 340
(methyl ester)
4 -f luoro-2-iodoto] .uene 0.883 260
methyl decanoate 0.873 870
a 5 Table 8.6 for the function of each test component in the
system performance evaluation.
bSoljd at room temperature.
Chap. 8 - 132
-------
(k) 5 mL gaseous HC1 in methanol (1.0 H)
(1) -tolysulfony1methylnitrosamide (Diazald®, Aldrich).
(in) 10 mL 60% KOH (w/v). Dissolve 6 g KOIt in 10 rnL reagent
water.
(n) Diethyl ether (peroxide free). To insure peroxide free
ethyl ether dry pack 25 g of Woelm W200 Basic Alumina
Super I into a glass column 100 cm X 20 mm i.d. Eulte with
500 mL of ethyl ether discarding the first 25 mL and collect-
ing the remaining ether in an amber glass bottle. Add
absolute ethanol (2% v/v) to preserve the solvent and cap
with a Teflon lined screw cap. Store cold (4°C).
(o) Reagent water - reagent water is defined as water that does
not produce a background interference at the limit of
detection. A commercial water purification system (Hillipore
Super-Q or equivalent) may be used to gen rate reagent
water.
(p) 5 mL phenolphthalein solution (0.05 g/50 niL ethanol + 50 niL
water).
(q) 1% formic acid solution in diethyl ether.
(r) Test external standard — 2.0 mg/mL of benzyl acetate in
MBE.
8.3 PREPARATION FOR ANALYSIS
8.3.1 Preparation of System Performance Standard (Table 8.1)
(1) Prepare individual stock solutions (12 mg/mI).
- solids — accurately weigh 0.120 g of pure material, dissolve
in methylene chloride, dilute to volume in a 10 niL volumetric
flask.
— liquids — with a 250 pL syringe accurately measure 120 IJL
of pure liquid; dissolve in methylene chloride, dilute to
volume in a 10 niL volumetric flask.
(2) Prepare a secondary standard. With a 1 mL graduated pipette
accurately measure 0.25 niL of each stock standard into a 10 niL
volumetric flask. For DFTPP measure 0.42 niL. Measure 0.83 niL
of methyl decanoate. Dilute to volume using methylene chloride.
Chap. 8 — 133
-------
This should give the concentrations shown in Table 8.1. Transfer
into a Teflon sealed screw cap bottle. Store at 4°C.
(3) If compound purity is 96% or greater, the weight can be used
without correction to calculate the concentration of stock
standards. If the compound is less than 96% pure, it cannot be
used as a standard. Transfer the stock standards into Teflon
sealed screw cap bottles, store at 4°C.
8.3.2 Cleaning of Materials
(1) All glassware to be used should be washed with Amway S-A-8
laundry compound (or equivalent), rinsed with deionized water
and heated for a minimum of 4 hours at 500 to 550°C. All cleaned
glassware should be capped immediately or covered with foil to
prevent contamination.
(2) Teflon liners and Teflon lined septa are sonicated for 10 minutes
in pesticide grade methanol followed bj 10 minutes in pesticide
grade pentane. The sonicated liners are vacuum-oven (—20 inches
of water) dried for 3 to 5 hours at 70° and stored in clean,
Teflon lined screw cap bottles.
(3) Anhydrous sodium sulfate is cleaned and dried prior to use.
Approximately 1000 g of material is needed to process nine
samples and is the minimum amount of material which should be
prepared. The following instructions describe procedures based
on 1000 g of material; however, it is possible to prepare larger
batches and store the prepared material in a sealed Erlenmeyer
flask. If a larger quantity of material is prepared, volumes
for rinsing should be adjusted accordingly.
Sodium sulfate (1000 g) is placed in a 1 L Erlenmeyer flask
with a ground glass joint. Methylene chloride (500 mL) is
added, the flask is swirled for 5 minutes, and the solvent
decanted. The procedure is repeated two additional times. To
dry the sodium sulfate, the cleaned material is heated in the
flask in a drying oven at 130°C overnight. The clean, dried
material can be stored in the drying oven, or stoppered and
stored in a dessicator.
Chap. 8 - 134
-------
To prepare drying columns, a small piece of pre-extracted
glass wool is placed in the bottom of the chromatography column,
13 cm of sodium sulfate is poured into the column, followed by
an additional plug of pre-extracted glass wool. Columns should
be used immediately or stored in a drying oven at 130°C. Rinse
the Na 2 SO 4 with 50 mL of extracting solvent just prior to using
the column.
(4) Glass wool is cleaned by placing in the extraction chamber of a
Soxhlet extractor and extracting overnight with metbylene chlo-
ride. The glass wool is placed in a wide mouth jar and the
solvent removed either under nitrogen stream or by warming. Cap
and store until needed.
8.3.3 GC/FID Performance Evaluation
Prior to analyzing procedural blanks, acceptable performance for the
GC/FID system must be demonstrated.
(1) Analyze the GC/HS system performance solution (SPS) as specified
in Table 8.2. Figure 8.2 shows a total ion chromatogram of this
mixture analyzed by GC/MS under similar conditions which may be
used to identify test components in the standard.
Table 8.2. GC/FID OPERATING CONDITIONS FOR
EXTRACTABLE SEMIVOLATILE STRONG ACIDS (ESSA)
GC Column 30 in DB-5 (1.0 p film thickness)
fused silica, wide bore (0.34 mm
I.D.), capillary column
GC Carrier Gas lleliuin
Carrier Gas Flow 1.6 inL/min column; 15:1 split
Temperature Program 50°C/5 mm to 250°C @ 4°/mm
Injector Temperature 250°C
Detector Temperature 260°C
Injection Volume 1.0 pL
Chap. 8 —135
-------
100.,
Cd
0
Cd
( -C
0)
4 )
rs
I’
4 )
f tC. t —
4) 1.—
4 )0
—
4 ) 4 )
4)
4.. 0.
4) ‘-4 ’-. .4 Cd 4)
E —. U
4) - 4)41 4)
C — I . .4
‘0 4 ) ) 0 ) Cd 0 41.4 >.. 4)
. 4 — u. - . 0. 0
a’ ‘ > Cd 410 0
C d 00 .C U . —
4) ‘4). 4. 4 0) C d l
o C .4 ,O 00) •0 4)
I C
.4 0 U ‘0 .4 Cd 0 C
i 4)
o C Cde’ 0.4 0 Cd i
0) .4 ‘0 d
. U I I 0) — 01 C
0. “I (‘4 , 0 4. 1 0 41
0.0 0 I I .41 LJ
4)4 ) l 0 (‘1 —, 4)fl 1.4
• U 0) 0
‘.1 Ii..
‘)Cd .0
CI I 1.4
-I I —
i 1
U) 44..
It l••’
‘0
io i:
.C 4) C I ..
II! k
Lt .L ___
_______ LJL
1 r— —T - - —-——r-————-r--——---— 1 -— —i——— r—-, , - • — —r —
1000 1500 2 00 SCAtI
16:40 25:O 33:2 41:40 UHF.
Figure 8.2. Total ion chromatogram of ESSA SPS.
-------
(2) Measure peak asymmetry or tailing for acetophenone of 1-tetrade-
canol using the percent peak asymmetry factor (PAF):
% PAF = x 100
where
B = the width of the back of a chromatographic peak to the
perpendicular from the peak measured at 10% above base-
line.
F = the width of the front of a chromatographic peak to the
perpendicular from the peak measured at 10% above base-
line.
PAF should measure less than 200% for both acetophenone and
l—tetradecanol.
(3) Check the acidity/basicity of the column by the peak area ratios
determined by integrator or triangulation of 2,6-dimethyiphenol
to acetophenone. A ratio of 0.7 to 1.3 is acceptable.
(4) Sensitivity is checked by measuring signal to noise ratios for
meatyl stearate. A signal-to-noise ratio of 10 is acceptable.
8.3.4 Procedural Blanks
For each lot of materials and reagents that are used, a set of three
procedural blanks are required. Table 8.3 identifies these blanks and
defines their purpose. These blanks must be processed and analyzed by
GC/FID prior to processing any samples. If a large number of samples are
to be processed, it is advantageous to prepare large lots of materials and
reagents thereby reducing the number of blanks which must be run. Proce-
dural blank 1 must also be run every time a batch of samples is processed
and analyzed.
(1) Prepare a separatory funnel rinse with 100 mL methylene chloride
and discard. Perform extraction with reagent water described in
step 8.4.1.5 through 8.4.1.20 and the derivatization procedure
in section 8.4.2.
(2) Procedural blank 2 is prepared from reagent water (920 mL)
placed in a solvent bottle and extracted as a sample.
(3) Procedural blank 3 is prepared in the same way as 2 except that
the reagent water is spiked with 1 mL each of the sulfuric acid,
Chap. 8 — 137
-------
Table 8.3. PROCEDURAL BLANKS
Blank
Description
Procedural
Blank
1
detects contamination in solvents
and glassware
Procedural
Blank
2
detects contamination in reagent
water
Procedural
Blank
3’
detects contamination in sulfuric
acid, sodium hydroxide, and sodium
thiosulfate solutions used during
sample collection and analysis
sodium hydroxide, and thiosulfate solution to be used in sample
collection. This blank is also extraction as a sample.
(4) Add 10 .iL of the GC external standard to each blank concentrate
after derivatization and concentration to 1 mL. Agitate the
K-D receiver to give a uniform distribution of standard in the
concentrates.
(5) Analyze the samples by GC/FID using the conditions in Table 8.2.
If significant contamination (>20% relative to external standard)
is present in procedural blank 1, reclean glassware and check
solvents and sodium sulfate. If contamination is present in
procedural blank 2 which is not in procedural blank 1 use another
source of reagent water. Procedural blank 3 contamination
should measure less than 20% relative to the external standard.
Excessive contamination of this blank requires procurement of
fresh solutions of sulfuric acid, sodium hydroxide, and sodium
thiosulfate. If the contamination persists, obtain materials
from another lot/source.
8.3.5 Preparation of Gaseous HC1:Methanol
HC1:methanol solution is usually prepared in large batches and may be
stored in the refrigerator up to 2 weeks between use. Because HC1 is
corrosive, the entire operation should be performed in a well-ventilated
hood. Methanol (500 mL) is added to a 1 L amber glass bottle, and the
bottle is chilled in an ice bath. Gaseous HC1 is bubbled through the
methanol as illustrated in Figure 8.3. HC1 is allowed to dissolve into
Chap. 8 - 138
-------
Pasteur pipet
gaseous HCI
magnetic
Stirrer
Teflon
tubing
HCI
lecture
bottle
I-
‘ .0
Safety trap
Teflon
tubing
mthano l
ice/H 20
Figure 8.3. Equipment for generating IJC1:mcthanol.
-------
the methanol for -15 minutes. The HC1 concentration of the methanol
solution is determined by adding 1 mL of the methanol solution to 20 mL of
distilled-deionized water, and titrating to the phenolphthalein endpoint.
A normality from 0.8 to 1.2 is acceptable for the analytical operation.
If the solution is stored, the normality should be checked before use.
Smaller amounts may be prepared in a similar manner.
8.3.6 Quality Control on the Derivatization Reaction
Before beginning the analysis of any samples, a procedural blank, a
derivatization control, and a derivatization blank are processed through
the procedure. The procedural blank is reagent water spiked with internal
standards processed through the procedure in 8.4. The derivatization
control is a 1.0 mL aliquot of a standard solution, (Table 8.4) with 0.30
mL of gaseous HC1 (1.0 M)/methanol added. The derivatization blank is 1.0
mL of NBE with gaseous HC1(l.0 N)/methanol added. These three samples are
derivatized by the procedure in 8.4.2 and analyzed by GC/FID to determine
the level of the background and the conversion of the acids to methyl
esters and ethers. An aliquot (25 ijL) of benzyl acetate (2.0 mg/niL in
MBE) is added to each sample before GC/FID analysis. The GC/FID response
ratio which must be attained for the derivatization control are given in
Table 8.4.
8.4 SAMPLE EXTRACTION A} D DERIVATIZATION
8.4.1 Sample Extraction
(1) Mark the menicus of the aqueous liquid on the sample bottle with
an indelible marker.
(2) Adjust the pH of the sample to “ .10 with 6 N NaOH using narrow
range pH paper to check the pH.
(3) Immediately, transfer the entire contents of the sample bottle
to the 2000 mL separatory funnel.
(4) Immediately, extract ( - 6O-120 cpm as required to avoid emulsions)
for 2 minutes with 100 mL of methylene chloride and discard the
methylene chloride.
(5) Immediately adjust the pH to <4 with concentrated HC1 using wide
range pH paper to check pH. Lapsed time from step 2 through
step 5 should not exceed 15 minutes. It is advisable to work
Chap. 8 - 140
-------
Table 8.4. RELATIVE GC/FID RESPONSES FOR ACID
SURROGATE COflPOU?4DS
Compound
Densities
(@ 20°C)
Concentrationa
(pg/mL)
Res’ponse Ratiob
Range
trichioroacetic acid
S
500
0.12 -
0.15
benzoic acid
S
500
0.75 -
0.88
n-octanoic acid
0.906
450
0.70 -
0.87
2,4-dichlorophenol
S
500
0.56 -
0.64
2-nitro-2-creso l
S
500
0.78 -
0.90
2,4-D
S
500
0.52 -
0.66
2,4,5-T
S
500
0.40 -
0.65
ap i of solution -
(1) Accurately weigh or volumetrically measure with a microliter
syringe each compound into a 50 mL volumetric flask (25 mg or
25 iL, respectively).
(2) Dissolve these materials in methanol and dilute to the mark.
b . ( Peak area compound)(pg benzylacetate )
Response Ratio =
(Peak area benzylacetate)(pg compound)
The range is defined by ± 3 standard deviations of triplicate determina-
tions. Response ratio computed using peak heights instead of areas.
with one sample at a time through these steps to minimize the
time which the sample is at an elevated pH.
(6) Add 200 g of NaCl and dissolve.
(fl Add concentrated HCl to make the final concentration 0.111 in
HC1. For a 1.0 L sample add 8.3 mL of concentrated HC1.
(8) Extract sample with 300 mL of methyl t-butyl ether.
(9) Drain aqueous layer into the sample bottle tapping the sides of
the separatory funnel to release droplets which may adhere to
the sides. Remove the aqueous layer as completely as possible.
(10) Drain the MEE through a glass wool plug into an amber bottle.
(11) Transfer the aqueous sample back into the separatory funnel and
repeat steps 8 through 10 combining extracts.
Chap. 8 — 141
-------
(12) Add —5 g of anhydrous Na 2 SO 4 to the combined extracts.
(13) Rinse Na 2 SO 4 drying column with 25 mL of ? [ BE.
(14) Transfer ‘4/2 the extract through the Na 2 SO 4 drying column into
a 500 mL K-D flask equipped with 4 mL concentrator tube containing
several boiling chips. The concentrator tube should be precali-
brated to compensate for the volume of the boiling chips.
Attach the three-ball macro-Snyder column. Place the K-D
apparatus on a warm water bath with the concentrator tube par-
tially immersed in water or on a steam bath. Adjust the tempera-
ture of the water bath such that evaporating solvent causes the
balls in the Snyder column to chatter actively but does not
flood the chambers of the column. Under these conditions evapo-
ration of 500 mL of MBE to 2 mL should take 20 to 30 minutes.
(15) When the liquid has reached an apparent volume of 2 mL remove
the K-D apparatus from the heat and allow to cool. Remove the
Snyder column.
(16) Transfer the remaining extract to the K-D apparatus through the
drying column and repeat steps 14 and 15.
(17) With 1 to 2 niL of solvent, rinse the Snyder column, the evapora-
tion flask, and its lower joint into the concentrator tube. The
final volume in the concentrator tube should be approximately 4
mL.
(18) Attach a modified micro-Snyder column to the K-D concentration
tube. Connect a transfer pipette by tubing to a nitrogen mani-
fold. Place the transfer pipette in the modified Snyder column
above the extract level (Figure 8.4). Gently blow a stream of
nitrogen above the extract surface until the volume is reduced
to approximately 1 niL. Rinse the sides of the concentrator tube
with approximately 0.5 mL of solvent. Reduce volume of extract
to 1.0 niL.
(19) Add 0.30 niL of gaseous HC1 in methanol (1.0 N) to each extract.
(20) Fill sample bottle to the mark with water. Transfer contents to
a graduated cylinder and record the volume to the nearest 5 niL.
Chap. 8 - 142
-------
Figure 8.4. Nitrogen blowdown with modified micro-Snyder column.
8.4.2 Derivatization
Caution! Diazomethane is toxic and prone to cause develapment of
specific sensitivity; in addition, it is potentially explosive. Hence one
should wear solvent impermeable gloves and goggles while performing this
experiment and should work behind a safety screen or a hood door with
safety glass. Also, it is recommended that ground glass joints and sharp
surfaces be avoided. Thus all glass tubes should be carefully fire-
polished, connections should be made with rubber stoppers or clear glass
seals, and separatory funnels should be avoided, as should etched or
scratched flasks. Furthermore, at least one explosion of diazomethane has
been observed at the moment crystals (sharp edges!) suddenly separated
from a supersaturated solution. Stirring by means of a Teflon—coated
magnetic stirrer is greatly to be preferred to swirling the reaction
mixture by hand, for there has been at least one case of a chemist whose
hand was injured by an explosion during the preparation of diazomethane in
a hand-swirled reaction vessel.
It is imperative that diazomethane solutions not be exposed to direct
sunlight or placed near a strong artificial light, because light is thought
to have been responsible for some of the explosions that have been encoun-
tered with diazomethane. Particular caution should be exercised when an
Chap. 8 - 143
-------
organic solvent boiling higher than ether is used. Because such a solvent
has a lower vapor pressure than ether, the concentration of diazomethane
in the vapor above the reaction mixture is greater and an explosion is
more apt to occur.
Most diazomethane explosions take place during its distillation.
When distilled diazomethane is required, the present procedure is particu-
larly good because at no time is much diazomethane present in the distilling
flask.
(1) Assemble the diazomethane
(Figure 8.5).
(2) Place 10 mL fresh 60% KOH
with 35 mL ethanol and 10
stirring bar.
(3) Place ice/salt mixture in
flask in a ice/salt bath.
Ic.IsaIt b.th
generation/distillation apparatus
(v/v in water) in the distilling flask
mL of diethyl ether. Add a magnetic
the condenser and place the collection
Chap. 8 - 144
t.hoi .r.
Id/salt
Vint
Figure 8.5. ZlO,85l—0 Macro Diazald® set (with Clear_Seal® joints).
-------
(4) Prepare a solution of 21.5 g (0.1 mole) of -tolylsu1fonylmethyl-
nitrosamide (Diazald®, Aldrich) in 125 mL of diethyl ether.
Larger amounts of ether may be needed to dissolve the reagent if
it is especially pure or the temperature is less than 20°C.
Place the Diazald® solution in the dropping funnel.
(5) The distilling flask is heated in a water bath at 70-75°C, the
stirrer is started.
(6) The Diazald® solution is added at a regular rate approximately
equal to the distillation rate over a 15-20 minute period. As
soon as this solution has been added, add 100 rnL of ether to the
dropping funnel and continue to add the ether at the same rate
used for the reagent.
(7) Continue distillation until the distillate is colorless.
(8) Cool the collection flask in an ice/salt bath during collection.
Remove the collection flask and keep in ice bath until used.
Destroy any residues of diazomethane in the apparatus by rinsing
with 1% formic acid in diethyl ether.
(9) With a Pasteur pipette add the distilled diazomethane-ether
solution to the sample extracts until a yellow color persists.
For highly colored samples add the diazomethane solution until
nitrogen evolution ceases. Additional diazomethane should be
prepared as needed. Excess diazomethane is evaporated from
samples using nitrogen blowdown. If excess diazomethane reagent
has been prepared it should be quenched with formic acid solution
(1% in ether) before discarding. The solution will be colorless
when fully quenched.
(10) Concentrate the derivatized samples to 1.0 mL in the test tubes
using nitrogen blowdown at ‘ 30°C. Since excess diazomethane is
still present in the samples this step must be performed in a
well ventilated hood.
(11) Store the samples at 0°C until analysis.
(12) Add an aliquot (25 pL) of external standard (4-fluoro-2-iodotolu—
ene) solution to the sample concentrate immediately before
analysis.
Chap. 8 - 145
-------
8.5 GC/MS/COHP ANALYSIS
Prior to beginning GC/MS/COMP analysis of the extractable acid fraction
the analyst should consult the general procedures described in Chapter 13
entitled “(GC) 2 /MS/COMP Analysis-General for All Protocols”.
8.5.1 Preparation for Analysis
8.5.1.1 ( GC) 2 /MS/COMP Operating Parameters
The recommended GC/MS operating parameters are given in Table 8.5 for
the analysis of the extractable semivolatile strong acid fractions (ESSA).
Gas chromatographic capillaries which meet the system performance criteria
may be substituted for the recommended fused silica (See Section 13.4). It
is recommended that this capillary be reserved for use only with sample
extracts containing methyl esters and ethers of strong acids, as it tends to
become acidic with continued analysis of such extracts.
Table 8.5. GC/1IS OPERATING CONDITIONS FOR
EXTRACTABLE STRONG ACIDS AS THEIR METHYL DERIVATIVES
GC Column 30 m DB-5 (1.0 p film thickness)
fused silica wide bore (0.34 mm
I.D.) capillary column
GC Carrier Gas Helium
Carrier Gas Flow 1.6 mL/min — column; 9:1 split
Carrier Gas Sweep Time 85 secs (50°C)
Temperature Program 50°C/5 mm; to 240°C @ 4°/mm
and hold
Injector Temperature 250°C
Transfer Line Temperature 255°C
Injection Mode Splitless - 0.4 mm/split
Injection Volume 1.0 pL
Ionizing Energy 70 eV
Ion Source Temperature 250°C
Scan Range 35-500
Chap. 8 - 146
-------
8.5.1.2 MS Calibration
The mass spectrometer is calibrated using the manufacturer’s recom-
mended approach (Chapter 13). The acceptability of the calibration results
is verified upon analysis of the system performance solution (SPS).
8.5.2 Analysis of SPS (Quality Control )
Using the prescribed CC/MS conditions the SPS is analyzed and all
necessary data is acquired and evaluated prior to proceeding to sample
analysis. The SPS is analyzed at the beginning of each day’s operation.
The SPS raw data are extracted from the run, test parameters calculated
and plotted or tabulated (Tables 8.6 and 8.7). Up to 14 days of SPS
analytical results can be historically recorded in Table 8.6. This allows
the analyst to follow subtle trends which may develop and anticipation of
GC or MS maintanence required. Calculation of test parameters and discus-
sion of their use is given in Chapter 13.
A check of RNRs on a daily basis is also calculated from SPS raw data
and tabulated (Table 8.7) and compared to that set developed for the his-
torical data bank. The usage of this data is also discussed in Chapter 13.
8.5.3 Determination of RMRs
RuBs for the User’s data bank (as opposed to the daily R 1R checks
discussed in the above paragraph) are determined by injecting a solution
containing the derivatized internal standards and analytes. Preparation
of standard solutions is the same as described in the footnote of Table 8.4.
The GC/HS/CONP operating parameters are given in Table 8.5. Table 13.8
may be used to tabulate RMR raw data.
8.5.4 Analysis of Field and Quality Control Samples
After meeting all specific criteria for GC and MS performance
(Table 8.6), then analysis of standard solutions (e.g., for RNR calcula-
tions) or (ESSA) sample extracts is conducted.
A set of sample extracts may have been derived from laboratory and
field blanks and controls, surrogates and collected field samples, and are
generally analyzed in that respective order.
Qualitative and quantitative procedures are described in Chapter 13.
Tables 8.8 may be used to tabulate sample analyte raw data prior to using
the MASQUANT software program or manual quantitation.
Chap. 8 - 147
-------
Table 8.6. GC-MS SYSTEM PERFORMANCE TEST FOR EXTRACTABLE
SEMIVOLATILE STRONG ACIDS (ESSA)
Dates:
Run Id Code
GC Column
and
Program:
Notebook
References:
PIS SYSTEM CHECKS
High/Low Mass Balance
Ion
51
68
70
127
197
198
199
275
365
441
442
443
68 70
30% to 60% of m/z 198
<2% of mfz 69
<2% of m/z 69
40% to 50% of m/z 198
<1% of m/z 198
100%
5-9% of rn/a 198
10% to 30% of rn/a 198
at least 1% of cs/a 198
present, but < rn/a 443
>40% of rn/a 198
17% to 23% of m/z 442
199 275 365 331 442
DYTPP
Relative Abundance Criteria
rn/a 51 — 127 197 198 — — — . il Pass/ Remedial
Abundance * ( ) j•J .L.l LI ..LJ. L .1 L_1. .LJ_ .LJ. j_ jJ jJ Fail Action
Criteria
*These abundances are initially established by the HAS user, and subsequently become guidelines for
acceptability of tune.
(continued)
Chap. 8 - 148
-------
Table 8.6 (cont’d.)
Test Compoaenti Criteria
Limit of
Detection Methyl Stearate S:N (m/z 74) >40:1
55
50
z
45
40
I I - I I I I I I I I I
Day
CC SYSTE)I HEcXS
Peak 1. Acetophenone (TIC) • VAP <300
Asynmetry 2. 1—Tetradecanol (TIC I JA2 <200
300 No.1
200 No.2
0.
p
100
I I I I I I I I I I 1 I p —
Day
1. Acetophenone (TIC)
Basicity* 2. 2,6-Dimethylphenol (TIC) Ratio 2:1 = 0.7 to 1.3
1.4
1.2
0
-a
0.8
0.6
I I I I I I I I I
Day
*Note. Acidity criteria are also easured in other MAS fractions.
(continued)
Chap. 8 - 149
-------
Table 8.6 (cont’d.)
Teat Cc ponents Criteria
Separation n-Eico.ane (rn/a 43)
Number n-Seueicosane (rn/a 43) SN ) 6
12
11
10
9
8
7
6
I I I I I I I I I I I I I
Day
n-Octadecane (TIC) R = rninimum 50%
Resolution 1—Octadecene (TIC) Valley
90
80
u
70
a
> 60
dP
40
I I I I I I I U I T I I I
Day
Inertness Methyl Stearate Ratio rn/a 74 to 298 < 14:1
20
18
0
—16
a
14
12
I I I I I I I I I I I I I
Day
(continued)
Chap. 8 - 150
-------
Test Cowpoaents Criteria
Capi 1 is ry
Capacity Methyl Decanoate (TIC) %PAF >70
100
90
80
70--
I I I I I I I I I I I I I
Day
Table 8.7. GC-MS SYSTEM PERFORMANCE TEST: RNR CHECK
FOR SEMIVOLATILE STRONG ACIDS(ESSA)
Date: Run ID Code:
DATA
Amount Ion Area Area
Standard MW (ng) pM (m/z) (Run 1) (Run 2)
4-Fluoro-2-Iodo- 236 109
toluene (FIT) 236
d 13 —heptanoic acid 143 77
91
d -benzoic acid 127 82
110
MATRIX OF STANDARD ION RMRs
Standard Ion 109 236 77 91 82 110
FIT 109
236
-
-
d 13 —heptanoic acid 77
91
—
—
d 5 -benzoic acid 82
110
—
A MW • A = ion area or height
RIIR X X y
x/y A ?Iw • ng ng = ng injected
y y x xana lyte
y = standard
COMMENTS:
Chap. 8 - 151
-------
Table 8.8. RAW ANALYTE DATA FOR ESSA FRACTION
Date:
Run I.D. Code:
Notebook Reference:
Vol. Water Processed
Sample Identification:
Vol. Water CL):
(to which i were added)
(mL):
STANDARDS
Spec-
I.D. trum
No. No.
4-Fluoro-2-Iodo-
Weight Ions
(ng ,) (m/z) Area
MW;
Ka
toluene
d 13 —Heptanoic
Acid
d 5 —Benzoic Acid
109,236:
77, 91: ,______
82,110:
.
,
,
,
ANALYTES
Class No.
I.D.
No.
Spectrum Ions
No. (mfz) Area
i .i a
, :
,
,
,
,
,
,
,
,
,
,
, :
,
,
a 1 f calculations are performed manually then this information is also
needed, whereas MASQUANT performs functions automatically.
A MW n A = ion area or height
ng = X A ng = ng recovered from volume
x/y y y of water processed
x = analyte
= y y = standard
A • MW
y y
Chap. 8 - 152
-------
CHAPTER 9
EXTRACTION AND ANALYSIS OF WEAK ACIDS, BASES AND NEUTRALS (WARN)
This section contains three extraction protocols (Parts A, B, and
C), one of which is selected for the extraction of organics from water
depending upon the water quality as assessed during sampling and sample
scouting. All of the protocols allow for the recovery of weak acids,
weak bases and neutral compounds (WARN) at pH 8.
The sorbent accumulator column (SC) protocol Is used for extracting
organics from drinking water samples. For other water samples, the emul-
sion index (determined just before sample processing) determines the
sample’s propensity for emulsification, and If that is the case, the
sample is extracted using the continuous flow—under (FIJ) protocol. Other-
wise, the batch liquid—liquid partitioning (BL) (separatory funnel)
protocol is employed. A clean—up protocol (Part D) is provided for both
FU and BL extracts; a screening test is prescribed in both extraction
protocols to determine whether clean—up is necessary. Part E of this
chapter is for GC/MS/COMP analysis of all WARN extracts.
Chap. 9 — 153
-------
A. SORBENT ACCUMULATOR COLUMN (WABN-SC)
9.1 INTRODUCTION
9.1.1 Principle of the Technique
This method describes the determination of pH 8-extractable (WABN)
organic compounds in drinking water samples. Resin materials are used to
adsorb and concentrate organic compounds from 11.5 L water samples. A
nominal 0.5 ppb detection limit is achieved using this size sample.
Although the method has been developed specifically for drinking water,
other water types, such as some surface waters may also be processed
depending upon desired sample size, organic content, and turbidity. The
sample pH is adjusted to 8 and the sample passed through a 10 mL Amberlite
XAD-4 macroreticular resin column. Excess water is aspirated off the
column and adsorbed organics are eluted using 12 mL of methanol followed
by 200 mL methylene chloride. The eluants are combined, then evaporated
to a volume of 4 niL using a Kuderna-Danish evaporator and further concen-
trated to 0.5 mL by nitrogen blowdown using a modified micro-Snyder column.
Neutral organics, weak acids, and bases are analyzed using this procedure.
9.1.2 Detection Limit
If the detection limit for GC/?IS analysis is estimated at 10 ng total
material for extractable organics, and samples are concentrated from an
original volume of 11.5 L to a final extract volume of 0.5 niL, the overall
nominal detection limit for this procedure would be 0.5 ppb for a 1 pL
injection.
Detection limits,may be decreased by further increasing sample size
to 50 to 100 L. If this approach is taken, column accumulation should be
performed in the field due to the prohibitive cost of shipping the samples.
Breakthrough characteristics using large water volumes are unknown and
recoveries may be significantly decreased.
9.1.3 Interferences
No interferences have been observed using the developed procedure. A
procedural blank must be run prior to processing any samples to assure
that contamination from resin materials, solvents, reagents, glassware,
and other sources is low.
Chap. 9 - 154
-------
9.1.4 Precision, Accuracy, and Scope
Table 1.5 in Chapter 1 presents average recoveries and standard
deviations for a variety of extractable organics. This method gives
acceptable recoveries for a variety of neutral organics, as well as, weak
and strong bases (i.e., trimethyl aniline, tributylamine, etc.), and weak
acids (i.e., t-butyl phenol).
9.2 APPARATUS AND REAGENTS
The following-materials are required for processing a set of nine
samples plus two procedural blanks. Nine is the maximum number of samples
which should be processed at a time. The quality control samples that
should be run during these analyses are listed in Table 4.1, Chapter 4.
The two procedural blanks should be run before any samples are collected
or processed. The extraction/concentration procedure will require two
working days to process nine samples.
(1) Nine glass accumulator columns —1 cm I.D. X 20 cm with a teflon
stopcock and a 24/40 ground glass joint at the top. Each
chromatography column should have an adaptor (Figure 9.1) for
siphoning the samples onto the resin.
(2) One vacuum aspirator to start sample flow through the resin
column.
(3) Nine separatory funnels (250 or 500 mL) with teflon stopcocks
and ground glass joints (24/40) to connect to the resin column
(Figure 9.2).
(4) One 10 mL graduated pipette with the tip cut off for measuring
and transferring resin material.
(5) Nine 500 mL amber bottles with Teflon lined screw caps.
(6) Glass wool ( ‘-‘50 mL) for resin. Glass wool should be precleaned
by extracting in a Soxhlet overnight with methylene chloride.
(7) Boiling chips - Hengar granules are ground with a mortar and
pestle and sieved to obtain a 60/80 mesh. These granules are
cleaned by extracting —‘5 g with 150 mL of methylene chloride.
(8) One Soxhlet extractor for cleaning resin material and glass
wool. A minimum 200 mL capacity is required. However, resins
can be processed in large batches and stored; therefore a larger
size preferred.
Chap. 9 - 155
-------
Tflon ibIn
24MC.1sU
nrmctaon
L
I
—1
Ld.
Teflon
typon bing to aphito?
Figure 9.1. Accumulator column with siphoning adaptor.
Chap. 9 - 156
-------
500 mL glass
dropping funnel
24/40
glass connection
Teflon stopcock
22cm
Figure 9.2. Accumulator column with separatory funnel.
T
— 1 cm i.d.
Teflon
stopcock
Chap. 9 - 157
-------
(9) Nine Kuderna-Danish apparatus. The apparatus consists of:
(a) Three-ball macro-Snyder columns (Kontes #K503000) - 300 mm
length with 24/40 joints.
(b) Evaporative flask (Kontes #K570002) - 500 mL with 24/40 top
joint and 12/22 lower joint.
Cc) Concentrator tube (Kontes #K570000) - 10 mL graduated, with
19/22 joint. Attach to evaporative flask with springs
(Kontes K503000-0232). Ground glass stopper (size 19/22
joints) is used to prevent evaporation of extracts.
(10) Nine modified micro—Snyder columns (Kontes #K569251) with 19/22
ground glass joint.
(11) One heated water bath or steam bath with concentric ring cover
capable of temperature control. Bath should be located in a
hood.
(12) One pH meter with a combination electrode.
(13) One manifold with temperature controlled water bath or aluminum
heating block for nitrogen blowdown. A manifold with nine
spaces is preferred but not essential.
(14) Pasteur pipettes.
(15) Twelve 1 dram glass vials with screw caps (Supelco 2-3213) and
Teflon lined rubber septa (Supelco 2-3216).
(16) Volumetric flasks for preparing standard solutions - 10 mL.
(17) Syringes for preparing standard solutions - 50 and 250 giL.
(18) Graduated pipettes for preparing standard solutions - 1 inL.
(19) Thirty 15 mL glass vials with Teflon lined screw caps for storing
standard solutions.
(20) One gas chromatograph suitable for capillary column chromatog-
raphy with flame ionization detection and all required accessories
including syringes, gases, and a strip chart recorders.
(21) One 30 m x 0.34 mm I.D. DB-l (1.0 p film thickness) fused silica
capillary column.
(22) Materials and Reagents
(a) Reagent water - reagent water is defined as a water source
that does not produce a background interference at the
limit of detection. A water purification system (Millipore
Chap. 9 — 158
-------
Super-Q or equivalent) may be used to generate reagent
water.
(b) 1.5 L methanol (Burdick and Jackson, pesticide grade).
(c) 500 mL ethyl acetate (Burdick and Jackson, pesticide grade).
(d) 3 L methylene chloride (Burdick and Jackson, pesticide
grade).
(e) 120 mL Amberlite XAD- 4 macroreticular resin (mesh 20-60,
Rohxn and Haas, Philadelphia, Pennsylvania).
(f) 100 mL 0.lN H 2 S0 4 in reagent water.
(g) 100 mL 0.1N NaOH in reagent water.
(h) 200 niL of 1.0 N pH 8.0 phosphate buffer. Weigh 74.4 g of
K 2 HPO 4 and 7.64 g of KH 2 PO 4 in a 200 niL volumetric flask
and dilute to the mark with reagent water.
(1) 10 mL Na 2 S 2 O 3 solution - 0.05 N in reagent water [ use the
same solution as specified in Chapter 4, section 4.2(14)(f)].
(j) 1 niL external standard solution — 4-fluoro-2-iodotoluene
(300 ng/10 pL).
(k) 1 niL GC/FID external standard solution — hexadecane (30
ng/lO pL) in C11 2 C1 2 .
(1) 10 mL system performance solution (Table 9.1).
9.3 PREPARATION FOR ANALYSIS
9.3.1 Preparation of WABN System Performance Solution
(1) Prepare individual stock standards (12 mg/mI).
— solids - accurately weigh 0.120 g of pure material, dissolve
in methylene chloride, dilute to volume in a 10 niL volumetric
flask.
— liquids — with a 250 pL syringe accurately measure 120 pL
of pure liquid, dissolve in methylene chloride, dilute to
volume in a 10 niL volumetric flask.
(2) Prepare a secondary standard. With a 1 mL graduated pipette
accurately measure 0.25 niL of each stock standard into a 10 mL
volumetric flask [ for quinoline and 6—t-butyl-rn-cresol use 0.8
niL, for 4-fluoro-2-iodotoluene use 125 pL (measure with a 250 pL
syringe), for 2,4,6-trimethylpyridine and rn-cresol use 25 pL
(measure with a 50 iL syringe), and for methyl stearate use
Chap. 9 - 159
-------
Table 9.1. SYSTEM PERFORMANCE SOLUTION FOR WARN FRACTIONa
Density
Compound (@ 20°C) Concentration (ng/ iL)
2,6-dimethylphenol S ’ 300
2,6-dimethylaniline 0.984 300
acetophenone 1.030 310
1—tetradecanol 0.823 240
1-octadecene 0.789 240
n—octadecane 0.777 230
DFTPP S 300
n—eicosane S 300
pyrene S 300
n-heneicosane S 300
methyl stearate S 10
d 10 -o-xylene 0.897 270
d 8 -naphtha lene S 300
d 5 -nitrobenzene 1.204 360
d 5 -phenylethanol 1.023 310
d 5 -propiophenone 1.009 300
d 5 -acetophenone 1.030 310
d 12 -perylene S 300
4-fluoro-2—iodotoluene 0.883 270
d 9 -acridine S 300
d 6 -phenol S 300
quinoline 1.095 1090
2,4,6—trimethylpyridine 0.917 23
rn-cresol 1.034 26
6-t-butyl-rn-cresol S 1000
aSee Table 9.11 for the function of each test component in the
system performance evaluation.
b
S solid.
Chap. 9 - 160
-------
10 i.JL (measure with a 50 i.iL syringe)]. Dilute to volume
using methylene chloride to give the concentrations in
Table 9.1. Transfer into a Teflon sealed screw cap bottle for
convenience in use. Store at 4°C.
(3) Transfer the stock standards into Teflon sealed screw cap bottles
for convenience in use. Store at 4°C.
(4) Fresh standard should be prepared every six months. If degrada-
tion or evaporation has occurred, fresh standards should be pre-
pared sooner.
(5) If compound purity is 96% or greater, the weight or volume can
be used without correction to calculate the concentration of
stock standards. If the compound is less than 96% pure, it
cannot be used as a standard.
9.3.2 Preparation of Resin Material
Amberlite XAD-4 resin material is cleaned prior to preparing the
resin columns. The minimum amount of resin that should be prepared is
120 mL which is the amount needed to process nine water samples plus two
procedural blanks. The following instructions describe procedures based
on 120 mL of resin material; however, it is possible to prepare larger
batches and store the prepared resin under methanol in a sealed glass jar.
If a larger volume of resin is prepared, volumes of cleaning solvents
should be adjusted proportionately.
Place the resin material in a 500 mL Erlenmeyer flask. Make a slurry
of the resin with 250 mL of distilled water. Stir the resin gently to
avoid fracturing beads. After allowing the resin to settle for 30 seconds,
remove fines by decantation. Repeat the slurry—decanatation procedure
three times. Then rinse the resin three times with 250 mL portions of
methanol. Place the resin in a Soxhlet extractor and extract with pesticide
analysis grade ethyl acetate for eight to sixteen hours (cycle time: —10
minutes). Then extract the resin with pesticide analysis grade methanol
for an additional eight to sixteen hours (cycle time: —10 minutes).
Store cleaned resin in a sealed glass jar under methanol with a Teflon
lined screw cap.
Chap. 9 - 161
-------
9.3.3 Cleaning of Materials
(1) Glassware to be used is washed with Aniway S-A-8 laundry compound
(or equivalent), rinsed with deionized water and baked for
a minimum of 4 hours at 500 to 550°C. All cleaned glassware is
immediately capped or covered with foil (precleaned with hexane)
to prevent contamination.
(2) Teflon liners and teflon lined septa are sonicated for 10 minutes
in pesticide grade methanol followed by 10 minutes in pesticide
grade pentane. The sonicated liners are vacuum-oven (-20 inches
of water) dried for 3 to 5 hours at 70° and stored in clean,
Teflon lined screw cap bottles.
9.3.4 GC/FID Performance Evaluation
Prior to analyzing procedural blanks, acceptable performance for the
GC/FID system must be demonstrated.
(1) Analyze the GC/HS performance standard (Table 9.1) as specified
in Table 9.2. Figure 9.3 shows a total ion chromatogram of this
mixture analyzed by GC/MS under similar conditions which may be
used to identify test components in the standard.
(2) Measure peak asymmetry or tailing for acetophenone and 1-tetra-
decanol using the percent peak asymmetry factor (PAP):
% p y = x 100
where
B = the width of the back half of a chromatographic peak
measured at 10% above baseline.
F = the width of the front half of a chromatographic peak
measured at 10% above baseline.
PAP should measure less than 200% for both acetophenone and
l-tetradecanol.
(3) Check the acidity/basicity of the column by the peak area ratios
determined by integrator or triangulation of 2,6-dimethylaniline
and 2,6-dimethylphenol to acetophenone. A ratio of 0.7 to 1.3
for both is acceptable.
Chap. 9 - 162
-------
i08.
all
sc, tl
BHE
w
‘- 4
0
w
U
-I
4 - i
i-i
0
w
C
-4
4)
p.
C
1 c0
•0
• .0
0 ”
l A)
‘-4
0
C
U
w
‘C
I .
4)
w
i -i
‘ -I
Figure 9.3.
Total ion chromatogram of WABN SPS (EA = experimental analyte).
-------
CC Column
CC Carrier Gas
Carrier Gas Flow
Temperature Program
Injector Temperature
Detector Temperature
Injection Volume
30 m DB—l (1.0 p film thickness)
fused silica, wide bore (0.34 mm
1.0.), capillary column
Helium
1.6 mL/min through column; 15:1
split injection
50°C/5 mm to 250°C @ 4°/mm
250°C
260°C
1.0 I.iL
(4) Sensitivity is checked by measuring signal-to-noise ratios for
2,4,6-trimethylpyridine, rn-cresol, and methyl stearate. A
signal-to-noise ratio of 10 to 1, or more for all three compounds
is acceptable.
9.3.5 Procedural Blanks
For each lot of materials and reagents that are used, a set of three
procedural blanks are required. Table 9.3 identifies these blanks and
defines their purpose. These blanks must be processed and analyzed by
GC/FID prior to processing any samples. If a large number of samples are
to be processed, it is advantageous to prepare large lots of materials and
reagents thereby reducing the number of blanks which must be run. Proce-
dural blank 1, however, must be run every time a new batch of samples is
processed and analyzed.
9.3.5.1 Procedural Blank 1--
(1)
Prepare a resin column as described in step 9.4.1, then follow
the extraction procedure as described in steps 9.4.6 to 9.4.15.
(2) With a 50 pL syringe, add 10 pL of the GC external standard
[ 9.2(22)(k)] to the blank concentrate. Agitate the K-D receiver
to give a uniform distribution of standard in the concentrate.
(3) Analyze samples by GC/FID using the conditions described in
Table 9.2. Contaminant peak heights should measure less than
20% relative to the internal standard.
Table 9.2. GC/FID OPERATING CONDITIONS FOR
EXTRACTABLE WEAK ACIDS, BASES AND NEUTRALS
Chap. 9 - 164
-------
Table 9.3. PROCEDURAL BLAN1(S
Blank Description
Procedural Blank 1 detects contamination in resin
material, solvents, and glass-
ware
Procedural Blank 2 detects contamination in
reagent water
Procedural Blank 3 detects contamination in sulfu-
ric acid, sodium hydroxide,
sodium thiosulfate, and phos-
phate buffer solutions used
during sample collection and
analysis
(4) If the blank is acceptable, add a 10 jiL aliquot of the PIS external
standard solution [ 9.2(22)(j)] using a 50 lJL syringe. Agitate
the K-D receiver to give a uniform distribution of standard in
the concentrate. Transfer a measured portion (0.3 to 0.4 mL) of
the concentrate to a 1 dram vial and cap. Store at 0°C until
GC/MS analysis.
(5) If significant contamination is present, the procedure should be
repeated using a blank chromatography column with no resin
added. If this blank is acceptable, then the resin is the
source of contamination and should be recleaned using the proce-
dure in Section 9.3.2. A procedural blank 1 should then be
repeated using the fresh resin material.
(6) If significant contamination is present in this blank, fresh
reagents should be used and the reagent blank (no resin) repeated.
When the reagent blank is acceptable, a procedural blank 1 must
be repeated. Procedural blanks 2 and 3 may be processed once
the procedural blank 1 is acceptable.
9.3.5.2 Procedural Blanks 2 and 3--
(1) Prepare a resin column as described in step 9.4.1. Attach a
separatory funnel to the top of the column. For procedural
blank 2, pour 100 mL of reagent water into the separatory funnel,
then drain the sample through the resin bed at a flow rate of
--10 mL/minute. For procedural blank 3 use 100 mL of reagent
Chap. 9 - 165
-------
water spiked with 1 mL each of the sulfuric acid, sodium hydrox-
ide, and sodium thiosulfate solutions, and 10 mL of the phosphate
buffer solution. The first three must be the same solutions
which were used when the samples were collected (Chapter 4).
See 9.2(22)(h) for phosphate buffer solution.
(2) Follow the extraction procedure as described in steps 9.4.6 to
9.4.15.
(3) With a 50 liL syringe, add 10 pL of the GC external standard
[ 9.2(22)(k)] to the blank concentrates. Agitate the K-D receiver
to give a uniform distribution of standard in the concentrates.
(4) Analyze the samples by GC/FID using the conditions in Table 9.2.
Peak heights for contaminants in Procedural blank 3 which are
not present in procedural blank 2 should measure less than 20%
relative to the internal standard.
(5) If significant contamination is present in procedural blank 3
which is not present in procedural blank 2, the procedure should
be repeated using fresh solutions of sulfuric acid, sodium
hydroxide, sodium thiosulfate, and phosphate buffer until an
acceptable blank is achieved.
9.4 SAIfPLE EXTRACTION
(1) Prepare resin column by placing a small glass wool plug in the
bottom of the chromatography column. Using a graduated pipette
with the tip cut off, pipette 10 mL (measure volume after resin
has settled) of cleaned XAD-4 resin into the open tubular chroma-
tography columns, allow the resin to settle, and rinse the
column with —50 mL of reagent water.
(2) Add 10 mL of the phosphate buffer solution to the sample. Mix
well. Check the pH of the sample using a pH meter. Adjust the
sample to 8 using 0.1N H 2 S0 4 or 0.1N NaOH.
(3) Attach siphoning adaptor (Figure 9.1) between sample containers
and resin column. Attach a piece of tygon tubing between the
end of the chromatography column and a water aspirator. To
start sample flowing through the resin, open column stopcock and
turn on aspirator. This should create sufficient suction to
Chap. 9 - 166
-------
pull the water sample through the siphon apparatus onto the
resin column. Once flow starts disconnect the aspirator.
(4) Using the stopcock at the base of the column, adjust flow through
the column to —10 iaL/minute. At this flow it should take —18
to 20 hours to pass the sample through the resin bed. Sample
may be discarded after elution through the column.
(5) After the sample has passed through the column, remove the
siphon adaptor from the column. If any sample remains in the
containers, transfer it to the separatóry funnel, attach the
funnel to the resin column as illustrated in Figure 9.2, and
pass remaining sample through the resin bed.
(6) Once all sample has passed through the column, remove the separa-
tory funnel and attach a piece of tygon tubing between the end
of the column and a water aspirator. Aspirate any remaining
water off the column (aspirate for 2 to 3 minutes). It is
important to remove all water during this procedure.
(7) Add 12 niL of methanol to the dried resin bed. Stopper the
column and shake to remove any air bubbles. Equilibrate the
column for 10 minutes.
(8) Rinse the containers for each sample with a total of 200 m l
methylene chloride. Pour the methylene chloride rinse into the
separatory funnel.
(9) Drain all of the methanol from the resin bed. Collect the
eluate in a 500 ml glass bottle with a Teflon lined screw cap.
(10) Add —15 niL methylene chloride to the resin bed, allow air bubbles
to escape. Place a glass wool plug on the top of the resin bed.
(11) Collect —5 niL of methylene chloride from the separatory funnel,
then return to the separatory funnel. This removes any entrained
water from the stopcock and connector joint.
(12) Attach the separatory funnel to the column. Elute the column
with the methylene chloride. Stop eluting when the methylene
chloride reaches the top of the resin bed. Do not elute any
water through the resin. Collect eluant in the 500 ml bottle
containing the methanol rinse. Eluant may be stored at 0°C
after this operation.
Chap. 9 — 167
-------
(13) Transfer the eluant to a 500 mL K-D flask equipped with a 10 mL
concentrator tube containing several glass boiling beads. Pre-
calibrate the concentrator tube to compensate for the volume of
the glass boiling beads. Rinse the bottle with three 5 rnL
portions of methylene chloride and transfer these rinses to the
K-D flask. Attach the three-ball macro-Snyder column. Place
the K-D apparatus on a warm water bath with the concentrator
tube partially immersed in water or on a steam bath. Adjust the
temperature of the water bath áuch that evaporating solvent
causes the balls in the Snyder column to chatter actively but
does not flood the chambers of the column. Under these conditions
evaporation of 500 mL of methylene chloride to 2 mL should take
15 to 20 minutes.
(14) When the liquid has reached an apparent volume of 2 mL remove
the K-D apparatus from the heat and allow to cool. Remove the
Snyder column. With 1 to 2 mL of solvent, rinse the evaporation
flask and its lower joint into the concentrator tube. The final
volume in the concentrator tube should be approximately 4 mL.
(15) Attach a modified micro-Snyder column to the K-D receiver.
Connect a transfer pipette to the nitogen manifold using Teflon
tubing. Place the pipette in the modified Snyder column above
the solvent level (Figure 9.4). Gently blow a stream of nitrogen
above the solvent surface until the volume is reduced to approxi-
mately 1 mL (CAUTION: contamination can occur if tygon tubing
is used to connect the manifold to the nitrogen source). Rinse
the sides of the concentrator tube with approximately 0.5 mL of
solvent. Reduce volume of eluant to 0.5 mL using nitrogen
blowdown. Record final volume.
(16) With a 50 IiL syringe, add 10 pL of MS external standard solution
to the sample concentrate. Agitate the K-D receiver to give an
uniform distribution of external standard in the sample concen-
trate. Transfer a measured portion (0.3 to 0.4 mL) of the
sample concentrate to a 1 dram vial and cap. Store at 0°C until
GC/MS analysis.
Chap. 9 - 168
-------
(17) Proceed to Section E - GC/NS/COMP ANALYSIS OF WARN-SC, WABN-BL,
or WABN-FU SANPLE EXTRACTS.
E n
i
II
III I
II S
I I
‘I,
p
—
—
Figure 9.4. Nitrogen blowdown with modified micro-Snyder column.
Chap. 9 - 169
-------
B. BATCH LIQUID-LIQUID EXTRACTION (WABN-BL)
9.5 INTRODUCTION
9.5.1 Principle of the Method
Batch liquid—liquid extraction is used to extract and concentrate
weak acids, some bases, and neutral (WARN) organic compounds from aqueous
samples that are not prone to emulsion formation, including surface waters
and some effluents and hazardous waste leachates. The 1 L sample is
collected in a 1 quart bottle with 60 mL of a keeper solvent (35% methylene
chloride in hexane). The extraction is performed by first transferring
the keeper solvent and sample to a 2 L separatory funnel, draining the
sample back into the sample bottle, transferring the keeper solvent to a 1
L bottle, and returning the aqueous sample to the separatory funnel. The
pH of the aqueous sample is adjusted to 8.0 and the sample extracted with
three 200 mL portions of methylene chloride. The methylene chloride
extracts are combined with the keeper solvent, dried by passing through an
anhydrous sodium sulfate column and evaporated to —4 mL using a macro
Kuderna-Danish evaporator and further concentrated to 0.5—1.0 mL by nitro-
gen blowdown using a modified micro-Snyder column. The concentrated
extract is screened to determine if clean-up will be required by packed
coltunn gas chromatography/flame ionization detection. If clean-up is not
required the extract is analyzed by capillary column gas chromatography!
mass spectrometry, otherwise the protocol for sample clean-up is to be
implemented.
9.5.2 Detection Limit and Sample Size
Since all water types covered by this protocol have the same initial
sample size, detection limits will depend upon the final volume of sample
concentrate. If the detection limit for CC/MS analysis of each extractable
organic is estimated at 10 zig and samples are concentrated from an original
volume of 1 L, then using a 1 pL injection size, nominal detection limits
for specific water types are as follows:
5 ppb in surface waters (0.5 mL final volume)
10 ppb in industrial and municipal vastewaters, and energy effluents
(1.0 mL final volume)
Chap. 9 — 170
-------
If the sample cannot be concentrated to the specified volume due to high
levels of organic components, nominal detection limits are proportionately
higher.
9.5.3 Interferences
A clean-up fractionation procedure is specified which is implemented
when the chromatographable organics are present in high levels and would
interfere with GC/MS analysis. A procedural blank must be run prior to
processing any samples tp assure that contamination is low.
9.5.4 Precision, Accuracy, and Scope
Table 1.5 in Chapter 1 presents average recovery and standard devia-
tions for a variety of extractable compounds.
9.6 APPARATUS AND REAGENTS
The following materials are required for processing a set of nine
samples plus two procedural blanks. Nine is the maximum number of samples
which should be processed at a time. The quality control samples that
should be run during these analyses are listed in Table 4.1 of Chapter 4.
The two procedural blanks should be run before any samples are collected
or processed. The extraction/concentration procedure will require two
working days for a set of nine samples.
(1) Nine separatory funnels (2000 mL) with Teflon stopcocks.
(2) Nine 1 L amber glass bottles with Teflon-lined screw caps for
collecting the extracting solvent prior to solvent evaporation.
(3) One 1 L graduated cylinder for measuring sample volumes.
(4) Nine chromatographic columns for sodium sulfate solvent drying.
300 mm X 10 mm i.d. with a glass wool plug at the bottom, contain-
ing about 85 g Na 2 SO 4 (Fig. 9.5).
(5) One Soxhiet extractor (50 mL capacity minimum) for extracting
glass wool.
(6) Eighteen glass funnels to fit chromatography columns and separa-
tory funnels.
(7) Pre—extracted glass wool ( —50 mL) for drying columns. Glass
wool should be precleaned by extracting in a Soxhlet overnight
with methylene chloride.
(8) Boiling chips - Hengar granules are ground with a mortar and
pestle and sieved to obtain a 60/80 mesh. These granules are
Chap. 9 — 171
-------
dium sulfate
(13 cm)
85g
glass wool
Figure 9.5. Sodiun sulfate drying column.
cleaned by extracting -‘5 g three times with —150 mL methylene
chloride.
(9) Water bath - Heated, with concentric ring cover, capable of
temperature control (+2°C). The bath should be used in a hood.
(10) Balance - analytical, capable of accurately weighing to the
nearest 0.0001 g.
(11) One pH meter.
(12) One manifold with a temperature controlled water bath for nitro-
gen blowdown. A manifold with nine spaces is preferred but not
essential.
(13) Pasteur pipettes.
(14) One S mL pipette for spiking water samples with internal standard
solution.
30cm
glass wool
Teflon
Chap. 9 - 172
-------
(15) Fifteen glass vials (1 dram) with 13 mm screw caps (Supelco 2—
3213) and Teflon lined rubber septa (Supelco 2-3216).
(16) One gas chromatography column (180 cm X 2 mm i.d.) packed with
3% OV-l on Chromosorb W HP (80/100 mesh) or equivalent.
(17) One gas chromatograph with flame ionization detector and strip
chart recorder.
(18) Five ring stands or ring supports suitable for supporting a 2000
mL separatory funnel.
(19) Counter top centrifuge capable of ‘40O0 rpm.
(20) Nine 10-15 niL centrifuge tubes with screw caps.
(21) Ten mL volumetric flasks for preparing standard solutions.
(22) Syringes for preparing standard o1utions.
(23) Graduated pipettes for preparing standard solutions.
(24) Thirty 15 niL glass vials with Teflon lined screw caps for storing
standard solutions.
(25) One gas chromatograph suitable for capillary column chromatography
with flame ionization detection and all required accessories
including syringes, gases, and a strip chart recorders.
(26) One 30 m x 0.34 mm I.D. DB-l (1.0 p film thickness) fused silica
capillary column.
(27) Nine Kuderna—Danish apparatus [ see 9.2(9)].
(28) Materials and Reagents
(a) 6 L methylene chloride (Burdick and Jackson, distilled in
glass).
(b) 100 mL H 2 S0 4 solution (1 + 1). Add 50 mL of concentrated
H 2 S0 4 (sp. gr. 1.84) slowly to 50 mL of reagent water.
Cc) 100 mL 6N NaOH. Weigh 24 g of NaOH into a 100 niL volumetric
flask and dilute to the mark with reagent water.
Cd) 1000 g anhydrous sodium sulfate powder.
(e) 1 niL external standard solution: 4-fluoro-2-iodotoluene
(30 mg) and 2-fluorobiphenyl (30 nig) in 10 niL CH 2 C1 2 .
(f) Alkane calibration standard - dissolve 50 mg each of
n-decane, n-undecane, n-dodecane, n-tridecane, n-tetra-
decane, n-pentadecane, n-hexadecane, and n-heptadecane in
10 niL methylene chloride.
Chap. 9 - 173
-------
(g) 1 mL WABN system performance solution (Table 9.1).
(h) 90 mL of 1.0 ZI pH 8.0 phosphate buffer. Weigh 15.0 g of
K 2 HPO 4 and 1.91 g of K11 2 P0 4 in a 100 mL volumetric flask
and dilute to the mark with reagent water.
(i) Reagent water - reagent water is defined as a water source
which does not produce a background interference at the
limit of detection. A water purification system (Nillipore
Super-.Q or equivalent) may be used to generate reagent
water.
9.7 PREPARATION FOR ANALYSIS
9.7.1 Preparation of WARN System Performance Solution
(1) Prepare individual stock standards (12 mg/mL) of the compounds
listed in Table 9.1.
- solids - accurately weigh 0.120 g of pure material, dissolve
in methylene chloride, dilute to volume in a 10 mL volumetric
flask.
- liquids - with a 250 pL syringe accurately measure 120 pL
of pure liquid; dissolve in methylene chloride, dilute to
volume in a 10 mL volumetric flask.
(2) Prepare secondary standard with a 1 mL graduated pipette accurate-
ly measure 0.25 mL of each stock standard into a 10 mL volumetric
flask (for quinoline and 6-t-butyl-rn-cresol use 0.8 mL, for 4-
fluoro-2-iodotoluene use 125 I.iL [ measure with a 250 pL syringe],
for 2,4,6- trimethylpyridine and rn-cresol use 25 pL [ measure
with a 50 pL syringe], and for methyl stearate use 10 IJL [ measure
with a 50 pL syringe]). Dilute to volume using methylene chloride
to give the concentrations in Table 9.1. Transfer into a Teflon
sealed screw cap bottle. Store at 4°C. Fresh standard should
be prepared every six months. If degradation or evaporation has
occurred, fresh standards should be prepared sooner.
(3) If compound purity is 96% or greater, the weight or volume can
be used without correction to calculate the concentration of
stock standards. If the compound is less than 96% pure, it
cannot be used as a standard. Transfer the stock standards into
Teflon sealed screw cap bottles for convenience. Store at 4°C.
Chap. 9 - 174
-------
9.7.2 Cleaning of ?laterials
(1) Glass glassware to be used is washed with Amway S-A-8 laundry
compound (or equivalent) rinsed with deionized water and baked
for a minimum of 4 hours at 500 to 550°C. All cleaned glassware
is immediately capped or covered with foil to prevent contamina-
tion.
(2) Teflon liners and Teflon lined septa are sonicated for 10 minutes
in pesticide grade methanol followed by 10 minutes in pesticide
grade pentane. The sonicated liners are vacuum-oven (‘-20 inches
of water) dried for 3 to 5 hours at 700 and stored in clean,
Teflon lined screw cap bottles.
(3) Anhydrous sodium sulfate is cleaned and dried prior to use.
Approximately 1000 g of material is needed to process eight
samples and is the minimum amount of material which should be
prepared. The-following instructions describe procedures based
on 1000 g of material; however, it is possible to prepare larger
batches and store the prepared material in a sealed Erlenmeyer
flask. If a larger quantity of material is prepared, volumes
for rinsing should be adjusted accordingly.
Sodium sulfate (1000 g) is placed in a 1 L Erlenmeyer flask
with a ground glass joint. Methylene chloride (600 mL) is
added, the flask is swirled for 5 minutes, and the solvent
decanted. The procedure is repeated two additional times. To
dry the sodium sulfate, the cleaned material is heated in the
flask in a drying oven at 130°C overnight. The clean dried
material can be stored in the drying oven, or stoppered and
stored in a dessicator.
To prepare drying columns, a small piece of pre—extracted
glass wool is placed in the bottom of the chromatography column,
13 cm of sodium sulfate is poured into the column, followed by
an additional plug of pre—extracted glass wool. Columns should
be used immediately or stored in a drying oven at 130°C. Rinse
the Na 2 SO 4 with 50 mL of extracting solvent just prior to using
the column.
Chap. 9 - 175
-------
(4) Glass wool is cleaned by placing in the extraction chamber of a
Soxhlet extractor and extracting overnight with distilled in
glass methylene chloride. The glass wool is placed in a wide
mouth jar and the solvent removed either under nitrogen stream
or by warming. Cap and store until needed.
9.7.3 GC/FID Performance Evaluation
Prior to analyzing procedural blanks, acceptable performance for the
GC/FID system must be demonstrated.
(1) Analyze the GC/MS performance standard (Table 9.1) as specified
in Table 9.2. Figure 9.3, (Part I), shows a total ion chromato-
gram of this mixture analyzed by GC/MS under similar conditions,
which may be used to identify test components in the standard.
(2) Measure peak asymmetry or tailing for acetophenone and 1-tetra-
decanol using the percent peak asymmetry factor (PAF):
% PAF = x 100
where
B = the width of the back of a chromatographic peak to the
perpendicular from the peak measured at 10% above
baseline.
F = The width of the front of the chromatographic peak to
the perpendicular from the peak measured at 10% above
baseline.
the PAl should measure less than 200% for both acetophenone and
1—tetradecanol. -
(3) Check the acidity/basicity of the column by the peak area ratios
determined by integrator or triangulation of 2,6—dimethylaniline
and 2,6-dimethyiphenol to acetophenone. A ratio of 0.7 to 1.3
for both is acceptable.
(4) Sensitivity is checked by measuring signal to noise ratios for
2,4,6-trimethylpyridine, rn-cresol, and methyl stearate. A
signal-to—noise ratio of 10 or greater for all three compounds
is acceptable.
Chap. 9 - 176
-------
9.7.4 Procedural Blanks
For each lot of materials and reagents that are used, a set of three
procedural blanks are required. Table 9.4 identifies these blanks and
defines their purpose. These blanks must be processed and analyzed by
GC/FID prior to processing any samples. If a large number of samples are
to be processed, it is advantageous to prepare large lots of materials and
reagents thereby reducing the number of blanks which must be run. Proce-
dural Blank 1, however, must also be run every time a new batch of samples
is processed and analyzed.
9.7.4.1 Procedural Blank 1-—
(1) Rinse separatory funnel with keeper solvent (60 mL of 35% methyl-
ene chloride in hexane) and drain into 1 L amber glass bottle.
Follow the extraction procedure as described in steps 4.2.4 to
4.2.14. (Use same lots of the keeper solvent as used during
sample collection.)
(2) Add 10 IJL of the GC external standard to the blank concentrate.
Agitate the K-D receiver to give a uniform distribution of
standard in the concentrate.
(3) Analyze samples by GC/FID using the conditions described in
Table 9.2. Contaminant peak heights should measure less than
20% relative to the external standard.
(4) If procedural blank 1 is acceptable, add an aliquot (10 pL) of
the MS external standard solution. Agitate the K-D receiver to
give a uniform distribution of standard in the concentrate.
Table 9.4. PROCEDURAL BLANXS
Blank Description
Procedural
Blank
1
detects contamination in solvents
and glassware
Procedural
Blank
2
detects contamination in reagent
water
Procedural
Blank
3
detects contamination in sulfuric
acid, sodium hydroxide, sodium thio-
sulfate, and phosphate buffer solu-
tions used during sample collection
and analysis
Chap. 9 - 177
-------
Transfer the concentrate to a 1 dram vial and cap. Store at 0°C
until GC/HS analysis.
(5) If significant contamination- is present, the procedure should be
repeated using freshly cleaned glassware.
(6) If significant contamination is still present in this blank,
fresh reagents (different lots or sources of solvents) should be
used. When procedural blank 1 is acceptable, procedural blanks
2 and 3 may be processed.
9.7.4.2 Procedural Blanks 2 and 3--
(1) Procedural blank 2 is prepared from reagent water (920 mL)
placed in a sample bottle. Cover with 60 inL of “keeper solvent”
(35% methylene chloride:65% hexane). Procedural blank 3 is
prepared in the same way as 2 except that the reagent water is
spiked with 1 mL each of the surfuric acid, sodium hydroxide,
and sodium thiosulfate to be used for sample collection (see
Chapter 4), and 10 mL of phosphate buffer [ 9.2(27)(i)J.
(2) Follow the extraction procedure as described in steps 9.8.2(2)
to 9.8.2(14).
(3) Add 10 pL of the GC external standard to the blank concentrate.
Agitate the K-D receiver to give a uniform distribution of
standard in the concentrate.
(4) Analyze the samples by GC/FID using the conditions in Table 9.2.
Peak heights for contaminants in procedural blank 3 which are
not present in procedural blank 2 should measure less than 20%
relative to the e cterna1 standard.
(5) If significant contamination is present in procedural blank 3
which is not present in procedural blank 2, the procedure should
be repeated using fresh solutions of sulfuric acid, sodium
hydroxide, sodium thiosulfate, and phosphate buffer until an
acceptable blank is achieved.
9.8 SANPLE EXTRACTION
9.8.1 Determination of Emulsion Prone Samples
All water samples except drinking water must be screened prior to
processing to separate emulsion prone samples from samples which can
adequately be extracted using the separatory funnel method. The scouting
Chap. 9 — 178
-------
sample (5 mL) is placed in a 10 mL glass centrifuge tube with screw cap
and the sample pH adjusted to pH 8 with 6N NaOH or 1:1 H 2 S0 4 . ?lethylene
chloride (2 ml) is added, the vial sealed and the mixture shaken for two
minutes (—60 cycles per minute). If the solvent and aqueous sample separate
into two discrete layers in less than 15 minutes then the separatory
funnel extraction may be used without modification. If two layers do not
form, centrifuge for 1 minute at —4000 rpm and inspect again. If two
layers have formed, mark samples as slightly emulsion prone. If no separa-
tion of layers occurs then use the flow-under extractor designed for
emulsion prone samples (Chapter 9, Part C).
9.8.2 Sample Extraction
(1) Hark the interface between the “keeper” solvent and water on the
sample bottle with an indelible marker.
(2) Transfer the entire contents of the sample bottle into a 2000 ml
separatory funnel and drain the lower aqueous layer back into
the sample bottle. Tap the separatory funnel several times to
remove water drops adhering to the sides. When all of the water
has been removed drain the solvent into a 1 L amber glass bottle
through a Na 2 SO 4 drying column.
(3) Return the sample to the separatory funnel and adjust the pH to
8.0 using 6N NaOH or 1:1 H 2 S0 4 . Check the pH with narrow range
pH paper. Add 10 mL of 1.0 M pH 8.0 phosphate buffer.
(4) Rinse the sample bottle with 200 mL of methylene chloride and
transfer to the separatory funnel.
(5) Shake the separatory funnel at —120 cpm for 2 minutes, venting
as required. For samples which are slightly emulsion prone
shake gently (30-45 cpm) for 5 minutes.
(6) Return separatory funnel to ring stand and allow phases to
separate for 30 minutes. Proceed with the extraction of other
samples (steps 1-6). Four or five samples can be processed
through this sequence with maximum utilization of time.
(7) Drain the methylene chloride through the same drying column
(step 2) into the 1 L bottle, combining it with the “keeper”
solvent.
Chap. 9 - 179
-------
(8) Add 200 niL of methylene chloride to the separatory funnel and
extract again as described in steps 5-7. Repeat for a third
extraction.
(9) Rinse Na 2 SO 4 drying column with 25 niL of methylene chloride to
complete extract transfer.
(10) Transfer —1/2 the dried extract to a 500 niL K-D flask equipped
with 4 niL concentrator tube containing several Hengar granules.
The concentrator tube should be precalibrated to compensate for
the volume of the granules. Attach the three-ball macro-Snyder
column. Place the K-D apparatus on a warm water bath with the
concentrator tube partially immersed in water, or on a steam
bath. Adjust the temperature of the water bath such that evapo-
rating solvent causes the balls in the Snyder column to chatter
actively but does not flood the chambers of the column. Under
these conditions evaporation of 500 niL of methylene chloride to
2 niL should take 15 to 20 minutes.
(11) When the liquid has reached an apparent volume of 2 niL remove
the K-D apparatus from the heat and allow to cool. Remove the
Snyder column.
(12) Transfer the remaining extract to the K-D apparatus and repeat
steps 10 and 11.
(13) With 1 to 2 niL of solvent, rinse the evaporation flask and its
lower joint into the concentrator tube. The final volume in the
concentrator tube should be approximately 4 niL.
(14) Attach a modified micro-Snyder column to the K-D receiver.
Connect a transfer pipette by Teflon tubing to a nitrogen mani-
fold. Place the transfer pipette in the modified Snyder column
above the liquid level (Figure 9.4). Gently blow a stream of
nitrogen above the liquid surface until the volume is reduced to
approximately 1 niL. Rinse the sides of the concentrator tube
with approximately 0.5 niL of solvent. Reduce volume of extract
to 0.5 for surface waters and to 1.0 niL for all other water
types. Record final volume.
(15) To determine whether the extract is so complex that it must be
cleaned up, a 1 pL injection is made on a gas chromatograph with
Chap. 9 — 180
-------
FID detection. The column (180 cm X 2 i.d.) is packed with
3% OV-1 on Chromosorb W HP. A temperature program from 30°C to
250°C at 10°/mm is used. The attenuation is set to give 30 to
70% of full scale deflection for the various peaks of the alkane
calibration standard.
(16) The chromatogram from step 15 is evaluated as follows: The
baseline rise (A) is estimated by connecting the valleys of the
chromatogram as shown in Figure 9.6. Draw a zero line (B)
horizontal to t the level of the baseline before injection. At
any point after the first 2 minutes of the chromatogram where A
is more than 5% of full scale above line B, begin an incremental
integration of the area between A and B. This is repeated in 5%
increments as indicated by the shaded areas in Figure 9.6.
Compute the area between A and B which is shaded (Area A-B).
Measure the area of the peaks produced by the alkane calibration
standard (Area C) by planimeter and compute the mg/mi equivalent
of the baseline rise MA by
MA — 40 mg/mi
AAB — Ac
Area - X 40 mg/mi
= AB
A Areas
A sample must be fractionated if > 50 mg/mi (go to Section D).
Otherwise it may be analyzed by GC/MS without further workup.
(17) Add 25 pL of uS external standard solution to the sample concen-
trate. Agitate the K-D receiver to give an uniform distribution
of external standard in the sample concentrate. Transfer a
measured portion of the sample concentrate to a one dram vial
and cap. Store at 0°C until GC/MS analysis.
(18) Proceed to SectionE - GC/MS/CONP ANALYSIS OF WARN-SC, WABN-BL, or
WARN-PU SAMPLE EXTRACTS.
Chap. 9 - 181
-------
Baseline rise equivalent to 285 mg/mt
Time (mm)
Figure 9.6.
OV-l packed column gas chromatogram of extract No. 8320 from a
timber industry wastewater (see text for conditions).
ro rd
p j
so
00
-------
- C. CONTINUOIJSLI.QUID-LIQUID EXTRACTION WITh FLOU-UNDER EXTRACTOR (WABN-FU) - —
9.9 INTRODUCTION
9.9.1 Principle of the Method
Weak acids, some bases, and neutral organic compounds are extracted
and concentrated from emulsion prone aqueous samples by using a continuous
liquid-liquid extraction technique with a flow-under extractor. (See
Part B, Section 9.8.1 for determination of emulsion prone samples.)
Because the flow-under extractor does not break the sample solvent inter-
face, it prevents the formation of emulsion. The composition of emulsion
prone wastevaters is extremely variable and these samples are subject to
much greater variations in recoveries due to matrix effects than are
wastewaters in general. For this reason the emulsion prone sample must be
analyzed in duplicate, with one of the samples spiked with the surrogate
compounds to determine recoveries.
The two samples are collected in 1 quart bottles and spiked with
internal standards; 60 mL of a keeper solvent (35% methylene chloride in
hexane). is then added and the sample shaken.
When ready for analysis, the keeper solvent is removed from the top
of the bottle using a transfer pipet. The samples are transferred to two
continuous liquid—liquid extractors (CLLE), adjusted to pH 8.0, and extrac-
ted with 500 mL of methylene chloride. After 3.5 hr of operation, the
methylene chloride is removed, combined with the keeper solvent, dried
over sodium sulfate, evaporated to a volume of 4 mL using Kuderna-Danish
evaporation techniques and further concentrated to 0.5 to 1.0 mL by nitro-
gen blowdown using a modified micro—Synder column. The extracts are
evaluated by packed coltimn GC/FID to determine if fractionation is required.
The concentrated extracts are analyzed by capillary column gas chromatog-
raphy/mass spectrome try.
9.9.2 Detection Limit and Sample Size
Since all water types covered by this protocol have the same initial
sample size, detection limit will depend upon the final volume of sample
concentrates. If the detection limit for GC/NS analysis is estimated at
10 ng total material for extractable organics and samples are concentrated
to 1.0 mL from an original volume of 1 L, then, using a 1 pL injection
Chap. 9 — 183
-------
size, the nominal detection limit for emulsion prone wastewaters is 10 ppb.
If the sample cannot be concentrated to the specified volume due to high
levels of organic components, nominal detection limits are proportionately
higher.
9.9.3 Interferences
A clean-up fractionation procedure is specified that is implemented
when the chromatographable organics are present in high levels and would
interfere with GC/MS analysis. A procedural blank must be run prior to
processing samples to assure that contamination is low.
9.9.4 Precision, Accuracy, and Scope
The precision and accuracy of this method are highly dependent upon
the particular matrix, so recoveries were determined during MAS development.
Recovery and reproducibility must be estimated for each sample matrix
through the use of surrogates and internal standards.
9.10 APPARATUS AND REAGENIS
The following materials are required for processing a set of four
samples (2 water samples and 2 surrogate spiked samples; however, only
one surrogate is required for each different sample matrix). Every set of
14 samples (7 water samples and 7 surrogate spiked samples) Dust contain
an appropriate number of procedural blanks and procedural controls (Chap-
ter 4, Table 4.1). The extraction/concentration procedure will require
one working day for four samples. The limiting equipment will probably be
the flow-under extractor. All other equipment and materials should be
scaled proportionately.
(1) Four flow-under extractors (RTI Extractor Schematic, Figure 9.7).
(2) Four mechanical stirrers (Eberback Model 58 or T-Line Model 101-
Talboys Engineering Company).
(3) Four heating mantles to hold a 500 mL round bottom flask.
(4) Four variable transformers.
(5) Four Friedrichs condensors (Corning 2640/with 24/40 inner joint
at bottom with drip tip and 24/40 female joint at vapor outlet).
(6) One 1 L graduated cylinder for measuring sample volume.
(7) Four 1 L Erylenmeyer flasks.
(8) Four 250 mL Erlenmeyer flasks.
Chap. 9 - 184
-------
10cm
T.TYPE TEFLON
STOPCOCK
r 30cm
—t
724/40 FEMALE
J GROUND GLASS
JOINT
X • NOT CRITICAL
DIMENSIONS
45!S0 FEMALE
GROUND GLASS
JOINT
I - f l
Figure 9.7. RTI extractor schematic.
-------
(9) Four long stem glass funnels.
(10) One 250 mL Erlenmeyer with ground glass joint.
(11) Four glass columns for sodium sulfate drying. Exact dimensions
are not critical; however, mass of sodium sulfate should be
approximately the same. Approximate dimensions are shown in
Figure 9.5.
(12) Four glass funnels to fit drying columns.
(13) Glass wool (—30 mL) for drying columns. Glass wool should be
precleaned by Soxhiet extracting overnight with methylene
chloride.
(14) Boiling chips - Hengar granules are ground with a mortar and
pestle and sieved to obtain a 60/80 mesh. These granules are
cleaned by extracting —5 g three times with —150 mL methylene
chloride.
(15) One Soxhiet extractor for cleaning glass wool. A minimum 50 ml
capacity is required. Glass wool can be processed in larger
batches and stored; therefore, a larger extractor is preferred.
(16) Four Kuderna-Danish apparatus. Each apparatus consists of
(a) Three-ball macro—Snyder columns (Kontes # K503000) - 300 mm
length with 24/40 joints,
(b) Evaporative flask (Kontes / K570002 — 500 ml with 24/40 top
joint and 19/22 lower joint,
(c) Concentrator tube (Kontes 11 K570000) - 4 ml graduated, with
19/22 joint. Attach to evaporative flask with springs
(Kontes 1(503000—0232. Ground glass stopper (size 19/22
joint) is used to prevent evaporation of extracts.
(17) Four modified micro-Synder columns (Kontes # 1(569251) with 19/22
joint.
(18) One heated water bath or steam bath with concentric ring cover
capable of temperature control. Bath should be located in a
hood.
One pH meter.
One manifold with a temperature controlled water bath or tempera-
ture-controlled aluminum block for nitrogen blowdown. A manifold
with eight spaces is preferred but not essential.
(19)
(20)
Chap. 9 - 186
-------
(21) Pasteur pipettes.
(22) Six 5 ml pipettes for spiking water samples with surrogate
compound standard solution.
(23) Four 1 dram glass vials with screw caps (Supelco 2—3213) and
Teflon lined rubber septa (Supelco 2-3216).
(24) One gas chromatograph suitable for capillary column chromatography
with flame ionization detection and all required accessories
including syringes, gases, and a strip chart recorder.
(25) One 30 m X 0.34 mm I.D. DB-l (1.0 p film thickness) fused silica
capillary column.
(26) One gas chromatograph for packed column chromatography with
flame ionization detection and all required accessories including
syringes, gases, and a strip chart recorder.
(27) One 180 X 0.2 cm i.d. glass gas chromatography column packed
with 3% OV-l on Chromosorb W HP (100/120 mesh) or equivalent.
(28) Materials and Reagents
(a) 3 1 methylene chloride (Burdick and Jackson, distilled in
glass).
(b) 100 mL H 2 S0 4 solution (1 + 1). Add 50 mL of concentrated
112504 (sp. gr. 1.84) slowly to 50 ml of reagent water.
Cc) 100 mL 6N NaO}1. Weigh 24 g of NaOH into a 100 ml volumetric
flask and dilute to the mark with reagent water.
(d) 50 g sodium sulfate, anhydrous powder.
(e) Internal standard solutions (Table 4.3, Chapter 4, or if
NBS Axnpoules are available, Table 4.7, Chapter 4).
(f) 1 ml MS external standard solution: 4-fluoro-2-iodotoluene
(30 mg).
(g) 5 ml surrogate standard solution (Table 9.5).
(h) 1 ml WABN system performance solution (Table 9.1).
(i) 4 ml of 1.0 M pH 8.0 phosphate buffer. Weigh 15.0 g of
K 2 HPO 4 and 1.91 g of KH 2 PO 4 in a 100 ml volumetric flask
and dilute to the mark with reagent water.
(j) Reagent water - reagent water is defined as a water source
which does not produce a background interference at the
limit of detection. A water purification system (Millipore
Chap. 9 - 187
-------
Table 9.5. SURROGATE STANDARD SOLUTION
Compoundsa
Pyridine 4-Chloro-3-methylphenol
a-Picoline -t-Butylphenol
2-Heptanone Phenyl carbamate
t-Butyl carbamate 1,2,4,5-Tetrachiorobeazene
2-n-Butoxyethanol Tridecane
t-Butylpropionate Nicotineb
Benzaldehyde Biphenyl b
Aniline 2,3 ,6-Trichlorophenol
Lutidine 2,6-Dinitrotoluene
2-Octanone 2, 4-Dimethyiquino line
Benzyl chloride Tetradecane
2,3 ,6-Trimethylpyridine Dimethyl phtha late
n-Decane Methyl- -toluene sulfonate
Salicylaldehyde 1, 8-Dimethylnaphthalene
Phenyl acetate Acenaphthene
Tolualdehyde 2, 4—Dinitrotoluene
2 ,3-Dihydrobenzofuran l-Naphthol
-Cresol Di-t-butyl-4-inethylphenol
Fenchone Ethyl-P-toluene sulfonate
3-Chlorobenzaldehyde N ,N-Diinethyldodecylamine
Isophorone n-Pentadecane
2-Bromo-l-chlorobenzene 2,4 ,5-Trimethylnaphthalene
o-Chloroanisole 1, 2-Dichloronaphthalene
4-Chlorobenzonitrile Fluorene
1, 4-Dicyanobutane 2-Aminobiphenyl
Di-t-butyldisulfide Diethylphtha late
2, 6-Dimethylaniline Diphenylamine
Benzyl acetate 4-Bromodiphenyl ether
2 ,4-Dichlorophenol n-Hexadecane
1,2, 4-Trichlorobenzene Tri-n-butyl phosphate
2 -Dibromobenzene Hexa chlorobenzene
2, 3-Dimethylphenol Dibenzylamine
rn-Chloroani line Atrazine
ci-Terpineol n-Heptadecane
Benzt hiozole Caffeine
o-Isopropy lphenol Benzyl sulfide
Quinoline Carbazole
Tributylamine n-Octadecane
Anisaldehyde 1, 8-Diaminonaplithalene
2-Nitrocresol Diphenylsulfone
Dimethyladipate Diphenyl mercury
Bis (2-chloroethoxy)ethane n-Nonadecane
3, 4-Dichlorobenzaldehyde Di-n-butyl phthalate
Indole Aldrin
n-Decanol n-Eicosane
2-Methylnaphthalene Pyrene
(continued)
Chap. 9 - 188
-------
9 , lO-Dimethylanthracene
Triphenylphosphate
n-Heneicosane
Methyl stea rate
Chrysene
Di(ethy lhexyl)phtha late
n-Docosane
Buty lbenzylphthalate
Tetraphenyl tin
Cholesterol
n-Tricosane
aEach compound is added to give approximately 100 .ig/mL as the
final concentration in methanol. Compounds are listed in elution
order on a DB-1 fused silica capillary column.
b
Coe lute.
Super-Q or equivalent) may be used to generate reagent
water.
(k) Alkane calibration standard - dissolve 50 mg each of
n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane,
n-pentadecane, n-hexadecane, and n-heptadecane in 10 mL
methylene chloride.
9.11 PREPARATION FOR ANALYSIS
9.11.1 Preparation of WARN System Performance Solution
(1) Prepare individual stock standards (12 mg/mL) of the compounds
listed in Table 9.1.
- solids - accurately weigh 0.120 g of pure material, dissolve
in methylene chloride, dilute to volume in a 10 mL volumetric
flask.
- liquids — with a 250 pL syringe accurately measure 120 pL
of pure liquid; dissolve in methylene chloride, dilute to
volume in a 10 mL volumetric flask.
(2) Prepare secondary standard - with a 1 mL graduated pipette accu-
rately measure 0.25 mL of each stock standard into a 10 m l
volumetric flask [ for quinoline and 6—t-butyl-rn-cresol use 0.8
mL, for 4-fluoro-2-iodotoluene use 125 pL (measure with a 250 pL
syringe), for 2,4,6-trimethylpyridine and rn-cresol use 25 pL
(measure with a 50 pL syringe), and for methyl stearate use 10
pL (measure with a 50 pL syringe)1. Dilute to volume using
Table 9.5 (cont’d.)
Compoundsa
Chap. 9 — 189
-------
methylene chloride to give the concentration in Table 9.1.
Transfer into a Teflon sealed screw cap bottle. Store at 4°C.
If degradation or evaporation has occurred, fresh standards
should be prepared sooner.
(3) If compound purity is 96% or greater, the weight or volume can
be used without correction to calculate the concentration of
stock standards. If the compound is less than 96% pure, it
‘cannot be used as a standard. Transfer the stock standards into
Teflon sealed screw cap bottles for convenience in use, store at
4°C.
9.11.2 Preparation of Surrogate Standard Solution
(1) Prepare individual stock standards (12 mg/mL) of the compounds
listed in Table 9.5.
— solids - accurately weigh 0.120 g of pure material, dissolve
in methylene chloride, dilute to volume in a 10 mL volumetric
flask.
- liquids - with a 250 pL syringe accurately measure 120 IlL
of pure liquid; dissolve in methylene chloride, dilute to
volume in a 10 mL volumetric flask. If the density of a
liquid is unknown, weigh an empty dry 250 LiL syringe, fill
with 120 IlL of the pure liquid and reweigh.
(2) Prepare a secondary standard by accurately pipetting 0.40 mL of
each stock standard into a 50 mL volumetric flask. Dilute to
the mark with methylene chloride and mix well. Transfer into a
Teflon sealed screw cap amber bottle and store at 4°C.
9.11.3 Cleaning of Naterials
(1) All glassware should be washed with Amway S-A-8 laundry compound
(or equivalent) rinsed with deionized water and heated for a
minimum of 4 hours at 500 to 550°C in a glassware oven. All
cleaned glassware is immediately capped or covered with foil to
prevent contamination.
(2) Anhydrous sodium sulfate is cleaned and dried prior to use.
Approximately 500 g of material is needed to process four samples
and is the minimum amount of material which should be prepared.
The following instructions describe procedures based on 500 g of
Chap. 9 - 190
-------
material; however, it is possible to prepare larger batches and
store the prepared material in a sealed Erlenmeyer flask. If a
larger quantity of material is prepared, volumes for rinsing
should be adjusted accordingly.
Sodium sulfate (500 g) is placed in a 1 L Erlenmeyer flask
with a ground glass joint. Methylene chloride (500 mL) is
added, the flask is swirled for 5 minutes, and the solvent
decanted. The procedure is repeated two additional times. To
dry the sodium sulfate, the cleaned material is heated in the
flask in a drying oven at 130°C overnight. The clean dried
material can be stored in the drying oven, or stoppered and
stored in a dessicator.
To prepare drying columns, a small piece of pre-extracted
glass wool is placed in the bottom of the chromatography column,
13 cm of sodium sulfate ( ‘-85 g) is poured into the column,
followed by an additional plug of pre-extracted glass wool.
Columns should be used immediately or stored in a drying oven at
130°C. Rinse the Na 2 SO 4 with 50 mL of extracting solvent just
prior to using the column.
(3) Teflon liners and teflon lined septa are sonicated for 10 minutes
in pesticide grade methanol followed by 10 minutes in pesticide
grade pentane. The sonicated liners are vacuum-oven (—20 inches
of water) dried for 3 to 5 hours at 70° and stored in clean,
Teflon lined screw cap bottles.
(4) Glass wool is cleaned by placing it in the extraction chamber of
a Soxhiet extractor and extracting overnight with pesticide
grade methylene chloride. The glass wool is placed in a wide
mouth jar and the solvent removed either under nitrogen stream
or by warming. Cap and store until needed.
9.11.4 GC/FID Performance Evaluation
Prior to analyzing procedural blanks, acceptable performance for the
GC/FID system must be demonstrated.
(1) Analyze the GC/MS system performance solution as specified in
Table 9.2. Figure 9.3 shows a total ion chromatogram of this
Chap. 9 — 191
-------
mixture analyzed by GC/MS under similar conditions which may be
used to identify test components in the standard.
(2) Measure peak asymmetry or tailing for acetophenone and 1-tetra-
decanol using the percent peak asymmetry factor (PAF):
% p y = X 100
where
B = the width from the back of the chromatographic peak to
the perpendicular from the peak measured at 10% above
baseline.
F = the width from the front of the chromatographic peak
to the perendicular from the peak measured at 10%
above baseline.
The PAF should measure less than 200% for both acetophenone and
1-tetradecanol.
(3) Check the acidity/basicity of the column by the peak area ratios
determined by integrator or triangulation of 2,6-dimethylaniline
and 2,6-dimethyiphenol to acetophenone. A ratio of 0.7 to 1.3
for both is acceptable.
(4) Sensitivity is checked by measuring signal to noise ratios for
2,4,6-trimethylpyridine, rn-cresol, and methyl stearate. A
signal- to-noise ratio of 10 or greater for all three compounds
is acceptable.
9.11.5 Procedural Blanks
If the BLLE procedure is also being performed with the same reagents,
solvents and materials only Procedural Blank 2 need be prepared. Otherwise,
for each lot of materials and reagents that are used, a set of three
procedural blanks are required. Table 9.4 identifies these blanks and
defines their purpose. These blanks must be processed and analyzed by
GC/FID prior to processing any samples. If a large number of samples are
to be processed, it is advantageous to prepare large lots of materials and
reagents thereby reducing the number of blanks which must be run.
9.11.5.1 Procedural Blank 1-—
(1) Prepare an extractor as described in step 9.12(4), then follow’
Chap. 9 — 192
-------
the extraction procedure as described in steps 9.12(6) to
9.12(20), except step 9.12(13). Run extractor for oniy 60 mm.
(2) Add 10 pL of the GC external standard to the blank concentrate.
Agitate the K-D receiver to give a uniform distribution of
standard in the concentrate.
(3) Analyze samples by GC/FID using the conditions described in
Table 9.2. Contaminant peak heights should measure less than
20% relative to the standard.
(4) If the blank is acceptable, add an aliquot (10 pL) of the MS
external standard solution. Agitate the K-D receiver to give a
uniform distribution of standard in the concentrate. Transfer
the concentrate to a 1 dram vial and cap. Store at 0°C until
GC/MS analysis.
(5) If significant contamination is present, the extraction apparatus
should be cleaned and the procedure repeated. If this blank is
acceptable, then the extractor was the source of contamination.
(6) If significant contamination is present in this blank, fresh
methylene chloride of a different lot or source should be used
and the reagent blank repeated. When the reagent blank is
acceptable, procedural blanks 2 and 3 may be processed.
9.11.5.2 Procedural Blanks 2 and 3--
(1) Prepare two extractors as described in step 9.l2(4).4. For
procedural blank 3, pour 1000 mL of reagent water into the
extractor and proceed with the extraction [ 9.12(5) and following].
For procedural blank 2 use 1000 mL of reagent water spiked with
1 mL each of the sulfuric acid, sodium hydroxide, and sodium
thiosulfate solutions to be used for sample collection (Chap-
ter 4), and 10 mL of the phosphate buffer solutions [ 9.10(28)(i)].
(2) Follow the extraction procedure as described in steps 9.12(5) to
9.12(20).
(3) Add 10 jiL of the GC external standard to the blank concentrates.
Agitate the K-D receiver to give a uniform distribution of
standard in the concentrates.
(4) Analyze the samples by GC/FID using the conditions in Table 9.2.
Peak heights for contaminants in Procedural blank 3 which are
Chap. 9 - 193
-------
not present in procedural blank 2 should measure less than 20%
relative to the external standard.
(5) If significant contamination is present in Procedural blank 3
which is not present in procedural blank 2, the procedure should
be repeated using fresh solutions of sulfuric acid, sodium
hydroxide, sodium thiosulfate, and phosphate buffer until an
acceptable blank is achieved.
9.12 SANPLE EXTRACTION
(1) Nark the interface between the keeper solvent and aqueous layer
on the sample bottle with an indelible mark.
(2) Transfer the keeper solvent to a clean 1 L Erlenmeyer flask
using a transfer pipet.
(3) Adjust the p11 to 8.0 using 6N NaOH or 1:1 11 2 S0 4 . Check the p11
with narrow range p11 paper. Add 10 mL of 1.0 N pH 8.0 phosphate
buffer.
(4) With the extractor stopcock closed, pour 275 mL of methylene
chloride into the extractor body.
(5) Slowly add the sample without disturbing the organic layer. Use
a glass funnel so that the sample slowly runs down the sides of
the extractor. Rinse the sample bottle with —10 mL of methylene
chloride and transfer to the extractor with the funnel tip
extending into the methylene chloride in the extractor.
(6) Determine the original sample volume by refilling the sample
bottle to the mark and transferring the liquid to a 1,000 mL
graduated cylinder. Record the sample volume to the nearest
5 mL.
(7) fount the motor and stirring rod above the extractor so that the
paddles on the stirring rod are just below the aqueous surface
(mark positions for reference) (Figure 9.8).
(8) With the stopcock closed, add 225 mL of methylene chloride to
the distillation flask along with cleaned boiling chips.
(9) Position the condenser, heating mantle, and variable transformer
(Figure 9.8).
Chap. 9 - 194
-------
ttype Teflon
stopcock
Figure 9.8.
condenser
24/40 female
ground glass
joint
Solvent return
arm
stirrer
motor
paddle
methylene
chloride
center
bottom
distillation
flaik
heating
mantle
Flow-under extractor with attachments.
Chap. 9 - 195
-------
(10) Adjust the variable transformer to give a 20 mL/min distillation
rate (the variable transformer should be calibrated before an
actual sample is extracted).
(11) Open the stopcock to allow solvent to cycle through the siphon
tube when the methylene chloride in the flask starts to distill
over.
(12) Turn on the stirring motor at a speed fast enough to suspend any
solids present but slow enough not to break the sample-solvent
interface.
(13) Extract sample for 3.5 hr.
(14) Close stopcock, turn off stirrer, and remove heating mantle.
(15) Remove solvent from distillation flask and transfer to the 1 L
Erlenmeyer flask containing the keeper solvent.
(16) Rinse the distillation flask three times with 5 mL portions of
methylene chloride and add rinse to the same Erlenrneyer flask.
(17) Remove water from the methylene chloride by adding -5 g of
cleaned and dried sodium sulfate. Swirl the flask for several
minutes. Pour the semi-dried extract through a sodium sulfate
drying column using a glass funnel to prevent spillage. Rinse
bulk sodium sulfate and Erlenmeyer flask and drying column with
an additional 5 niL of solvent and add rinsings to eluate.
(18) The eluate and rinse solvent are collected in a 500 mL K-D
flask equipped with 4 niL concentrator tube containing several
boiling chips. Pre-calibrate the concentrator tube to compensate
for the volume of the boiling chips. Attach the three-ball
macro-Synder column. Place the K-D apparatus on a warm water
bath with the concentrator tube partially immersed in water or
on a steam bath. Adjust the temperature of the water bath such
that evaporating solvent causes the balls in the Synder column
to chatter actively but does not flood the chambers of the
column. Under these conditions evaporation of 500 niL of methylene
chloride to 2 niL should take 20 to 30 minutes.
(19) When the liquid has reached an apparent volume of 2 niL remove
the K-D apparatus from the heat and allow to cool. Remove the
Snyder column. With 1 to 2 niL of solvent, rinse the evaporative
Chap. 9 - 196
-------
flask and its lower joint into the concentrator tube. The final
volume in the concentrator tube should be approximately 4 mL.
(20) Attach a modified micro-Snyder column to the K-D receiver.
Connect a transfer pipette by Teflon tubing to the nitrogen
manifold. Place the pipette in the modified Synder column above
the solvent level (Figure 9.4). Gently blow a stream of nitrogen
above the solvent surface until the volume is reduced to approxi-
mately 1. mL. Rinse the sides of the concentrator tube with
approximately 0.5 mL of solvent. Reduce volume of extract to
1.0 mL. Record final volume.
(21) To determine whether the extract is so complex that it must be
cleaned up, a 1 pL injection is made on a gas chromatograph with
FID detection. The 180 cm X 2 mm i.d. column is packed with 3%
OV-l on Chromosorb W HP. A temperature program from 30°C to
250°C at 10°/mm is used. The attenuation is set to give 30 to
70% of full scale deflection for the various peaks of the alkane
calibration standard [ 9.l0(28)(k)]. The area produced by all
eight alkanes is Area C. Areas may be measured by planimeter.
(22) The extract chromatogram from step 21 is evaluated as follows:
The baseline rise (A) is estimated by connecting the valleys of
the chromatogram as shown in Figure 9.6. Draw a zero line (B)
horizontal to the level of the baseline before injection. At
any point after the first 2 minutes of the chromatogram where
line A is more than 5% of full scale above line B begin an
incremental integration of the area between A and B. This is
repeated in 5% increments a indicated by the shaded areas in
Figure 9.6. Compute the area between A and B which is shaded
(Area A-B). Measure the area of the peaks produced by the
alkane calibration standard (Area C) and compute the mg/mL
equivalent of the baseline rise MA by
Area X 60 mg/mI
N = AB
A Area
A sample must be fractionated if MA > 50 mg/mI (go to Section D).
Otherwise it may be analyzed by GC/MS without further workup.
Chap. 9 - 197
-------
(23) If no fractionation is required add 25 IJL of external standard
solution to the sample concentrate. Agitate the K-D receiver to
give a uniform distribution of external standard in the sample
concentrate. Transfer a measured portion of the sample concen-
trate to a 1 dram vial and cap. Store at 0°C until analysis by
GC/MS.
(24) Proceed to Section E -GC/NS/COMP ANALYSIS OF WABN-SC, WABN-BL,
OR WARN-ru SANPLE EXTRACTS.
Chap. 9 — 198
-------
D. CLEANUP/FRACTIONATION OF WABN FRACTIONS
9.13 INTRODUCTION
This protocol describes a procedure for fractionating WARN extracts
identified by the scouting test [ 9.8(15—16) and 9.12(21—22)] as containing
high levels of chromatographable organics. These high levels of organic
substances would prevent the identification and quantitation of analytes
at the levels specified for the overall methods.
The fractionation is performed using a 5 g silica gel open column.
The silica gel is activated at 150°C and deactivated with 14 mL of water
per 100 g of silica gel. The sample is then fractionated into three
fractions consisting of pentane (20 mL), 20% methylene chloride in pentane
(50 mL) and 6% methanol in methylene chloride (100 mL). The second two
fractions may be recombined for GC/MS/DS analysis.
The m an percent recoveries and coefficients of variation for this
procedure alone are given in Table 9.6.
9.14 APPARATUS AND REAGENTS
The following materials are required for processing a set of nine
sample extracts. The fractionation of nine sample extracts will require
one and a half working days including solvent evaporations.
(1) Nine chromatography columns 220 mm X 10 mm i.d. with Teflon
stopcocks and 24/40 ground glass joints at the top (Figure 9.9).
(2) Nine 250 mL dropping funnels with 24/40 ground glass joints and
Teflon stopcocks.
(3) Nine glass funnels to fit dropping funnels.
(4) Pre—extracted glass wool ( ‘-50 mL) for columns. Glass wool
should be precleaned by Soxhiet extracting overnight with methyl-
ene chloride.
(5) One Soxhiet extractor (50 mL capacity minimum) for extracting
glass wool.
(6) Nine Kuderna-Danish apparatus. Each apparatus consists of
(a) Three-ball macro—Snyder columns (Kontes #1 (503000) - 300 nun
length with 24/40 joints,
(b) Evaporative flask (Kontes #K570001) - 250 mL with 24/40 top
joint and 19/22 lower joint,
Chap. 9 — 199
-------
Table 9.6. RECOVERIES 8 AND ELUTION PROFILES FO
CLEANUP/FRACTIONATION BY SILICA GEL OPEN-COLUMN
c . d
Compound Fraction 1 Fraction 2
Pyridinee
a-Picoline 75 ± 13
2-Heptanone 61 ± 30
o-Xylene e 82 ± 5.6
t-Butyl carbamate
2-n-Butoxyethanol 108 ± 15
t-Butyl propionate 51 ± 14
Benzaldehyde 84 ± 23
Aniline 75 ± 8.5
Lutidenee
2-Octanone 78 ± 27
Phenol 61 ± 18
Benzyl chloride 69 ± 5.5 trace
2,3,6-Trimethyl pyridine 80 ± 19
Decane f 84 ± 3.2
Salicylaldehyde
Acetophenone 88 ± 6.6
Phenyl acetate 62 ± 19
Phenyl ethanol 87 ± 7.9
Nitrobenzene -67 ± 22
Tolualdehyde 64 ± 16
2,3-Dihydrobenzofuran 44 ± 5.2
—Creso1 86 ± 7.4
Fenchone 85 ± 10
3-Chiorobeuzaldehyde 99 ± 17
Isophorone 87 ± 13
2-Bromo-l-ch lorobenzene 76 ± 5.5
o-Chloroanisole 41 ± 8.0
4-Chlorobenzonitrile 46 ± 17
1,4-Dicyanobutane 53 ± 18
Di—t-buty ldisulfide 77 ± 4.5
2,6-Dimethylaniline 88 ± 7.0
Benzyl acetate 77 ± 10
2,4-Dichiorophenol 64 ± 14
l,2,4—Trichlorobenzene 76 ± 5.1
Naphthalene 79 ± 4.9
-Dibromobenzene 87 ± 5.4
2,3-Dimethyiphenol 85 ± 9.5
rn—Ch loroani line 150 ± 5.1
a-Terpineo l 81 ± 11
Benzthiozole 87 ± 9.6
o-Isopropy lphenol 77 ± 5.6
Quinoline 69 ± 14
Tributylamine 54 ± 6
Anisaldehyde 86 ± 13
(continued)
Chap. 9 - 200
-------
Table 9.6 (cont’d.)
. c . d
Compound Fraction 1 Fraction 2+3
2—Nitro-rn—cresol 49 ± 22
Dimethyladipate 79 ± 11
Bis(2-chloroethoxy)ethane 84 ± 4.0
3,4-Dichlorobenzaldehyde 54 ± 14
Indole 72 ± 10
Decanol 86 ± 6.7
2-Methy lnaphthalene 67 ± 9.0 trace
4-Chloro-3-methyl phenol 87 ± 14
-t-Butylpheno1 95 ± 9.5
Phenyl carbamate 120 ± 36
l,2,4,5-Tetrachlorobenzene 77 ± 9.0
Tridecane 87 ± 6.2
Njcotjne 8
2,3,6-Trichlorophenol 46 ± 12 35 ± 3.7
2,6-Dinitrotoluene 80 ± 9.0
2,4-Dimethy lquino line 81 ± 7.9
Tetradecane 93 ± 5.9
Dimethyl phthalate e ± 10
Hethy1- -to1uene sulfonate
1,8-Dimethy lnaphtha lene 68 ± 3.1
Acenaphthene 63 ± 7.1 22 ± 14
2,4-Dinitrotoluene 77 ± 12
1—Naphtho l 83 ± 16
Di-t-butyl-4-methyl phenol 71 ± 5.2 12 ± 8.3
Ethyl-2-toluene sulfonate 110 ± 19
N,N-Dimethyldodecyl amine 75 ± 12
Pentadecane 91 ± 4.5
2,3,5-Trimethylnaphthalene 57 ± 11.2 31 ± 15
1,2-Dichloronaphthalene 88 ± 7.3
Fluorene 27 ± 21 58 ± 7.9
2-Aminobiphenyl 82 ± 1.5
Diethyl phthalate 100 ± 10
Diphenyl amine 78 ± 6.7
(4-Bromodiphenyl) ether 31 ± 21 55 ± 8.0
Hexadecane 99 ± 3.3
Tri-n-butyl phosphate 110 ± 10
Hexachlorobenzene 95 ± 5.9
Dibenzylamine 70 ± 14
Atrazine 87 ± 16
Heptadecane 99 ± 5.5
Acridine 92 ± 6.0
Anthracene 50 ± 9.0 48 ± 8.8
Caffeine 74 ± 18
Benzyl sulfide 82 ± 11
Carbazole 100 ± 15
(continued)
Chap. 9 - 201
-------
Table 9.6 (cont’d.)
Compound
C
Fraction 1
. a
Fraction 2+3
Octadecane
98 ± 6.7
1,8-Diaminonaphtha lene
30 ± 15
Diphenyl sulfone
71 ± 9.0
Diphenyl mercury
82 ± 8.8
Nonadecane
103 ± 3.6
Di-n-butyl phthalate
110 ± 12
Aidrin
96 ± 2.9
Eicosane
110 ± 6.9
Pyrene
34 ± 9.1
62 ± 17
9,10-Dimethyl anthracene
17 ± 25
85 ± 1.0
Heneicosane
99 ± 11
Nethyl stearate
70 ± 6
Docosane
92 ± 9.3
Butylbenzyl phthalate
79 ± 12
Tricosane
89 ± 8.3
Triphenyl phosphate
86 ± 5.6
Chrysene
71 ± 5.0
Di(ethyl hexyl) phthalate
104 ± 18
Tetraphenyl tin
trace
54 ± 20
Perylene
98 ± 21
Cholesterol
71 ± 27
aMean ± coefficient of variation (n=3), nominal spiking level was 10 ppb.
b 5 g of silica gel (100/120 mesh) activated overnight at 150°C,
deactivated by 14 mL deionized water/l00 g silica gel.
cExtract solution (10 mL vol. —1% methylene chloride iii pentane)
+ 10 additional mL pentane.
mL 20% methylene chloride in pentane and 100 mL 6% methanol
in methylene chloride.
e.
Did not chromatograph wider conditions used.
Interference prevented quantitation.
recovered.
(c) Concentrator tube (Kontes #K570000) - 4 niL graduated, with
19/22 joint. Attach to evaporative flask with springs
(Kontes #K50300-0232). Ground glass stopper (size 19/22
joint) is used to prevent evaporation of extracts.
(7) Eighteen modified micro-Snyder columns (Kontes #K569251) with
19/22 joint.
Chap. 9 - 202
-------
500 mL glass
separatory funnel
Figure 9.9.
24/40
glass connection
-lcmi.d.
Teflon
stopcock
Teflon stopcock
I
22cm
Chromatography column with sample reservoir (exact
dimensions of column are not critical).
Chap. 9 - 203
-------
(8) Eighteen concentrator tubes (Kontes #1(570050-25) — 25 mL gradua-
ted, with 19/22 joint.
(9) Boiling chips - Hengar granules are ground with a mortar and
pestle and sieved to obtain a 60/80 mesh. These granules are
cleaned by extracting —5 g three times with —150 mL methylene
chloride.
(10) Water bath - Heated, with concentric ring cover, capable of
temperature control (±2°C). The bath should be used in a hood.
(11) One manifold with a temperatu e controlled water bath for nitro-
gen blowdown. A manifold with nine spaces is preferred but not
essential.
(12) Nine glass vials (1 dram) with 13 mm screw caps (Supelco 2—3213)
and Teflon lined rubber septa (Supelco 2-3216).
(13) One gas chromatography column (180 cm X 2 mm i.d.) packed with
3% OV-l on Chromosorb W HP (80/100 mesh) or equivalent.
(14) One gas chromatograph with flame ionization detector.
(15) One laboratory oven.
(16) Materials and Reagents
(a) 1.5 L methylene chloride (Burdick and Jackson, distilled in
glass).
(b) 0.5 L pentane (Burdick and Jackson, distilled in glass).
Cc) 50 mL methanol (Burdick and Jackson, distilled in glass).
(d) 100 g silica gel 100/120 mesh (Fisher reagent grade).
Ce) 1 mL column performance standard (Table 9.7).
(f) Reagent water — reagent water is defined as a water source
which does not produce a background interference at the
Table 9.7. COLUMN PERFORMANCE STANDARD
Concentration
Compound
mg/b
a
Naphthalene
10
Anthracene
20
aN hl chloride solvent.
Chap. 9 - 204
-------
limit of detection. A water purification system (Millipore
Super-Q or equivalent) may be used to generate reagent
water.
9.15 PREPARATION FOR FRACTIONATION
(1) Cleaning of Glassware
All glassware to be used should be washed with Amway S-8-A, rinsed
with distilled deionized water and heated to 500 to 550°C in a glassware
oven for a minimum of 4 hours. Cleaned glassware is immediately capped or
covered with foil to prevent contamination.
(2) Activation of the Silica Gel
Silica gel (100 g) is placed in a wide mouth jar in an oven at 105°C
for a minimum of 14 hr. Immediately prior to use cap the jar and place in
a desiccator to cool. When cool add 14 niL of reagent water and agitate
until evenly distributed. Allow to equilibrate for at least 2 hrs.
(3) Cleaning Glass Wool
Glass wool is cleaned by placing in the extraction chamber of a
Soxhlet extractor and extracting overnight with pesticide grade methylene
chloride. The glass wool is placed in a wide mouth jar and the solvent
removed either under nitrogen stream or by warming. Cap and store until
needed.
9.16 SAMPLE FRACTIONATION
9.16.1 Determination of Silica Gel Activity
(1) A chromatography column containing 5.00 ± 0.05 g of silica gel
is prepared by layering the silica gel on top of a plug of glass
wool and tapping the column gently to settle the sorbent.
(2) Dilute 0.5 mL of the column performance standard (Table 9.7) to
10 niL with pentane and mix gently.
(3) Transfer the diluted column performance mixture to the silica
gel column and allow the solution to elute through the column
until the solvent reaches the top of the silica gel. Collect
this eluate.
(4) Elute the column with an additional 10 niL of pentane and collect
this eluate as above (3) in the same container. This should be
—15 niL.
Chap. 9 - 205
-------
(5) Dilute 0.5 inL of column performance standard with 15 mL of
pentane and mix.
(6) Inject the column eluate (4) on the 180 cm X 2 mm.i.d. 3% OV-1
on Chromosorb W liP packed GC column. Compare peak areas (or
heights) directly against those obtained from an equal volume
injection of the diluted performance standard (5).
(7) If the naphthalene recovery is <70% and no anthracene is observed,
reduce the amount of silica gel in the column (1) by 0.50 g and
repeat steps 1-6. If the naphthalene recovery is >70% and the
anthracene recovery is >50%, increase the amount of silica gel
by 0.50 8 and repeat steps 1-6. If the naphthalene recovery is
>70% and the anthracene recovery is <50% proceed with samples.
9.16.2 Fractionation of Samples
(1) Adjust extract volumes to 1.0 ± 0.2 mL with methylene chloride.
(2) Transfer the sample extracts with 9 niL of pentane to a 25 niL
K-D concentrator tube and evaporate to 1.0 mL under a nitrogen
stream with a modified micro-Snyder column. Connect a transfer
pipette by Teflon tubing to a nitrogen manifold. Place the
transfer pipette in the modified Snyder column above the liquid
level (Figure 9.4). Gently blow a stream of nitrogen above the
liquid surface until the volume is reduced to approximately
1 niL. Rinse the sides of the concentrator tube with approximate-
ly 0.5 niL of solvent.
(3) Redilute the extracts with 9.0 mL of pentane.
(4) Prepare nine columns using the amount of silica gel determined
in Section 9.16.1. The silica gel is layered over a glass wool
plug and the column tapped to settle the sorbent.
(5) The rediluted extract is transferred to a column and the solvent
eluted until it reaches the surface of the silica gel. Collect
this eluate in a 25 niL K-D concentrator tube.
(6) Rinse the extract container in 2 niL portions with a total of
10.0 niL of pentane transferring each portion to the column.
Elute this solvent until the liquid level reaches the sorbent
and close the stopcock. Collect this eluate with that from
Chap. 9 - 206
-------
step 5 and label as Fraction I. The volume of eluate should be
—15 niL.
(7) Transfer the eluate to a 250 mL K-D evaporator flask fitted with
a 4 niL graduated concentrator tube.
(8) Measure 50 mL of 20% methylene chloride, 80% pentane (v/v).
Rinse the sample extract container (from step 2) three times
with 2 niL portions of this solvent and transfer to the column.
Add remaining solvent to column using a droping funnel (Fig-
ure 9.9). Elute the column until the solvent reaches the top of
the silica gel. Collect in a 250 niL K-D flask equipped with
4 niL concentrator tube containing several boiling chips. The
concentrator tube should be precalibrated to compensate for the
volume of the boiling chips.
(9) Measure 100 niL of 6% methanol in methylene chloride. Rinse the
sample extract container three times with 2 niL portions of this
solvent and transfer to the column. Add remaining solvent to
the column using a dropping funnel. Elute the column until the
solvent reaches the top of the silica gel. Label the combined
eluates from steps 8 and 9 as Fraction II.
(10) Evaporate Fraction I to 1.0 niL by nitrogen blowdown with a
modified micro-Snyder column as described in step 1.
(11) Evaporate the eluate labeled Fraction II. Attach the three-
ball macro-Snyder column. Place the K-D apparatus on a warm
water bath with the concentrator tube partially immersed in
water or on a steam bath. Adjust the temperature of the water
bath such that evaporating solvent causes the balls in the
Snyder column to chatter actively but does not flood the chambers
of the column. Under these conditions evaporation of 150 niL of
solvent to 2 mL should take 10 to 15 minutes.
(12) When the liquid has reached an apparent volume of 2 niL remove
the K-D apparatus from the heat and allow to cool. Remove the
Snyder column. With 1 to 2 mL of solvent, rinse the evaporation
flask and its lower joint into the concentrator tube. The final
volume in the concentrator tube should be approximately 4 mL.
Chap. 9 — 207
-------
(13) Attach a modified micro—Snyder column to the concentrator tube
and concentrate to 1.0 mL as described in step 2.
(14) Add 25 pL of MS external standard solution [ Part III, Section
9.l0(28)(f)] to each sample concentrate. Agitate the K-D
receiver to give an uniform distribution of external standard in
the sample concentrate. Transfer a measured portion of the
sample concentrate to a one dram vial and cap. Store at 0°C
until GC/1IS analysis.
(15) Proceed to Section E - GC/MS/COMP ANALYSIS OF WABN’SC, WABN-BL,
OR WABN-FU SAMPLE EXTRACTS.
Chap. 9 - 208
-------
E. GC/MS/COlIP ANALYSIS OF WABN-SC, WABN-BL, OR WABN-IU SAMPLE EXTRACTS
9.17 INTRODUCTION
Sample extracts containing weak acids, weak bases, and neutrals
(pH 8.0 extractables) obtained from protocols I, II, III or IV are analyzed
using the conditions prescribed here. Prior to beginning GC/MS/COMP
analysis of these fractions the analyst should be familiar with the proce-
dures described in Chapter 13.
9.18 PREPARATION FOR ANALYSIS
9.18.1 GC/NS/COHP Operating Parameters
The recommended GC/MS operating parameters are given in Table 9.10.
It is also recommended that the capillary used here not be used for the
analysis of fractions from other protocols.
9.18.2 MS Calibration
The mass spectrometer is calibrated using the manufacturer’s recom-
mended approach (Chapter 13). The acceptability of the calibration results
is verified with the analysis of the system performance solution (SPS).
9.19 GC/MS/COMP ANALYSIS
9.19.1 SPS (Quality Control )
Using the prescribed GC/MS conditions the SPS (Table 9.1) is analyzed
and all necessary data is acquired and evaluated prior to proceeding to
sample analysis. The SPS is analyzed at the beginning of each day of
operation.
The SPS raw data is extracted from the run, and test parameters
calculated and plotted or tabulated (Tables 9.11 and 9.12). Up to 14 days
of SPS analytical results can be historically recorded in Table 9.11 for
day-to-day comparisons. This allows the analyst to anticipate GCIMS
maintenance requirements. Calculations of test parameters and discussion
of their use are given in Chapter 13.
A check of RNRs on a daily basis is also calculated from SPS raw data
and tabulated (Table 9.12). The R}lRs are compared to those obtained
during the development of the historical data bank. The usage of this
data is discussed in Chapter 13.
Chap. 9 - 209
-------
Table 9.10. GC/MS OPERATING CONDITIONS
FOR EXTRACTABLE WEAK ACIDS, BASES AND NEUTRALS
CC Column 30 m DB-1 (1.0 p film thickness)
fused silica wide bore (0.34 mm
I.D.) capillary column
GC Carrier Gas Helium
Carrier Gas Flow 1.6 mL/min through column; 9:1
split injection
Carrier Gas Sweep Time 85 sec (50°C)
Temperature Program 50°C/S mm; to 250°C @ 4°/mm and
hold
Injector Temperature 250°C
Transfer Line Temperature 255°C
Injection Mode Splitless, 0.4 mm/split
Injection Volume 1.0 pL
Ionizing Energy 70 eV
Ion Source Temperature 250°C
Scan Range 35-600
Scan Speed Scan 0.95 sec, hold 0.05 sec
9.19.2 Determination of RNRs
RMRs for the User’s data bank (as opposed to the daily RMR checks
discussed above) are determined by injecting a solution containing the
internal standards and analytés.
The GC/MS/COMP operating parameters are given in Table 9.10.
Table 13.8 may be used to tabulate RIIR raw data.
9.19.3 Quality Control and Field Samples
After all specified criteria for GC and MS performance (Table 9.11)
have been met, analysis of quality control or sample extracts is conducted.
A set of sample extracts may have been derived from laboratory and
field blanks, surrogates, and collected field samples, and are generally
analyzed in that respective order.
Qualitative and quantitative procedures are described in Chapter 13.
Table 9.13 may be used to tabulate sample analyte raw data prior to using
the MASQUANT software program or manual interpretation.
Chap. 9 - 210
-------
Table 9.11. CC-MS SYSTEM PERFORMANCE TEST FOR WEAK ACIDS,
BASES AND NEUTRALS (WABN, pH 8 EXTRACTABLES)
Dates:
Run Id Code
GC Column
and
Program:
Notebook References:
MS SYSTE1I CHECKS
rn/a 51 - -
Abundance * ( ) j ( ) Li Li Li. LJ L1 Li ___ ____
Criteria
>‘
*These abundances are initially established by the HAS user, and subsequently become guidelines for
acceptability of tune
(continued)
DflPP
Relative Abundance Criteria
High/Low Mass Balance
Ion
51
68
70
127
197
198
199
275
365
441
442
443
68 70 127
30% to 60% of m/z 198
<2% of rn/z 69
<2% of m/z 69
40% to 60% of m/z 198
(1% of rn/a 198
100%
5-9% of rn/a 198
10% to 30% of rn/a 198
at least 1% of m/z 198
present, but < rn/a 443
>40% of rn/a 198
17% to 23% of rn/a 442
197 198 199 275 365 331 442 3 Remedial
Fail Action
Chap. 9 - 211
-------
Table 9.11 (cont’d.)
Test Components Criteria
1. 2,4,6—Tri etby1pyridine • S:N (a/z 122) ) 75 1
La. it of 2. —Creso1 x S:N (e/z 109) > 6:1
Detect ion 3. Methyl Stearate S:N (w/z 74) > 40:1
(1) (2) (3)
90 12 .55
8 5 10 .50
‘ 80 8 45
6 40
I I I I I I U I I I I I
Day
GC SYSTEM CHECKS
Peak 1. Acetophenone (TIC) • %PAF <300
Asynetry 2. 1-Tetradecanol (TIC) • %PAF <200
300 No. 1
200 No.2
.
0.
d l 100
I I U I U I I U I I U I
Day
1. Acetophenone (TIC)
Acidity/ 2. 2,6-Dimethylphenol (TIC) • Ratio 2:1 = 0.7 to 1.3
Basicity 3. 2,6-Dusethylaniline (TIC) I Ratio 3:1 0.7 to 1.3
1.4
1.2
0
‘I
‘I
C s
0.8
0.6
I I I I J I I I
Day
(continued)
Chap. 9 - 212
-------
Table 9.11 (cont’d.)
Test Components Criteria
Separation n—Eicosane ( /z 43)
Number n-Heneicosane (m/z 43) SN > 6
12 -
11
10
z
u,
8
7.
6--
I I I I I I I I I I I I I
Day
n-Octadecane (TIC) R = minimum 50%
Resolution 1—Octadecene (TIC) Valley
90
80
- 70
60
>
d
40
I i I I I I I I I I I I
Day
Inertness flethyl Steerate Ratio m/z 74 to 298 < 14:1
20
18
2 16
14
12
I I u i I I I
Day
(continued)
Chap. 9 - 213
-------
Table 9.11 (cont’d.)
Test Components Criteria
Capillary 1. 6-t-butyl—rn—creaol (TIC) ‘
Capacity 2. Quinoline (TIC) I PAF >70
100
90
I ’
80
dS
70
I I I I I I I I I I I I I
Day
Relative
Retention 1. Heneicosane
Shift 2. Pyrene RRS = 0.95—1.05
1.10
. , 1.05 --
1.0
0.95 •--
0.90
I I I I I I I I T I I I I
Day
Chap. 9 - 214
-------
Table 9.12. GC-MS SYSTEM PERFORMANCE TEST: RMR CHECK FOR WABN EXTRACTABLES (pH 8.0)
Date:
Run ID Code:
DATA
Standard
MW
Amount
(pg)
Ion
(m/z)
Area
(Run 1)
Area
(Run 2)
Area
(Run 3)
4-Fluoro-2-Iodotoluene
(FIT)
236
109
236
d 10 -o-xy lene
116
98
116
d 8 -naphtha lecie
136
136
d -nitrobenzene
128
82
128
d 5 -pheny letbano l
112
84
112
d 5 -propiopheoone
110
82
110
d 5 —acetopbenone
125
110
125
d 12 -pery lene
264
264
r)
p
d 9 -acridine
188
188
.
o
d 5 -pheno l
99
I MATRIX OF STANDARD ION RHRs
F ’.)
Standard Ion 109 236 98 116 136 82 128 84 112 82 110 110 125 264 188 99
FIT 109
236
d 10 -o-xy lene 98
116
d 8 -naphtha lene 136
d 5 -nitrobenzegie 82
128
d 5 -phenylethanol 84
112
d 5 -proptophenone 82
110
d 5 -acetophenone 110
125
d 12 -pery lene 264
d 9 -acridine 188
t JrIrIrI1 La
-------
Table 9.13. RAW DATA FOR WARN (-SC,-BL, OR-FU) FRACTION
Date:
Run I.D. Code:
Notebook Reference:
Vol. Water Processed
Sample Identification:
Vol. Water (L):
(to which i were added)
(mL):
STANDARDS
Spec-
I.D. trwn
No. No.
4-Fluoro-2-Iodo-
Weight Ions
(ng ) (m/z) Area
MW;
Ka
toluene
d 10 -o-Xylene
d 8 -Naphthalene
d 5 -Nitrobenzene
d 5 —Phenylethanol
d 5 —Propiophenone
d 5 —Acetophenone
d 12 -Perylene
d 9 -Acridine
d 5 -Pheno l
109,236: ,______
98,116: ,
136: ,
82,128: ,
84,112: ,
82,110: ,______
110,125: ,______
264: ,______
188: ,______
99: ,______
,
,
,
,
,
,
,
,
,
,
ANALYTES
Class No.
1.0.
No.
Spectrum Ions
No. (m/z) Area
HWa
•
,
,
,
,
,
,
, :
,
—
—
a 1 f calculations are performed manually then this information is also
needed, whereas MASQUANT performs functions automatically.
A MW ng A = ion area or height
— x x _____
ng X
x — RMR A . NW g = ng recovered from volume
x/y y y of water processed
x = analyte
K = ng y = standard
A .
y y
Chap. 9 - 216
-------
CHAPTER 10
ANION-EXCHANGE, DISTILLATION AND ANALYSIS OF VOLATILE STRONG ACIDS (VOSA)
10.1 INTRODUCTION
10.1.1 Principle of the Technique
This technique describes the determination of volatile carboxylic
acids (C 3 to C 8 acids) in drinking water, surface waters, and treated
municipal and industrial wastewater effluents. Low molecular weight
carboxylic acids are polar, water soluble compounds which will not easily
extract into organic solvents. In addition, these acids in their protonated
form are sufficiently volatile to give significant losses during solvent
evaporation procedu res. The procedure utilizes a strong anion exchange
resin to concentrate these acids from the aqueous sample. The acids are
eluted from the resin using sodium bisulfate in an acetone:water solution.
Bisulfate both protonates acids to their nonionic forms which are then
rinsed off the resin with acetone:water, and displaces acid salts from the
resin material. Volatile acids are separated from the sodium bisulfate
and nonvolatile sample components by djstillation. Carboxylic acids are
derivatized prior to GC/MS analysis to prevent adsorptive losses during
chromatography. Benzyl bromide was selected as the derivatizing reagent
for several reasons; carboxylic acids are derivatized quantitatively; the
derivatizing reagent adds a large nonvolatile group to the acids to separate
derivative from the solvent front; and derivatives are easily detectable
during GC/IIS analysis.
10.1.2 Detection Limits
If an average detection limit for GC/MS analysis for derivatized car-
boxylic acids is estimated as 10 ng per component, and the final volume of
derivatized sample is 0.3 niL, then detection limits for a 1 pL injection
are directly related to sample size.
1 ppb for a 2.5 L sample (drinking water);
5 ppb for a 500 niL sample (surface water);
Chap. 10 - 217
-------
10 ppb for a 250 rnL sample (industrial and municipal wastewater
effluents); and
50 ppb for a 50 niL sample (e.g., complex energy effluents).
10.1.3 Interferences
Background from the ion exchange resin can cause interferences for
the analysis of acetic and propanoic acids. The measured levels of acetic
and propanoic acids found in procedural blanks must be subtracted from the
amount found in water samples to determine actual concentrations. Chloride
ion on the ion exchange resin will interfere with eluant concentration and
derivatization. Methods for removing chloride ion from the resin material
are given.
10.1.4 Precision, Accuracy, and Scope
Table 1.7 in Chapter 1 presents average recoveries and standard
deviations for the range of carboxylic acids determined in drinking water
and municipal/industrial wastewater. This method gives acceptable recover-
ies for short chain carboxylic acids (C 3 -C 8 )
10.2 APPARATUS AIW REAGENTS -
The following materials are required for processing a set of nine
samples plus three procedural blanks. Nine is the maximum number of
samples which should be processed at a time, and includes certain quality
control samples as listed in Table 5.1, Chapter 5. The tt ree -proc edura1
blanks should be run before any samples are collected or processed. It
will require two working days to process nine quality control and field
samples.
(1) Nine glass chromatography columns, —l cm I.D. X 22 cm equipped
with 500 mL sample reservoir (Figure 10.1). The size of the
sample reservoir is not critical; however a 500 niL reservoir
will hold the entire sample for all water types except drinking
water. For drinking water an adaptor (Figure 10.2) for siphoning
the sample onto the resin column should be used in lieu of a
sample reservoir. This allows unattended flow through the
sample for a 14 to 16 hr period such that the sample can be
concentrated overnight. A vacuum aspirator is needed to start
sample flow through the siphoning adaptor and resin column.
Chap. 10 — 218
-------
Figure 10.1.
24/40
glass connection
Teflon
stopcock
500 mL glass
separatory funnel
Teflon stopcock
-lcmi.d.
22cm
]
Chromatography column with sample reservoir (exact
dimensions of column are not critical - must hold
10 mL of resin).
Chap. 10 - 219
-------
Ld.
Titian
rtopcock
I
tygon tubing to aspirator
Figure 10.2. Chromatography coluimi with siphoning adaptor for
drinking water.
1 gallon
i.mple
bottle
Teflon tubing
24/40 glass
nn.cdon
Chap. 10 - 220
-------
(2) One 10 mL graduated pipette with the tip cut off for measuring
and transferring resin material.
(3) One graduated cylinder for measuring sample volume. The size of
the the cylinder will depend upon the results of conductivity
measurements. In general, they may be:
1000 niL for drinking water
500 niL for surface water
250 niL for industrial waste effluent
50 mL for energy wastewater effluents
The cylinder may be reused for each sample if it is rinsed 5
times with deionized water.
(4) One conductivity bridge - range 1 to 30,000 ohms.
(5) Eighteen 250 niL round bottom flasks (24/40).
(6) Five distillation apparatus (Figure 10.3) consisting of:
(a) one distilling head (Lab Glass ?IL—135—700).
(b) one long path condenser (Lab Glass ML-560-700). Jacket of
column should be approximately 10 cm in length.
(c) one condenser adapter (Lab Glass ML-390—700).
Cd) one enlarging adapter (14/24 to 24/40) (Ace Glass 9092-24).
Ce) tygon tubing to connect cooling water source to condenser.
(f) one 250 mL round bottom flash with 14/24 joint.
(7) Five variable transformers.
(8) Five heating mantles to fit 250 niL round bottom flasks.
(9) Micro—porous boiling chips (Todd Scientific Co. 3006).
(10) One pH meter with a combination electrode.
(11) One rotary evaporator. If more than one is available up to four
samples can be evaporated at one time. Samples must be watched
closely at the start but after acetone has evaporated, the
samples require little attention.
(12) Fifteen small (15 niL) round bottom test tubes with 15 mm screw
caps (Supelco 3-3112) and Teflon lined rubber septa (Supelco
3—3115). The specific materials must be used to prevent solvent
evaporation during derivatization.
Chap. 10 - 221
-------
stopper or thermometer
14/24
ground glass
joint
glass joint
24/40 ground gtass joint
14/24 ground glass joint
water
inlet
14/24 ground glass joint
C)
0
I ’ )
pressure equalization
/
250 mL
round bottom
flask
Figure 10.3.
Distillation apparatus.
-------
(13) One Soxhiet extractor for cleaning ion exchange resin. A minimum
150 ml capacity is required. Resins can be processed in large
batches and stored; therefore a 500 mL to 1000 mL size would be
preferred.
(14) Ten ml volumetric flasks for preparing standard solutions.
(15) Fifteen vials (15 ml) with Teflon lined screw caps for storing
standard solutions.
(16) Syringes for preparing standard solutions - 50 and 250 1.JL.
(17) Graduated pipettes for preparing standard solutions - 1 and
2 ml.
(18) One large chromatography column for preparing resin material.
The dimensions of this column are not critical, it should hold a
minimum of 160 ml of resin material; for large batches of resin,
a larger column is preferred. The flow through the column
should not exceed 50 mI/minute.
(19) One heating block capable of maintaining 60°C with at least nine
spaces for the 15 niL test tubes.
(20) One manifold with a temperature controlled water bath or heating
block for nitrogen blowdown. A manifold with nine spaces is
preferred but not essential.
(21) One gas chromatograph suitable for capillary column chromatography
and all required accessories including syringes, gases, flame
ionization detector, and a strip chart recorder.
(22) One 30 m X 0.34 mm I.D. DB—l (1.0 p film thickness) fused silica
capillary column.
(23) ?faterials and reagents
(a) 120 niL AG l-X8 (50-100 mesh) ion exchange resin (Biorad
Laboratories, Richmond CA) in Cl form.
(b) Reagent water - reagent water is described as a water
source which does not produce a background interference at
the limit of detection, with a conductivity (1 ohm. A
water purification system (Millipore Super-Q or equivalent)
may be used to generate reagent water.
(c) 1 L acetonitrile (Burdick and Jackson, pesticide analysis
grade).
Chap. 10 - 223
-------
(d) 1L methanol (Burdick and Jackson, pesticide analysis grade).
Ce) 200 mL NaOH - 0.1N in reagent water (use the same solution
as specified in Chapter 5, Section 5.2.14).
(f) 200 mL H 2 S0 4 - 0.1W in reagent water (use the same solution
as specified in Chapter 5, Section 5.2.14).
(g) 100 mL Na 2 S 2 O 3 solution - 0.05 H in reagent water (use the
same solution as specified in Chapter 5, Section 5.2.14).
(h) 1.2 L iN NaOH and 200 mL 0.1N KOR in reagent water.
Ci) 2.5 L NaHSO 4 — 0.55M in 50:50 acetone:water. This solution
should be prepared just prior to use.
(j) 100 niL K 2 C0 3 - iN in reagent water.
(k) 100 niL benzyl bromide reagent - 5% w/v in acetone.
(1) 1 niL MS external standard solution - 2—fluorobiphenyl and
4-fluoroiodotoiuene (300 ng/l0 liL each) in CH 2 C1 2 .
(in) 1 niL GC external standard solution - hexadecane (30 ag/b i.iL)
in CH 2 C1 2 .
(a) 1 mL system performance standard solution (Table 10.1).
10.3 PREPARATION FOR ANALYSIS
10.3.1 Preparation of System Performance Standard
(1) Prepare individual stock standards as specified in Table 10.2.
- solids — accurately weigh 0.120 g of pure material, dissolve
in the specified solvent, dilute to volume in a 10 niL
volumetric flask.
— liquids — with a 250 pL syringe accurately measure 120 pL
of pure liquid, dissolve in the specified solvent, dilute
to volume in a 10 niL volumetric flask.
(2) Prepare a secondary neutral standard. With a 1 niL graduated
pipette, measure 0.5 niL of each stock standard in group A into a
10 niL volumetric. Dilute to volume using pesticide grade methyl-
ene chloride. Transfer into a Teflon sealed screw-cap bottle.
Store at 4°C.
(3) Prepare a secondary acid standard. With 2 niL gradaute pipette,
measure 0.5 niL of the d 7 -butyric acid, 1.65 niL of the hexanoic
acid, and 25 pL of the crotonic acid (use a 50 iL syringe) stock
standards into a 10 inL volumetric flask. Dilute to volume using
Chap. 10 - 224
-------
Table 10.1. SYSTEM PERFORMANCE SOLUTION FOR
VOLATILE STRONG ACIDS (VOSA)
Compounds
Concentration (ng/pL)
2, 6—dimethyiphenol
300
acetophenone
310
1-tetradecanol
240
1-octadecene
240
n-octadecane
230
DFTPP
300
n-eicosane
300
pyrene
300
n-heneicosane
300
methyl stearate
10
2-fluorobiphenyl
300
d 7 —butyric acida (benzyl ester)
crotonic acida,b (benzyl ester)
n-hexanoic acida ,c (benzyl ester)
300
15
1000
a -
Concentration as the underivatized acid in solution.
bFor determining limit of detection.
CFor determining capillary capacity.
pesticide grade acetone. Transfer into a Teflon sealed screw
cap bottle. Store at 4°C.
(4) Fresh standards should be prepared every six months. If degrada-
tion or evaporation has occurred, standards should be prepared
sooner.
(5) If compound purity is 96% or greater, the weight or volume can
be used without correction to calculate the concentration of
stock standards. Compounds which are less than 96% pure cannot
be used for standards.
(6) With a 1 mL graduated pipette, measure 0.5 mL of the secondary
acid standard into a 15 mL round bottom test tube. Add 0.1 mL
of aqueous 111 K 2 C0 3 . Mix well. Evaporate to dryness using
nitrogen blowdown at an elevated temperature ( —60°C). Add an
Chap. 10 - 225
-------
Table 10.2. STOCK STANDARDS FOR SYSTEM PERFORMANCE SOLUTION
Compounds
Density
(@ 20°C)
Concentr
mg/lO
ation
mL
Group Aa
2,6-dimethyiphenol
s”
120
acetophenone
1.033
124
1-tetradecanol
0.823
98
1-octadecene
0.789
95
n-octadecane
0.777
93
DFTPP
S
120
n-eicosane
S
120
pyrene
S
120
n-heneicosane
S
120
methyl stearate
S
120
2-fluorobiphenyl
S
120
Group BC
d 7 -butyric acid
S
120
crotonic acid
S
120
n-hexanoic acid
S
120
aSolvent is methylene chloride.
b
S = solid.
CSlt is acetone.
aliquot (0.5 mL) of benzyl bromide reagent to the residue. Cap
the tubes, shake, and react the solution overnight at 60°C.
(7) With a 1 niL graduated pipette, add 0.5 niL of the secondary
neutral standard to give the SPS as described in Table 10.1.
Cap the test tube. Store at 0°C until GC/MS analysis.
10.3.2 Preparation of Resin Material
Ion-exchange resin is cleaned and converted to its OH form prior to
preparing the resin columns. One hundred and twenty niL of resin material
is needed to process 9 samples and 4 procedural blanks and is the minimum
amount of resin which should be prepared. The following instructions
Chap. 10 - 226
-------
describe procedures based on 120 niL of resin material; however, it is
possible to prepare larger batches and store the prepared resin. If a
larger volume of resin is prepared, volumes for rinsing the resin should
be adjusted accordingly.
Biorad AG1-X8 (50-100 mesh) in the Cl form is extracted with redis-
tilled, pesticide analysis grade methanol in a Soxhiet extractor overnight
followed by a 24 hr extraction with redistilled pesticide analysis grade
acetonitrile. The resin is placed in a column and rinsed with 10 bed
volumes (1.2 L) of deionized water and 20 bed volumes (2.4 L) of iN NaOH.
The resin is then rinsed with reagent water until the eluate water is at
pH 6 to 7.
Residual chlorine on the resin material will interfere with sample
derivatization. To assure that chlorine and other interferents have been
removed a procedural blank should be run using the cleaned resin material
prior to processing any samples, as specified in Section 10.3.6.3 and
Section 10.4, step 8.
10.3.3 Cleaning of Materia3.s
(1) All glassware to be used is washed with Amway S-A-8 laundry
compound (or equivalent) rinsed with deionized water and baked
for a minimum of 4 hours at 500 to 550°C. All cleaned glassware
is immediately capped or covered with foil (precleaned with
hexane) to prevent contamination.
(2) Teflon liners and Teflon lined septa are sonicated for 10 minutes
in pesticide grade methanol followed by 10 minutes in pesticide
grade pentane. The sonicated liners are vacuum-oven dried for 3
to 5 hours at 70°C, - 28 inches of water, and stored in clean,
Teflon lined screw-cap bottles.
10.3.4 Maximum Sample Size
Conductivity measurements must be determined for each sample to
assure that the total concentration of anions in the sample does not
exceed the resin capacity. Prior to measuring conductivity, check the pH
of the sample using a pH meter. Adjust the sample pH to 7.5 to 8.5 using
0.lN KOH or 0.1N H 2 S0 4 if necessary. Table 10.3 lists the sample size
which should be used for a given conductivity, regardless of water type.
Corresponding detection limits are also included.
Chap. 10 - 227
-------
Table 10.3 ACCEPTABLE SAMPLE SIZE AND CORRESPONDING DETECTION
LIMITS FOR SAMPLE CONDUCTIVITY RANGES
Conductivity Range
(ohm)
Sample Size
(mL)
Detection Limit
(ppb)
<150
2500
1
150-300
1000
3
300-600
500
6
600-1,200
250
12
1,200-3,000
100
30
3,000-6,000
50
60
6,000-12,000
25
120
12,000-30,000
10
300
30,000-60,000
5
600
10.3.5 GC/FID Performance Evaluation
Prior to analyzing procedural blanks, acceptable performance for the
GC/FID system must be demonstrated.
(1) Analyze the GC/NS system performance standard (Table 10.1) as
specified in Table 10.4. Figure 10.4 shows a total ion chromato-
gram of a similar mixture analyzed by GC/1IS under similar condi-
tions that may be used to identify test components in the stand-
ard.
(2) Measure peak asymmetry or tailing for acetophenone and 1-tetra-
decanol using the percent peak asymmetry factor (PAF):
% p p = x 100
where
B = the width of the back half of a chromatographic peak
measured at 10% above baseline
F = width of the front half of the chromatographic peak
measured at 10% above baseline.
PAP should measure less than 200% for both acetophenone and
1—tetradecanol.
Chap. 10 - 228
-------
0
F ..)
t• . )
‘I
w
‘I
N
w
-4
U
U
-I
0
U
-I
U
0.
- 4
0
0
-l
‘4. 4
c 1
1 4
U
4 . 4
U
N
U
-U
‘I
c i
U
14
U
4.4
U
U
—I
N
U
.0
U
1.4
U
oil..
U 4.4
01 001
IU
‘0 l-4
U
o ic.c
U 01101 ,4 . 4
4 OLCOI
0
r -— - _______- _______-— -- ______- u - — r - — -
( I00 800 11)00 1200 HUll IGOO
6:4 ( 1 10:01* 13:20 16.10 20:00 23:21) 26:41)
T!I
Figure 10.4. Total ion chromatogram for VOSA SPS.
-------
(3) Sensitivity is checked by measuring the signal-to-noise ratio
for crotonic acid. A signal-to-noise ratio of 10 to 1 or greater
is acceptable.
10.3.6 Derivatization and Procedural Blanks and Controls
Prior to processing any samples a derivatization blank and a deriva-
tization control must be run and analyzed by GC/FID to assure that back-
ground contamination during derivatization is low and that derivatization
yield is acceptable. After validating the derivatization step, three
procedural blanks must be processed and analyzed by GC/FID to detect
contamination and artifacts from the resin material, solvents, reagents,
dirty glassware, and other sources.
10.3.6.1 Derivatization Blank --
(1) Add 0.1 mL aqueous iN K 2 C0 3 to a 15 mL round bottom test tube.
Derivatize as described in step 10.4(10).
(2) With a 50 pL syringe, add 10 pL of the GC external standard (hexadecane)
solution to the derivatized solution.
(3) Analyze the solution by GC/PID using the conditions described in
Table 10.4. Contaminant (other than the derivatizing reagents)
peak heights should be less than 10% relative to the peak height
of the external standard, hexadecane.
(4) If significant contamination is present, rerun derivatization
blanks using fresh glassware and reagents (acetone, benzyl
bromide, and K 2 C0 3 ). Reagents may be distilled or taken from
another source.
10.3.6.2 Derivatization Control - -
(1) Prepare a control solution. With a 50 pL syringe, measure 25 iJL
of each stock solution in group B (Table 10.2) into a 10 mL
volumetric flask and dilute to volume using pesticide grade
acetone. (Unlabeled butyric acid may be used instead of
d 7 -butyric acid.) Transfer into a Teflon sealed screw cap
bottle and store at 4°C.
(2) With a 1 mL graduated pipette, measure 0.5 niL of this control
solution into a 15 niL round bottom test tube. Add 0.1 niL of
aqueous 1M K 2 C0 3 . Mix well. Evaporate to dryness using nitrogen
Chap. 10 - 230
-------
Table 10.4. GC/FID OPERATING CONDITIONS FOR
VOLATILE STRONG ACIDS
GC Column 30 m DB-l (1.0 p film thickness)
fused silica, wide bore (0.34 mm
i.d.), capillary column
CC Carrier Gas Helium
Carrier Gas Flow 1.6 mL/min through the column;
15:1 split injection
Temperature Program 80 to 250°C @ 5°/mm
Injector Temperature 250°C
Detector Temperature 260°C
Injection Volume 1.0 pL
blowdown at an elevated temperature (-60°C). With a 1 mL gradu-
ated pipette, add an aliquot (0.3 mL) of benzyl bromide reagent
to the residue. Cap the tube, shake, and react the solution
overnight at 60°C.
(3) With a 50 i.iL syringe, add 10 pL of the GC external standard to
the derivative mixture.
(4) Analyze samples by GC/FID as described in Table 10.4. Peak
heights of the three control acid derivatives measured relative
to the external standard should fall within the range of 0.9-1.1.
(5) If derivative yield is low (as measured by relative peak heights),
this step must be repeated using fresh reagents and glassware.
Reagents may be distilled or taken from another source.
10.3.6.3 Procedural Blanks ——
For each batch of materials and reagents that are used, a set of
three procedural blanks are required. Table 10.5 identifies these blanks
and defines their purpose. These blanks must be processed and analyzed by
GC/FID prior to processing any samples. If a large number of samples are
to be processed, it is advantageous to prepare large batches of materials
and reagents thereby reducing the number of blanks which must be run.
Chap. 10 - 231
-------
Table 10.5. PROCEDURAL BLANKS
Blank Description
Procedural
Blank
1
Detects contamination in resin
material, solvents, and glassware
Procedural
Blank
2
Detects contamination in reagent
water
Procedural
Blank
3
Detects contamination in sulfuric
acid, sodium hydroxide, and
sodium thiosulfate solutions used
during sample collection and
analysis
Procedural blank 1 must also be run every time a set of samples is processed
and analyzed.
10.3.6.3.1 Procedural Blank 1- -
(1) Prepare a resin column as described in step 10.4(1).
(2) Follow the procedure for volatile acid analysis as described in
steps 10.4(5) to 10.4(10).
(3) If more than 3 mL of the K 2 C0 3 solution is needed to neutralize
the distillate, residual chlorine has not been removed from the
resin material. Additional rinsing with iN NaOH is required
before using the resin material.
(4) With a 50 pL syringe, add 10 pL of the GC external standard to
each derivatized mixture.
(5) Analyze samples by GC/FID as described in Table 10.4. Contami-
nant (other than the derivatizing reagent, benzyl acetate, and
benzyl propionate) peak heights should measure less than 25%
relative to the standard.
(6) If significant contamination is present, the procedure should be
repeated using a blank chromatography column with no resin
added. If this blank is acceptable, then the resin is the
source of contamination and should be recleaned using the proce-
dure in Section 10.3.2. A procedural blank 1 should then be
repeated using the fresh resin material.
Chap. 10 - 232
-------
(7) If significant contamination is present in this blank, fresh
reagents and clean glassware should be used and the reagent
blank repeated. When the reagent blank is acceptable, a proce-
dural blank 1 must be repeated. Procedural blanks 2 and 3 may
be processed once the procedural blank 1 is acceptable.
10.3.6.3.2 Procedural Blanks 2 and 3- -
(1) Prepare a resin column as described in step 10.4(1). Attach a
separatory funnel to the top of the column. For procedural
blank 2, pour 100 mL of reagent water into the separatory funnel,
then drain the sample through the resin bed at a flow rate of
—10 mL/ minute. For procedural blank 3, use 100 mL of reagent
water spiked with 1 mL each of the sulfuric acid, sodium hydrox-
ide, and sodium thiosulfate solutions. These must be the same
solutions which are to be used when the samples are collected
(Chapter 5, section 5.2.14).
(2) Follow the extraction procedure as described in steps 10.4(5) to
10.4(10).
(3) With a 50 pL syringe, add 10 jiL of the GC external standard to
each derivative mixture.
(4) Analyze the samples by GC/FID using the conditions in Table 10.4.
Peak heights for contaminants in procedural blank 3 which are
not present in procedural blank 2 should measure less than 20%
relative to the external standard.
(5) If significant contamination is present in Procedural blank 3
which is not present in procedural blank 2, the procedure should
be repeated using fresh solutions of sulfuric acid, sodium
hydroxide, and sodium thiosulfate until an acceptable blank is
achieved.
10.4 ION-EXCRANGE SEPARATION AND DERIVATIZATION
(1) Prepare ion exchange column by placing a small glass wool plug
in the bottom of the chromatography column (Fig. 10.1). Using a
graduated pipette with the tip cut off, pipette 10 mL (measure
volume after resin has settled) of cleaned Biorad AG 1-X8 resin
into the open tubular chromatography column, allow the resin to
settle, and rinse resin bed with —10 mL of reagent water.
Chap. 10 - 233
-------
(2) If the sample is turbid, allow it to stand undisturbed for 4
hours or overnight prior to analysis to allow particulates to
settle.
(3) Gently pour the aqueous portion of the sample (equilibrated to
room temperature) into a graduated cylinder to measure sample
volume. Both total volume of sample collected and the volume of
sample analyzed should be recorded.
(4) For surface waters and effluents, transfer sample water to the
reservoir on top of the column and allow it to drain through the
resin bed. Flow is gravity controlled and should be approximate-
ly 5 mL/min (NOTE: The glass wool plug can restrict flow if
packed too tightly.) For drinking water, transfer sample to a
one gallon container. Attach siphoning adaptor (Fig. 10.2)
between sample container and column, and drain sample through
the resin bed. Attach a piece of tygon tubing between the end
overnight accumulation. If particulate matter in the sample
plugs the column, positive head pressure can be used to restore
flow.
(5) Rinse the resin with 25 mL of deionized water. Flow is gravity
controlled (—5 mL/minute).
(6) Elute organics from the column using 150 mL of O.55N NaHSO 4 in
acetone:water (1:1). Acids should be eluted immediately after
all of the sample has passed through the resin column. Collect
the eluant solvent in a 250 mL round bottom flask. Add a boiling
chip to the flask.
of the chromatography column and a
sample flowing through the resin,
chromatography column and turn on
create sufficient suction to pull
siphon adaptor and onto the resin
after water is flowing through the
at the base of the column, adjust
—2.5 mL/minute. At this flow, it
sample to drain through the resin
water aspirator. To start
open the stopcock on the
the aspirator. This should
the water sample through the
column. Disconnect suction
resin bed. Using the stopcock
flow through the column to
will require —16 hours for the
bed, which is suitable for
Chap. 10 - 234
-------
(7) Connect the flask containing the eluant solution to the distil-
lation apparatus (Figure 10.3). Heat the flask and allow the
distillation to proceed until all liquid has distilled into the
250 mL round bottom flask. Distillation should be complete in
e 30 minutes. The sample should be heated gradually to avoid
bumping at the beginning of the distillation. Samples should be
distilled immediately after elution.
(8) Volatile acids in the distillate are converted to their nonvola-
tile salts by adding sufficient aqueous 111 K 2 C0 3 to the distillate
to raise the pH to 8 or higher. (If more than 2 mL of the K 2 C0 3
solution is needed to raise the pH of the procedural blank,
residual chloride has not been removed from the resin material,
see Section 10.3.6.3.1). Samples can be stored refrigerated
anytime after this step is complete.
(9) Concentrate the distillate to —2 mL using rotary evaporation at
an elevated temperature (—60°C). Some industrial effluents may
foam during rotary evaporation. If this occurs the entire
sample may be concentrated using nitrogen blowdown at an elevated
temperature (-70°C).
(10) Transfer the concentrate to a 15 mL, round bottom, screw cap,
test tube. Rinse the evaporation flask with two 2.5 mL portions
of distilled water and combine with the concentrate. Evaporate
the concentrate to dryness using nitrogen blowdown at an elevated
temperature (—60°C). With a 1 mL graduated pipette, add an
aliquot (0.3 niL) of benzyl bromide reagent solution to the
residue. The tubes are capped, shaken, and the solution is
allowed to react at 60°C overnight. Since the derivatizing
reagent is the solvent, no further manipulations are needed. If
samples evaporate to dryness during derivatization, they must be
discarded. If they partially evaporate, adjust to a known
volume before GC/MS analysis.
(11) After derivatization add 10 pL of the MS external standard
solution to each derivatized sample concentrate using a 50 pL
syringe. Store the derivatized samples at 0°C until GC/tIS
analysis.
Chap. 10 - 235
-------
10.5 GC/MS/COEIP ANALYSIS
Prior to beginning GC/MS/CONP analysis of the volatile strong acids
fraction the analyst should be familiar with the procedures described i 4
Chapter 13, “GC/NS/COHP Analysis - General for all Protocols.”
10.5.1 Preparation for Analysis
10.5.1.1 GC/MS/COMP Operating Parameters--
The recommended GC/MS operating parameters are given in Table 10.6
for the analysi s of volatile strong acids (VOSA). Other GC capillaries
which meet the system performance criteria may be substituted for the
recommended fused silica (See Section 13.4). It is also recommended that
the capillary used here be used only with sample extracts containing methyl
esters of strong acids.
10.5.1.2 MS Calibration-—
The mass spectrometer is calibrated using the manufacturerts recom-
mended approach (Chapter 13). The acceptability of the calibration results
is verified with the analysis of the system performance solution (SPS).
Table 10.6. GC/MS OPERATING CONDITONS FOR VOLATILE STRONG
ACIDS AS THEIR BENZYL ESTERS
GC Column 30 m DB-l (1.0 p film thickness)
fused silica wide bore (0.34 mm
I.D.) capillary column
GC Carrier Gas Helium
Carrier Gas Flow 1.6 mL/min through the column;
9:1 split injection
Carrier Gas Sweep Time 85 sec (50°C)
Temperature Program 65°C/5 mm; to 250°C @ 4°/mm and
hold
Injector Temperature 250°C
Transfer Line Temperature 255°C
Injection Mode Splitless 0.4 mm/split
Injection Volume 1.0 pL
Ionizing Energy 70 eV
Ion Source Temperature 250°C
Scan Range 35-500
Scan Rate 0.9 s, 0.1 sec hold
Chap. 10 - 236
-------
10.5.2 Analysis of SPS (Quality Control )
Using the prescribed GC/HS conditions the SPS is analyzed and all
necessary data is acquired and evaluated prior to proceeding to sample
analysis or determination of R fRs. The SPS is analyzed at the beginning
of each day’s operation.
The SPS raw data is extracted from the run, test parameters calculated
and plotted or tabulated (Tables 10.7 and 10.8). Up to 14 days of SPS
analytical results can be historically recorded in Table 10.7 for day-to-day
comparisons. This allows the analyst to anticipate GC/NS maintenance
requirements. Calculation and usuage of these data are discussed in
Chapter 13.
As part of additional QC/QA procedures, a check of RMRs is made on a
daily basis (Table 10.8). The RlfRs are compared to those obtained during
the development of the historical data bank. The usage of these data is
also discussed in Chapter 13.
10.5.3 Determination of R11Rs
RMRs for the User’s data bank (as opposed to the daily RMR checks
discussed in the above paragraph) are determined by injecting a solution
containing the derivatized internal standards and analytes.
The GC/HS/COMP operating parameters are given in Table 10.6.
Table 13.8 may be used to tabulate RMR raw data.
10.5.4 Analysis of Field and Quality Control Samples
After all specified criteria for GC and HS performance (Tables 10.7
and 10.8) have been met, analysis of VOSA sample extracts is conducted.
A set of VOSA sample extracts may have been derived from procedural
blanks, laboratory and field controls and blanks, surrogates, and collected
field samples, and are generally analyzed in that respective order.
Qualitative and quantitative procedures are described in Chapter 13.
Table 10.9 may be used to tabulate sample analyte raw data prior to using
the ?IASQUANT software program or manual calculations.
Chap. 10 - 237
-------
Table 10.7. GC-MS SYSTEM PERFORMANCE TEST FOR VOLATILE STRONG ACIDS (VOSA)
Dates:
Run Id Code
GC Column
and
Program:
Notebook References:
MS SYSTE1I CHHCKS
High/Low Mass Balance
Ion
51
68
70
127
197
198
199
275
365
441
442
443
68 70 127
DFrPP
Relative Abundance Criteria
30% to 60% of m/z 198
<2% of ic/z 69
<2% of m/z 69
40% to 60% of m/z 198
<1% of in/z 198
100%
5—9% of m/z 198
10% to 30% of mfz 198
at least 1% of w/z 198
present, but < m/z 443
>40% of m/z 198
17% to 23% of m/z 442
275 365 331 442 443
Paas/ Remedial
Fail Action
51
Abundance * ( )
Criteria
( )
(1
_____ 197 198 199 -
_— LL L1 L1_LI Li L1 Li Li - - - - __
a
*These abundances are initially established by the HAS user, and subsequently becoise guidelines for
acceptability of tune.
(continued)
Chap. 10 - 238
-------
Table 10.7 (cont t d.)
Test Components Criteria
Limit of
Detection Crotonic Acid (Benayl Ester) S:N (m/z 91) > 6:1
12
10
z
,, 8
6
‘ I I I I I I I I I I I I
Day
GC SYSTEM CHECKS
Peak 1. Acetophenone (TIC) • %PAF (300
Asy etry 2. 1-Tetradecanol (TIC) • tPAF (200
300 No. 1
200 No.2
0.
100
I I I I I I I I I I I I I
Day
I I 1 I I I
Day
(continued)
1. Acetophenone (TIC)
Basicity 2. 2,6-Dimethylphenol (TIC)
1.4
1.2
0
-I
4..
0.8
0.6
Ratio 2:1 = 0.7 to 1.3
Chap. 10 - 239
-------
Teat Components Criteria
Separation n-Eicosane (w/z 43)
Number n-Heneicosane (m/z 43) SN > 6
12
11
10
Z 9
In
8
7.
6--
I I I I I I I I I I I I I
Day
!-Octadecane (TIC) 9 = minimum 50%
Resolution 1—Octadecene (TIC) Valley
90
80’
70
> 60
50 --
40
I I I I I I I I I I I I I
Day
Day
Chap. 10 - 240
1 I I I •
(continued)
Table 10.7 (cont’d.’)
Inertness ethyl Stearate
20
18
0
— 16
14
12
Ratio m/z 74 to 298 12.1 to 16:1
-------
Table 10.7 (cont’d.)
Teat Components Criteria
Capillary
Capacity I4exanoic Acid (Benzyl Ester) (TIC) %PAJ’ ‘70
100
90
I I I I I I I I I I I I I
Day
Relative
Retention 1. Heneicosane
Shift 2. Pyrene RRS = 0.95-1.05
1.10
1.05 --
1.0
0.95 --
0.90
I I I I I I J I I I I I I
Day
Chap. 10 - 241
-------
Table 10.8. GC-HS SYSTEM PERFORMANCE TEST:
RHR CHECK FOR VOLATILE STRONG ACIDS (VOSA)
Date: Run ID Code:
DATA
Amount Ion Area Area
Standard MW (rig) pM (m/z) (Run 1) (Run 2)
2-Fluorobiphenyl 172 172
d 7 —butyric acida 109 50
109
MATRIX OF STANDARD ION RNRs
Standard Ion 172 50 109
2-Fluorobiphenyl 172
-
d 7 -butyric acida 50
109
-
aAS methyl esters. A = ion area or height
ng = ng injected
A •NW Y xana lyte
x x
= A MW y standard
x
COMMENTS:
Chap. 10 - 242
-------
Table 10-9. RAW ANALYTE DATA FOR VOSA FRACTION
Date:
Run I.D. Code;
Notebook Reference:
Vol. Water Processed
Sample Identification:
Vol. Water CL):
(to which i were added)
(niL):
STANDARDS
Spec-
I.D. trum
No. No.
2-Fluorobiphenyl
d 7 —Butyric Acid
Weight
(ng )
Ions
(m/z) Area
NW
a
K
172: ,______
50,109:
,
,
ANALYTES
Class No.
I.D.
No.
Spectrum
No.
Ions
(m/z) Area
MWa
,
,
,
, :
,
,
,
,
,
,
,
,
,
,
,
a 1 f calculations are performed manually then this information is also
needed, whereas ?IASQUANT performs functions automatically.
A NW n A = ion area or height
ng = X A ng = ng recovered from volume
x/y y y of water processed
x analyte
K y standard
A NW
y y
Chap. 10 - 243
-------
CHAPTER 11
ANION-EXCHANGE AND ANALYSIS OF NONVOLATILE ACIDS (NOVA)
11.1 INTRODUCTION
11.1.1 Principle of the Method
This method describes the determination of nonvolatile organic acids
in drinking water, surface waters, and effluents. Since strong acids such
as strong phenols and sulfonic, phosphonic and dicarboxylic acids are too
ionic to efficiently extract from water into organic solvents, the procedure
utilizes ion-exchange resins to concentrate them from the aqueous matrix.
Acids are eluted from the resin using HC1 in methanol. The mineral acid
protonates the organic acids to their nonionic forms, which are then
rinsed off the resin with methanol. Both HC1 and methanol are removed
using rotary evaporation without significant losses of the nonvolatile
acids. These organic acids must be derivatized prior to gas chromatography
in order to increase compound volatility and reduce adsorptive losses.
Diazoinethane was the only reagent tested which successfully derivatized
sulfonic and phosphonic acids to chromatographable products. Since diazo-
methane will methylate only free acids, a small volume of HC1:methanol is
added to sample concentrates just prior to derivatization. The samples
are concentrated using nitrogen blowdown to remove excess derivatizing
reagent, HC1 and solvent.
11.1.2 Detection Limits
If an average detection limit for GC/MS analysis for methylated
nonvolatile acids is estimated as 10 ag per component, and the final
volume of derivatized sample is 0.5 mL, then nominal detection limits for
a 1 pL injection are directly related to sample size:
2 ppb for a 2.5 L sample (drinking water);
10 ppb for a 500 rnL sample (surface water);
20 ppb for a 250 mL sample (industrial and municipal wastewater); and
100 ppb for a 50 mL sample (energy effluents).
Chap. 11 - 244
-------
11.1.3 Interferences
Background from thermal decomposition of humic acids which are ex-
tracted from the sample by the ion-exchange resin during gas chromatographic
analysis may cause interferences for surface waters and municipal wastewater
effluents. A procedural blank must be run prior to processing any samples
to assure that contamination and artifacts from resin material, solvents,
reagents, glassware, and other sources is low.
11.1.4 Precision, Accuracy, and Scope
Table 1.8 in Chapter 1 presents average recoveries and standard
deviations for a variety of organic acids. This method gives acceptable
recoveries for sulfonic, dicarboxylic, and certain aromatic
carboxylic acids, as well as pentachlorophenol. Long chain fatty acids
are not recovered well.
11.2 APPARATUS AND REAGENTS
The following materials are required for processing a set of nine
samples plus three procedural blanks. Nine is the maximum number of samples
(including field and quality control) which should be processed at a time.
The corresponding quality control samples which should be run during these
analyses are listed in Table 5.1, Chapter 5. The three procedural blanks
must be run before any samples are collected or processed. It will
require approximately one working day to process nine samples.
(1) Nine glass chromatography columns, —l cm 1.0. X 22 cm, equipped
with 500 mL sample reservoir (Figure 11.1) and a ground glass
stopper. The size of the sample reservoir is not critical;
however a 500 mL reservoir will hold the entire sample for all
water types except drinking water. For drinking water, an
adaptor (Figure 11.2) for siphoning the sample onto the resin
should be used in lieu of a sample reservoir. This allows
unattended flow through the sample for a 14 to 16 hour period
such that the sample can be concentrated overnight. A vacuum
aspirator is needed to start sample flow through the siphon
adaptor and resin column.
(2) One 10 mL graduated pipette with the tip cut off for measuring
and transferring resin material.
Chap. 11 - 245
-------
24/40
glass connection
1
- 22cm
.1
500 mL glass
separatory funnel
Figure 11.1. Chromatography column with sample reservoir (exact
dimensions of column are not critical - must hold
10 mL of resin).
Teflon stopcock
lcmi.d.
Teflon
stopcock
Chap. 11 - 246
-------
connectof
—I i
14.
Teflon
itopoock
Figure 11.2. Chromatography column with siphoning adaptor for drinking
water.
1 gallon
ample
bottle
Teflon tubing
24140 —
connection
I
tygon tubing to aspirator
Chap. 11 - 247
-------
(3) One graduated cylinder for measuring sample volume. The size of
the sample, thus, the cylinder will depend upon the results of
conductivity measurements. In general they may be:
1000 mL for drinking water;
500 mL for surface water;
250 mL for industrial and municipal wastewater effluents; and
50 oiL for energy samples.
The cylinder may be reused for each sample if it is rinsed 5
times with deionized water.
(4) One conductivity bridge - range 1 to 30,000 ohm.
(5) Nine 200 oiL round bottom flasks (24/40).
(6) One rotary evaporator. If more than one is available up to four
samples can be evaporated at a time. Care should be taken to
avoid bumping; however since concentrates can go to dryness, the
samples require little attention.
(7) One vacuum aspirator for pulling water through the resin column.
(8) Fifteen 15 mL round bottom centrifuge tubes with Teflon lined
screw caps.
(9) One Soxhlet extractor for cleaning ion-exchange resin. A minimum
200 oiL capacity is required. Resin can be processed in large
batches and stored; therefore a 500 oiL to 1000 oiL size would be
preferred.
(10) One large chromatography column for preparing resin material.
The dimensions of this column are not critical, it should hold a
minimum of 120 oiL of resin material; for large batches of resin,
a larger column is preferred. The flow through the column
should not exceed 50 oiL/minute.
(11) One manifold with a temperature controlled water bath or heating
block for nitrogen blowdown. A manifold with nine spaces is
preferred but not essential.
(12) One p11 meter with a combination electrode.
(13) 10 oiL volumetric flasks for preparing standard solutions.
(14) 15 oiL vials with Teflon lined screw caps for storing standard
solutions.
(15) Syringes for preparing standard solutions - 50 and 250 pL.
Chap. 11 — 248
-------
(16) Graduated pipettes for preparing standard solutions •- 1 mL.
(17) One well ventilated hood or toxic lab for working with diazometh-
ane.
(18) Diazomethane generation/distillation apparatus (Aldrich Macro
Diazald set Zl0,85l-0) with all clear glass seals (Caution the
presence of ground glass or scratched glass may catalyze an
explosive reaction of dizaomethane).
(19) One gas chromatograph suitable for capillary column chromatography
with flame ionization detection and all required accessories
including syringes, gases, and a strip chart recorder.
(20) One 30 m x 0.34 mm I.D. DB—5 (1.0 p film thickness) fused silica
capillary column.
(21) Materials and reagents
(a) 120 mL AG l-X8 (50-100 mesh) ion exchange resin (Biorad
Laboratories, Richmond, CA) in C1 form.
(b) Reagent water - reagent water is described as a water
source that does not produce a background interference at
the limit of detection and that has a conductivity of
<1 ohm. A water purification system (?lillipore Super Q or
equivalent) may be used to generate reagent water.
Cc) 1 gallon methanol (Burdick and Jackson, pesticide analysis
grade).
(d) one lecture bottle containing gaseous HC1.
(e) 1 L methyl-t—butyl ether (Burdick and Jackson, pesticide
analysis grade).
(f) 5.0 L lN KOH in reagent water.
(g) 240 mL iN formic acid in reagent water.
(h) 250 mL 0.lN NaOH in reagent water (use the same solution as
specified in Chapter 5, section 5.2.14).
(i) 250 mL O.lN HC1 in reagent water (use the same solution as
specified in Chapter 5, section 5.2.14).
(j) 100 mL 0.05M Na 2 S 2 O 3 in reagent water (use the same solution
as specified in Chapter 5, section 5.2.14).
(k) 5 mL phenolphthalein solution (0.05 g in 50 mL ethanol +
50 mL reagent water).
Chap. 11 - 249
-------
(1) 50 mL diethyl ether (Nallinkrodt, pesticide analysis grade,
peroxide free).
(m) —100 mg p-tolylsulfonyl methylnitrosamide (Diazald®,
Aldrich Co.).
(a) 10 mL 60% KOH (w/v). Dissolve 6 g KOH in 10 mL reagent
water.
(o) 1 mL MS external standard solution - 4-fluoro-2-iodotoluene
(300 ng/l0 pL) and 2-fluorobiphenyl (300ng/l0 ML) in
CH 2 C1 2 .
(p) 1 mL GC external standard solution - hexadecane (30 ng/lO ML)
in CH 2 C1 2 .
(q) 1 mL system performance standard solution for nonvolatile organic
acids (Table 11.1).
Table 11.1. GC/MS SYSTEM PERFORMA}ICE SOLUTION
FOR NONVOLATILE ACIDS (NOVA)
Compound
Concentration (ng/pL)
2, 6-dimethylphenol
300
acetophenone
310
1-tetradecanol
240
1-octadecene
240
n-octadecane
230
DFIPP
300
n—eicosane
300
pyrene
300
n-heneicosane
300
methyl stearate
300
dimethyl adipate
1000 a
methyl 2,4,5-trichiorophenoxyacetate
18 a
2-fluorobiphenyl
300
4—fluoro-2-iodotoluene
130
2—naphthalene-sulfonic acid-d 7 , methyl ester 300 a
of underivatized acid.
Chap. 11 - 250
-------
11.3 PREPARATION FOR ANALYSIS
11.3.1 Preparation of System Performance Standard
(1) Prepare individual stock standards as specified in Table 11.2.
- solids — accurately weight 0.120 g of pure material, dissolve
in the specified solvent, dilute to volume in a 10 mL
volumetric flask.
Table 11.2. STOCK STANDARD SOLUTIONS
Compounds
D
(@
ensity
20°C)
Concentr
mg/1O
ation
mL
Group Aa
2,6-dimethyiphenol
sb
120
acetophenone
1.030
124
1—tetradecanol
0.823
98
1-octadecene
0.789
95
n-octadecane
0.777
93
DFTPP
S
120
n-eicosane
S
120
pyrene
S
120
n-heneicosane
S
120
methyl stearate
S
120
2-fluorobiphenyl
S
120
4-f luoro-2-iodotoluene
0.883
110
Group BC
2-naphthalene-sulfonic acid-
S
120
d 7 H 2 0
2,4,5-trichiorophenoxyacetic
S
120
acid
adipic acid
S
400
aSolvent is methylene chloride.
b
S = solid.
is methanol.
Chap. 11 - 251
-------
- liquids - using a 250 pL syringe, accurately measure 120 IJL
of pure liquid, dissolve in the specified solvent, dilute
to volume in a 10 mL volumetric flask.
(2) Prepare a secondary neutral standard. With a 1.0 mL graduated
pipette measure 0.5 mL of each stock standard in group A (for
4-fluoro-2-iodotoluene measure 0.25 mL) into a 10 mL volumetric
flask. Dilute to volume using pesticide grade methylene chloride.
Transfer into a Teflon sealed screw cap bottle. Store at. 4°C.
(3) Prepare a secondary acid standard. With a 1.0 mL graduated
pipette measure 0.5 mL of the group B standards (for 2,4,5-tn-
chlorophenoxy acetic acid measure 30 I.iL with a 50 pL syringe)
into a 10 mL volumetric flask. Dilute to volume using pesticide
quality methanol. Transfer into a Teflon sealed screw cap
bottle. Store at 4°C.
(4) Transfer the stock standards into Teflon sealed screw cap bottles.
Store at 4°C.
(5) Fresh standards should be prepared every six months. If degrada-
tion or evaporation has occurred, standards should be prepared
sooner.
(6) If compound purity is 96% or greater, the weight or volume can
be used without correction to calculate the concentration of
stock standards. Compounds which are less than 96% pure cannot
be used for standards.
(7) With a 1 niL graduated pipette, measure 0.5 mL of the secondary
acid standard into a 15 niL round bottom test tube. Evaporate to
—0.1 niL using nitrogen blowdown at 40°C. Resuspend the sample
in 0.2 niL of lN HC1:methanol solution (See Section 11.3.4 for
preparation of HC1:methanol).
(8) Derivatize the sample using diazomethane as described in Sec-
tion 11.4.2, steps 1 to 10.
(9) Evaporate the sample to 0.5 niL using nitrogen blowdown at 30°C.
With a 1 niL graduated pipette, add 0.5 niL of the secondary
neutral standard to the derivatized mixture and mix well. This
gives the concentrations shown in Table 11.1. Cap the test
tube. Store at 4°C until GC/MS analysis.
Chap. 11 - 252
-------
11.3.2 Preparation of Resin Material
Ion-exchange resin is cleaned prior to preparing the resin columns.
One hundred and twenty mL of resin material is needed to process 9 samples
and three procedural blanks and is the minimum amount of resin which
should be prepared. The following instructions describe procedures based
on 120 mL of resin material; however, it is possible to prepare larger
batches and store the prepared resin wet in a sealed glass jar. If a
larger volume’of resin is prepared, volumes for rinsing the resin should
be adjusted accordingly.
Biorad AG l-X8 (50-100 mesh) in the C1 form is extracted with pesti-
cide grade methanol in a Soxhlet apparatus overnight. The resin is placed
in a column and rinsed with 10 bed volumes of deionized water (1.2 L), 20
bed volumes of iN NaOH (2.4 L), and 2 bed volumes of lN formic acid
(240 mL). The resin is then rinsed with deionized water until the eluant
water is at pH 5 to 6. Resin which is not used immediately may be stored
wet, sealed in glass jars.
11.3.3 Cleaning of Materials
(1) All glassware to be used is washed with Amway S-A-8 laundry
compound (or Isoclean or other nondetergent) rinsed with deionized
water and baked for a minimum of 4 hours at 500 to 550°C. All
cleaned glassware is immediately capped or covered with foil
(precleaned with hexane) to prevent contamination.
(2) Teflon liners and teflon lined septa are sonicated for 10 minutes
in pesticide grade methanol followed by 10 minutes in pesticide
grade pentane. The sonicated liners are vacuum-oven (- ‘20 inches
of water) dried for 3 to 5 hours at 70° and stored in clean,
Teflon lined screw-cap bottles.
11.3.4 Preparation of Gaseous HC1:Methanol
iN HC1:methanol solution is usually prepared in large batches and
stored in the refrigerator for up to 2 weeks between use. Because HC1 is
corrosive, the entire operation should be performed in a well-ventilated
hood. Pesticide analysis grade methanol (500 mL) is added to a 1 L amber
glass bottle and the bottle is chilled in an ice bath. Gaseous HC1 is
bubbled through the methanol as illustrated in Figure 11.3. HC1 is allowed
to dissolve into the methanol for ‘45 minutes. The HC1 concentration of
Chap. 11 - 253
-------
Teflon
tubing
methanol
ice/H 2 0 bath
Pasteur pipet
gaseous HCI
magnetic
stirrer
Tif Ion
tubing
C)
p .,
U’
HCI
lecture
bottle
trap
Figure 11.3. Equipment for generating IIC1:methanol.
-------
the methanol solution is determined by adding 1 mL of the methanol solution
to 20 mL of deionized water, and titrating to the phenolphthalein endpoint
with iN KOH. A normality from 0.9 to 1.1 is acceptable for the analytical
operation. If the solution is stored, the normality should be checked
before use. Smaller amounts may be prepared in a similar manner.
11.3.5 Maximum Sample Size
Conductivity measurements must be determined for each sample to
assure that the total concentration of anions in the sample does not
exceed the resin capacity. Prior to measuring conductivity, check the pH
of the sample using a pH meter. Adjust the sample pH to —8 using 0.1N KOll
or 0.1N 11 2 S0 4 if necessary. Table 11.3 lists the sample size which should
be used for a given conductivity regardless of water type. Corresponding
detection limits are also included.
11.3.6 GC/FID Performance Evaluation
Prior to analyzing procedural blanks, acceptable performance for the
GC/FID system must be demonstrated.
(1) Analyze the GC/NS system performance standard as specified in
Table 11.4. Figure 11.4 shows a total ion chromatogram of this
mixture analyzed by GC/MS under similar conditions, which may be
used to identify test components in the standard.
Table 11.3. ACCEPTABLE SAMPLE SIZE AND CORRESPONDING DETECTION
LIMITS FOR SAMPLE CONDUCTIVITY RANGES
Conductivity Range
(ohm)
Sample Size
(m l)
Detection Limit
(ppb)
<150
2500
1.5
150-300
1000
5
300-600
500
10
600-1,200
250
40
1,200—3,000
100
50
3,000-6,000
50
100
6,000—12,000
25
200
12,000-30,000
10
500
30,000-60,000
5
1000
Chap. 11 — 255
-------
Table 11.4. GC/FID OPERATING CONDITIONS
FOR NONVOLATILE ACIDS
GC Column 30 in DB-5 (1.0 j film thickness)
fused silica, wide bore (0.34 mm
i.d.), capillary column
GC Carrier Gas Helium
Carrier Gas Flow 1.6 mL/min through column; 15:1
split injection
Temperature Program 35 to 180°C @ 5°/mm
Injector Temperature 250°C
Detector Temperature 260°C
Injection Volume 1.0 pL
(2) Measure peak asymmetry (tailing) for acetophenone, 1-tetradecanol,
and methyl d 7 -naphthalene sulfonate using the percent peak
asymmetry factor (PAP):
% p p = x 100
where
B = the width of the back half of a chromatographic peak
measured at 10% above baseline
F = width of the front half of the chromatographic peak
measured at 10% above baseline
PAP should measure less than 200% for acetophenone, 1—tetra-
decanol, and methyl d 7 -naphthalene sulfonate.
(3) Check the acidity/basicity of the column by the peak area ratios
determined by integrator or triangulation of 2,6-dimethylphenol
to acetophenone. A ratio of 0.7 to 1.3 for both is acceptable.
(4) Sensitivity is checked by measuring signal-to—noise ratio for
the methyl ester of 2,4,5-trichlorophenoxy acetic acid. A
signal—to-noise ratio of 10 to 1 is acceptable.
Chap. 11 - 256
-------
U
C
SCAN
tit lE
lee.
-4
C
C )
-c
. 0
0
0
14.1
pJ
C)
C
C)
0
0
-ø
0
r 4
0
k
0
(4 ;)
C ,
a)
1
P4
c - n
-4
C)
C
0
C
C)
-C
a
0
4. ,
C)
U
(C
C)
C
( CC)
w( IJ
0 cC
C) U I-.
C —‘ (C
(C C)C)
V I C) CI-’
o c Cflfl
o C)
__( t_(
C)
a
C
-4
0
C
( C
U
C)
•0
( C
S.
4-.
C)
I ;.
-4
‘ V
33:20
Figure 11.4. Total ion chromatogram for nonvolatile acids GC/MS performance standard.
-------
11.3.7 Derivatization and Procedural Blanks and Controls
Prior to processing any samples a derivatization blank and a derivati-
zation control must be run and analyzed by GC/FID to assure that background
contamination is low and that derivatization yield is acceptable. After
validating the derivatization step, three procedural blanks must be proc-
essed and analyzed by GC/FID to detect contamination and artifacts from
the resin material, solvents, reagents, dirty glassware, and other sources.
11.3.7.1 Derivatization Blank-—
(1) Add 0.2 mL of iN HC1:methanol solution to a 15 mL round
bottom test tube. Derivatize as described in steps 1 to
10, Section 11.4.2.
(2) With a 50 pL syringe, add 10 pL of the GC external standard (hexadecane)
solution to the derivatized solution.
(3) Analyze by GC/FID using the conditions described in
Table 11.4. Contaminant peak heights should be less the
20% relative to the peak height of the axternal standard
(hexadecane).
(4) If significant contamination is present, repeat the deriva—
tization blank using fresh glassware and reagents. Reagents
and solvents may be distilled or taken from another source.
11.3.7.2 Derivatization Control--
(1) Prepare a control solution. With a 50 pL syringe, measure
25 I.iL of each stock solution in group B (Table 11.2) into a
10 mL volumetric flask and dilute to volume using pesticide
grade acetone. Transfer into a Teflon sealed screw cap
bottle and store at 4°C until use.
(2) With a 1 mL graduated pipette, measure 0.5 mL of this
solution into a 15 mL round bottom test tube. Evaporate to
-0.1 mL using nitrogen blowdown at 40°C. Resuspend the
sample in 0.2 m l. of iN HC1:methanol solution.
(3) Derivatize the sample using diazomethane as described in
steps 1 to 10, Section 11.4.2.
(4) With a 50 pL syringe, add 10 pL of the GC external standard
to the derivative mixture.
Chap. 11 - 258
-------
(5) Analyze samples by GC/FID as described in Table 11.4.
Peaks heights of the control acids relative to the ex ternal
standard should fall within the values given in Table 11.5.
(6) If derivative yield is low (as measured by relative peak
heights) this step must be repeated using fresh reagents
and glassware.
11.3.7.3 Procedural Blanks--
If the derivative blank and derivative control sample are acceptable,
then a procedural blank must be run.
For each batch of materials and reagents that are used, a set of
three procedural blanks are required. Table 11.6 identifies these blanks
and defines their purpose. These blanks must be processed and analyzed by
GC/FID prior to processing any samples. If a large number of samples are
to be processed, it is advantageous to prepare large batches of materials
and reagents thereby reducing the number of blanks which must be run.
Procedural blank 1 must be run every time a set of samples is processed
and analyzed.
11.3.7.3.1 Procedural Blank 1- -
(1) Prepare a resin column as described in step 11.4.1.1.
(2) Follow the procedure for nonvolatile organic acid analysis as
described in steps 11.4.1.5 to 11.4.2.10.
(3) With a 50 pL syringe, add 10 pL of the GC external standard to
the derivatized mixture.
Table 11.5. RELATIVE PEAK IIEIGHTSa (RPH) OF CONTROL ACIDS
Acid
Acceptable RPH Range
d 7 —naphthalene sulfonic acid,
ester
methyl
.15-.20
adipic acid, dimethyl ester
.50- .60
2,4,5—tricklorophenoxyacetic
acid,
.50- .60
methyl ester
a f 11 — peak height of derivatized acid
peak height of external standard
Chap. 11 - 259
-------
Table 11.6. PROCEDURAL BLA 1(S
Blank Description
Procedural
Blank
1
Detects contamination in resin
material, solvents, and glassware
Procedural
Blank
2
Detects contamination in reagent
water
Procedural
Blank
3
Detects contamination in sulfuric
acid, sodium hydroxide, and
sodium thiosulfate solutions used
during sample collection and
analysis
(4) Analyze samples by GC/FID as described in Table 11.4. Contaminant
peak heights should measure less than 20% relative to the external
standard.
(5) If significant contamination is present, the procedure should be
repeated using a blank chromatography column with no resin
added. If this blank is acceptable, then the resin is the
source of contamination and should be recleaned using the proce-
dure in Section 11.3.2. A procedural blank 1 should then be
repeated using the fresh resin material.
(6) If significant contamination is present in this blank, fresh
reagents and recleaned glassware should be used and the reagent
blank repeated. When the reagent blank is acceptable, a proce-
dural blank 1 must be repeated.
(7) Procedural blanks 2 and 3 may be processed once procedural
blank 1 is acceptable.
11.3.7.3.2 Procedural Blanks 2 and 3- —
(1) Prepare a resin column as described in step 11.4.1. Attach a
separatory funnel to the top of the column. For Procedural
blank 2, pour 100 mL of reagent water into the separatory funnel,
then drain the sample through the resin bed at a flow rate of
—10 mL/min. For procedural blank 3 use 100 mL of reagent water
spiked with 1 mL each of the sulfuric acid, sodium hydroxide and
sodium thiosulfate solutions. These must be the same solutions
Chap. 11 - 260
-------
which are to be used when the samples are collected (Chapter 5,
section 5.2.14).
(2) Follow the extraction procedure as described in steps 11.4.1(5)
to 11.4.2(10).
(3) With a 50 pL syringe, add 10 I.iL of the GC external standard to
each derivative mixture.
(4) Analyze the samples by GC/FID using the conditions in Table 11.4.
Peak heights for contaminants in procedural blank 3 which are
not present in procedural blank 2 should measure less than 20%
relative to the external standard.
(5) If significant contamination is present in procedural blank 3
which is not present in procedural blank 2, the procedure should
be repeated using fresh solutions of sulfuric acid, sodium
hydroxide, and sodium thiosulfate, until an acceptable blank is
achieved.
11.4 ION EXCHANGE SEPARATION AND DERIVATIZATION
11.4.1 Ion-Exchange Separation
(1) Prepare ion-exchange columns by placing a small glass wool plug
in the bottom of the chromatography columns. Using a graduated
pipette with the tip cut off, pipette 10 mL (measure volume
after resin has settled) of cleaned Biorad AG 1—X8 resin into
the open tubular chromatography columns, allow the resin to
settle, and rinse the resin bed with ‘-lO mL reagent water.
(2) If the sample is turbid after pH adjustment/conductivity measure-
ment, allow it to stand undisturbed for 4 hours or overnight
prior to analysis to allow particulates to settle.
(3) Gently pour the aqueous portion of the sample (equilibrated to
room temperature) into a graduated cylinder to measure sample
volume. Both total volume of sample collected and the volume of
sample analyzed should be recorded.
(4) For surface waters and effluents, transfer sample water to
reservoirs on top of the column and allow it to drain through
the resin bed. Flow is gravity controlled and should be approxi-
mately 5 mL/min (NOTE: The glass wool plug can restrict flow if
packed too tightly). For drinking water, transfer sample to a
Chap. 11 - 261
-------
one gallon container. Attach siphoning adaptor between sample
container and column. Attach a piece of tygon tubing between
the end of the chromatography column and a water aspirator. To
start sample flow through the resin bed, open stopcock on the
chromatography column and turn on aspirator. This should create
sufficient suction to pull the water sample through the siphon
adaptor and onto the resin column. Disconnect suction after
water is flowing through the resin bed. Using the stopcock at
the base of the resin bed, adjust flow to 2.5 mL/minute for
a 2500 mL sample volume. At this flow rate, it will require —16
hours for the sample to drain through the column, which is
suitable for overnight accumulation. Allow the water to drain
out of the resin bed. If particulate matter in the sample plugs
the column, positive head pressure can be used to restore flow.
(5) Rinse the resin with 25 mL deionized water. Allow the water to
drain out of the resin bed, aspirate off remaining water from
the bottom of the resin column. Rinse the resin with 100 mL
methanol. First, pass a fraction of the methanol (—50 mL)
through the column, stop solvent flow, remove the reservoir,
stopper the column with a ground glass stopper and shake the
column to remove air bubbles. Pass remaining methanol through
the resin. Stop flow when methanol reaches the top of the resin
column. Rinse at —5 mL/minute, discard rinse.
(6) Elute acids from the column using 100 mL of iN HC1:methanol.
Collect the eluant in a 200 niL round bottom flask. Elute at
—5 niL/minute. -
(7) Remove all of the eluant solvent, except any water residual
using rotary evaporation at 30°C. Residual water is removed by
adding 50 niL of methyl-t-butyl ether to the flask and evaporating
at —40°C. If any water still remains, add a second 50 niL portion
of methyl-t-butyi ether and repeat the concentration step.
Evaporation should require 15 to 20 minutes per sample (NOTE:
sample must evaporate to dryness to remove }ICl).
(8) Resuspend the sample residue in 3 niL of methanol and transfer to
a 15 niL test tube. Rinse the flask with two 2 niL portions of
Chap. 11 - 262
-------
methanol and transfer to the test tube. Flasks must be rinsed
thoroughly to avoid loss of sample components.
(9) Evaporate the solvent to dryness using nitrogen blowdown at
40°C.
(10) Resuspend the sample in 0.2 mL of iN HC1:methanol solution.
Sample may be stored at this point.
(ii) Derivatize using diazomethane.
11.4.2 Derivatization
CAUTION: Diazomethane is toxic and prone to cause development of
specific sensitivity; in addition, it is potentially explosive. Hence one
should wear heavy gloves and goggles while performing this experiment and
should work behind a safety screen or a hood door with safety glass.
Also, it is recommended that ground joints and sharp surfaces be avoided.
Thus all glass tubes should be carefully fire-polished, connections should
be made with rubber stoppers or clear glass seals, and separatory funnels
should be avoided, as should etched or scratched flasks. Furthermore, at
least one explosion of diazomethane has been observed at the moment crystals
(sharp edges!) suddenly separated from a supersaturated solution. Stirring
by means of a Teflon coated magnetic stirrer is preferred to swirling the
reaction mixture by hand, for there has been at least one case of a chemist
whose hand was injured by an explosion during the preparation of diazometh-
ane in a hand-swirled reaction vessel.
It is imperative that diazomethane solutions not be exposed to direct
sunlight or placed near a strong artificial light, because light is thought
to have been responsible for some of the explosions that have been encount-
ered with diazomethane. Particular caution should be exercised when an
organic solvent boiling higher than ether is used. Because such a solvent
has a lower vapor pressure than ether, the concentration of diazomethane
in the vapor above the reaction mixture is greater and an explosion is
more apt to occur.
Most diazomethane explosions take place during its distillation.
When distilled diazomethane is required, the present procedure is particu-
larly good because at no time is much diazomethane present in the distilling
flask.
Chap. 11 - 263
-------
(1) Assemble the diazometha e generation/distillation apparatus
(Figure 11.5).
(2) Place 10 mL fresh 60% KOH (w/v) in the distilling flask with
35 mL ethanol and 10 mL of diethyl ether (CAUTION: flammable;
forms explosive peroxides). Add a magnetic stirring bar.
(3) Place ice/salt mixture in the condenser and place the collection
flask in a ice/salt bath.
(4) Prepare a solution of 21.5 g (0.1 mole) of -tolylsulfony1methyl-
nitrosamide (Daizald®, Aldrich) in 125 mL of ether. Larger
amounts of ether may be needed to dissolve the reagent if it is
especially pure or the temperature is less than 20°C. Place the
Diazald® reagent in the dropping funnel.
(5) The distilling flask is heated in a water bath at 70-75°C, the
stirrer is started.
(6) The Diazald® solution is added at a regular rate over a 15-20 min-
ute period. As soon as this solution has been added, add 100 mL
of ether to the dropping funnel and continue to add the ether at
the same rate used for the reagent.
(7) Continue distillation until the distillate is colorless.
(8) Cool the distillation flask. Remove the collection flask and
keep cold until used. Destroy any residues of diazomethane in
the apparatus and distilling flask by rinsing with 1% formic
acid in ether.
(9) With a Pasteur pipette add the distilled & .azometl ane—ether solution to
the sample extracts until a yellow color persists. For highly colored
samples add the diazomethane solution until nitrogen evolution
ceases. Additional diazomethane should be prepared as needed.
Excess diazometbane is evaporated from samples during nitrogen
blowdown. If excess diazomethane reagent has been prepared it
should be quenched with formic acid solution (1% in ether)
before discarding. The solution will be colorless when fully
quenched.
(10) Concentrate the derivatized samples to 0.5 mL in the test tubes
using nitrogen blowdown at —30°C. Since excess diazomethane is
Chap. 11 - 264
-------
T.IIor EOC9SI —
Figure 11.5. Z1O,851-0 Macro Diazald® set (with I Clear_Seal® joints).
still present in the samples this step must be performed in a
well ventilated hood.
(11) Store the samples at 0°C until analysis.
(12) With a 50 pL syringe, add a 10 pL aliquot of the MS external
standard solution to the sample concentrate immediately before
analysis.
11.5 GC/MS/CONP ANALYSIS
Prior to beginning GC/MS/CONP analysis of the nonvolatile acids
(NOVA), the analyst should be familiar with procedures described in Chap-
ter 13.
11.5.1 Preparation for Analysis
11.5.1.1 GC/HS/COFIP Operating Parameters--
The recommended CC/MS operating parameters for the analysis of NOVA
fractions are given in Table 11.7. (This capillary column should not be
used for the analysis of any fractions from other protocols.)
Tsfloii
StOOcock
Chap. 11 — 265
-------
Table 11.7. GC/NS OPERATING CONDITIONS FOR
NONVOLATILE ACIDS AS THEIR METHYL ESTERS
GC Column 30 m DB-5 (1.0 p film thickness)
fused silica wide bore (0.34 mm)
capillary column
GC Carrier Gas Helium
Carrier Gas Flow 1.6 mL/min through column; 9:1
split injection
Carrier Gas Sweep Time 85 sec (50°C)
Temperature Program 50°C/5 mm; to 250°C @ 4°/mm and
hold
Injector Temperature 250°C
Transfer Line Temperature 255°C
Injection Mode Splitless, 0.4 mm/split
Injection Volume 1.0 I.iL
Ionizing Energy 70 eV
Ion Source Temperature 250°C
Scan Range 35-500
Scan Rate 2 sec (1.9 sec scan, 0.1 sec
settling)
11.5.1.2 MS Calibration--
The mass spectrometer is calibrated using the manufacturer’s recom-
mended approach (Chapter 13). The acceptability of the calibration results
is verified by the analysis of the NOVA system performance solution (SPS).
11.5.2 Analysis of SPS (Quality Control )
Using the above prescribed GC/MS conditions the SPS (Table 11.1) is
analyzed and all necessary data is acquired and evaluated prior to proceed-
ing to sample analysis. The SPS is analyzed at the beginning of each
day’s operation.
The SPS raw data is extracted from the run, and test parameters
calculated and plotted or tabulated (Tables 11.8 and 11.9). Up to 14 days
of SPS analytical results can be historically recorded in Table 11.8 for
day-to-day comparisons. This allows the analyst to follow subtle trends
that may develop in anticipation of GC or MS maintenance requirements.
Chapter 13 explains calculation and usage of these data.
Chap. 11 - 266
-------
Table 11.8. GC-MS SYSTEM PERFORMANCE TEST
FOR NONVOLATILE ACIDS (NOVA)
Dates: Run Id Code
CC Column and Program:
Notebook References:
MS SYSTEII CHECKS
lfigb/Low Mass Balance
Ion
51
68
70
127
197
198
199
275
365
441
442
443
DFTPP
Relative Abundance Criteria
30% to 60% of /z 198
<2% of m/z 69
<2% of m/z 69
40% to 60% of m/z 198
<1% of m/z 198
100%
5-9% of a/z 198
10% to 30% of m/z 198
at least 1% of m/z 198
present, but < m/z 443
‘40% of m/z 198
17% to 23% of m/z 442
275 6S 311 442 443
- Pass/ Remedial
Fail Action
a/z 51 68 70 127 197 198 199 -
Abundance * ( ) j LI Li LI LI LI LI LI LI LI ___ ____
Criteria
*These abundances are initially established by the MAS user, and subsequently become guidelines for
acceptability of tune.
(continued)
Chap. 11 - 267
-------
Table 11.8 (cont’d.)
Test Components Criteria
Limit of Methyl 2,4,5-Trichioro
Detection phenoxy Acetate S. (m/: 3) > 3 1
55
50
z
; 45,
40
I I I I I I I U I I p I I V
Day
GC SYSTEJI 1 CKS
Peak 1. Acetophenone (TIC) • PAF (300
Asyetry 2. 1-Tetradecanol (TIC) S %PAF (200
300-- No 1
200 No 2
100
u u u i I I I I I I U I
Day
I. Acetophenone (TIC)
Basacity 2. 2,6—Dimethylphenol (TIC) Ratio 2:1 0.7 to 1.3
1.4
1.2
0
‘a
0.8
0.6
I I I I I I I I I I I I
Day
(continued)
Chap. 11 - 268
-------
Table 11.8 (c’ont’d.)
Test Components Criteria
Separation !-Eicosane ( /z 43)
Number - n-Hecejcosane ( /z 43) SN > 6
12
11
10
Z 9.
U)
8
7,
6
I I I 1 I I I I I I I I I
Day
n—Octadecane (TIC) R = mini.eun 50%
Resolution 1—Octadecene (TIC) Valley
90
80-
70
e
> 60
50
40
I I I I I I I I I I I I
Day
Inertness Nethyl Stearate Ratio m/ 74 to 29S 12 1 to 1(’ I
20
18
I I I I I I I —
Day
(continued)
Chap. 11 - 269
-------
Table 11.8 (cont’d.)
Test Cowponents Criteria
Capillary
Capacity Ditncthyl Adipate (TIC) %PAF ‘70
100
90
U.
.
C.
..
70
I I I I I I I 1 I I I I I
Day
Relative
Retention 1. ileneicosane
Shift 2. Fyrene RRS = 0.95-1.05
1.10
1.05
1.0
0.95
0.90
I I I I I I T I I I I I I
Day
Chap. 11 - 270
-------
Table 11.9. GC-IIS SYSTEM PERFORMANCE TEST:
RMR CHECK FOR NONVOLATILE ACIDS (NOVA)
Date: Run ID Code:
DATA
Amount Ion Area Area
Standard MW (ng) pM (m/z) (Run 1) (Run 2)
2-Fluorobiphenyl i 2 172
4-Fluoro-2—Iodo- 236 109
toluene (FIT) 236
d 7 -Naphthalene 229 134
sulfonic acid 229
MATRIX OF STANDARD ION RMRs
Standard Ion 172 109 236 134 229
2-Fluorobiphenyl 172
-
FIT 109
236
—
-
d 7 -Naphtha lene 134
sulfonic acid 229
A MW n
= g , A = ion area or height
x/y A • 1W . ng ng = ng injected
y y x xanalyte
COMMENTS: y = standard
Chap. 11 — 271
-------
Additional QC/QA procedures include a check on the RMRs on a daily
basis (Table 11.9). These RMR data are compared to those obtained during
the development of the historical data bank. The usage of these data are
also discussed in Chapter 13.
11.5.3 Determination of RNRs
RMRs for the User’s data bank (as opposed to the daily RMR checks
discussed in the above paragraph) are determined by injecting a solution
containing the derivatized internal standards and analytes.
The GC/MS/COLIP operating parameters are given in Table 11.7. Table
13.8 may be used to, tabulate RNR raw data.
11.5.4 Analysis of Field and Quality Control Samples
After all specified criteria for GC and MS performance (Tables 11.8
and 11.9) have been met, analysis of standards, QA/QC, or NOVA sample
extracts is conducted.
Qualitative and quantitative procedures are described in Chapter 13.
Table 11.10 may be used to tabulate RIIR and sample analyte raw data, prior
to using the MASQUANT software program or manual quantitative calculation.
Chap. 11 - 272
-------
Table 11.10. RAW ANALYTE DATA FOR NOVA FRACTION
Date:
Run I.D. Code:
Notebook Reference:
Vol. Water Processed
Sample Identification: .
Vol. Water (L):
(to which i were added)
(mL):
STANDARDS
Spec-
I.D. trum
No. No.
2-Fluorobiphenyl
4-Fluoro—2-Iodo—
Weight Ions
(ng ) (in/z) Area
MW
Ka
172:
,
toluene
d 7 -Naphtha lene
sulfonic acid
109,236: ,______
134,229: ,
,
,
ANALYTES
Class No.
I.D.
No.
Spectrum Ions
No. (mfz) Area
MWa
,
,
,
,
,
,
,
,
,
, : ,
,
,
, :
a 1 f calculations are performed manually then this information is also
needed, whereas MASQIJANT performs functions automatically.
A MW ng A ion area or height
= X A ng = ng recovered from volume
x/y y y of water processed
x = analyte
= y y = standard
A MW
y y
Chap. 11 - 273
-------
CHAPTER 12
CATION-EXCHANGE AND ANALYSIS OF STRONG MINES (SAN)
12.1 INTRODUCTION
12.1.1 Principle of the Technique
This technique describes the determination of volatile primary,
secondary, and tertiary amines in drinking water, surface waters, and
treated wastewater effluents. Although some amines can be extracted from
water with organic solvents, ion-exchange concentration offers several
important advantages. First, the recovery of primary amines and low
molecular weight secondary amines may be low during solvent extraction.
Second, samples tend to emulsify at alkaline pH during solvent extraction.
Finally, larger volumes of water can be processed when utilizing accumulator
ion-exchange columns thereby giving lower detection limits.
This procedure utilizes cation-exchange resins to concentrate amine
salts from the aqueous matrix. Amines are eluted from the resin material
using NaOH in an acetonitrile:water solution. The base deprotonates the
amine cation and the free amines are then rinsed off the resin with the
acetonitrile:water solution. The amines are then converted to their
nonvolatile HC1 salts and the solution is evaporated to dryness. The
residue is resuspended in 3 mL water, and sufficient NaOH is added to give
a pH greater than 13, which converts the amines to their free base form.
Amines are then extracted into 6 mL of methyl- t-butyl ether. Amines
should be derivatized prior to GC/MS analysis to prevent adsorptive losses
during chromatography. Primary amines are derivatized with pentafluoroben-
zaldehyde (SAN-PT fraction). Secondary amities are derivatized with penta-
fluorobenzylbromide (SAN-S fraction). Since the volatile amines have been
converted to less volatile derivatives, derivatized samples may be concen-
trated using nitrogen blowdown prior to analysis. Because the primary and
secondary amities are not derivatized with the same derivatizing reagent,
the sample must be split prior to derivatization. If the conductivity of
the sample extract water is low, a larger sample volume may be used and
Chap. 12 - 274
-------
the sample split immediately before derivatization. However, for samples
with high conductivity, the sample must be split prior to ion-exchange
concentration to achieve the specified detection limits.
Tertiary amines are also separated by this protocol and quantified
(underivatized) in the primary amine fraction.
12.1.2 Detection Limits
If an average detection limit for GC/MS analysis is estimated as 10
ng for each sample component for derivatized amines, and the final volume
is 0.3 mL, then nominal detection limits for the various water types using
a 1 pL injection are directly related to sample size. Detection limits
for a sample (split before ion—exchange concentration) are:
1 ppb for a 2.5 L sample (drinking water);
5 ppb for a 500 ml. sample (surface water);
10 ppb for a 250 ml. sample (industrial and municipal wastewater
effluents); and
50 ppb for a 50 ml. sample (energy effluents).
12.1.3 Interferences
By-products from the derivatizing reagent can cause interferences
during the analysis of secondary amines if excess KOH is present during
derivatization. The primary amines are derivatized with a different
reagent which does not form chromatographable by-products. A procedural
blank must be run prior to processing any samples to assure that contamina-
tion from resin material, solvents, reagents, glassware, and other sources
is low.
12.1.4 Precision, Accuracy, and Scope
Table 1.9 in Chapter 1 presents average recoveries and standard
deviations for a variety of primary, secondary, and tertiary amines. The
procedure gives acceptable recoveries for low molecular weight aliphatic
amines. Piperidine and morpholine do not give acceptable recoveries.
Certain weak bases may be detected in these fractions, but are measured in
the pH 8 extractable fraction (WABN) where they are extracted more effi-
ciently.
12.2 APPARATUS AND REAGENTS
The following materials are required for processing a set of nine samples
plus three procedural blanks. Nine is the maximum number of samples
Chap. 12 - 275
-------
(field and quality control) which should be processed at a time. The
quality control samples that should be run during these analyses are
listed in Table 5.1, Chapter 5. The three procedural blanks must be run
before any samples are collected or processed. When the samples are split
after concentration, nine samples can be processed for both primary and
secondary amines. When the samples are split prior to ion-exchange concen-
tration, the nine samples can be processed for either primary or secondary
amines. It will require two to three days to process nine samples, depend-
ing upon the sample size.
(1) Nine glass chromatography columns, —‘1 cm I.D. X 22 cm with 500
mL sample reservoir (Figure 12.1). The size of the sample
reservoir is not critical; however, a 500 ml reservoir will hold
the entire sample for all water types except drinking water.
For drinking water an adaptor (Figure 12.2) for siphoning the
sample onto the resin should be used in lieu of a sample reser-
voir. This allows unattended flow through the sample for a 14
to 16 hr period such that the sample can be concentrated over-
night. A vacuum aspirator is needed to start sample flow through
the siphoning adaptor and resin column.
(2) One 10 ml graduated pipette with the tip cut off for measuring
and transferring resin material.
(3) One graduated cylinder for measuring sample volume. The size of
the sample, thus, the cylinder will depend upon the results of
conductivity measurements. In general it may be:
1000 ml for drinking water
500 mL for surface water
250 mL for industrial and municipal wastewater effluents
50 ml for energy wastewater effluents
The cylinder may be reused for each sample if it is rinsed 5
times with distilled-deionized water.
(4) One conductivity bridge — range 1 to 30,000 ohm.
(5) Nine 200 ml round bottom flasks (24/40).
(6) One rotary evaporator. If more than one is available up to four
samples can be evaporated at a time. Samples may bump initially
Chap. 12 - 276
-------
500 mL glass
separatory funnel
24/40
glass connection
Teflon stopcock
22cm
Figure 12.1. Chromatography column with sample reservoir (exact
dimensions of column are not critical - must hold
10 mL of resin).
lcmi.d.
Teflon
stopcock
Chap. 12 — 277
-------
glass
connector
Teflon
stopcock
Figure 12.2. Chromatography column with siphoning adaptor for
drinking water.
—1
Ld.
1 gallon
sample
bottle
Teflon tibing
24/40 glass
confliction
L
.1
tygon tubing to aspirator
Chap. 12 - 278
-------
but after acetonitrile has evaporated the samples require little
attention.
(7) Small (15 mL) round bottom test tubes with 15 mm screw caps
(Supelco 3-3112) and Teflon lined rubber septa (Supelco 3-
3115). Specified materials must be used to prevent solvent
evaporation during derivatization. Sixty tubes (2 for each
sample) are required if the sample is split after concentration.
Thirty tubes (1 for each sample) are required if the sample is
split prior to ion-exchange concentration. An additional twenty
test tubes will be needed for processing primary amines.
(8) One Soxhiet extractor for cleaning ion-exchange resin. A minimum
150 mL capacity is required. However, resins can be processed
in large batches and stored; therefore a 500 niL or 1000 niL size
would be preferred.
(9) One large chromatography column for preparing resin material.
The dimensions of this column are not critical, it should hold a
minimum of 160 mL of resin material; for large batches of resin
a larger column is preferred. The flow through the column
should not exceed 50 niL/minute.
(10) 10 niL volumetric flasks for preparing standard solutions.
(11) 15 niL vials with Teflon lined screw caps for storing standard
solutions.
(12) Syringes for preparing standard solutions - 50 and 150 pL.
(13) Graduated pipettes for preparing standard solutions - 1 niL.
(14) One heating block capable of maintaining 60°C with at least ten
spaces for 15 mL round bottom tubes.
(15) One manifold with a temperature controlled water bath or heating
block for nitrogen blowdown. A manifold with nine to eighteen
spaces is preferred but not required.
(16) One vortex mixer - vortex Genie, Fisher Scientific or equivalent.
(17) One clinical centrifuge capable of —4000 rpm.
(18) One pH meter with a combination electrode.
(19) One gas chromatograph suitable for capillary column chromatography
and all required accessories including syringes, gases, flame
ionization detector and a strip chart recorder.
Chap. 12 — 279
-------
(20) One 30 in x 0.34 mm I.D. DB-1 (1.0 p film thickness) fused
silica capillary column.
(21) Materials and Reagents
(a) 160 mL AG 50 W-X8 (50-100 mesh) cation-exchange resin (Biorad
Laboratories, Richmond, CA) in the form.
(b) Reagent water - reagent water is described as a water
source that does not produce a background interference at
the limit of detection and which has a conductivity of
<1 ohm. A water purification system (Millipore Super Q or
equivalent) may be used to generate reagent water.
Cc) Methanol (pesticide analysis grade).
(d) 1 L 0.57N NaOH in acetonitrile:reagent water (10:25).
Ce) 200 ml concentrated HC1.
(f) 10 mL lON NaOH in reagent water.
(g) 200 ml 0.1N NaOH in reagent water (use the same solution as
specified in Chapter 5, section 5.2.14).
(h) 200 ml 0.1N H 2 S0 4 in reagent water (use the same solution
as specified in Chapter 5, section 5.2.14).
(i) 200 ml 0.0511 Na 2 S 2 0 3 in reagent water (use the same solution
as specified in Chapter 5, section 5.2.14).
(j) 100 ml methyl-t—butyl ether (pesticide analysis grade).
(k) Reagents for derivatizing primary amines.
- 50 niL pentafluorobenzaldehyde in methanol (2.5 g/lO ml).
Recently purchased reagent does not require distillation
before use.
(1) Reagents for derivatizing secondary amines.
- 50 ml pentafluorobenzylbromide in acetone (5% w/v). Re-
cently purchased reagent does not require distillation
before use. (Available from Aldrich as a-bromo-2,3,4,5,6-
pentafluorotoluene).
- 1 ml triethylamine.
(m) 1 ml MS external standard solution - 4-fluoro-2-iodoto luene
(300 ng/lO pL) and 2-fluorobiphenyl (300 ng/lO .iL) in
CH 2 C1 2 .
Chap. 12 - 280
-------
(n) 1 mL GC external standard solution - hexadecane (30 ng/l0 i.JL)
in CH 2 C1 2 .
(o) 1 mL system performance standard solution for the primary amines
(Table 12.1) and the secondary amines (Table 12.2).
12.3 PREPARATION FOR ANALYSIS
12.3.1 Preparation of System Performance Solutions
(1) Prepare individual stock standards as specified in Table 12.3.
— solids - accurately weight 0.120 g of pure material, dissolve
in the specified solvent, dilute to volume in a 10 mL
volumetric flask.
Table 12.1. GC/MS SYSTEM PERFORMANCE SOLUTION
FOR PRIMARY AND TERTIARY ANINES (SAN-PT)
Compounds Concentration (ng/pL)
2,6-diinethylaniline 300
acetophenone 310
1-tetradecanol 240
1-octadecene 240
n-octadecane 230
DFTPP 300
n-eicosane 300
pyrene 300
n-heneicosane 300
methyl stearate 10
2-fluorobiphenyl 300
4-fluoro—2-iodotoluene 270
n-butylamine-d 9 (PFB Schif? base) 29 0 b
phenylethylamine-d 4 (PFB SChlffa base) 300 b
allylamine (PFB SChlffa base) 16 b
n-hexylamine (PFB Schif? base) 990 b
aDerivatized with pentafluorobenzaldehyde.
bCtti of underivatized amine.
Chap. 12 - 281
-------
Table 12.2. GC/MS SYSTEM PERFORMANCE SOLUTION FOR
SECONDARY ANINES (SAN-S)
Compounds
Concentration (ng/pL)
2,6—dimethylaniline
290
acetophenone
310
1-tetradecanol
240
1-octadecene
240
n-octadecane
230
DFTPP
300
n-eicosane
300
pyrene
300
n-heneicosane
300
methyl stearate
10
N-ethyl-2-fluorobenzylamine (PFBa
derivative)
300 b ,c
2-fluorobiphenyl
300
4-f luoro-2-iodotoluene
270
a
diallylamine (PFB derivative)
c
16
morpholine (PFBa derivative)
990 c
aDerivatized with pentafluorobenzylbromide.
b
No adjustment made for density.
CCtti of underivatized amine.
- liquids — with a 250 iL syringe, accurately measure 120 pL
of pure liquid, dissolve in the specified solvent, dilute
to volume in a 10 mL volumetric flask. To determine the
quantity of N-ethyl-2-fluorobenzylamine added to the solu-
tion, weigh the dry syringe, then weigh the syringe contain-
ing the neat compound. The difference between these two
weights is the weight of material delivered to the solution.
The syringe must be rinsed well with solvent to assure that
all of the neat material is delivered.
(2) Prepare a secondary, neutral standard. With a 1 mL graduated
pipette, measure 0.5 mL of each stock standard in group A (for
Chap. 12 - 282
-------
Table 12.3. STOCK STANDARD SOLUTIONS
De
Compounds (@
nsi ty
200C)
Concentration
mg/lO mL
a
Standard A
2,6-dimethylaniline 0.984 120
acetophenone 1.030 120
1-tetradecanol 0.823 98
1-octadecene 0.789 95
n-octadecane 0.777 93
DFTPP S 120
n-eicosane S 120
pyrene S 120
n-heneicosane S 120
methyl stearate S 120
2-fluorobiphenyl S 120
4-f luoro-2—iodotoluene 0.883 120
b
Standard B
n-buty lamine-d 7 0.740 89
allylamine 0.761 91
n-hexylainine 0.766 92
2-pheny lethyl—1,1,2,2— 0.965 120
d 4 -amine
b
Standard C
diallylamine 0.787 94
morpholine 0.999 120
_ethy1_2_f1uorobenzy1amineC unknown 12 Oc
aSolvent is methylene chloride.
bSolvent is methyl-t-butyl ether.
CNO adjustment made for compound density.
Chap. 12 - 283
-------
methyl stearate measure 17 iiL with a 50 pL syringe) into a 10 mL
volumetric. Dilute to volume using pesticide grade methylene
chloride. Transfer into a Teflon sealed screw cap bottle.
Store at 4°C.
(3) Prepare a secondary standard for the primary amines. With a 1 niL
graduated pipette, measure 0.65 niL of the n—butylamine, 0.5 niL of
the phenylethylamine, and 2.15 niL of the hexylamine standards into
a 10 niL volumetric flask. With a 50 jiL syringe add 35 .iL of the
allylamine stock standard to the same flask. Dilute to volume
using pesticide grade methyl—t—butyl ether. Transfer into a
Teflon sealed screw cap bottle. Store at 4°C.
(4) Prepare a secondary standard for the secondary amines. With a
1 mL graduated pipette, measure 0.5 mL of the N-ethyl-2-fluoro-
benzylamine and 1.65 niL of the morpholine standards into a 10 niL
volumetric flask. With a 50 IJL syringe, add 35 I.iL of the diallyl-
amine stock standard to the same flask. Dilute to volume using
pesticide grade methyl-t-butyl ether. Transfer into a Teflon
sealed screw cap bottle. Store at 4°C.
(5) Fresh standards should be prepared every six months. If degrada-
tion or evaporation has occurred, fresh standards should be
prepared sooner.
(6) If compound purity is 96% or greater, the weight or volume can
be used without correction to calculate the concentration of
stock standards. Compounds which are less than 96% pure cannot
be used for standards.
(7) For the primary amine SPS (Table 12.1), with a 1 mL graduated
pipette, measure 0.5 niL of the primary amine secondary standard
solution into a 15 niL round bottom test tube. Add 5 niL methyl-
t-butyl ether. Derivatize and extract as described in step
15(a), section 12.4. Evaporate the sample to 0.5 niL, then
with a 1 niL graduated pipette, add 0.5 niL of the secondary
neutral standard solution. Cap the test tube. Store at 0°C
until GC/MS analysis.
(8) For the secondary amine SPS (Table 12.2), with a 1 niL graduated
pipette, measure 0.5 niL of the secondary amine secondary standard
Chap. 12 — 284
-------
solution into a 15 mL round bottom test tube. Add 5 mL methyl-
t-butyl ether and derivatize as described in step 15(b), Sec-
tion 12.4. Evaporate the derivatized sample to 0.5 mL, then
with a 1 mL graduated pipette, add 0.5 mL of the secondary
neutral standard solution. Cap the test tube. Store at 0°C
until GC/MS analysis.
12.3.2 Preparation of Resin Material
Ion-exchange resin is cleaned prior to preparing the resin columns.
One hundred and twenty niL of resin material is needed to process 9 samples
and three procedural blanks and is the minimum amount of resin which
should be prepared. The following instructions describe procedures based
on 120 niL of resin material; however, it is possible to prepare larger
batches and store the prepared resin. If a larger volume of resin is
prepared, volumes for rinsing the resin should be adjusted accordingly.
Biorad AG SOW-X8 (50-100 mesh) in the form is extracted with
redistilled pesticide analysis grade methanol in a Soxhlet extractor
overnight. The resin is placed in a column and rinsed with 10 bed volumes
(1.2 L) of deionized water. Resin which is not used immediately may be
stored wet, sealed in glass jars.
12.3.3 Cleaning of Materials
(1) All glassware to be used is washed with Amway S-A-8 laundry
compound (or isoclean or other nonionic detergent) rinsed with
deionized water and baked for a minimum of 4 hours at 500 to
550°C. All cleaned glassware is immediately capped or covered
with foil (precleaned with hexane) to prevent contamination.
(2) Teflon liners and teflon lined septa are sonicated for 10 minutes
in pesticide grade methanol followed by 10 minutes in pesticide
grade pentane. The sonicated liners are vacuum-oven dried for 3
to 5 hours at 70°, —28 inches of water, and stored in clean,
Teflon lined screw-capped bottles.
12.3.4 £4a cimum Sample Size
Conductivity measurements must be determined for each sample to
assure that the total concentration of cations in the sample will not
exceed the resin capacity. Prior to measuring conductivity, check the pH
of the sample using a pH meter. Adjust the sample pH to 4 to 5 using O. 1N
Chap. 12 - 285
-------
KOH or 0.1N H 2 S0 4 . Because the primary and secondary amines are not
derivatized under similar conditions, the sample must be split prior to
derivatization. If the conductivity of the sample water is low, a larger
sample volume may be used and the sample split immediately before deriva-
tization. However, for samples with high conductivity, smaller volumes
must be used. The sample is split prior to ion-exchange concentration to
achieve the specified detection limits. Table 12.4 list the sample size
which should be used for a given sample conductivity regardless of water
type. Corresponding detection limits both with and without sample split
prior to derivatization are given for each sample size.
12.3.5 GC/FID Performance Evaluation
Prior to analyzing procedural blanks, acceptable performance for the
GC/FID system must be demonstrated.
(1) Analyze the GC/NS system performance standards as specified in
Table 12.5. Figures 12.3 and 12.4 show total ion chromatograms
of both mixtures analyzed by GC/HS under similar conditions
which may be used to identify test components in the standards.
(2) ?leasure peak asymmetry or tailing for acetophenone and 1-tetrade-
canol using the percent peak asymmetry factor (PAP):
% p y = x 100
where
B = the width of the back half of a chromatographic peak
measured at 10% above baseline.
F = width of the front half of the chromatographic peak
measured at 10% above baseline.
PAP should measure less than 200% for acetophenone and 1-tetrade-
canol.
(3) Check the acidity of the column by the peak area ratios determined
by integrator or triangulation of 2,6—dimethylaniline to aceto-
phenone. A ratio of 0.7 to 1.3 for both is acceptable.
(4) Sensitivity is checked by measuring signal-to-noise ratios for
the derivatives of allyl- and diallylamine. A signal-to-noise
ratio of 10 to 1 for both compounds is acceptable.
Chap. 12 — 286
-------
Table 12.4 ACCEPTABLE SAMPLE SIZE AND CORRESPONDING DETECTION
LIMITS FOR SAMPLE CONDUCTIVITY RANGES
Detection Limit (ppb)
a Split Prior To
Conductivity Range Sample Size Ion Exchange b Split Prior to
(ohm) (mL) Concentration Derivatization’
<150 2500 1 2
150-300 1000 2 4
300-600 500 5 10
600-1,200 250 10 20
1,200-3,000 100 25 50
3,000-6,000 50 50 100
6,000-12,000 25 100 200
12,000-30,000 10 250 500
aVolume of sample passed through the ion exchange resin.
bSampie concentrate is not split prior to derivatization (split prior
to ion exchange concentration).
CSample concentration split immediately prior to derivatization.
Table 12.5 GC/FID OPERATING CONDITIONS FOR STRONG ANINES
GC Column 30 m DB-l (1.0 p film thickness)
fused silica, wide bore (0.34 mm
i.d.), capillary column
GC Carrier Gas Helium
Carrier Gas Flow 1.6 mL/min through column; 15:1
split injection
Temperature Program 80 to 250°C @ 5°/mm
Injector Temperature 250°C
Detector Temperature 260°C
Injection Volume 1.0 pL
Chap. 12 - 287
-------
41
U
41
I .
.4
41
U
41
41
41
41
1
0
0. .
—I
0
41
0.
-4
>‘
t1
41
H
‘4
.7
‘0
(‘4
-4
0
C
41
U
41
.41
. 4
I.
U
41
‘a
‘-4
41
C
41
41
0
U
‘4
41
C
41
400
6:40
I • I • I
800 I 0 140 1 1 16U1 1
13.20 20:00 23:29
Figure 12.3. Total ion chromatogram of SAM-PT SPS.
lUtE
-------
V
.4
‘I
4,
.4
1.
4’
•0
-l
N
U
4’
.0
4 ’
‘4
-4
0
U.
1.
0
a
V
.
-I
‘I
4,
>
-I
1.
4’
0
-4
>‘
II
U
4 ’
4,
U
‘4
4’ -4
U
.4
.-I
-4
.4
4,
U
U . 4
- 4, 0
LLLJ
200 .
3;29 6:40 10:00
4,
>
.4
‘I
4,
.4
14
4,
•0
-4
N
U
4,
. 0
U
I
I • I — — I • -.— I
800 1Q00 1700 1400 1603
13:20 16:40 20:00 23:20 26:10
lee..
RIC.
I-’
I. . ,
4 ’
U
4,
I i
4,
4, 4,
U ‘I
4, 4 ,
U
4,
.4’
4, 4,
U V U
U U 4’
(5 14
UI 4,
0 U.
U V
.4
4,
U
-4
C
U
5 1
4,
(4
Li
V
‘a
-4
se.*u
TI lIE
Figure 12.4. Total ion chromatogram of SAM-S SPS.
-------
12.3.6 Derivatization and Procedural Blanks and Controls
Prior to processing any samples, a derivatization blank and a deriva-
tization control must be run and analyzed by GC/FID to assure that back-
ground contamination is low and that derivatization yield is acceptable.
After validating the derivatization step, procedural blanks must be proc-
essed and analyzed by GC/FID to detect contamination and artifacts from
the resin material, solvents, reagents, dirty glassware, and other sources.
12.3.6.1 Derivatization Blanks--
(1)
For the primary amine blank, add 6 mL methyl t-butyl ether to a
15 mL round bottom test tube. Derivatize and extract as described
in step 15(a), Section 12.4.
(2) For the secondary amine standard, measure
ether to a 15 mL round bottom test tube.
trate as described in step 15(b), Section
(3) With a 50 j.iL syringe, add 10 I.IL of the GC
solution to each derivatized solution.
(4) Analyze by GC/FID using the conditions described in
Contaminant (other than derivatizing reagents) peak
should be less than 20% relative to the peak height
external standard (hexadecane).
(5) If significant contamination is present, repeat the
blank using fresh glassware and reagents. Reagents
may be distilled or taken from another source.
12.3.5.2 Derivatization Controls--
(1)
For the primary amines, prepare a control solution. With a
500 I.iL syringe, measure 250 pL each of the stock solutions
(Table 12.3) of allylamine, n-butylamine, phenylethylamine, and
hexylamine into a 10 mL volumetric flask and dilute to volume
using pesticide grade acetone. Transfer into a Teflon sealed
screw cap bottle and store at 4°C until use. For derivatization,
with a 1 mL graduated pipette, measure 0.1 mL of this solution
into a 15 mL round bottom test tube, add 6 mL methyl— t-butyl
ether, and derivatize as described in step 15(a), Section 12.4.
(2) For the secondary amines, prepare a control solution. With a
500 pL syringe, measure 250 pL each of the stock solutions
6 mL methyl t-butyl
Derivatize and concen-
12.4.
external standard (hexadecane)
Table 12.5.
heights
of the
derivatization
and solvents
Chap. 12 - 290
-------
(Table 12.3) of diallyamine, morpholine, and N-ethyl-2-fluoroben-
zylamine into a 10 ml volumetric flask and dilute to volume
using pesticide grade acetone. Transfer into a Teflon sealed
screw cap bottle and store at 4°C until use. For derivatization,
with a 1 mL graduated pipette, measure 0.1 mL of the control
solution into a 15 ml round bottom test tube, add 6 mL rnethyl-t-
butyl ether, and derivatize as described in step 15(b), Section
12.4.
(3) With a 50 iL syringe, add 10 pL of the GC external standard to
each derivative mixture.
(4) Analyze samples by GC/FID as described in Table 12.5. Peak
heights of the derivatized amines measured relative to the
external standard should fall within the values given in
Table 12.6.
(5) If derivative yield is low (as measure by relative peak heights)
this step must be repeated using fresh reagents and glassware.
Reagents may be distilled or taken from another source.
12.3.6.3 Procedural Blanks--
For each batch of materials and reagents that are used, a set of
three procedural blanks are required. Table 12.7 identifies these blanks
and defines their purpose. These blanks must be processed and analyzed by
GC/FID prior to processing any samples. If a large number of samples are
to be processed, it is advantageous to prepare large batches of materials
and reagents, thereby reducing the number of blanks which must be run.
Procedural blank 1 must also be run every time a set of samples is processed
and analyzed.
12.3.6.3.1 Procedural Blank 1— -
(1) Prepare a resin column as described in step 12.4(1).
(2) Follow the procedure for amine analysis as described in
step 12.4(5) to 12.4(15).
(3) With a 50 pL syringe, add 10 pL of the GC external standard to
each derivative mixture.
(4) Analyze samples by GC/FID as described in Table 12.5. Contaminant
(other than the derivatizing reagent) peak heights should measure
less than 25% relative to the e, erna1 standard.
Chap. 12 - 291
-------
Table 12.6. RELATIVE PEAK HEIGHTS (RPH) OF CONTROL AIIINES
Amine
Acceptable RPH Range
n-butylamine
0.8-1.2
allylamine
0.8-1.2
hexylamine
0.8-1.2
diallylamine
1.5-2.0
morpholine
1.8-2.2
N-ethyl-2-f luorobenzylamine
1.5-2.0
Table 12.7.
PROCEDURAL BLANKS
Blank
Description
Procedural Blank 1
Detects contamination in resin
material, solvents, and glassware
Procedural Blank 2
Detects contamination in reagent
water
Procedural Blank 3
Detects contamination in sulfuric
acid, sodium hydroxide, and
sodium thiosulfate solutions used
during sample collection and
analysis
(5) If significant contamination is present, the procedure should be
repeated using a blank chromatography column. If this blank is
acceptable, then the resin is the source of contamination and
should be recleaned using the procedure in Section 12.3.2.
Procedural blank 1 should then be repeated using the fresh resin
material.
(6) If significant contamination is present in this blank, fresh
reagents and glassware should be used and the reagent blank
repeated. When the reagent blank is acceptable, procedural
blank 1 must be repeated.
(7) Procedural blanks 2 and 3 may be processed once procedural
blank 1 is acceptable.
Chap. 12 - 292
-------
12.3.6.2.2 Procedural Blanks 2 and 3- -
(1) Prepare a resin column as described in step 12.4(1). Attach a
separatory funnel to the top of the column. For procedural
blank 2, pour 100 mL of reagent water into the separatory funnel,
then drain the sample through the resin bed at a flow rate of
l0 mL/minute. For procedural blank 3, use 100 mL of reagent
water spiked with 1 mL each of the sulfuric acid, sodium hydroxide
and sodium thiosulfate solutions. These must be the same solu-
tions which are to be used when the samples are collected
(Chapter 5, section 5.2.14).
(2) Follow the extraction procedure as described in steps 12.4(5) to
12.4(15).
(3) With a 50 pL syringe, add 10 iL of the GC external standard to
each derivative mixture.
(4) Analyze the samples by GC/FID using the conditions in Table 12.5.
Peak heights for contaminants in procedural blank 3 which are
not present in procedural blank 2 should measure less than 20%
relative to the terna1 standard.
(5) If significant contamination is present in procedural blank 3
which is not present in procedural blank 2, the procedure should
be repeated using fresh solutions of sulfuric acid, sodium
hydroxide, and sodium thiosulfate until an acceptable blank is
achieved.
12.4 ION-EXCHANGE SEPARATION AND DERIVATIZATION
(1) Prepare ion-exchange resins by placing a small glass wool plug
in the bottom of the chromatography column. Using a graduated
pipette with the tip cut off, pipette 10 mL (allow resin to
settle prior to measuring) of cleaned Biorad AG 50W-X8 resin
into the open tubular chromatography column, allow the resin to
settle, and rinse the column with 10 mL of deionized water.
(2) Mix sample well (split here if necessary, see section 12.3.4).
If the sample is turbid, it should stand undisturbed for 4 hours
or overnight to allow particulates to settle.
Chap. 12 - 293
-------
(3) Gently pour the aqueous portion of the sample (equilibrated to
room temperature) into a graduated cylinder to measure sample
volume. ?lethylene chloride used as sample preservative should
not be transferred. The total volume of sample collected and
the volume of sample used should be recorded.
(4) For surface waters and effluents, transfer sample water to
reservoir on top of the column and allow it to drain through the
resin bed. Flow is gravity controlled and should be approximately
5 mL/min (NOTE: the glass wool plug can restrict flow if packed
too tightly). For drinking water, transfer sample to a one
gallon container. Attach siphoning adaptor between sample
container and resin column. Attach a piece of tygon tubing
between the end of the chromatography column and a water aspirator.
To start sample flowing through the resin, open stopcock on the
chromatography column and turn on aspirator. Discon.nect suction
after water is flowing through the resin bed. Using the stopcock,
adjust flow to 2.5 mL/minute if a 2500 mL sample volume is used
or 1.25 mL/minute if a 1250 mL sample volume is used. At these
flow rates, it will require —16 hours for the sample to drain
through the resin bed, which is suitable for overnight accumula-
tion. If particulate matter stops flow through the column,
positive head pressure can be used to restore flow.
(5) Rinse the resin bed with 25 mL of reagent water followed by
100 mL acetonitrile. Discard these rinses.
(6) Elute the amines with 80 mL of 0.57N NaOH in 10:25 acetonitrile:
water. Collect the eluate in a 200 mL round bottom flask con-
taining 3 mL of concentrated HC1 diluted with l5 mL of reagent
water.
(7) Adjust pH to <2 using additional 6N HC1 if necessary. Samples
can be stored refrigerated after this.
(8) Evaporate the eluate to dryness using rotary evaporation at an
elevated temperature (60°C). This should take —20 minutes.
Sample must be evaporated to dryness to remove excess lid.
(9) Resuspend the residue in 5 m l. of reagent water and transfer the
concentrate to a small test tube (10-15 ml). Rinse the flask
Chap. 12 - 294
-------
with two additional 2.5 mL portions of water. Transfer rinses
to the test tube. Concentrates are split at this point, if
necessary (see Section 12.3.4). To split concentrates mix well.
Pipette a 5 mL portion into each of two small screw cap test
tubes. Similarly, split each of two rinses equally between the
two test tubes. Treat one fraction as described for primary
amines and one fraction as described for secondary amines.
Samples can be stored refrigerated after transfer.
(10) Concentrate the solution to —3 niL using nitrogen blowdown at an
elevated temperature (60°C). Samples can be stored refrigerated
at this point.
(11) Add 6 mL methyl-t-butyl ether to the test tube.
(12) Convert the amines to their free form by adding 0.2 niL of lON
NaOH. Check the pH (pH paper or meter) of the aqueous phase.
Add additional NaOH until the pH of the aqueous phase is >13.
(13) Cap the tubes, and shake for 5 minutes. Vortex the tubes to
remove salt particles which might cling to the sides of the
tube. Centrifuge the tubes to separate the phases.
(14) Transfer the ether phase (top layer) to a second test tube for
derivatization using a pasteur pipette (NOTE: Water or NaOH in
the ether layer will give a significantly decreased derivative
yield and cause formation of derivative by-products. Special
care should be taken to avoid entraining water during this
step).
(15) Derivatize samples.
(a) Primary amines
- Add 0.5 niL of the pentafluorobenzylaldehyde reagent to
the methyl-t-butyl ether solution, cap the tubes with
septum seal screw caps, and allow the reaction to
proceed at 60°C for 90 minutes.
- Allow the reaction mixture to cool.
— Add 5 niL of 0.1N NaOH to destroy excess reagent, shake
the mixture for 5 minutes, allow the layers to separate,
and transfer the ether (top layer) to a small round
Chap. 12 - 295
-------
bottom test tube (8 niL Size). Evaporate the sample to
approximately 0.3 niL using nitrogen blowdown.
(b) Secondary amines
- Add 1 niL of pentafluorobenzyl bromide reagent plus 50
pL triethylamine to the methyl-t-butyl ether extract,
cap the tubes with septum seal screw caps and allow
the reaction to proceed at 60°C overnight. Allow the
reaction mixture to cool, then evaporate the solvent
to approximately 0.3 niL using nitrogen blowdown at
30°C. Since the derivatizing reagent also serves as a
solvent, no further manipulations are needed. (Caution:
Pentafluorobenzyl bromide is a severe lacyramator and
should be used in a well ventilated hood. Although
the toxicity of this reagent is unknown, exposure
should be avoided).
(16) With a 50 IiL syringe, add 10 iL of the MS external standard
solution to each derivatized sample. Store derivatized samples
at 0°C until GC/MS analysis.
12.5 GC/MS/COMP ANALYSIS
Prior to beginning GC/NS/COMP analysis of th primary and tertiary
amine (SAN-PT) and secondary amine (SAN-S) fractions the analyst should be
familiar with the procedures described in Chapter 13.
12.5.1 Preparation for Analysis
12.5.1.1 GC/MS/COMP Operating Parameters--
The recommended GC/HS operating parameters for the analysis of SAN-PT
and SAN-S fractions are identical. They are given in Table 12.8. (The
same capillary column can be used for their analysis; however, it is also
recommended that the capillary used here not be used for the analysis of
fractions from other protocols.)
12.5.1.2 MS Calibration--
The mass spectrometer is calibrated using the manufacturer’s recom-
mended approach (Chapter 13). The acceptability of the calibration results
is verified for each SAN fraction by analyzing the appropriate system
performance solution (SPS).
Chap. 12 - 296
-------
Table 12.8. GC/MS OPERATING CONDITIONS
FOR PRIMARY (AS THEIR SCuFF BASES), SECONDARY (PFB DERIVATIVES),
AND TERTIARY STRONG AMINES
GC Column 30 m DB-l (1.0 p film thickness)
fused silica wide bore (0.34 mm
I.D.) capillary column
GC Carrier Gas Helium
Carrier Gas Flow 1.6 mL/min through column; 9:1
split injection
Carrier Gas Sweep Time 85 sec (50°C)
Temperature Program 60°C to 250°C @ 4°/mm and hold
Injector Temperature 250°C
Transfer Line Temperature 255°C
Injection Mode Splitless 0.4 mm/split
Injection Volume 1.0 liL
Ionizing Energy 70 eV
Ion Source Temperature 250°C
Scan Range 35-500
Scan Rate 2 sec (1.9 sec scan, 0.1 sec
settling)
12.5.2 Analysis of SPS (Quality Control )
12.5.2.1 SPS for Primary/Tertiary Amines--
Using the prescribed GC/MS conditions the SPS is analyzed and all
necessary data is acquired and evaluated prior to proceeding to sample
analysis. The SPS is analyzed at the beginning of each day of operation
or prior to starting the analysis of this fraction for the first time.
12.5.2.2 SPS for Secondary Amines-—
Approach is identical as 12.5.2.1.
12.5.2.3 SPS Data Analysis-—
The SPS data is extracted from the run, and test parameters calcula-
ted, and plotted or tabulated (Tables 12.9 and 12.10). Up to 14 days of
SPS analytical results can be historically recorded on Tables 12.9 or
12.10 for each amine fraction set. This provides for day-to-day compari-
sons, and allows the analyst to follow subtle trends that may develop in
Chap. 12 — 297
-------
Table 12.9. GC-MS SYSTEM PERFORMANCE TEST
FOR PRIMARY AND TERTIARY AMINES (SAN-PT)
Dates:
Run Id Code
GC Column
and
Program:
Notebook References:
HS SYSTEN CHEEKS
High/Low Mass Balance
Ion
51
68
70
127
197
198
199
275
365
461
442
443
68 70 127 197 i95 199
DFTPP
Relative Abundance Criteria
30% to 60% of m/z 198
(2% of m/z 69
<2% of m/z 69
40% to 60% of m/z 198
<1% of rn/s 198
100%
5-9% of m/z 198
10% to 30% of rn/s 198
at least 1% of rn/s 198
present, but < rn/s 443
‘40% of rn/z 198
17% to 23% of rn/s 442
275 365 331. 442 443
Pass/ Remedial
Fail Action
In/s •51
Abundance * ( )
Criteria
( )
_ — LI LI L1 LI LI LI LI LI L - - - - __
*These abundances are initially established by the HAS user, and subsequently become guidelines for
acceptability of tune.
(continued)
Chap. 12 - 298
-------
Table 12.9 (cont’d.)
Test Components Criteria
12
10
8
6
CC S’iSTE9 CHECKS
Day
I I
No
• No 2
kcidity
14
12
0
10
08
06
1 Acetophenone (TIC)
2. 2,6—Diinethylaniline (TIC)
I
Day
Ratio 2 1 0 7 to 1 3
(cont i nued)
Limit of
Detection Ally]ainifle (as Schiff’s Base)
S:N (m/z 207) > 6:1
Peak
Asymmetry
1. Acetophenone (TIC) •
2 1—Tetradecanol (TIC) I
300
200
100
%PAF <300
%PAF <200
Chap. 12 — 299
-------
Table 12.9 (cont’d.)
Test Coaponents Criteria
Separation o-Eicossne (a/s 43)
Nuaber -Reneicossne (a/s 43) SN > 6
12
11
10
z 9
U)
8
7
6
, I I I I I I U I U I I I
Day
n-Octadecane (TIC) R ini u 50%
Resolution 1-Octadecene (TIC) Valley
90
80• -
.2 70
60
50
40
I I I U I I U I U I I I U
Day
Inertness Hetbyl Stearate Ratio mIx 74 to 29S 1 I to ](‘ 1
20
IS
.2 16
14
12
I U I I I I I I I — I I —
Day
(contioued)
Chap. 12 - 300
-------
Table 12.9 (cont’d.)
Test Components Criteria
Capillary
Capacity Nexylamine (as Schiff’s Base) (TIC) % 4 >70
100
90
I .
80
dP
70
I I I I I I I I I I I 1
Day
Relative
Retention 1. Heneicosane
Shift 2. Pyrene RRS 0 95-1.05
1.10
1.05 --
1.0
0.95 --
0.90
I I I I I I I I I I I I I
Day
Chap. 12 — 301
-------
Table 12.10. GC-MS SYSTEM PERFORMANCE TEST
FOR SECONDARY AI4INES (SAN-S)
Dates:
Run Id Code
GC Column
and
Progra i:
Notebook References:
MS SYSTEM CHECKS
High/Low Mass Balance
Ion
51
68
70
127
197
198
199
275
365
441
442
443
68 70
DF PP
Relative Abundance Criteria
ai/z
Abundance *
Criteria
30% to 60% of m/z 198
<2% of m/z 69
<2% of /z 69
40% to 60% of m/z 198
<1% of m/z 198
100%
5—9% of m/z 198
10% to 30% of m/z 198
at least 1% of e/z 198
present, but < m/z 443
>40% of w/z 198
17% to 23% of /z 442
in.. _i z_ J12 _am_ — — — — - — Pass/ Remedial
Fail Action
51
( )
( )
(
( )
( )
( )
( )
( )
( )
( )
( )
( )
>‘
*These abundances are initially established by the HAS user,
acceptability of tune.
and subsequently become guidelines for
(continued)
Chap. 12 — 302
-------
Table 12.10 (cont’d 1
Test Components Criteria
Limit of
Detection DlalI>lanine (as PFB Derivative) 5.\ (m/: 250) > I
12
10
z
U,
6--
T I I I I I I
Day
GC SYSTEM C ECXS
Peak 1. Acetophenone (TIC) • %PAF <300
AsymmetrY 2 1-Tetradecanol (TIC) I %PAF (200
300 -- i o I
200-- No 2
100
I I I I I I I I I I I I
Da’
1 Acetophenone (TIC)
Acidity 2. 2,6—Dimethylanillne (TIC) Ratio 2 1 0 7 t 1 3
1.4
1.2
C
10
0.8
0.6
I I I I I I I
Day
(cont inue 1
Chap. 12 - 303
-------
Table 12.10 (cont’d.)
Test Co poneots Criteria
Separation n-Eicosane (./z 43)
Number D-Heneico.ane (rn/a 43) SN > 6
12
11
10
2 9.
V)
8 ’
7,
6--
I I 1 1 I I I I I I I I I
Day
n-Octadecane (TIC) R = mlnimu 50%
Resolution 1—Octadecene (TIC) Valley
90
80
70
15
> 60
50 --
40
I I I I I I I I I I I I I
Day
Inert ness fletbyl Stearate Ratio mix 74 to 9S I .I to I C ’ I
20
18
.9 16
14
12
I I I I I I
Day
(continued)
Chap. 12 - 304
-------
Table 12.10 (cont’d.)
Test Cos ponents Criteria
Capillary
Capacity Morpholine (as PFB Derivative) (TIC) tJAF ‘70
100
90
U.
80
V.
70
i I I I I I I I I I I I I
Day
Relative
Retention I. fleneicosane
Shift 2. Pyrene RJ S = 0 95-1.05
1.10
1.05
10
0.95
0.90
I I I I I I V I I I I I I
Day
Chap. 12 - 305
-------
anticipation of GC or MS maintenance requirements. Chapter 13 explains
calculations and usage of these data.
Daily RMR checks are also made and recorded in Table 12.11 or 12.12,
depending on the amine fraction undergoing analysis. These RMRs are
compared to those obtained during the development of the historical data
bank. The usage of these data is also discussed in Chapter 13.
12.5.3 Determination of RNRs
RMRs for the User’s data bank (as opposed to the daily RJ.fR checks
discussed in the above paragraph) are determined by injecting a solution
containing the derivatized internal standards and analytes.
Table 12.11. GC4IS SYSTEM PERFORNANCE TEST: R 1R CHECK
FOR PRIMARY AND TERTIARY MINES
Date: Run ID Code:
DATA
Amount Ion Area Area
Standard MW (ng) pM (m/z) (Run 1) (Run 2)
4-Fluoro-2-iodo- 236 109
toluene (FIT) 236
2-Fluorobiphenyl 172 172
d 9 -butylamine 261 210
212
d 4 -phenylethylamine 304 304
MATRIX OF STANDARD ION RNRs
Standard Ion 109 236 172 210 212 304
FIT 109
236
-
-
2-Fluorobiphenyl 172
-
d 9 -butylamine 210
212
-
d 4 -phenylethylamine 304
-
A MW n
g A = ion area or height
x/y A MW ng ng ng injected
y y x xana lyte
COMMENTS: standard
Chap. 12 - 306
-------
Table 12.12 GC-MS SYSTEM PERFORMANCE TEST:
RNR CHECK FOR SECONDARY AJIINES (SAH-S)
Date: Run ID Code:
DATA
Amount Ion Area Area
Standard MW (ng) pM (m/z) (Run 1) (Run 2)
2-Fluorobiphenyl 172 172
4—F luoro-2-iodo- 236 109
toluene (FIT) 236
N-Ethyl-2-fluoro- 314 314
benzylamine
MATRIX OF STANDARD ION RrlRs
Standard Ion 172 109 236 314
2-Fluorobiphenyl 172
-
FIT 109
236
-
-
N-Ethyl-2-fluoro— 314
benzylamine
-
A MW ng,, A = ion area or height
J fl X X ng ng injected
x,y A l ng x = analyte
y = standard
COMMENTS:
The GC/MS/COIIP operating parameters are given in Table 12.8.
Table 13.8 may be used to tabulate RMR raw data.
12.5.4 Analysis of Field and Quality Control Samples
After all specified criteria for GC and MS performance (Table 12.9 or
12.10 and 12.11 or 12.12) have been met, analysis of standard solutions,
QA/QC samples, (e.g., for RMR calculations) or SAN—PT/SAM-S sample extracts
is conducted.
Qualitative and quantitative procedures are described in Chapter 13.
Tables 12.13 and 12.14 may be used to tabulate sample analyte raw data for
the SAN-PT and SAN-S fractions, respectively, prior to using the MASQUANT
software program or manual quantitative calculations.
Chap. 12 - 307
-------
Table 12.13. RAW ANALYTE DATA FOR SAN-PT FRACTION
Date:
Run 1.0. Code:
Notebook Reference:
Vol. Water Processed
Sample Identification:
Vol. Water (L):
(to which i were added)
(mL):
STANDARDS
Spec-
I.D. truin
No. No.
4—F luoro-2—iodo-
Weight Ions
(ng ) (m/z) Area
MW
a
K
toluene
2—Fluorobiphenyl
d 9 —Buty lamine
d 4 -Phenylethyl-
amine
109,236: ,______
172: ,
210,212: ,______
304: ,______
,
,
,
,
ANALYTES
Class No.
I.D.
No.
Spectrum Ions
No. (m/z) Area
?lWa
, • ,
,
,
,
,
,
,
,
, : ,
,
,
,
—
a 1 f calculations are performed manually then this information is also
needed, whereas MASQUANT performs functions automatically.
A MW ng A = ion area or height
ng = RMR X A ng = ng recovered from volume
x/y y y of water processed
x = analyte
K — ng y = standard
A MW
y y
Chap. 12 - 308
-------
Table 12.14. RAW ANALYTE DATA FOR SAN-S FRACTION
Date:
Run I.D. Code:
Notebook Reference:
Vol. Water Processed
Sample Identification:
Vol. Water (L):
(to which i were added)
(mL):
STANDARDS
Spec—
I.D. trum
No. No.
2-Fluorobiphenyl
4-Fluoro-2-iodo-
Weight Ions
(n 5 ) (m/z) Area
MW
Ka
172:
,
toluene
N-Ethy l-2-fluoro-
109,236:
,
benzy lamine
314: ,______
,
ANALYTES
Class No.
I.D.
No.
Spectrum Ions
No. (m/z) Area
MWa
,
,
,
,
,
,
,
,
,
,
,
, :
,
—
a 1 f calculations are performed manually then this information is also
needed, whereas MASQUANT performs functions automatically.
A MW A = ion area or height
x x ____
ng = x A • MW ng = ng recovered from volume
x/y y y of water processed
x = analyte
nR
K = -‘y y = standard
A • MW
y y
Chap. 12 - 309
-------
CHAPTER 13
GC/MS/COMP ANALYSIS PROCEDURES - GEL ERAL INSTRUCTIONS FOR ALL ROTOCOLS
13.1 INTRODUCTION
This chapter discusses the general approach and procedures for quali-
tative and quantitative analysis of sample fractions derived from each MAS
analytical protocol. Also, an example of calculations of the concentra-
tions of analytes in a water sample are given. Once the analyst has
become acquainted with this chapter and skillful with the instrumental
analysis steps, he must consult the individual analytical protocol to
obtain further detailed information specific to the fraction (extract) to
be analyzed.
The application of a good quality assurance/quality control (QA/QC)
program is essential to ensure the validity of data obtained from the
GC/MS/COMP system. As part of the QA/QC program, the Master Analytical
Scheme incorporates the use of system performance standards (SPS) to
assure the proper operation of the GC/MS/COMP system. The instrument
characteristics, especially those of the capillary column, are tested with
the SPS using established procedures (Grob, K., et al., J. Chromatogr.,
156, 1, 1978). The criteria given for column performance in each analyt-
ical protocol are based on “safe” limits. As the Scheme is used for a
variety of water types and sample fractions, the information gathered will
permit the user to adjust the boundaries of the limits for each criterion
according to his own application needs.
13.2 APPARATUS AND MATERIALS
This section describes the general apparatus and materials which will
be required to perform instrumental analysis of fractions produced by each
of the protocols in the Master Analytical Scheme. These are:
(1) Combined gas chromatograph/mass spectrometer with the following
capabilities:
Chap. 13 — 310
-------
(a) scan range of 35-600 daltons (maximum, range needed varies with
specific protocol);
(b) scan cycle of 2 sec or faster (start-to-start);
Cc) at least unit resolution through the mass range;
Ce) GC injector (Grob or Grob-type) capable of split and split/
splitless injection modes;
(f) capability of direct coupling of capillary column to the
ion source or of coupling through a separator with make-up
gas; and
(g) computer with, as a minimum, a software package capable of
acquiring continuous mass scans, extracting selected ion
current, quantifying ions by peak area (preferable) or peak
height (acceptable) and equipped with an ANSI Fortran
compiler.
(2) Gas chromatographic column — fused silica or glass capillary
column, wide bore, with thick film. The exact length, internal
diameter, stationary phase coating and thickness are prescribed
in each analytical protocol.
(3) System Performance Solution - contains a series of compounds to
assess the tuning and chromatographic performance of the GCIMS
system. Each SPS is tailored to the specific fraction generated
by an analytical protocol. An example is given in Table 13.1
for the ESSA fraction.
(4) Solutions for generating RMRs for all HAS fractions, containing
target compounds of interest:
(a) —‘400 ng/IJL in methylene chloride, for all compounds except
very volatile VO and NEWS;
(b) -‘400 ng/ JL in water, for elevated temperature purge and
trap (NEWS) compounds;
(c) in gas-phase mixture, for very volatile VO organics (those
which cannot be put into methylene chloride solution).
(5) Ultra—pure helium, associated clean lines and two—stage stainless
steel diaphragm pressure regulator.
(6) 10 IJL syringe.
Chap. 13 - 311
-------
Table 13.1. GC/MS SYSTEM PERFORMANCE SOLUTION FOR ESSA
Density
Compound (@ 20°C)
Concentration
pg/mL in Methanol
2,6-dimethyiphenol S 300
acetophenone 1.030 310
1-tetradecanol 0.823 250
1-octadecene 0.789 230
n-octadecane 0.777 231
DFTPP S 500
n-eicosane S 300
pyrene S 300
n-heneicosane S 300
methyl stearate S 10
d 13 -heptanoic acid 0.948 280
(methyl ester)
d 5 -ben.zoic acid 1.13 340
(methyl ester)
4-f luoro-2-iodoto luene 0.883 260
methyl decanoate 0.873 870
a
Solid.
13.3 OPERATIONAL PARE METERS
13.3.1 Mass Spectrometer Operating Conditions and Tuning
For a quadrupole mass spectrometer, a 0.1 sec hold at low mass and a
scan cycle of l sec (0.9 sec scan, 0.1 sec settling time at low mass) for
the Finnigan 4021 (2 msec/mass integration time) and 2 sec (1.9 sec scan,
0.1 sec settling time at low mass) for the Finnigan 3300 (4 msec/mass
integration) are used. All spectra are recordçd at 70 eV. The transfer
line (if capillary is not directly coupled to the ion source) is maintained
at 280°C (or at a temperature to allow satisfactory transmission of pyrene).
Chap. 13 - 312
-------
The complete mass spectra are recorded from mass 40 to mass 450. All data
recorded are archived on IBM-compatible 9—track magnetic tapes for future
reference.
The system is given an initial tune each day of operation (or as
needed) to obtain a good signal level, acceptable peak shape, at least unit
resolution through the mass range, and calibration of the mass range according
to the MS manufacturer’s recommended procedure. The system can be given
this preliminary tune by the use of perfluorotributylamine, perfluorQkerosene,
tris(perfluoroheptyl)-s-triazine or any other standard which is in routine
use in a given laboratory. The standard should be introduced through a
molecular leak or some other type of reservoir system which will maintain a
stable low steady-state concentration of the material in the ion source
(similar to pressures achieved under GC conditions) during the time adjust-
ments to the system are being made. The mass spectrometer inlet system used
for the introduction of the tuning compound should allow an easily reprodu-
cible quantity of the standard compound to be introduced in order to avoid
any possible discrimination effects which could arise from differences in
source pressures on different days.
13.3.1.1 Purgeable Protocols (VO and NEWS fractions)--
The ability of the tuned system to meet specific HAS criteria for
analysis of volatile organics can be evaluated by the introduction of
perfluorotoluene (PFT) and l-bromo-4-fluorobenzene (BFB) through the same
inlet system used for perfluorokerosene (or other tuning standard). This
tune, however, is ultimately confirmed by the spectrum of PFT and BIB, which
are components of the system performance solution, obtained when the system
performance solution is chromatographed. Mean relative abundances of major
ions of the PIT and BFB spectra obtained under one set of conditions are
shown in Table 13.2 and 13.3, respectively. The user should be able to tune
his instrument to a stable point within the percent relative standard devia—
tion and thereafter maintain his tune to within ±15%. The exact position of
stable tune on any user’s instrument must be determined experimentally. The
resulting PET or BFB ion distribution should be attainable under normal
operating conditions and the instrument must be stable (±15%) for at least
an eight-hour day. (If a double or triple shift day is used, tune stability
Chap. 13 — 313
-------
Table 13.2. RELATIVE INTENSITIES OF IONS IN pEIffLUOROTOLUENIEa
rn/z Relative Intensities (CV)
69 33 (5)
79 11 (10)
93 16 (8)
117 43 (8)
167 15 (7)
186 59 (5)
217 100
236 66 (4)
237 5
aData obtained on Finnegan 4021 GC/!IS/DS; 60 m wide-bore (0.35 mm)
thick film (1 i ) fused silica DB-1 column; isothermal hold, 5 mm
at 40°C, then programmed to 200°C at 4°C/mm.
Table 13.3. RELATIVE ABUNDANCES OF BFB IONS
Ion Relative Abundance
50 15-40% of mass 95
75 30-60% of mass 95
95 100%
96 5-9% of mass 95
173 <2% of mass 174
174 >50% of mass 95
175 5—9% of mass 174
176 >95%, <101% of mass 174
177 5—9% of mass 176
Chap. 13 - 314
-------
should be checked every eight hours, until sufficient data are accumulated
to show that such frequent checking is unnecessary.)
13.3.1.2 Other Protocols--
The ultimate check on the tune of the system for all fractions except
VO and NEWS is made using decafluorotriphenylphosphine (DFTPP) as indicated
in the Federal Register, 44, 69536 (12/3/79). DFTPP must be chromatographed
in order to assess correspondence of the observed relative abundances of
ions with those reported in the Federal Register, and presented in Table
13.4. Many methods can be used to obtain the mass spectrum from a chromato-
graphic peak: a spectrum may be taken at the top of the peak, a background
(before or after the peak) may be subtracted, an averaged spectrum (by
several different techniques) may be used, or an averaged spectrum with
subtracted background may be obtained. The exact technique used in obtaining
the spectrum is less important than ensuring that the same method is used
consistently for a given instrument. Each MAS system performance solution
Table 13.4. RELATIVE ABUNDANCES OF DFTPP IONSa
Ion Relative Abundance
51 30-60% of mass 198
68 less than 2% of mass 69
70 less than 2% of mass 69
127 40—60% of mass 198
197 less than 1% of mass 198
198 base peak, 100% relative abundance
199 5-9% of mass 198
275 10—30% of mass 198
365 at least 1% of mass 198
441 present, but less than mass 443
442 greater than 40% of mass 198
443 17-23% of mass 442
aFederal Register, 44, 69536 (12/3/79).
Chap. 13 - 315
-------
(except those for VO and NEWS fractions) contains DFTPP and is used to
obtain and check the DFTPP spectrum. The relative abundances of the ions of
DFTPP are to conform to the ranges presented in Table 13.4. (An example is
given in Table 13.5.) Precision for the user’s ion abundances should be
+15% within one eight—hour day’s operation.
13.3.2 GC Operating Conditions
The specific capillary column to be used with each protocol is given
under Section 13.5 of each protocol. The GC operating conditions are also
specifically stated for analysis of extracts/fractions produced by a
protocol.
If glass capillaries that meet performance specifications are employed
rather than fused silica, then the following guidelines are given for
straightening their ends for installation:
(1) a low flow (1-2 mL/min) of an inert gas is passed through the
column toward the end to be straightened;
Table 13.5. MEAN RELATIVE ABUNDANCES OF MAJOR IONS OF DFTPP
OBSERVED IN A SYSTEM PERFORMANCE SOLUTION a
Ion Relative Abundance C.V.
51 31 3
68 0.3 8
69 31 3
70 0.6 7
77 40 2
110 23 1
127 41 2
197 0.3 12
198 100
199 8 7
206 17 1
255 30 1
275 19 2
365 2 15
441 3 29
(continued)
Chap. 13 — 316
-------
Table 13.5 (cont’d.)
Ion
Relative Abundance
C.V.
442
45
4
443
7
4
aData obtained on Finnegan 4021 GC/MS/DS; column, 30 m DB-l wide-bore
(0.32 mm) thick film (1 p) fused silica capillary; 50°C for 5 mm,
then programmed to 240°c at 4°C/mm.
(2) the column is held so that the portion to be straightened will
point down when straightened;
(3) a yellow flame (from, e.g., the base of a Bunsen burner), just
hot enough to allow the glass to straighten but not melt, is
passed along the column from the point where the straight portion
is to start to the end of the column, allowing the weight of the
glass to straighten the end as it is heat’ed;
(4) a 0.1% Carbowax 20N solution in CH 2 C1 2 is injected into the
straightened end of the capillary (using a 50 lJL syringe connect-
ed to the column end with Teflon tubing) against the inert gas
flow until it fills the portion of the column that has been
heated in the flame;
(5) the 0.1% Carbowax 20M solution is withdrawn and the syringe dis-
connected;
(6) the inert gas flow is maintained until any remaining solvent is
evaporated;
(7) the inert gas flow is stopped; and
(8) the last 2-3 cm of the column is broken off.
After installing the capillary column, the carrier flow is optimized
according to the capillary column manufacturer’s instructions. A typical
flow rate would be 2.0 mL/min (—18 cm/sec) for a 0.32 mm i.d. fused silica
capillary. See each MAS protocol for specific flow rates.
The GC injection system should be capable of split and split/splitless
modes. The split/splitless mode must be optimized for each fraction as
per manufacturer’s instructions. Splitter and carrier gas flows must, of
Chap. 13 — 317
-------
course, be adjusted when the GC/ZIS system is set up for a given HAS proto-
col. At the initial stages of operation in a given protocol, the accuracy
of the gas flows should be verified for every 24 Ii period. After the user
has established the stability of flows, it may be sufficient to check less
frequently (perhaps weekly).
Au injection size of up to 2.0 liL is used for a split injection and,
for spli.tless, up to 1.0 IlL. The injection port is maintained at 250°C.
Approximately once a month, during the analysis of a SPS, the GC oven
temperature readout should be verified using an alternate measuring device
such as a thermometer or digital thermometer. The rate of rise of the
oven temperature at the various temperature program rates should also be
verified.
13.4 ANALYSIS OF SYSTEM PERFORMANCE SOLUTION (SPS) AND SAMPLES
After establishing mass spectrometer tuning and GC conditions, the
first GC/HS analysis performed each day is on the appropriate SPS. The
data acquired from the SPS analysis are examined to determine whether the
MS and GC performance is acceptable for sample analysis. Then, extracts!
fractions are analyzed. The extracts/fractions are grouped as sets (e.g.,
all extractable acid, ESSA extracts from one series of water samples is a
set) and analyzed by GC/MS.
This section provides general comments and procedures, which are
prescribed more specifically in each of the analytical protocols, for the
analysis of SPS and sample extracts. Specific case examples are given.
13.4.1 SPS - Data Acquired and Calculations
The following information is needed from the SPS analysis for subse-
quent calculations:
(1) Perfluorotoluene (PFT), 1—bromo—4—fluorobenzene or decafluorotri—
phenyiphosphine (DFTPP) spectrum, as specified by the analytical protocol;
(2) Reconstructed total or extracted ion current profiles for the
other SPS components;
(3) Retention time (spectrum no.) for each SPS component; (Spectrum
number is used because this is the parameter used by MASQUANT to
determine which of the internal standards elutes closest to the
target compound of interest. No generation of relative retention
Chap. 13 - 318
-------
times (RRT) as a separate data base is being done. If the user
desires an RRT data base, he may use ratios of spectrum numbers
or absolute retention times at peak tops.);
(4) Areas for selected ions of certain components (usually the
internal and external standards) for calculation of RMRs to
correct for tuning differences (see Section 13.4.2.2.3) if MS
tune cannot meet criteria.
13.4.1.1 Acceptability of MS Tune--
The abundance of each appropriate ion for PFT, BFB, or DFTPP (depending upon
fraction to be analyzed) that is obtained from analyzing the SPS is tabulated for
inspection and a historical record (for example, Table 13.6). The analyst is to
initially establish the precise abundance for these ions on his instrument by
analyzing PFT, BFB, or DFTPP a minimum of six times, preferably over several
days of operation. This mean average abundance for each ion is recorded as,
for example, on the chart at the bottom of Table 13.6 in the “blank” paren-
theses (line labeled “Abundance”). Subsequently, the analysis of the SPS
(prior to analysis of MAS fractions) is conducted daily the PFT or DFTPP
ion abundances listed for each day’s operation.. Acceptability of tune is
demonstrated by the maintenance of ion abundance within ±15% relative
standard deviation of the analyst’s established guidelines.
An example of data obtained for the SPS of extractable acids (ESSA)
is given in Table 13.6.
Failure to meet criteria requires one or all of the following:
(a) retuning; (b) cleaning the source (and rods, if necessary); or
(c) other maintenance to reestablish acquisition of data within performance
specifications. All reasonable efforts should be made to bring the instru-
ment within performance specifications. If, for some reason (time con-
straints, sample storage deadlines), the system cannot be made to meet
specifications, see Section 13.4.2.2.3 for a method for correcting relative
molar response values.
After criteria of ion abundance for PFT or DFTPP have been met the
chromatographic performance characteristics for the SPS analysis are
examined.
Chap. 13 — 319
-------
Table 13.6. CC-MS SYSTEM PERFORMANCE TEST FOR
EXTRACTABLE SENIVOLATILE STRONG ACIDS (ESSA)
Dates: 74J J14j
.
•
Run Id Code E5 4-I E55-12.
e-.-eI
155A-2.
g.g4g
1-n -1I
E A-g
2-ia-2J
-I5-ti
5-i -2a
ci - iô
GC Column and Progran:
DB-I
i.,i4e
bore ,
S.b .i.
film 5o-aMb
Wotebook References:
l1 i— 1 g
MS SYSTEM C CXS (Injection of 1 iL of SPS)
I Reproduced from
best available copy.
Migh/Lov Mass Balance
DFIPP
Ion
51
Relative Abundance Criteria
30% to 60% of m/z
198
68
<2% of m/z 69
70
<2% of c/i 69
127
40% to 60% of /z
198
197
<1% of n/z 198
198
100%
199
5—9% of m/z 198
275
10% to 30% of dr
198
365
at least 1% of dr
198
441
present, but ( dr
443
442
>40% of c/a 198
443
17% to 23% of m/z
442
( fl* (J. )
(q )
31 68 70 127 197 195 1gb 275 365 331 442 443
(.?)
(i&, ) (7)
(2 ) (LI) (1) (Vv) (4
Pass/ Re;ed al
h 1 Action
rn/a
Abundance
$ 7 I i. # i. W 1 iO ‘7 , ,I ‘7 I s . —
j p —
e-5a9 I.&, /.T39 .4, IOC(a 29/.3 7 .3f
$-$ AL.. ..5 . ?° 1 P —
sq ‘• ‘• 39 . 27 1.2 ‘7 3V F
-q 30 M .!. q .. .1 —
1-Id 30 I .7 4W .1 leo 1 ‘ 1.1 7 9 P —
s-°’ ,_.y . 7 ?’
5. .’5 3 / 41 ,2 .1 /00 1.2. 1 ‘:; P —
,-Ib L. -- . —
5- 17 3 , 5 /,p VI .7 bc 7 ,./ g y’/ q p —
*These abundances are initially established by the MAS user, and subsequently becowe guidelines for
acceptability of tune.
(continued)
Chap. 13 — 320
-------
Table 13.6 (cont’d.)
Test Cosponenti Criteria
Linit of
Detection Methyl Stearate S:N (nlz 74) ) 40:1
,:, ,:, e ..o 1 i $ 4 b
Day
CC SYSTEII CHECKS
Peak 1. Acetophenone (TIC) %PAF <300
Asy etry 2. 1-Tetradecanol (TIC t3AI <200
::
1 ’1 $0 11 1 1 -95 t-lb $-M ‘ ‘ ‘ I I
Day
Baaicity*
1.4
1.2
1.0
0.8
0.6
0
1. I
1. Acetophenone (TIC)
2. 2,6—Dinethyiphenol (TIC) Ratio 2:1 0.7 to 1.3
s., $ .$ $9 1 :j a. 9-is 9-jo 9—,,
Day
*Note: Acidity criteria are also neasured in other MAS fractions.
(continued)
IReproduced from
best available copy.
Chap. 13 - 321
-------
Inertness
Table 13.6 (cont’d.)
Methyl Stearate
Ratio ID/Z 74 to 298 < 14 1
s’i g $ , i:se
Day
(continued)
Test Cosiponeats Criteria
Separation u-Eicosane (./z 43)
Nuiber D-Reneicosane a/z 43) SN > 6
“““
,.1 t-Y 8-’ f-to S-di s -ss - s - I l
Day
n-Octadecane (TIC) R = minimum 50%
Resolution 1-Octadecene (TIC) Valley
100
> 60
50 --
40
,: sq s .Io ‘ ‘ ‘ ‘
Day
10
11
0
— 12
‘I
13
14
Chap. 13 - 322
-------
Table 13.6 (cont’d.)
Test Co iponenti Criteria
Capillary
Capacity Methyl Decaøoate (TIC) VAF >70
.,.
100
90
80
:
70 .
S 1 $- fO -‘ii 9- s 8 lé $ . g7 ‘ I I I
13.4.1.2 Peak Asymmetry--
The peak asymmetry or tailing is measured by the percent peak asymmetry
factor (PAY):
% PAP = x 100
where B = the width of the back half of a chromatographic peak mea-
sured at 10% above baseline
F = width of the front half of the chromatographic peak measured
at 10% above baseline
An example of this measurement is given in Figure 13.1 for acetophe-
none. Specific criteria are given with each analytical protocol, e.g.,
see Table 13.6.
13.4.1.3 Acidity/Basicity-—
The acidity/basicity of the column is checked by the peak area ratios
(determined by computer or triangulation) of 2,6-dimethylaniline and/or
2,6-dimethyiphenol to acetophenone. A ratio of 0.7 to 1.3 for both is
acceptable. A typical measurement is given in Figure 13.2. Specific
criteria are given with each analytical protocol, e.g., see Table 13.6.
13.4.1.4 Separation Number—-
The separation number (SN) is a measure of the separation efficiency
of the column and is determined as follows:
SN = ÷ -l
Chap. 13 — 323
-------
IIC lAIAs 4ZV0997 SI
BI/II/8$ 0 $7:SS CAUi 4 1 lb I l
SAn LEI ULI riwsil
IUICE G I.S4$ LAlais,4.sswIz s.s..us &ua. 3
cMIc 115$ 1 i s i s
l- )
(A)
S • 2.30 Ca
p • a.as c.
I PAP .
2 PAP 100
I PAP • 124
I5’o.
178$ • AN
29z4 5
Figure 13.1. Reconstructed total ion current profile of acetophenone used in calculating
percent peak asymmetry factor.
-------
348672
IN.
2 6—DimethylphenoL
A - 3160090
ace Lophenone
2987450 • area
2 ,6—Dttnethylaniline
A
1835
-a
A)
‘A)
“I
2051
1811 l IO 2111 2 10 1 2211 SCAIS
3611 31*4 1 33 2 1 35*11 36*41 1l1
Figure 13.2. Reconstructed total ion current profile for acetophenone, 2,6-dimethylaniline
and 2,6-dimethylphenol used for measuring acidity/basicity.
-------
where
D = the distance between two peaks, measured between perpendiculars
dropped from the peak apices
= widths at 1/2 height
Typically two hydrocarbons are used for this measurement (e.g., eicosane
and heneicosane in Table 13.1). The specific ions of the two components
in the SPS to be used for measurement of the SN and the acceptable cri-
teria are given with each analytical protocol (e.g., see Table 13.6). An
example of this measurement is depicted in Figure 13.3.
13.4.1.5 Resolution--
An example of calculating peak resolution or percent valley is shown
in Figure 13.4. Minimal criteria and specific compounds used for this
measurement are given in each analytical protocol. Percent valley is
defined as:
valley
% valley =
peak height
where
valley height of valley relative to height of first peak
peak height = height of the first peak of the doublet.
13.4.1.6 Inertness--
Inertness of the chromatographic system’s interface is determined
from the methyl stearate peak. Methyl stearate ion ratios (m/z 74 to 298;
see Table 13.6) as well as the acidity/basicity ratios (Section 13.4.1.3)
are used to assess system inertness.
13.4.1.7 Changes in Column Polarity and/or Stationary Phase Film--
Retention time shift (absolute or relative) of components in the SPS
(for example, eicosane and heneicosane in Table 13.1) is used to determine
deterioration of chromatographic performance as a capillary column ages
from usage. Exact guidelines for acceptability are GC/MS system-dependent
and must be established by the analyst for his own system. This change
may reflect an alteration in polarity or stationary film thickness. The
performance criteria are given in each analytical protocol.
Chap. 13 — 326
-------
$CA G 4 I 10 5I
3t 6O.
• •.5
1II
Figure 13.3.
Extracted ion current profile of rn/z 43 of eicosane
and heneicosane used for measuring SN.
SIASS OIt IflOC8AJ1
ivitiua 0. w.ug
SAtltI1 liii
IAMG i C
bATAi 4 e997 II
CAUi V11 1910 12
111.4.1 W$IA III AStiU2S. 3
43
D — LA.SS
— .55 c
D —1
w 2
L4. 5 5
SM — +
SN — 13 —1
SN — 12
V 2 — .
-------
TAi 997 IS
CW1 17 7 75 62
]&IS TD 3 S
3
v.1 ioy
Z v.11.y — p..k b.&ghi
12.6
I ..11.y — x 100
I v.11 — 94
Figure 13.4.
octadecane
‘ IC
SI/Still 8 S7N
SAIIPI.Is I J ?UI}STh
laISG&i G 1.5469 LANU; 0. 4. CIUNi 4 5.9
(A)
p. .)
Reconstructed total ion current profile of octadecene and
used for measuring resolution (percent valley).
WAN
uI
-------
13.4.1.8 GC/ZIS/COMP System Sensitivity and Dynamic Range--
Two components in each SPS are designated at levels which provide,
upon analysis, information concerning sensitivity of the GC/NS/CONP system
and potential overloading of the capillary column (dynamic range of analy-
sis). The compounds selected, for example, methyl stearate and methyl
decanoate (Table 13.1), are intended to be representative of the types of
functionalities present in the extract/fraction to be analyzed. Example
charts are provided in Table 13.6 for limit of detection (system sensitiv-
ity) of methyl stearate and capillary capacity (% PAP) for methyl decano-
ate. Specific criteria are given with each analytical protocol.
13.4.1.9 Presentation of System Performance Information--
Table 13.6 is a hypothetical example of the formatted information
acquired from the SPS designated in the “extractable acid” (ESSA) analyt-
ical protocol. The information acquired is displayed for historical
reference over a period of 14 days. The analyst can not only assess prior
to analysis whether performance specifications have been met for that
day’s operation, but can also determine trends in performance to anticipate
potential problems in the near future. Blank forms such as used for
Table 13.6, but tailored for each analytical protocol, are provided with
each protocol; the analyst can duplicate them for his own use.
Adherence to acquiring and assessing the system performance informa-
tion is an essential facet of the quality control procedures in the MAS.
13.4.1.10 Failure to Meet Chromatographic Criteria--
A new capillary column should be well within the specifications
provided for each analytical protocol’s SPS. When one or more of the
criteria can no longer be met, the capillary column should no longer be
used. Rejuvenation may be attempted by removing 2 coils from each end of
the capillary. If the coils are removed from a glass (not fused silica)
column, the ends must be straightened, and the straightened ends should be
recoated with a 0.1% Carbowax 20M/methylene chloride solution (Section
13.3.2).
13.4.2 Sample Analysis
After establishing that the MS tuning and chromatographic criteria
have been met, analysis of sample extracts may begin. The analysts
Chap. 13 - 329
-------
conditions for each sample extract are prescribed in each analytical protocol
and are identical to those for the SPS. If more than one day’s CC/MS
running time is required for a set of sample extracts from an analytical
protocol, the system performance solution must again be analyzed as the
first analysis of a new day.
When sample extracts from a different analytical protocol are to be
analyzed, then the operating conditions prescribed for that protocol are to
be set up and the system performance solution for that protocol analyzed
first.
A set of sample extracts is defined as extracts derived from field
samples, surrogate samples, blanks, controls, standards used for retention
time and RMR determination, etc., that are all associated with a single
analytical protocol. The recommended analysis order is laboratory and
field blanks and controls, surrogates and collected field samples, respec-
tively.
13.4.2.1 Compound Identification--
At the Master Analytical Scheme user’s discretion, the identities of
the compounds in the sample extracts are established by computer search
and/or manually. Table 13.7 gives suggested levels of confidence for the
identifications, depending on the approach(es) employed.
13.4.2.2 Quantification-—
13.4.2.2.1 General Description of Approach--The general approach to
quantification employed by the Master Analytical Scheme is to determine
the peak area ratios of the major ion(s) of the analyte relative to those
of a deuterated internal standard and, using a known detector response
ratio for the analyte and standard, known concentration of standard, and
recovery factor for the analyte, calculate the amount of analyte present
in the original water sample processed.
Once the analyst has identified the components, he may wish to use
the relative molar response (RHR) factors supplied with the Master Analyt-
ical Scheme protocols (Appendix B) or determine his own response factors
on the GC/MS/COMP system employed for sample extract analysis. The latter
will be, of course, more accurate. In addition, the hAS user may frequent-
ly analyze for target compounds not in Appendix B, in which case he should
Chap. 13 - 330
-------
Table 13.7. SUGGESTED LEVELS OF CONFIDENCE FOR COMPOUND IDENTIFICATION
Level I Computer Interpretation . The raw data generated from the analy-
sis of samples are subjected to computerized deconvolution/
library search. Compound identification made using this compu-
terized approach has the lowest level of confidence. In general
Level I is reserved for only those cases where verification of
tentative compound identification is the primary intent of the
qualitative analysis.
Level II Manual Interpretation . The plotted mass spectra are manually
interpreted by a skilled interpreter and compared to those spec-
tra in a data compendium. In general a minimum of five to eight
masses and intensities (±20%) should match between the unknown
and library spectrum. This level does not utilize any further
information such as retention time since, for many compounds, the
authentic compound may not be available for establishing reten-
tion times.
Level III Manual Interpretation Plus Retention Time/Boiling Point of
Compound . In addition to the criteria attained under Level II,
the retention time of the compound is compared to the retention
time of a standard which has been derived from previous chromat-
ographic analysis. Also the boiling point of the identified
compound is compared to the boiling points of other compounds
in the near vicinity of the one in question when a capillary
coated with a non-polar phase has been used.
Level IV Manual Interpretation Plus Retention Time and Matching Spectrum
of Authentic Compounds . Under this level, the authentic com-
pound has been chromatographed on the same capillary column
using identical operating conditions and the mass spectrum and
retention time of the authentic compound are compared to that of
the unknown. Major peaks of the mass spectrum should correspond
±15% and retention times should correspond ±10%. The ultimate
confirmation of identification consists of coinjection of authen-
tic standard and observation of no change in mass spectrum or
retention time.
Level IVa Independent Confirmation Techniques . This Level utilizes other
physical methods of analysis such as GC/Fourier transform IR,
GC/high resolution mass spectrometry, or NNR analysis in the
absence of authentic standard. This level constitutes the high-
est degree of confidence in the identification of organic com-
pounds when no standard is available.
Chap. 13 - 331
-------
establish his own RflR data bank. To determine RMR factors, solutions
containing precisely-known amounts of target compounds and standards must
be analyzed in replicate. Compound concentrations and ion peak areas are
obtained from the replicate analyses to allow calculation of RIIRs.
The quantification process requires that the peak areas (or heights
if areas are unavailable) of the ions listed for the analyte in the data
bank (Appendix B) be determined. Generally two ions are listed. The
second ion is provided in case the more intense ion is saturated, overlaps
with an ion of the same mass from another eluent, or cannot produce an
unambiguous area for some other reason. The peak areas for the internal
standard ions listed are to be determined also. Retention times (as scan
numbers) of the analytes and standard are to be recorded.
The quantification procedure is preferably to be performed by computer,
and a computer program (MASQUANT) has been developed for this purpose.
However, an understanding of the process used is essential and a detailed
description is presented here. This section includes procedures for
determination of response factors as well as for quantification of analytes.
13.4.2.2.2 Target Compound RHR Determination--If target compounds
and internal standards have been analyzed as part of an SPS or for the
purpose of determining response factors for a data base, the response
factors on a molar basis (RNRs) are to be determined from equation 1.
A
sought—for sought—for 6 standard
RI1R eqn. 1
target/standard A MW
standard standard sought-for
where
A = ion area (or height)
g = grams injected
11W = gram-molecular weight (if compounds are derivatized, use the
molecular weight of the derivative).
Specific instructions for determining RMRs are included in each ana-
lytical protocol. Table 13.8 is a form that may be used for tabulating
raw RNR data.
13.4.2.2.3 RMR Correction Factors-—If the tune of the instrument
when the samples are analyzed differs from the tune of the instrument when
Chap. 13 - 332
-------
Table 13.8. RMR RAW DATA - MAS
Date(s): Run identification code:
HAS fraction:
CC column and program:
Notebook reference:
Compound
HWa
Amount
(ng)
Micro—
moles
Ion
Area
( )C
Area
( )
Area
( )
Area
( )
In case of derivative, use MW of derivative.
ng of material in solution ready for GC-MS.
CDesignate code number of each GC-MS run; if run on different dates, give
dates.
Chap. 13 - 333
-------
the R fRs were determined, errors will be introduced into the quantifica-
tion. With a change in tune, the historical RNRs are no longer correct
when applied to the calculation of compound quantities. Depending on the
exact nature and magnitude of the tuning variance, the effect on the RMRs
can range from insignificant to order—of-magnitude. An empirically
derived method of determining a correction factor which will reduce (not
eliminate) these errors has been developed. It is suggested that this
correction factor be applied if the relative abundances of the major ions
of PFT or DFTPP observed during the analysis of the SPS differ by more
than ±15% from those observed when the RMRs were determined. This figure
is currently an arbitrary figure and may be raised or lowered at the
discretion of the analyst. For this reason the relative abundances for
the major ions of PFT and DFTPP observed when the MASQUANT data base
historical RIIRs were determined are given in Appendix B.
RMR data obtained from the analysis of internal and external standards
in the SPS solution may be used to correct for tuning differences, and,
therefore, RNRs, between the current and historical data file (Appendix B).
The current RHRs are calculated from selected ion peak areas of the inter-
nal and external standards using equation 1, and tabulated on forms
provided in each analytical protocol (see Table 13.9, for example). The
RMRs determined under the different tuning conditions can then be correlat-
ed using a linear regression analysis. To determine the slope and intercept
for the linear regression, the series of current RNRs for each standard ion
of the internal and external standards (Table 13.9, for example), are compared
to the RMRs in the Appendix. (The Appendix contains tables of the matrix
Chap. 13 - 334
-------
Table 13.9. CC-MS SYSTEM PERFORMANCE TEST: RMR CHECK
FOR SE1 IIVOLATILE STRONG ACIDS (ESSA)
Date:
Run ID Code:
DATA
Standard
MW
Amount
(ng)
Ion Area
pM (m/z) (Run 1)
Area
(Run 2)
4-Fluoro-2-Iodo—
toluene (FIT)
236
109 49,903
236 63,372
49,351
64,051
d 13 -heptanoic acid
143
77 46,584
56,538
91 13,314
15,465
d 5 —benzoic acid
127
82 28,756
31,509
110 57,351
61,935
MATRIX OF STANDARD
Standard
ION RMRs
Ion
109
236 77 91 82
110
FIT
d 13 —heptanoic acid
109
—
0.78 2.77 9.56 3.51
1.77
236
1.28
- 3.55 12.25 4.50
2.27
77
91
0.36
0.10
0.28 - 3.47 1.26
0.08 0.29 - 0.37
0.64
0.19
d —benzoic acid
82
110
0.28
0.56
0.22 0.79 2.72 —
0.44 1.56 5.39 1.98
0.51
-
A . MW
x x
RMR =
x/y A •MW
y y
• n
‘-y
A =
ng =
x
y =
ion area or height
..
ng injected
analyte
standard
•ng
x
COMMENTS:
of historical standard ion RNRs.) A linear regression analysis of the
current responses as versus the corresponding historical responses
(Appendix) as x is performed. The slope (m) and intercept (b) are used to
modify the historical (data bank) RHRs for current use by the equation:
Y(pJ4J( today) — (data bank RMR) ‘ b
An example for one standard ion of the sample follows:
The Appendix gives the matrix of standard ions for the ESSA fraction
when the historical RNRs were determined (Finnegan 4021). Table 13.9
gives the RNRs of the ions of the internal and external standards relative
Chap. 13 - 335
-------
to one another observed in the SPS for ESSA run on a typical day. The
linear regression analysis of the SPS RMR values of the internal standards
relative to m/z 236 of FIT as versus the corresponding historical RMR
values from the Appendix as x gives a correlation coefficient of 0.995
with a slope of 1.31 and an intercept of -0.13. Thus, an RNR value of
0.81 from the data bank (Appendix) would be corrected as follows;
y = i + b
x = 0.81
m = 1.31
b = -0.13
y = 1.31 (0.81) + —.13
y = 0.93 (corrected RHR)
Several caveats should be mentioned concerning the use of correction
factors: 1) as the correlation coefficient decreases from 0.9999.. .9 the
accuracy decreases; 2) the fewer the x,y pairs the less accurate the
correction factor (a minimum of 5 pairs is recommended); 3) this correction
factor is meant to help reduce the error in R1 1Rs and is only an alternate
technique to determining the RNRs interspersed with samples. The latter
approach is more accurate but may not be practical with very complex
samples and an extensive list of sought-for compounds. The MAS computer
program MASQUANT includes a subroutine to perform the linear regression
analysis and calculate the corrected R 1R using x and y data provided by
the analyst.
13.4.2.2.4 Quantification of Compounds Not In MASQUANT Data Base--In
most sample extracts there will be detected and identified components that
are not in the ?IASQUANT data base, or even in the user’s own special data
bank, and for which there are no RuB or recovery data. To obtain an
estimate of concentration, which would be accurate to within a factor of
ten in most cases, the R R and recovery factors of a structurally similar
compound in the data base may be used.
13.4.2.2.5 Determination of Analyte Concentration--A flow diagram
depicting the steps in this procedure is given in Figure 13.5. The follow-
ing steps are performed:
(1) The amount of internal standard (I.S.) added to the initial
volume of water sample multiplied by the fraction of the initial
Chap. 13 - 336
-------
I Calculate I.S. added (forward amountjr
[ hoose E.S. to calculate I.S.obsI
4,
Calculate I.S.obs amoun ]
ICompa re I . S. forward asiount and I S. obs amoun ]
lè
Choose [ I S•obs amount closest to (I.S. forward amount),
use the ion giving this I.S. 0 b 5 in subsequent calculations
I I.S. 0 , 8 >12o% I.S. forward amount?1
veal \ No
[ ti.sd = (1.2) x (l.S. forward amount .S.obs<4O% I.S. forward aoount?I
Yes
Yes No’ tDo not calculate further ,
choose another I.S
Use this amount for 8 std in further calculations
For each analyte, choose appropriate I.S.
Calculate 8 analyte using appropriate ions and
Correct 8 analyte for recove ]
Calculate quantity of analyte per liter of water
Figure 13.5. Schematic of steps in quantification
procedure. (obs = observed; E.S. =
external standard; and I.S. = internal
standard)
Chap. 13 - 337
-------
water analyzed is corrected for historical recovery by multiply-
ing by the recovery factor in the Appendix and is called the
forward amount.
(2) If more than one external standard (E.S.) is used, the external
standard eluting in closest proximity to each internal standard
is determined from the ratio of the spectrum number of the
internal standard to the spectrum number of the external standard.
(3) The amount of internal standard recovered, relative to the
external standard, is calculated on the basis of equation 2
which is derived from equation 1. Standard ion R fRs are given
in each analytical protocol and the Appendix.
g. Ajfltstd•l lWintstd•8extstd eqn. 2
int.std. = A •I 1W R1IR
ext.std. ext.std. int.std./ext.std.
where
g. = grams of internal standard recovered from the
int.std.
amount of water processed
A. = area of the ion peak selected for the internal
int.std.
standard
int.std. = molecular weight of the internal standard (of the
derivative if the compound is derivatized)
= grams of external standard added to the extract
immediately before GC/MS analysis
A area of the ion peak selected for the external
ext. std.
standard
MW = molecular weight of the external standard
ext. std.
RMRittd/d = RZIR of the internal standard ion relative to the
external standard ion
From two to four calculated amounts can be generated depending
on the number of standard ions used in the external and internal
standards. Only ions that clearly originate with the standard
under consideration should be used for calculations; avoid
interferences.
Chap. 13 - 338
-------
(4) The calculated amount closest to the forward amount is selected.
This amount and the ion of the internal standard giving rise to
this amount are used in the quantification oL the analytes.
(a) If the calculated amount exceeds the forward amount by 21%
or more, 1.2 times the forward amount is used. (Internal
standard recovery has been observed to be 80-120%. If the
calculated amount is outside this range, this standard
should not be used to calculate analyte quantities because
it has not been recovered properly.)
(b) If the calculated amount is less than 40% of the amount of
internal standard added, this internal standard should not
be used for quantification of the analytes because of poor
recovery. Calculate recovery of another internal standard.
(5) For each analyte the internal standard (giving adequate recovery)
eluting in closest proximity is determined from the ratio of the
spectrum number of the internal standard to the spectrum number
of the analyte.
(6) The analytes are quantified from equation 3, derived from equa-
tion 1.
A NW g.
— analyte analyte int.std . eqn. 3
ye_A •NW •RMR
mt. std. in std. analyte/int . std.
where
g = grams of analyte recovered from the amount of
analyte
water processed
A = area of the ion peak selected for the analyte
analyte
NW = molecular weight of the analyte (of the derivative
analyte
if the compound is derivatized)
g. = grams of internal standard recovered from the
int.std.
amount of water processed
Aintstd = area of the ion peak selected for the internal
standard
NWintstd = molecular weight of the internal standard (of the
derivative if the compound is derivatized)
Chap. 13 — 339
-------
RNR . = RMR of the analyte ion relative to the internal
analyte/int. std.
standard ion.
The base peak of the mass spectrum of the analyte is the ion of
choice to use in quantification. The ion of the internal stand-
ard as well as the g. to use have been determined in
int.std.
steps 1-4. The RMR to use is given in the Appendix or determined.
(7) The calculated g (which is the amount calculated for the
analyte
volume of sample processed) is corrected for recovery by dividing
by the recovery factor listed in the Appendix. (If the listed
recovery factor is 0.4 or less, the compound should be analyzed
by a more appropriate protocol (e.g., ESSA rather than WARN).
(8) The amount of analyte corrected for recovery is divided by the
amount of water analyzed in liters to yield the amount per
liter.
An example of the steps involved in a typical calculation for an
analyte in the ESSA fraction is given in Table 13.10. The computer software
program (MASQUANT), however, executes these steps as the input information
is provided via a dialogue. Appendix C gives the operating instructions
for use of the computer program. Forms are provided in each analytical
protocol for logging the raw GC/MS data necessary for calculation of
analyte concentration.
Chap. 13 — 340
-------
Table 13.10. STEPS INVOLVED IN A TYPICAL QUANTITATIVE CALCULATION
(1) Analyze sample (see analytical proto-
col for conditions). In this example,
a 200 mL water sample was collected and
processed through the protocol.
Results:
4-Fluoro-2-iodo- 236 198
toluene (external)
d 5 -Benzoic Acid, 141 250
Methyl Ester
(internal)
(data bank recovery 0.92)
RNR 821109 = 0.28
RflR 1101109 = 0.56
Analyte MW
-To1uic Acid, 150
Methyl Ester
(data bank recovery = 0.91)
= 0.22
RHR11O/236 = 0.44
Spectrum
No. Ions Area
1044 119 77,374
150 27,412
(2) Determine amount of internal standard
from ion areas, external standard
amount (198 ng) and RNRs, using
eqn. 2.
— AJSMWIS8ES
— AE.S. MWE.S. ‘ I.S./E.S.
d 5 -benzoic acid, methyl ester
2-iodotoluene has two ions.
answers:
rn/z 82 and rn/z 109 = 131 ng
rn/z 110 and rn/z 109 = 172 ng
rn/z 82 and rn/z 236 222 ng
rn/z 110 and rn/z 236 = 287 ng
has two ions and 4-fluoro-
Therefore, there are four
Step
Data
Spect rum
Standards NW ng No .
Ion Area
878
734
109
236
82
110
30,065
17,990
19,354
39,349
(continued)
------- |