&EPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington, DC 20460
EPA/600/R-92/129
August 1992
Methods for the
Determination of
Organic Compounds in
Drinking Water
Supplement II
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ADDENDUM
The following sections will replace Section 14.2 and Section 15 in all
methods published in the EPA/600/R-92/129 methods manual entitled, "Methods
for the Determination of Organic Compounds in Drinking Water - Supplement II."
14.2 For information about pollution prevention that may be applicable to
laboratory operations, consult "Less is Better: Laboratory Chemical
Management for Waste Reduction" available from the American Chemical
Society's Department of Government Relations and Science Policy, 1155
16th Street N.W., Washington, D.C. 20036.
15. WASTE MANAGEMENT
15.1 It is the laboratory's responsibility to comply with all federal, state,
and local regulations governing the waste management, particularly the
hazardous waste identification rules and land disposal restrictions. It
is also the laboratory's responsibility to protect the air, water, and
land by minimizing and controlling all releases from fume hoods and bench
operations. Compliance is also required with any sewage discharge
permits and regulations. For further information on waste management,
consult "The Waste Management Manual for Laboratory Personnel," also
available from the American Chemical Society at the address in Section
14.2.
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EPA-600/R-92/129
AUGUST 1992
METHODS FOR THE DETERMINATION
OF ORGANIC COMPOUNDS
IN DRINKING WATER
SUPPLEMENT II
Environmental Monitoring Systems Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
Printed on Recycled Paper
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DISCLAIMER
This manual has been reviewed by the Environmental Monitoring Systems
Laboratory - Cincinnati, U.S. Environmental Protection Agency, and approved
for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
ii
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FOREWORD
Environmental measurements are required to determine the quality of
ambient waters and the character of waste effluents. The Environmental
Monitoring Systems Laboratory - Cincinnati (EMSL-Cincinnati) conducts research
to:
o Develop and evaluate analytical methods to identify and measure the
concentration of chemical pollutants in drinking waters, surface
waters, groundwaters, wastewaters, sediments, sludges, and solid
wastes.
o Investigate methods for the identification and measurement of viruses,
bacteria and other microbiological organisms in aqueous samples and to
determine the responses of aquatic organisms to water quality.
o Develop and operate a quality assurance program to support the
achievement of data quality objectives in measurements of pollutants
in drinking water, surface water, groundwater, wastewater, sediment
and solid waste.
o Develop methods and models to detect and quantify responses in aquatic
and terrestrial organisms exposed to environmental stressors and to
correlate the exposure with effects on chemical and biological indica-
tors.
This publication, "Determination of Organic Compounds in Drinking Water
Supplement II," was prepared to gather together under a single cover a set of
eight new or improved laboratory analytical methods for organic compounds in
drinking water. EMSL-Cincinnati is pleased to provide this manual and believe
that it will be of considerable value to many public and private laboratories
that wish to determine organic compounds in drinking water for regulatory or
other reasons.
Thomas A. Clark, Director
Environmental Monitoring Systems
Laboratory - Cincinnati
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ABSTRACT
Eight analytical methods covering 133 organic contaminants which may be
present in drinking water or drinking water sources are described in detail.
These methods will give the laboratory analyst the capability to accurately
and precisely determine organic compounds currently regulated in drinking
water, designated for regulation by the Office of Ground Water and Drinking
Water in the near future, or are potential candidates for regulatory concern.
Five of the methods in this manual, Methods 515.2, 524.2 Revision 4.0,
548.1, 549.1, and 552.1, replace older versions of these methods. The older
versions were numbered 515.1, 524.2 Revision 3.0, 548, 549, and 552, respec-
tively, and appeared in either of two previous organic methods manuals . The
new versions employ new analytical techniques, such as liquid-solid extrac-
tion, to improve method performance and reduce the use of organic solvents.
Three are new methods for the determination of semi volatile compounds: Method
554 for ozonation disinfection by-products, Method 555 for phenoxyacid herbi-
cides using the novel approach of in-line extraction/high performance liquid
chromatography (HPLC), and Method 553 for nonvolatile compounds using particle
beam HPLC/MS.
1 "Methods for the Determination of Organic Compounds in Drinking Water,"
EPA/600/4-88/039, December 1988, revised July 1991, or "Methods for the
Determination of Organic Compounds in Drinking Water - Supplement I,"
EPA/600/4-90/020, July 1990; Environmental Monitoring Systems Laboratory -
Cincinnati.
IV
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TABLE OF CONTENTS
Method
Number Title Revision Page
Foreword j -j ^
Abstract jv
Acknowledgment vj
Analyte - Method Cross Reference . vii
Introduction . . 1
524.2 Measurement of Purgeable Organic Compounds in
Water by Capillary Column Gas Chromatography/
Mass Spectrometry 4.0 ... 5
515.2 Determination of Chlorinated Acids in Water
Using Liquid-Solid Extraction and Gas
Chromatography With an Electron Capture
Detector 1.0 ... 51
548.1 Determination of Endothall in Drinking Water
by Ion Exchange Extraction, Acidic Methanol,
Methylation Gas Chromatography/Mass
Spectrometry 1.0 ... 89
549.1 Determination of Diquat and Paraquat in
Drinking Water by Liquid-Solid Extraction
and HPLC with Ultraviolet Detection 1.0 ... 119
552.1 Determination of Haloacetic Acids and
Dalapon in Drinking Water by Ion Exchange
Liquid-Solid Extraction and Gas
Chromatography With Electron Capture
Detection 1.0 ... 143
553 Determination of Benzidines and Nitrogen-
Containing Pesticides in Water by Liquid-
Liquid Extraction or Liquid-Solid Extraction
and Reverse Phase High Performance Liquid
Chromatography/Particle Beam/Mass Spectrometry 1.1 ... 173
554 Determination of Carbonyl Compounds in
Drinking Water by DNPH Derivatization and High
Performance Liquid Chromatography 1.0 ... 213
555 Determination of Chlorinated Acids in Water
by High Performance Liquid Chromatography With
a Photodiode Array Ultraviolet Detector 1.0 ... 237
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ACKNOWLEDGMENT
This methods manual was prepared and assembled by the Organic Chemistry
Branch of the Chemistry Research Division, Environmental Monitoring Systems
Laboratory - Cincinnati. Special thanks and appreciation are due to Glenora
F. Green for providing outstanding secretarial and word processing support,
for format improvements in presentation of the material in the manual, and for
coordinating the final assembly of the manual.
In addition, William L. Budde, Director of the Chemistry Research
Division, is recognized for his significant contributions. James W.
Eichelberger supervised the development of the methods, reviewed and edited
each of the individual methods, and directed the publication of the manual.
Dr. Jimmie Hodgeson managed the research which led to the successful develop-
ment of three of the methods and prepared the method descriptions. Thomas A.
Bellar, Thomas D. Behymer, and James S. Ho conducted the research in particle
beam high performance liquid chromatography (HPLC) mass spectrometry which led
to the development of Method 553. Jean W. Munch conducted the research which
led to the addition of 24 new volatile organic compounds to the analyte list
in Method 524.2 Revision 4.0. Jeffery D. Collins, David Becker, and Winslow
J. Bashe, Technology Applications, Inc., performed the major portion of the
laboratory support necessary to develop five of the methods. Appreciation is
also extended to the scientists in the Technical Support Division of the
Office of Ground Water and Drinking Water for their constructive and benefi-
cial review of the analytical methods contained in this manual.
The Quality Assurance Research Division of the Environmental Monitoring
Systems Laboratory - Cincinnati also assisted by providing sound constructive
reviews of many of the methods.
Finally, all the method authors and contributors wish to thank the
administrators and managers of the Environmental Protection Agency, who
supported the development and preparation of this manual. Special apprecia-
tion is due to Thomas A. Clark, Director of the Environmental Monitoring
Systems Laboratory - Cincinnati, for his cooperation and support during this
project.
vi
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ANALYTE - METHOD CROSS REFERENCE
Analyte Method No.
Acetaldehyde 554
Acetone 524.2
Acifluorofen 515.2, 555
Acrylonitrile 524.2
Ally! chloride 524.2
Bentazon 515.2, 555
Benzene 524.2
Benzidine 553
Benzoylprop ethyl 553
Bromobenzene 524.2
Bromochloroacetic acid 552.1
Bromochloromethane 524.2
Bromodichlorobenzene 524.2
Bromoform 524.2
Bromomethane 524.2
Butanal 554
2-Butanone 524.2
n-Butylbenzene 524.2
sec-Butyl benzene 524.2
tert-Butylbenzene 524.2
Carbaryl 553
Carbon disulfide 524.2
Carbon tetrachloride 524.2
Chloramben 515.2, 555
Chloroacetonitrile 524.2
Chlorobenzene 524.2
1-Chlorobutane 524.2
Chloroethane 524.2
Chloroform 524.2
Chioromethane 524.2
2-Chlorotoluene 524.2
4-Chlorotoluene 524.2
Crotonaldehyde 554
Cyclohexanone 554
Dalapon 552.1
DCPA (Dacthal) and metabolites 515.2, 555
Decanal 554
Dibromoacetic acid 552.1
Dibromochloromethane 524.2
l,2-Dibromo-3-chloropropane 524.2
1,2-Dibromoethane 524.2
Dibromomethane 524.2
Dicamba 515.2, 555
Dichloroacetic acid 552.1
1,2-Dichlorobenzene 524.2
1,3-Dichlorobenzene 524.2
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Analvte Method No.
1,4-Dichlorobenzene 524.2
3,3'-Dichlorobenzidine 553
3,5-Dichlorobenzoic acid 515.2, 555
2,4-DB (2,4-dichlorobutanoic acid) 515.2, 555
trans-l,4-Dichloro-2-butene 524.2
Dichlorodifluoromethane 524.2
1,1-Dichloroethane 524.2
1,2-Dichloroethane 524.2
1,1-Dichloroethene 524.2
cis-l,2-Dichloroethene 524.2
trans-l,2-Dichloroethene 524.2
2,4-D (2,4-dichlorophenoxyacetic acid) 515.2, 555
1,2-Dichloropropane 524.2
1,3-Dichloropropane 524.2
2,2-Dichloropropane 524.2
1,1-Dichloropropene 524.2
1,1-Dichloropropanone 524.2
cis-l,3-Dichloropropene 524.2
trans-l,3-Dichloropropene 524.2
Dichlorprop 515.2, 555
Di ethyl ether 524.2
3,3'-Dimethoxybenzidine 553
3,3'-Dimethy1benzidine 553
Dinoseb 515.2, 555
Diquat 549.1
Diuron 553
Endothall 548.1
Ethyl benzene 524.2
Ethyl methacryl ate 524.2
Formaldehyde 554
Heptanal 554
Hexachlorobutadiene 524.2
Hexachloroethane 524.2
Hexanal 554
2-Hexanone 524.2
5-Hydroxydicamba 515.2, 555
I sopropyl benzene 524.2
4-Isopropyltoluene 524.2
Linuron (Lorox) 553
Methacryl onitrile 524.2
Methyl acryl ate 524.2
Methyl ene chloride 524.2
Methyl iodide 524.2
Methyl methacryl ate 524.2
4-Methyl-2-pentanone 524.2
Methyl -t-butyl ether 524.2
Monobromoacetic acid 552.1
Monochloroacetic acid 552.1
viii
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Analvte Method No.
Monuron 553
Naphthalene 524.2
Nitrobenzene . 524.2
4-Nitrophenol 515.2, 555
2-Nitropropane 524.2
Nonanal 554
Octanal 554
Paraquat 549.1
Pentachloroethane 524.2
Pentachlorophenol (PCP) 515.2, 555
Pentanal 554
Picloram 515.2, 555
Propanal 554
Propionitrile 524.2
n-Propylbenzene 524.2
Rotenone 553
Siduron 553
2,4,5-TP (silvex) 515.2, 555
Styrene 524.2
Tetrachloroethene 524.2
1,1,1,2-Tetrachloroethane 524.2
1,1,2,2-Tetrachloroethane 524,2
Tetrahydrofuran 524.2
Toluene 524.2
Trichloroacetic acid 552.1
1,2,3-Trichlorobenzene 524.2
1,2,4-Trichlorobenzene 524.2
Trichloroethene 524.2
1,1,1-Trichloroethane 524.2
1,1,2-Trichlorbethane 524.2
Trichlorofluoromethane 524.2
2,4,5-T (2,4,5-trichlorophenoxyacetic acid) 515.2, 555
1,2,3-Trichloropropane 524.2
1,2,4-Trimethyl benzene 524.2
1,3,5-Trimethyl benzene 524.2
Vinyl chloride 524.2
m-Xylene 524.2
o-Xylene 524.2
p-Xylene 524.2
rx
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INTRODUCTION
An integral component of the role of the Environmental Protection Agency
(EPA) in protecting the quality of the Nation's water resources is the
provision of means for monitoring water quality. In keeping with this role,
EPA develops and disseminates analytical methods for measuring chemical and
physical parameters affecting water quality, including chemical contaminants
that may have potential adverse effects upon human health. This manual
provides eight analytical methods for 133 organic contaminants, which may be
present in drinking water or drinking water sources. In December 1988, EPA
published "METHODS FOR THE DETERMINATION OF ORGANIC COMPOUNDS IN DRINKING
WATER," EPA/600/4-88/039, a manual containing 13 methods for approximately 200
potential drinking water contaminants. This original manual was revised and
reprinted in July 1991. Supplement I, containing nine methods to determine 54
compounds, was published in July 1990.
This manual is a second supplement to the July 1991 revision of the
earlier 1988 manual. This manual provides methods to determine analytes that
appear at a later time in the regulatory framework, and technology that
supports the EPA Pollution Prevention Policy. Efforts have been made to
provide a manual that is consistent with the earlier versions.
REGULATORY BACKGROUND
Analytical methodology for monitoring water quality serves a number of
related purposes, including occurrence studies in community water systems,
health effects studies, and the determination of the efficacy of various water
treatment approaches. These activities, in turn, form the supporting basis
for water quality regulations, and the support of these regulations is the
ultimate purpose of the analytical methods. Limitations on the levels of
specific contaminants are codified in proposed and promulgated Federal
regulations developed in response to the Safe Drinking Water Act (SDWA) of
1974 and the SDWA amendments of 1986. The Act requires EPA to promulgate
regulations for drinking water contaminants that may cause adverse health
effects and which are known or anticipated to occur in public water systems.
The 1986 amendments require regulations to include Maximum Contaminant Levels
(MCLs) with compliance determined by regulatory monitoring or by the applica-
tion of an appropriate treatment, when adequate analytical methodology is not
available. In addition, the 1986 amendments specified 83 contaminants,
originally scheduled for regulation by June 19, 1989. The amended Act also
required EPA to develop a priority list of additional contaminants, to propose
25 more of these by January 1988 for subsequent regulation, and to continue
this process by the addition of 25 from the priority list on a triennial basis
thereafter.
Of the original 83 pollutants, regulations for eight volatile organic
chemicals (VOCs) were promulgated in June 1987 (see 52 FR 25690 and 51 FR
11396). Analytical methods for these eight as well as other unregulated VOCs
were published in the December 1988 manual (EPA Methods 502.1, 502.2, 503.1,
524.1 and 524.2). Regulations for 30 organic chemicals, 10 volatile compounds
and 20 pesticide and related compounds, were finalized and published in
1
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January 1991. This group included six compounds which, by authority of
provisions in the 1986 amendments, were substituted into the original list of
83 in January 1988 (53 FR 1892): namely, aldicarb sulfoxide, aldicarb
sulfone, ethylbenzene, heptachlor, heptachlor epoxide and styrene. With the
exception of lindane, analytical methods for all 30 compounds are by the VOC
methods above or SOC methods also included in the 1988 manual.
Supplement I provides analytical methods for many of the remaining
contaminants on the original list of 83: namely, adipates, diquat, endothall,
glyphosate, polycyclic aromatic hydrocarbons (PAHs), phthalates and dioxin.
Phase V of EPA regulations for 3 volatile compounds, 9 pesticides, and 6 other
organics from the list of 83 was promulgated on July 17, 1992.
GENERAL COMMENTS
Supplement II provides methods, which are cast in the same terminology as
the December 1988 manual, the July 1991 revision, and Supplement I. The
introductions to the earlier manuals discuss general method features on
format, sample matrices, method detection limits (MDLs), and calibration and
quality control samples. These same comments apply herein. In particular,
these methods are written in standardized terminology in a stand-alone format,
requiring no other source material for application. The methods in this
manual, unlike previous manuals, are assembled in the format recommended by
the Agency's Environmental Monitoring Management Council (EMMC). The methods
are designed primarily for drinking water and drinking water sources.
However, some performance data are included for more complex matrices such as
wastewater. The MDLs provided were determined by replicate analyses of
fortified reagent water over a relatively short period of time. As such,
these are somewhat idealized limits; nevertheless, provide a useful index of
method performance. Reporting limits for reliable quantitative data may be
considerably higher.
The quality control sections are uniform and contain minimum requirements
for operating a reliable monitoring program — initial demonstration of
performances routine analyses of reagent blanks, analyses of fortified reagent
blanks and fortified matrix samples, and analyses of quality control (QC)
samples. Other QC practices are recommended and may be adopted to meet the
particular needs of monitoring programs; e.g., the analyses of field reagent
blanks, instrument control samples and performance evaluation samples. Where
feasible, surrogate analytes have been included in the methods as well as
internal standards for calibration. Surrogate recoveries and the internal
standard response should be routinely monitored as continuing checks on
instrument performance, calibration curves and overall method performance.
THE ANALYTICAL METHODS
This manual contains eight methods. These methods utilize new sample
preparation technology such as disk or cartridge liquid-solid extraction, or
use new relatively harmless methylating reagents; therefore, directly support
the Environmental Protection Agency's Policy on Pollution Prevention. Methods
515.2, 524.2 Revision 4.0, 548.1, 549.1, and 552.1 replace older versions of
these methods. The older versions were numbered 515.1, 524.2 Revision 3.0,
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548, 549, and 552 respectively, and appeared in either of the two previously
published organic methods manuals ("Methods for the Determination of Organic
Compounds in Drinking Water," EPA/600/4-88/039, December 1988, revised July
1991, or "Methods for the Determination of Organic Compounds in Drinking Water
- Supplement I," EPA/600/4-90/020, July 1990; Environmental Monitoring Systems
Laboratory - Cincinnati). The Environmental Protection Agency's Office of
Ground Water and Drinking Water believes that only one version of any analyti-
cal method should be approved for compliance with drinking water regulations.
Thus, EPA will quickly approve the new versions of these five methods. Until
these methods are promulgated by EPA, the older versions should be retained
for compliance purposes. The remaining three are new methods for the determi-
nation of semi volatile or nonvolatile compounds: Method 553 for nonvolatile
organics using a high performance liquid chromatograph interfaced to a mass
spectrometer through a particle beam interface, Method 554 for ozonation
disinfection by-products, and Method 555 for phenoxyacetic acid herbicides
using the novel approach of in-line liquid-solid extraction and high perfor-
mance liquid chromatography.
Method 524.2, Revision 4.0 contains 24 new target analytes, which are
marked in the analyte list with an asterisk, bringing the total number of
method analytes to 84 compounds. Initial studies were conducted to evaluate
48 candidate VOCs of environmental interest for possible inclusion into this
method. These candidate compounds included many polar, water soluble com-
pounds which are very difficult to remove from the water matrix. Results
indicated that only 24 of these candidates were stable in water over a 14-day
holding time and could be efficiently purged and trapped from water with
acceptable accuracy and precision. MDLs for these newly added compounds are
generally 1 fig/I or lower.
Method 515.2 is an improved method to determine chlorinated herbicides in
water. This method utilizes a new liquid-solid disk extraction procedure.
Some of the phenolic herbicides are very difficult to derivatize, and still
require the use of the stronger reagent, diazomethane. The disk extraction
replaces the cumbersome liquid-liquid extraction and the Florisil cleanup in
Method 515.2. Dalapon, a method analyte in Method 515.1, is no longer a method
analyte in this new method. This compound is now determined usinq Method
552.1.
Method 548.1 is an improved method for the determination of endothall and
is intended to replace the older Method 548. This method utilizes an interme-
diate strength amine anion exchange sorbent to extract the endothall from a
100 ml sample aliquot, and forms the dimethyl derivative quickly and easily
using acidic methanol as the methylating reagent. Dimethyl endothall is then
identified and measured with gas chromatography/mass spectrometry (GC/MS). A
flame ionization detector may be used if a second dissimilar column is used
for corroboration. Method 548.1 has a MDL approximately ten times lower than
the older 548.
Method 549.1 is an improvement over the existing Method 549 in that it
now utilizes liquid-solid disk extractions in addition to the cartridges. The
expanded liquid-solid extraction technology significantly reduces the amount
of organic solvent required to carry out the extraction. Using less poten-
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tially harmful solvents directly supports the Environmental Protection
Agency's Policy on Pollution Prevention in the laboratory. Comparable MDLs
below a part per billion are achieved with both disks and cartridges.
Method 552.1 is a liquid-solid extraction method to determine haloacetic
acid disinfection by-products and the chemically similar chlorinated herbi-
cide, dalapon, in water. This method was designed as a simplified alternative
to the cumbersome Method 552 which employs liquid-liquid extraction. This
method provides a much superior technique for Dalapon over the complex
herbicide procedure described in Method 515.1. The sample is extracted with a
miniature anion exchange column, and the analytes are methylated directly in
the eluant using acidic methanol instead of diazomethane. MDLs using this
method for matrices which pose no analyte losses due to matrix effects are
generally ,1 /zg/L or lower.
Method 553 is a new method for the determination of nonvolatile organic
compounds, including benzidines and nitrogen containing pesticides, in water.
This method employs reverse phase high performance liquid chromatography
(HPLC) interfaced to a mass spectrometer through a particle beam interface.
This new technology provides the analyst the ability to determine a large, new
scope of nonvolatiles heretofore extremely difficult or impossible to deter-
mine in a water matrix. Among the compounds on the analyte list for this
method are two regulated compounds, aldicarb sulfone regulated in January 1991
and carbofuran regulated in May 1992.
Method 554 is a new HPLC method optimized for the determination of
selected carbonyl compounds in finished drinking water and raw source water.
These carbonyl compounds are either known or suspected disinfection by-
products from the ozonation disinfecting process. This method also utilizes
cartridge liquid-solid extraction technology in support of the Agency's
Pollution Prevention Policy. MDLs for the analytes in this method range from
3 to 69 /Kj/L.
Method 555 is a new method utilizing HPLC with a conventional photodiode
array HPLC detector to determine the same chlorinated herbicides on the
analyte lists in Methods 515.1 and 515.2. This method requires no derivatiza-
tion procedure which completely eliminates the need for diazomethane or even
acidic methanol. This method utilizes a new extraction approach, in-line
concentration of the analytes on a concentrator column, which requires only
the HPLC mobile phase as the extracting solvent. The entire amount of
herbicide contained in a 20-mL sample aliquot is introduced into the analyti-
cal system giving the method the needed sensitivity. This method directly
supports the Pollution Prevention Policy, and completely eliminates the
possible exposure of the analyst to harmful extracting solvent vapors, and to
the possibility of a diazomethane explosion.
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METHOD 524.2. MEASUREMENT OF PURGEABLE ORGANIC COMPOUNDS IN WATER BY
CAPILLARY COLUMN GAS CHROMATOGRAPHY/MASS SPECTROMETRY
Revision 4.0
August 1992
A. AT ford-Stevens, J. W. Eichelberger, and W. L. Budde
Method 524, Revision 1.0 (1983)
R. W. Slater, Jr.
Revision 2.0 (1986)
J. W. Eichelberger, and W. L. Budde
Revision 3.0 (1989)
J. W. Eichelberger, J. W. Munch, and T. A.Bellar
Revision 4.0 (1992)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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METHOD 524.2
MEASUREMENT OF PURGEABLE ORGANIC COMPOUNDS IN WATER BY
CAPILLARY COLUMN GAS CHROMATOGRAPHY/MASS SPECTROMETRY
1. SCOPE AND APPLICATION
1.1 This is a general purpose method for the identification and simulta-
neous measurement of purgeable volatile organic compounds in surface
water, ground water, and drinking water in any stage of treatment
(1,2). The method is applicable to a wide range of organic com-
pounds, including the four trihalomethane disinfection by-products,
that have sufficiently high volatility and low water solubility to be
removed from water samples with purge and trap procedures. The
following compounds can be determined by this method.
Compound
Acetone*
Acrylonitrile*
Ally! chloride*
Benzene
Bromobenzene
Bromochloromethane
Bromodichloromethane
Bromoform
Bromomethane
2-Butanone*
n-Butylbenzene
sec-Butyl benzene
tert-Butylbenzene
Carbon disulfide*
Carbon tetrachloride
Chioroacetoni tri1e*
Chlorobenzene
1-Chlorobutane*
Chloroethane
Chioroform
Chioromethane
2-Chlorotoluene
4-Chlorotoluene
Di bromochloromethane
1,2-Di bromo-3-chloropropane
1,2-Dibromoethane
Dibromomethane
1,2-Di chlorobenzene
1,3-Di chlorobenzene
1,4-Di chlorobenzene
trans-1,4-Di chloro-2-butene*
Di chlorodi f1uoromethane
Chemical Abstract Service
Registry Number
67-64-1
107-13-1
107-05-1
71-43-2
108-86-1
74-97-5
75-27-4
75-25-2
74-83-9
78-93-3
104-51-8
135-98-8
98-06-6
75-15-0
56-23-5
107-14-2
108-90-7
109-69-3
75-00-3
67-66-3
74-87-3
95-49-8
106-43-4
124-48-1
96-12-8
106-93-4
74-95-3
95-50-1
541-73-1
106-46-7
110-57-6
75-71-8
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1,1-Dichloroethane 75-34-3
1,2-Dichloroethane 107-06-2
1,1-Dichloroethene 75-35-4
cis-l,2-Dichloroethene 156-59-4
trans-l,2-Dichloroethene 156-60-5
1,2-Dichloropropane 78-87-5
1,3-Dichloropropane 142-28-9
2,2-Dichloropropane 590-20-7
1,1-Dichloropropene 563-58-6
1,1-Dichloropropanone* 513-88-2
cis-l,3-Dichloropropene 10061-01-5
trans-l,3-Dichloropropene 10061-02-6
Diethyl ether* 60-29-7
Ethyl benzene 100-41-4
Ethyl methacrylate* 97-63-2
Hexachlorobutadiene 87-68-3
Hexachloroethane* 67-72-1
2-Hexanone* 591-78-6
Isopropylbenzene 98-82-8
4-IsopropyTtoluene 99-87-6
Methacrylonitrile* 126-98-7
Methylacrylate* 96-33-3
Methylene chloride 75-09-2
Methyl iodide* 74-88-4
Methylmethacrylate* 80-62-6
4-Methyl-2-pentanone* 108-10-1
Methyl-t-butyl ether* 1634-04-4
Naphthalene 91-20-3
Nitrobenzene* 98-95-3
2-Nitropropane* 79-46-9
Pentachloroethane* 76-01-7
Propionitrile* 107-12-0
n-Propylbenzene 103-65-1
Styrene 100-42-5
1,1,1,2-Tetrachloroethane 630-20-6
1,1,2,2-Tetrachloroethane 79-34-5
Tetrachloroethene 127-18-4
Tetrahydrofuran* 109-99-9
Toluene 108-88-3
1,2,3-Trichlorobenzene 87-61-6
1,2,4-Trichlorobenzene 120-82-1
1,1,1-Trichloroethane 71-55-6
1,1,2-Trichloroethane 79-00-5
Trichloroethene 79-01-6
Trichlorofluoromethane 75-69-4
1,2,3-Trichloropropane 96-18-4
1,2,4-Trimethylbenzene 95-63-6
1,3,5-Trimethylbenzene 108-67-8
Vinyl chloride 75-01-4
o-Xylene 95-47-6
m-Xylene 108-38-3
p-Xylene 106-42-3
New Compound in Revision 4.0
7
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1.2 Method detection limits (MDLs) (3) are compound, instrument and
especially matrix dependent and vary from approximately 0.02 to 1.6
/*g/L. The applicable concentration range of this method is primarily
column and matrix dependent, and is approximately 0.02 to 200 /ig/L
when a wide-bore thick-film capillary column is used. Narrow-bore
thin-film columns may have a capacity which limits the range to about
0.02 to 20 /ig/L. Volatile water soluble, polar compounds which have
relatively low purging efficiencies can be determined using this
method. Such compounds may be more susceptible to matrix effects,
and the quality of the data may be adversely influenced.
1.3 Analytes that are not separated chromatographically, but which have
different mass spectra and noninterfering quantitation ions (Table
1), can be identified and measured in the same calibration mixture or
water sample as long as their concentrations are somewhat similar
(Sect. 11.6.2). Analytes that have very similar mass spectra cannot
be individually identified and measured in the same calibration
mixture or water sample unless they have different retention times
(Sect. 11.6.3). Coeluting compounds with very similar mass spectra,
typically many structural isomers, must be reported as an isomeric
group or pair. Two of the three isomeric xylenes and two of the
three dichlorobenzenes are examples of structural isomers that may
not be resolved on the capillary column, and if not, must be reported
as isomeric pairs. The more water soluble compounds (> 2% solubili-
ty) and compounds with boiling points above 200°C are purged from the
water matrix with lower efficiencies. These analytes may be more
susceptible to matrix effects. g^
2. SUMMARY OF METHOD
2.1 Volatile organic compounds and surrogates with low water solubility
are extracted (purged) from the sample matrix by bubbling an inert
gas through the aqueous sample. Purged sample components are trapped
in a tube containing suitable sorbent materials. When purging is
complete, the sorbent tube is heated and backflushed with helium to
desorb the trapped sample components into a capillary gas chromatog-
raphy (GC) column interfaced to a mass spectrometer (MS). The column
is temperature programmed to facilitate the separation of the method
analytes which are then detected with the MS. Compounds eluting from
the GC column are identified by comparing their measured mass spectra
and retention times to reference spectra and retention times in a
data base. Reference spectra and retention times for analytes are
obtained by the measurement of calibration standards under the same
conditions used for samples. The concentration of each identified
component is measured by relating the MS response of the quantitation
ion produced by that compound to the MS response of the quantitation
ion produced by a compound that is used as an internal standard.
Surrogate analytes, whose concentrations are known in every sample,
are measured with the same internal standard calibration procedure.
3. DEFINITIONS
3.1 INTERNAL STANDARD (IS) — A pure analyte(s) added to a sample,
extract, or standard solution in known amount(s) and used to measure
8
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the relative responses of other method analytes and surrogates that
are components of the same sample or solution. The internal standard
must be an analyte that is not a sample component.
3.2 SURROGATE ANALYTE (SA) — A pure analyte(s), which is extremely
unlikely to be found in any sample, and which is added to a sample
aliquot in known amount(s) before extraction or other processing and
is measured with the same procedures used to measure other sample
components. The purpose of the SA is to monitor method performance
with each sample.
3.3 LABORATORY DUPLICATES (LD1 and LD2) — Two aliquots of the same
sample taken in the laboratory and analyzed separately with identical
procedures. Analyses of LD1 and LD2 indicates precision associated
with laboratory procedures, but not wit'h sample collection, preserva-
tion, or storage procedures.
3.4 FIELD DUPLICATES (FD1 and FD2) -- Two separate samples collected at
the same time and place under identical circumstances and treated
exactly the same throughout field and laboratory procedures. Analy-
ses of FD1 and FD2 give a measure of the precision associated with
sample collection, preservation and storage, as well as with labora-
tory procedures.
3.5 LABORATORY REAGENT BLANK (LRB) ~ An aliquot of reagent water or
other blank matrix that is treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The LRB
is used to determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or the appara-
tus.
3.6 FIELD REAGENT BLANK (FRB) — An aliquot of reagent water or other
blank matrix that is placed in a sample container in the laboratory
and treated as a sample in all respects, including shipment to the
sampling site, exposure to sampling site conditions, storage, preser-
vation, and all analytical procedures. The purpose of the FRB is to
determine if method analytes or other interferences are present in
the field environment.
3.7 LABORATORY PERFORMANCE CHECK SOLUTION (LPC) ~ A solution of one or
more compounds (analytes, surrogates, internal standard, or other
test compounds) used to evaluate the performance of the instrument
system with respect to a defined set of method criteria.
3.8 LABORATORY FORTIFIED BLANK (LFB) — An aliquot of reagent water or
other blank matrix to which known quantities of the method analytes
are added in the laboratory. The LFB is analyzed exactly like a
sample, and its purpose is to determine whether the methodology is in
control, and whether the laboratory is capable of making accurate and
precise measurements.
3.9 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) — An aliquot of an environ-
mental sample to which known quantities of the method analytes are
-------
added in the laboratory. The LFM is analyzed exactly like a sample,
and its purpose is to determine whether the sample matrix contributes
bias to the analytical results. The background concentrations of the
analytes in the sample matrix must be determined in a separate
aliquot and the measured values in the LFM corrected for background
concentrations.
3.10 STOCK STANDARD SOLUTION (SSS) — A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed
reference materials or purchased from a reputable commercial source.
3.11 PRIMARY DILUTION STANDARD SOLUTION (PDS) — A solution of several
analytes prepared in the laboratory from stock standard solutions and
diluted as needed to prepare calibration solutions and other needed
analyte solutions.
3.12 CALIBRATION STANDARD (CAL) — A solution prepared from the primary
dilution standard solution or stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.13 QUALITY CONTROL SAMPLE (QCS) — A solution of method analytes of
known concentrations which is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally prepared
test materials. ,
4. INTERFERENCES
4.1 During analysis, major contaminant sources are volatile materials in
the laboratory and impurities in the inert purging gas and in the
sorbent trap. The use of Teflon tubing, Teflon thread sealants, or
flow controllers with rubber components in the purging device should
be avoided since such materials out-gas organic compounds which will
be concentrated in the trap during the purge operation. Analyses of
laboratory reagent blanks provide information about the presence of
contaminants. When potential interfering peaks are noted in labora-
tory reagent blanks, the analyst should change the purge gas source
and regenerate the molecular sieve purge gas filter. Subtracting
blank values from sample results is not permitted.
4.2 Interfering contamination may occur when a sample containing low
concentrations of volatile organic compounds is analyzed immediately
after a sample containing relatively high concentrations of volatile
organic compounds. A preventive technique is between-sample rinsing
of the purging apparatus and sample syringes with two portions of
reagent water. After analysis of a sample containing high concentra-
tions of volatile organic compounds, one or more laboratory reagent
blanks should be analyzed to check for cross-contamination.
4.3 Special precautions must be taken to determine methylene chloride.
The analytical and sample storage area should be isolated from all
10
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atmospheric sources of methylene chloride, otherwise random back-
ground levels will result. Since methylene chloride will permeate
Teflon tubing, all GC carrier gas lines and purge gas plumbing should
be constructed of stainless steel or copper tubing. Laboratory
worker's clothing should be cleaned frequently since clothing previ-
ously exposed to methylene chloride fumes during common liquid/liquid
extraction procedures can contribute to sample contamination.
4.4 Traces of ketones, methylene chloride, and some other organic sol-
vents can be present even in the highest purity methanol; This is
another potential source of contamination, and should be assessed
before standards are prepared in the methanol.
5. SAFETY
5.1 The toxicity or carcinogenicity of chemicals used in this method has
not been precisely defined; each chemical should be treated as a
potential health hazard, and exposure to these chemicals should be
minimized. Each laboratory is responsible for maintaining awareness
of OSHA regulations regarding safe handling of chemicals used in this
method. Additional references to laboratory safety are available
(4-6) for the information of the analyst.
5.2 The following method analytes have been tentatively classified as
known or suspected human or mammalian carcinogens: benzene, carbon
tetrachloride, 1,4-dichlorobenzene, 1,2-dichlorethane, hexachloro-
butadiene, 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane, chloro-
form, l,2-dibromoethane,tetrachloroethene, trichloroethene, and vinyl
chloride. Pure standard materials and stock standard solutions of
these compounds should be handled in a hood. A NIOSH/MESA approved
toxic gas respirator should be worn when the analyst handles high
concentrations of these toxic compounds.
6. EQUIPMENT AND SUPPLIES
6.1 SAMPLE CONTAINERS — 40-mL to 120-mL screw cap vials each equipped
with a Teflon faced silicone septum. Prior to use, wash vials and
septa with detergent and rinse with tap and distilled water. Allow
the vials and septa to air dry at room temperature, place in a 105°C
oven for 1 hr, then remove and allow to cool in an area known to be
free of organics.
6.2 PURGE AND TRAP SYSTEM — The purge and trap system consists of three
separate pieces of equipment: purging device, trap, and desorber.
Systems are commercially available from several sources that meet all
of the following specifications.
6.2.1 The all glass purging device (Figure 1) should be designed to
accept 25-mL samples with a water column at least 5 cm deep.
A smaller (5-mL) purging device is recommended if the GC/MS
system has adequate sensitivity to obtain the method detec-
tion limits required. Gaseous volumes above the sample must
be kept to a minimum (< 15 mL) to eliminate dead volume
11
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effects. A glass frit should be installed at the base of the
sample chamber so the purge gas passes through the water
column as finely divided bubbles with a diameter of < 3 mm at
the origin. Needle spargers may be used, however, the purge
gas must be introduced at a point about 5 mm from the base of
the water column. The use of a moisture control device is
recommended to prohibit much of the trapped water vapor from
entering the GC/MS and eventually causing instrumental prob-
lems.
6.2.2 The trap (Figure 2) must be at least 25 cm long and have an
inside diameter of at least 0.105 in. Starting from the
inlet, the trap should contain 1.0 cm of methyl silicone
coated packing and the following amounts of adsorbents: 1/3
of 2,6-diphenylene oxide polymer, 1/3 of silica gel, and 1/3
of coconut charcoal. If it is not necessary to determine
dichlorodifluoromethane, the charcoal can be eliminated and
the polymer increased to fill 2/3 of the trap. Before ini-
tial use, the trap should be conditioned overnight at 180°C
by backflushing with an inert gas flow of at least 20 mL/min.
Vent the trap effluent to the room, not to the analytical
column. Prior to daily use, the trap should be conditioned
for 10 min at 180°C with backflushing. The trap may be
vented to the analytical column during daily conditioning;
however, the column must be run through the temperature
program prior to analysis of samples. The use of alternative
sorbents is acceptable, depending on the particular set of
target analytes or other problems encountered, but the new
trap packing must meet all quality control criteria described
in Sect. 9.
6.2.3 The use of the methyl silicone coated packing is recommended,
but not mandatory. The packing serves a dual purpose of
protecting the Tenax adsbrbant from aerosols, and also of
insuring that the Tenax is fully enclosed within the heated
zone of the trap thus eliminating potential cold spots.
Alternatively, silanized glass wool may be used as a spacer
at the trap inlet.
6.2.4 The desorber (Figure 2) must be capable of rapidly heating
the trap to 180°C either prior to or at the beginning of the
flow of desorption gas. The polymer section of the trap
should not be heated higher than 200°C or the life expectancy
of the trap will decrease. Trap failure is characterized by
a pressure drop in excess of 3 Ib/in2 across the trap during
purging or by poor bromoform sensitivities. The desorber
design illustrated in Fig. 2 meets these criteria.
6.3 GAS CHROMATOGRAPHY/MASS SPECTROMETER/DATA SYSTEM (GC/MS/DS)
6.3.1 The GC must be capable of temperature programming and should
be equipped with variable-constant differential flow control-
lers so that the column flow rate will remain constant
throughout desorption and temperature program operation. If
the column oven is to be cooled to 10°C or lower, a subam-
bient oven controller will likely be required. If syringe
12
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injections of 4-bromofluorobenzene (BFB) will be used, a
split/splitless injection port is required.
6.3.2 Capillary GC Columns. Any gas chromatography column that
meets the performance specifications of this method may be
used (Sect, 10.2.4.1). Separations of the calibration mix-
ture must be equivalent or better than those described in
this method. Four useful columns have been evaluated, and
observed compound retention times for these columns are
listed in Table 2.
6.3.2.1 Column 1 — 60 m x 0.75 mm ID VOCOL (Supelco, Inc.)
glass wide-bore capillary with a 1.5 /im film thick-
ness.
Column 2 -- 30 m x 0.53 mm ID DB-624 (J&W Scien-
tific, Inc.) fused silica capillary with a 3 /am film
thickness.
Column 3 — 30 m x 0.32 mm ID DB-5 (J&W Scientific,
Inc.) fused silica capillary with a 1 /un film thick-
ness.
Column 4 — 75 m x 0.53 mm id DB-624 (J&W Scien-
tific, Inc.) fused silica capillary with a 3 pi film
thickness.
6.3.3 Interfaces between the GC and MS. The interface used depends
on the column selected and the gas flow rate.
6.3.3.1 The wide-bore columns 1, 2, and 4 have the capacity
to accept the standard gas flows from the trap
during thermal desorption, and chromatography can
begin with the onset of thermal desorption. Depend-
ing on the pumping capacity of the MS, an additional
interface between the end of the column and the MS
may be required. An open split interface (7) or an
all-glass jet separator is an acceptable interface.
Any interface can be used if the performance speci-
fications described in this method (Sect. 9 and 10)
can be achieved. The end of the transfer line after
the interface, or the end of the analytical column
if no interface is used, should be placed within a
few mm of the MS ion source.
6.3.3.2 When narrow bore column 3 is used, a cryogenic
interface placed just in front of the column inlet
is suggested. This interface condenses the desorbed
sample components in a narrow band on an uncoated
fused silica precolumn using liquid nitrogen cool-
ing. When all analytes have been desorbed from the
trap, the interface is rapidly heated to transfer
them to the analytical column. The end of the ana-
lytical column should be placed within a few mm of
the MS ion source. A potential problem with this
interface is blockage of the interface by frozen
13
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water from the trap. This condition will result in
a major loss in sensitivity and chromatographic
resolution.
6.3.4 The mass spectrometer must be capable of electron ionization
at a nominal electron energy of 70 eV. The spectrometer must
be capable of scanning from 35 to 260 amu with a complete
scan cycle time (including scan overhead) of 2 sec or less.
(Scan cycle time = Total MS data acquisition time in seconds
divided by number of scans in the chromatogram.) The spec-
trometer must produce a mass spectrum that meets all criteria
in Table 3 when 25 ng or less of 4-bromofluorobenzene (BFB)
is introduced into the GC. An average spectrum across the
BFB GC peak may be used to test instrument performance.
6.3.5 An interfaced data system is required to acquire, store,
reduce, and output mass spectral data. The'computer software
should have the capability of processing stored GC/MS data by
recognizing a GC peak within any given retention time window,
comparing the mass spectra from the GC peak with spectral
data in a user-created data base, and generating a list of
tentatively identified compounds with their retention times
and scan numbers. The software must allow integration of the
ion abundance of any specific ion between specified time or
scan number limits. The software should also allow calcula-
tion of response factors as defined in Sect. 10.2.6 (or
construction of a linear or second order regression calibra-
tion curve), calculation of response factor statistics (mean
and standard deviation), and calculation of concentrations of
analytes using either the calibration curve or the equation
in Sect. 12.
6.4 SYRINGE AND SYRINGE VALVES
6.4.1 Two 5-mL or 25-mL glass hypodermic syringes with Luer-Lok tip
(depending on sample volume used).
6.4.2 Three 2-way syringe valves with Luer ends.
6.4.3 Micro syringes - 10, 100 /zL.
6.4.4 Syringes - 0.5, 1.0, and 5-mL, gas tight with shut-off valve.
6.5 MISCELLANEOUS
6.5.1 Standard solution storage containers — 15-mL bottles with
Teflon lined screw caps.
7. REAGENTS AND STANDARDS
7.1 TRAP PACKING MATERIALS
7.1.1 2,6-Diphenylene oxide polymer, 60/80 mesh, chromatographic
grade (Tenax GC or equivalent).
14
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7.1.2 Methyl silicons packing (optional) — OV-1 (3%) on Chromosorb
W, 60/80 mesh, or equivalent.
7.1.3 Silica gel — 35/60 mesh, Davison, grade 15 or equivalent.
7.1.4 Coconut charcoal — Prepare from Barnebey Cheney, CA-580-26
lot #M-2649 by crushing through 26 mesh screen.
7.2 REAGENTS
7.2.1 Methanol — Demonstrated to be free of analytes.
7.2.2 Reagent water — Prepare reagent water by passing tap water
through a filter bed containing about 0.5 kg of activated
carbon, by using a water purification system, or by boiling
distilled water for 15 min followed by a 1-h purge with inert
gas while the water temperature is held at 905C. Store in
clean, narrow-mouth bottles with Teflon lined septa and screw
caps.
7.2.3 Hydrochloric acid (1+1) — Carefully add measured volume of
cone. HC1 to equal volume of reagent water.
7.2.4 Vinyl chloride -- Certified mixtures of vinyl chloride in
nitrogen and pure vinyl chloride are available from several
sources (for example, Matheson, Ideal Gas Products, and Scott
Gases).
7.2.5 Ascorbic acid — ACS reagent grade, granular.
7.2.6 Sodium thiosulfate — ACS reagent grade, granular.
7.3 STOCK STANDARD SOLUTIONS -- These solutions may be purchased as
certified solutions or prepared from pure standard materials using
the following procedures. One of these solutions is required for
every analyte of concern, every surrogate, and the internal standard.
A useful working concentration is about 1-5 mg/mL.
7.3.1 Place about 9.8 ml of methanol into a 10-mL ground-glass
stoppered volumetric flask. Allow the flask to stand,
unstoppered, for about 10 min or until all alcohol-wetted
surfaces have dried and weigh to the nearest 0.1 mg.
7.3.2 If the analyte is a liquid at room temperature, use a 100-/JL
syringe and immediately add two or more drops of reference
standard to the flask. Be sure that the reference standard
falls directly into the alcohol without contacting the neck
of the flask. If the analyte is a gas at room temperature,
fill a 5-mL valved gas-tight syringe with the standard to the
5.0-mL mark, lower the needle to 5 mm above the methanol
meniscus, and slowly inject the standard into the neck area
of the flask. The gas will rapidly dissolve in the methanol.
15
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7.3.3 Reweigh, dilute to volume, stopper, then mix by inverting the
flask several times. Calculate the concentration in ng/iil
from the net gain in weight. When compound purity is certi-
fied at 96% or greater, the weight can be used without cor-
rection to calculate the concentration of the stock standard.
7.3.4 Store stock standard solutions in 15-mL bottles equipped with
Teflon lined screw caps. Methanol solutions of acryloni-
trile, methyl iodide, and methyl acrylate are stable for only
one week at 4°C. Methanol solutions prepared from other
liquid analytes are stable for at least 4 weeks when stored
at 4°C. Methanol solutions prepared from gaseous analytes
are not stable for more than 1 week when stored at < 0°C; at
room temperature, they must be discarded after 1 day.
7.4 PRIMARY DILUTION STANDARDS — Use stock standard solutions to prepare
primary dilution standard solutions that contain all the analytes of
concern in methanol or other suitable solvent. The primary dilution
standards should be prepared at concentrations that can be easily
diluted to prepare aqueous calibration solutions that will bracket
the working concentration range. Store the primary dilution standard
solutions with minimal headspace and check frequently for signs of
deterioration or evaporation, especially just before preparing
calibration solutions. Storage times described for stock standard
solutions in Sect. 7.3.4 also apply to primary dilution standard
solutions.
7.5 FORTIFICATION SOLUTIONS FOR INTERNAL STANDARD AND SURROGATES
7.5.1 A solution containing the internal standard and the surrogate
compounds is required to prepare laboratory reagent blanks
(also used as a laboratory performance check solution), and
to fortify each sample. Prepare a fortification solution
containing fluorobenzene (internal standard), 1,2- dichloro-
benzene-d4 (surrogate), and BFB (surrogate) in methanol at
concentrations of 5 jig/mL of each (any appropriate concentra-
tion is acceptable). A 5-/iL aliquot of this solution added
to a 25-mL water sample volume gives concentrations of 1 pg/L
of each. A 5-/iL aliquot of this solution added to a 5-mL
water sample volume gives a concentration of 5 ng/L of each.
Additional internal standards and surrogate analytes are
optional. Additional surrogate compounds should be similar
in physical and chemical characteristics to the analytes of
concern.
7.6 PREPARATION OF LABORATORY REAGENT BLANK (LRB) -- Fill a 25-mL (or
5-mL) syringe with reagent water and adjust to the mark (no air
bubbles). Inject an appropriate volume of the fortification solution
containing the internal standard and surrogates through the Luer Lok
valve into the reagent water. Transfer the LRB to the. purging
device. See Sect. 11.1.2.
16
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7.7 PREPARATION OF LABORATORY FORTIFIED BLANK — Prepare this exactly
like a calibration standard (Sect. 7.8). This is a calibration
standard that is treated as a sample.
7.8 PREPARATION OF CALIBRATION STANDARDS
7.8.1 The number of calibration solutions (CALs) needed depends on
the calibration range desired. A minimum of three CAL solu-
tions is required to calibrate a range of a factor of 20 in
concentration. For a factor of 50, use at least four stan-
dards, and for a factor of 100 at least five standards. One
calibration standard should contain each analyte of concern
at a concentration of 2-10 times the method detection limit
(Tables 4, 5, and 7) for that compound. The other CAL stan-
dards should contain each analyte of concern at concentra-
tions that define the range of the method. Every CAL solu-
tion contains the internal standard and the surrogate com-
pounds at the same concentration (5 /jg/L suggested for a 5-mL
sample; 1 /zg/L for a 25-mL sample).
7.8.2 To prepare a calibration standard, add an appropriate volume
of a primary dilution standard containing all analytes of
concern to an aliquot of acidified (pH 2) reagent water in a
volumetric flask. Also add an appropriate volume of internal
standard and surrogate compound solution from Sect. 7.5.1.
Use a microsyringe and rapidly inject the methanol solutions
into the expanded area of the filled volumetric flask.
Remove the needle as quickly as possible after injection.
Mix by inverting the flask three times only. Discard the
contents contained in the neck of the flask. Aqueous stan-
dards are not stable in a volumetric flask and should be
discarded after 1 hr unless transferred to a sample bottle
and sealed immediately.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 SAMPLE COLLECTION, DECHLORINATION, AND PRESERVATION
8.1.1 Collect all samples in duplicate. If samples, such as fin-
ished drinking water or waste water, are suspected to contain
residual chlorine, add about 25 mg of ascorbic acid per 40 mL
of sample to the sample bottle before filling. If the resid-
ual chlorine is likely to be present > 5 mg/L, a determina-
tion of the amount of the chlorine may be necessary.
Diethyl-p-phenylenediamine (DPD) test kits are commercially
available to determine residual chlorine in the field. Add
an additional 25 mg of ascorbic acid per each 5 mg/L of
residual chlorine. If compounds boiling below 25°C are not
to be determined, sodium thiosulfate may be used to reduce
the residual chlorine. Fill sample bottles to overflowing,
but take care not to flush out the rapidly dissolving ascor-
bic acid. No air bubbles should pass through the sample as
the bottle is filled, or be trapped in the sample when the
bottle is sealed. Adjust the pH of the duplicate samples to
17
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< 2 by carefully adding two drops of 1:1 HC1 for each 40 ml
of sample. Seal the sample bottles, Teflon face down, and
shake vigorously for 1 min. Do not mix the ascorbic acid or
sodium thiosulfate with the HC1 prior to sampling.
8.1.2 When sampling from a water tap, open the tap and allow the
system to flush until the water temperature has stabilized
(usually about 10 min). Adjust the flow to about 500 mL/min
and collect duplicate samples from the flowing stream.
8.1.3 When sampling from an open body of water, such as surface
water, waste water, and possible leachate samples, partially
fill a 1-quart wide-mouth bottle or 1-L beaker with sample
from a representative area. Fill a 60 ml or a 120 ml sample
vial with sample from the larger container, and adjust the pH
of the sample to about 2 by adding 1+1 HC1 dropwise while
stirring. Check the pH with narrow range (1.4 to 2.8) pH
paper. Record the number of drops of acid necessary to
adjust the pH to 2. To collect actual samples, refill the
large container with fresh sample and pour sample into sample
vials. Follow filling instructions in Sect. 8.1.1. Add the
appropriate number of drops of 1+1 HC1 to each sample to
adjust the pH to about 2. If samples are suspected to con-
tain residual chlorine, add ascorbic acid or sodium thiosul-
fate according to Sect. 8.1.1.
8.1.4 The samples must be chilled to about 4°C when collected and
maintained at that temperature until analysis. Field samples
that will not be received at the laboratory on the day of
collection must be packaged for shipment with sufficient ice
to ensure that they will arrive at the laboratory with a
substantial amount of ice remaining in the cooler.
8.1.5 If a sample foams vigorously when HC1 is added, discard that
sample. Collect a set of duplicate samples but do not acidi-
fy them. These samples must be flagged as "not acidified"
and must be stored at 4°C or below. These samples must be
analyzed within 24 hr of collection time.
8.2 SAMPLE STORAGE
8.2.1 Store samples at < 4°C until analysis. The sample storage
area must be free of organic solvent vapors and direct or
intense light.
8.2.2 Analyze all samples within 14 days of collection. Samples
not analyzed within this period must be discarded and re-
placed.
8.3 FIELD REAGENT BLANKS (FRB)
8.3.1 Duplicate FRBs must be handled along with each sample set,
which is composed of the samples collected from the same
18
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general sample site at approximately the same time. At the
laboratory, fill field blank sample bottles with reagent
water and sample preservatives, seal, and ship to the sam-
pling site along with empty sample bottles and back to the
laboratory with filled sample bottles. Wherever a set of
samples is shipped and stored, it is accompanied by appropri-
ate blanks. FRBs must remain hermetically sealed until
analysis.
8.3.2 Use the same procedures used for samples to add ascorbic acid
and HC1 to blanks (Sect. 8.1.1). The same batch of ascorbic
acid and HC1 should be used for the field reagent blanks in
the field.
9. QUALITY CONTROL
9.1 Quality control (QC) requirements are the initial demonstration of
laboratory capability followed by regular analyses of laboratory
reagent blanks, field reagent blanks, and laboratory fortified
blanks. Each laboratory must maintain records to document the
quality of the data generated. Additional quality control practices
are recommended.
9.2 Initial demonstration of low system background. Before any samples
are analyzed, it must be demonstrated that a laboratory reagent blank
(LRB) is reasonably free of contamination that would prevent the
determination of any analyte of concern. Sources of background
contamination are glassware, purge gas, sorbents, and equipment.
Background contamination must be reduced to an acceptable level
before proceeding with the next section. In general, background from
method analytes should be below the method detection limit.
9.3 Initial demonstration of laboratory accuracy and precision. Analyze
five to seven replicates of a laboratory fortified blank containing
each analyte of concern at a concentration in the range of 0.2-5 /ig/L
(see appropriate regulations and maximum contaminant levels for
guidance on appropriate concentrations).
9.3.1 Prepare each replicate by adding an appropriate aliquot of a
quality control sample to reagent water. If a quality con-
trol sample containing the method analytes is not available,
a primary dilution standard made from a source of reagents
different than those used to prepare the calibration stan-
dards may be used. Also add the appropriate amounts of
internal standard and surrogate compounds. Analyze each
replicate according to the procedures described in Sect. 11,
and on a schedule that results in the analyses of all repli-
cates over a period of several days.
9.3.2 Calculate the measured concentration of each analyte in each
replicate, the mean concentration of each analyte in all
replicates, and mean accuracy (as mean percentage of true
19
-------
value) for each analyte, and the precision (as relative
standard deviation, RSD) of the measurements for each
analyte. Calculate the MDL of each analyte using the equa-
tion described in Sect. 13.2 (3).
9.3.3 For each analyte, the mean accuracy, expressed as a percent-
age of the true value, should be 80-120% and the RSD should
be < 20%. Some analytes, particularly the early eluting
gases and late eluting higher molecular weight compounds, are
measured with less accuracy and precision than other
analytes. The MDLs must be sufficient to detect analytes at
the required levels according to the SDWA Regulations. If
these criteria are not met for an analyte, take remedial
action and repeat the measurements for that analyte to demon-
strate acceptable performance before samples are analyzed.
9.3.4 Develop and maintain a system of control charts to plot the
precision and accuracy of analyte and surrogate measurements
as a function of time. Charting surrogate recoveries is an
especially valuable activity because surrogates are present
in every sample and the analytical results will form a sig-
nificant record of data quality.
9.4 Monitor the integrated areas of the quantitation ions of the internal
standards and surrogates (Table 1) in all samples, continuing cali-
bration checks, and blanks. These should remain reasonably constant 4m
over time. An abrupt change may indicate a matrix effect or an ^^
instrument problem. If a cryogenic interface is utilized, it may
indicate an inefficient transfer from the trap to the column. These
samples must be reanalyzed or a laboratory fortified duplicate sample
analyzed to test for matrix effect. A more gradual drift of more
than 50% in any area is indicative of a loss in sensitivity, and the
problem must be found and corrected.
9.5 LABORATORY REAGENT BLANKS (LRB) — With each batch of samples pro-
cessed as a group within a work shift, analyze a LRB to determine the
background system contamination. A FRB (Sect. 9.7) may be used in
place of a LRB.
9.6 With each batch of samples processed as a group within a work shift,
analyze a single laboratory fortified blank (LFB) containing each
analyte of concern at a concentration as determined in Sect. 9.3. If
more than 20 samples are included in a batch, analyze one LFB for
every 20 samples. Use the procedures described in Sect. 9.3.3 to
evaluate the accuracy of the measurements, and to estimate whether
the MDLs can be obtained. If acceptable accuracy and MDLs cannot be
achieved, the problem must be located and corrected before further
samples are analyzed. Add these results to the ongoing control
charts to document data quality.
9.7 With each set of field samples a field reagent blank (FRB) should be
analyzed. The results of these analyses will help define contamina-
20
-------
lion resulting from field sampling and transportation activities. If
the FRB shows unacceptable contamination, a LRB must be measured to
define the source of the impurities.
9.8 At least quarterly, replicate LFBs should be analyzed to determine
the precision of the laboratory measurements. Add these results to
the ongoing control charts to document data quality.
9.9 At least quarterly, analyze a quality control sample (QCS) from an
external source. If measured analyte concentrations are not of
acceptable accuracy, check the entire analytical procedure to locate
and correct the problem source.
9.10 Sample matrix effects have not been observed when this method is used
with distilled water, reagent water, drinking water, or ground water.
Therefore, analysis of a laboratory fortified sample matrix (LFM) is
not required unless the criteria in Section 9.4 are not met. If
matrix effects are observed or suspected to be causing low recover-
ies, analyze a laboratory fortified matrix sample for that matrix.
The sample results should be flagged and the LFM results should be
reported with them.
9.11 Numerous other quality control measures are incorporated into other
parts of this procedure, and serve to alert the analyst to potential
problems.
10. CALIBRATION AND STANDARDIZATION
10.1 Demonstration and documentation of acceptable, initial calibration is
required before any samples are analyzed and is required intermit-
tently throughout sample analysis as dictated by results of continu-
ing calibration checks. After initial calibration is successful, a
continuing calibration check is required at the beginning of each 8
hr. period during which analyses are performed. Additional periodic
calibration checks are good laboratory practice.
10.2 INITIAL CALIBRATION
10.2.1 Calibrate the mass and .abundance scales of the MS with cali-
bration compounds and procedures prescribed by the manufac-
turer with any modifications necessary to meet the require-
ments in Sect. 10.2.2.
10.2.2 Introduce into the GC (either by purging a laboratory reagent
blank or making a syringe injection) 25 ng or less of BFB and
acquire mass spectra for m/z 35-260 at 70 eV (nominal). Use
the purging procedure and/or GC conditions given in Sect. 11.
If the spectrum does not meet all criteria in Table 3, the MS
must be retuned and adjusted to meet all criteria before
proceeding with calibration. An average spectrum across the
GC peak may be used to evaluate the performance of the sys-
tem.
10.2.3 Purge a medium CAL solution, (e.g., 10-20 ftg/l) using the
procedure given in Sect. 11.
21
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10.2.4 Performance criteria for the medium calibration. Examine the
stored GC/MS data with the data system software. Figures 3
and 4 shown acceptable total ion chromatograms.
10.2.4.1 GC performance. Good column performance will pro-
duce symmetrical peaks with minimum tailing for most
compounds. If peaks are unusually broad, or if
peaks are running together with little vallies be-
tween them, the wrong column has been selected or
remedial action is probably necessary (Sect.10.3.6).
10.2.4.2 MS sensitivity. The GC/MS/DS peak identification
software should be able to recognize a GC peak in
the appropriate retention time window for each of
the compounds in calibration solution, and make
correct tentative identifications. If fewer than
99% of the compounds are recognized, system mainte-
nance is required. See Sect. 10.3.6.
10.2.5 If all performance criteria are met, purge an aliquot of each
of the other CAL solutions using the same GC/MS conditions.
10.2.6 Calculate a response factor (RF) for each analyte and isomer
pair for each CAL solution using the internal standard fluor-
obenzene. Table 1 contains suggested quantitation ions for
all compounds. This calculation is supported in acceptable
GC/MS data system software (Sect. 6.3.5), and many other
software programs. RF is a unitless number, but units used
to express quantities of analyte and internal standard must
be equivalent.
RF
(Ax)(Qis)
(Ais)(Qx)
IS
where: Ax = integrated abundance of the quantitation ion
of the analyte.
integrated abundance of the quantitation ion
of the internal standard.
Qx = quantity of analyte purged in nanograms or
concentration units.
Qis - quantity of internal standard purged in ng or
concentration units.
10.2.6.1 For each analyte and surrogate, calculate the mean
RF from analyses of CAL solutions. Calculate the
standard deviation (SO) and the relative standard
deviation (RSD) from each mean: RSD - 100 (SD/M).
If the RSD of any analyte or surrogate mean RF
exceeds 20%, either analyze additional aliquots of
appropriate CAL solutions to obtain an acceptable
RSD of RFs over the entire concentration range, or
22
-------
take action to improve GC/MS performance Sect.
10.3.6). Surrogate compounds are present at the
same concentration on every sample, calibration
standard, and all types of blanks.
10.2.7 As an alternative to calculating mean response factors and
applying the RSD test, use the GC/MS data system software or
other available software to generate a linear or second order
regression calibration curve.
10.3 CONTINUING CALIBRATION CHECK — Verify the MS tune and initial
calibration at the beginning of each 8-hr work shift during which
analyses are performed using the following procedure.
10.3.1 Introduce into the GC (either by purging a laboratory reagent
blank or making a syringe injection) 25 ng or less of BFB and
acquire a mass spectrum that includes data for m/z 35-260.
If the spectrum does not meet all criteria (Table 3), the MS
must be retuned and adjusted to meet all criteria before
proceeding with the continuing calibration check.
10.3.2 Purge a medium concentration CAL solution and analyze with
the same conditions used during the initial calibration.
10.3.3 Demonstrate acceptable performance for the criteria shown in
Sect. 10.2.4.
10.3.4 Determine that the absolute areas of the quantitation ions of
the internal standard and surrogates have not decreased by
more than 30% from the areas measured in the most recent
continuing calibration check, or by more than 50% from the
areas measured during initial calibration. If these areas
have decreased by more than these amounts, adjustments must
be made to restore system sensitivity. These adjustments may
require cleaning of the MS ion spyrce, or other maintenance
as indicated in Sect. 10.3.6, and recalibration. Control
charts are useful aids in documenting system sensitivity
changes.
10.3.5 Calculate the RF for each analyte of concern and surrogate
compound from the data measured in the continuing calibration
check. The RF for each analyte and surrogate must be within
30% of the mean value measured in the initial calibration.
Alternatively, if a linear or second order regression is
used, the concentration measured using the calibration curve
must be within 30% of the true value of the concentration in
the medium calibration solution. If these conditions do not
exist, remedial action must be taken which may require re-
calibration.
23
-------
10.3.6 Some possible remedial actions. Major maintenance such as
cleaning an ion source, cleaning quadrupole rods, etc. re-
quire returning to the initial calibration step.
10.3.6.1 Check and adjust GC and/or MS operating conditions;
check the MS resolution, and calibrate the mass
scale.
10.3.6.2 Clean or replace the splitless injection liner;
silanize a new injection liner. This applies only
if the injection liner is an integral part of the
system.
10.3.6.3 Flush the GC column with solvent according to manu-
facturer's instructions.
10.3.6.4 Break off a short portion (about 1 meter) of the
column from the end near the injector; or replace GC
column. This action will cause a slight change in
retention times. Analyst may need to redefine
retention windows.
10.3.6.5 Prepare fresh CAL solutions, and repeat the initial
calibration step.
10.3.6.6 Clean the MS ion source and rods (if a quadrupole).
10.3.6.7 Replace any components that allow analytes to come
into contact with hot metal surfaces.
10.3.6.8 Replace the MS electron multiplier, or any other
faulty components.
10.3.6.9 Replace the trap, especially when only a few com-
pounds fail the criteria in Sect. 10.3.5 while the
majority are determined successfully. Also check
for gas leaks in the purge and trap unit as well as
the rest of the analytical system.
10.4 Optional calibration for vinyl chloride using a certified gaseous
mixture of vinyl chloride in nitrogen can be accomplished by the
following steps.
10.4.1 Fill the purging device with 25.0 ml (or 5-mL) of reagent
water or aqueous calibration standard.
10.4.2 Start to purge the aqueous mixture. Inject a known volume
(between 100 and 2000 pL) of the calibration gas (at room
temperature) directly into the purging device with a gas
tight syringe. Slowly inject the gaseous sample through a
septum seal at the top of the purging device at 2000 /iL/min.
If the injection of the standard is made through the aqueous
24
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sample inlet port, flush the dead volume with several ml of
room air or carrier gas. Inject the gaseous standard before
5 min of the 11-min purge time have elapsed.
10.4.3 Determine the aqueous equivalent concentration of vinyl
chloride standard, in /jg/L, injected with the equation:
S = 0.102 (C)(V)
where S - Aqueous equivalent concentration
of vinyl chloride standard in 0g/L;
C = Concentration of gaseous standard in mg/L (v/v);
V = Volume of standard injected in ml.
11. PROCEDURE
11.1 SAMPLE INTRODUCTION AND PURGING
11.1.1 This method is designed for a 25-mL sample volume, but a
smaller (5 ml.) sample volume is recommended if the GC/MS
system has adequate sensitivity to achieve the required
method detection limits. Adjust the helium purge gas flow
rate to 40 mL/min. Attach the trap inlet to the purging
device and open the syringe valve on the purging device.
11.1.2 Remove the plungers from two 25-mL (or 5-mL depending on
sample size) syringes and attach a closed syringe valve to
each. Warm the sample to room temperature, open the sample
bottle, and carefully pour the sample into one of the syringe
barrels to just short of overflowing. Replace the syringe
plunger, invert the syringe, and compress the sample. Open
the syringe valve and vent any residual air while adjusting
the sample volume to 25.0-mL (or 5-mL). To all samples,
blanks, and calibration standards, add 5-/tL (or an appropri-
ate volume) of the fortification solution containing the
internal standard and the surrogates to the sample through
the syringe valve. Close the valve. Fill the second syringe
in an identical manner from the same sample bottle. Reserve
this second syringe for a reanalysis if necessary.
11.1.3 Attach the sample syringe valve to the syringe valve on the
purging device. Be sure that the trap is cooler than 25°C,
then open the sample syringe valve and inject the sample into
the purging chamber. Close both valves and initiate purging.
Purge the sample for 11.0 min at ambient temperature.
11.1.4 Standards and samples must be analyzed in exactly the same
manner. Room temperature changes in excess of 10°F may
adversely affect the accuracy and precision of the method.
25
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11.2 SAMPLE DESORPTION
11.2.1 Non-cryogenic interface ~ After the 11-min purge, place the
purge and trap system in the desorb mode and preheat the trap
to 180°C without a flow of desorption gas. Then simultan-
eously start the flow of desorption gas at a flow rate suit-
able for the column being used (optimum desorb flow rate is
15 mL/min) for about 4 min, begin the GC temperature program,
and start data acquisition.
11.2.2 Cryogenic interface — After the 11-min purge, place the
purge and trap system in the desorb mode, make sure the
cryogenic interface is a -150°C or lower, and rapidly heat
the trap to 180°C while backflushing with an inert gas at
4 mL/min for about 5 min. At the end of the 5 min desorption
cycle, rapidly heat the cryogenic trap to 250°C, and simulta-
neously begin the temperature program of the gas chromato-
graph, and start data acquisition.
11.2.3 While the trapped components are being introduced into the
gas chromatograph (or cryogenic interface), empty the purging
device using the sample syringe and wash the chamber with two
25-mL flushes of reagent water. After the purging device has
been emptied, leave syringe valve open to allow the purge gas
to vent through the sample introduction needle.
11.3 GAS CHROMATOGRAPHY/MASS SPECTROMETRY — Acquire and store data over
the nominal mass range 35-260 with a total cycle time (including scan
overhead time) of 2 sec or less. If water, methanol, or carbon
dioxide cause a background problem, start at 47 or 48 m/z. If
ketones are to be determined, data must be acquired starting at m/z
43. Cycle time must be adjusted to measure five or more spectra
during the elution of each GC peak. Suggested temperature programs
are provided below. Alternative temperature programs can be used.
11.3.1 Single ramp linear temperature program for wide bore column 1
and 2 with a jet separator. Adjust the helium carrier gas
flow rate to within the capacity of the separator, or about
15 mL/min. The column temperature is reduced 10°C and held
for 5 min from the beginning of desorption, then programmed
to 160°C at 6°C/min, and held until all components have
eluted.
11.3.2 Multi-ramp temperature program for wide bore column 2 with
the open split interface. Adjust the helium carrier gas flow
rate to about 4.6 mL/min. The column temperature is reduced
to 10°C and held for 6 min from the beginning of desorption,
then heated to 70°C at 10°/min, heated to 120°C at 5°/nrin,
heated to 180° at 8°/min, and held at 180° until all com-
pounds have eluted.
26
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11.3.3 Single ramp linear temperature program for narrow bore column
3 with a cryogenic interface. Adjust the helium carrier gas
flow rate to about 4 mL/min. The column temperature is
reduced to 10°C and held for 5 min from the beginning of
vaporization from the cryogenic trap, programmed at 6°/min
for 10 min, then 15°/min for 5 min to 145°C, and held until
all components have eluted.
11.3.4 Multi-ramp temperature program for wide bore column 4 with
the open split interface. Adjust the helium carrier gas flow
rate to about 7.0 mL/min. The column temperature is - 10°C
and held for 6 min. from beginning of desorption, then heated
to 100°C at 10°C/min, heated to 200°C at 5°C/min and held at
200°C for 8 min or until all compounds of interest had elut-
ed.
11.4 TRAP RECONDITIONING — After desorbing the sample for 4 min, recondi-
tion the trap by returning the purge and trap system to the purge
mode. Wait 15 sec, then close the syringe valve on the purging
device to begin gas flow through the trap. Maintain the trap temper-
ature at 180°C. Maintain the moisture control module, if utilized,
at 90°C to remove residual water. After approximately 7 min, turn
off the trap heater and open the syringe valve to stop the gas flow
through the trap. When the trap is cool, the next sample can be
analyzed.
11.5 TERMINATION OF DATA ACQUISITION — When all the sample components
have eluted from the GC, terminate MS data acquisition. Use appro-
priate data output software to display full range mass spectra and
appropriate plots of ion abundance as a function of time. If any ion
abundance exceeds the system working range, dilute the sample aliquot
in the second syringe with reagent water and analyze the diluted
aliquot.
11.6 IDENTIFICATION OF ANALYTES ~ Identify a sample component by compari-
son of its mass spectrum (after background subtraction) to a refer-
ence spectrum in the user-created data base. The GC retention time
of the sample component should be within three standard deviations of
the mean retention time of the compound in the calibration mixture.
11.6.1 In general, all ions that are present above 10% relative
abundance in the mass spectrum of the standard should be
present in the mass spectrum of the sample component and
should agree within absolute 20%. For example, if an ion has
a relative abundance of 30% in the standard spectrum, its
abundance in the sample spectrum should be in the range of 10
to 50%. Some ions, particularly the molecular ion, are of
special importance, and should be evaluated even if they are
below 10% relative abundance.
11.6.2 Identification requires expert judgment when sample compo-
nents are not resolved chromatographically and produce mass
spectra containing ions contributed by more than one analyte.
27
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When GC peaks obviously represent more than one sample compo-
nent (i.e., broadened peak with shoulder(s) or valley between
two or more maxima), appropriate analyte spectra and back-
ground spectra can be selected by examining plots of charac-
teristic ions for tentatively identified components. When
analytes coelute (i.e., only one GC peak is apparent), the
identification criteria can be met but each analyte spectrum
will contain extraneous ions contributed by the coeluting
compound. Because purgeable organic compounds are relatively
small molecules and produce comparatively simple mass spec-
tra, this is not a significant problem for most method
analytes.
11.6.3 Structural isomers that produce very similar mass spectra can
be explicitly identified only if they have sufficiently
different GC retention times. Acceptable resolution is
achieved if the height of the valley between two peaks is
less than 25% of the average height of the two peaks. Other-
wise, structural isomers are identified as isomeric pairs.
Two of the three isomeric xylenes and two of the three di-
chlorobenzenes are examples of structural isomers that may
not be resolved on the capillary columns. If unresolved,
these groups of isomers must be reported as isomeric pairs.
11.6.4 Methylene chloride, acetone, carbon disulfide, and other
background components appear in variable quantities in labo-
ratory and field reagent blanks, and generally cannot be
accurately measured. Subtraction of the concentration in the
blank from the concentration in the sample is not acceptable
because the concentration of the background in the blank is
highly variable.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Complete chromatographic resolution is not necessary for accurate and
precise measurements of analyte concentrations if unique ions with
adequate intensities are available for quantitation.
12.1.1 Calculate analyte and surrogate concentrations.
C = (AX)(Q,-S)
(Ais) RF V
where: Cx = concentration of analyte or surrogate in /jg/L
in the water sample.
Ax = integrated abundance of the quantitation ion
of the analyte in the sample.
A- = integrated abundance of the quantitation ion
1 of the internal standard in the sample.
Q, - total quantity (in micrograms) of internal
standard added to the water sample.
V - original water sample volume in ml.
RF - mean response factor of analyte from the
initial calibration.
28
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12.1.2 Alternatively, use the GC/MS system software or other
available proven software to compute the concentrations of
the analytes and surrogates from the linear or second order
regression curves.
12.1;3 Calculations should utilize all available digits of precis-
ion, but final reported concentrations should be rounded to
an appropriate number of significant figures (one digit of
- uncertainty). Experience indicates that three significant
figures may be used for concentrations above 99 ng/L, two
significant figures for concentrations between 1- 99 /xg/L,
and one significant figure for lower concentrations.
12.1.4 Calculate the total trihalomethane concentration by summing
the four individual trihalomethane concentrations.
13. METHOD PERFORMANCE
.13.1-Single laboratory accuracy and precision data were obtained for the
method analytes using laboratory fortified blanks with analytes at
concentrations between 1 and 5ng/L. Results were obtained using the
four columns specified (Sect. 6.3.2.1) and the open split or jet
separator (Sect. 6.3.3.1), or the cryogenic interface (Sect.
6.3.3.2). These data are shown in Tables 4-8.
13.2 With these data, method detection limits were calculated using the
formula (3): ,
MDL = S tin-l.l-alfha - 0.99)
where:
t(n-ifi-aiPha = 0.99) " Student's t value for the 99% confidence
level with n-1 degrees of freedom,
n * number of replicates
S = the standard deviation of the
replicate analyses.
14. POLLUTION PREVENTION
14.1 No solvents are utilized in this method except the extremely small
volumes of methanol needed to make calibration standards. The only
other chemicals used in this method are the neat materials in prepar-
ing standards and sample preservatives. All are used in extremely
small amounts and pose no threat to the environment.
15. WASTE MANAGEMENT
15.1 There are no waste management issues involved with this method. Due
to the nature of this method, the discarded samples ai*e chemically
less contaminated than when they were collected.
29
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16. REFERENCES
1. A. Alford-Stevens, J.W. Eichelberger, W.L. Budde, "Purgeable Organic
Compounds in Water by Gas Chromatography/Mass Spectrometry, Method
524." Environmental Monitoring and Support Laboratory, U.S.
Environmental Protection Agency, Cincinnati, Ohio, February 1983.
2. C. Madding, "Volatile Organic Compounds in Water by Purge and Trap
Capillary Column GC/MS," Proceedings of the Water Quality Technology
Conference, American Water Works Association, Denver, CO, December
1984.
3. J.A. Glaser, D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde,
"Trace Analyses for Wastewaters, "Environ. Sci.. Techno!.. 15, 1426,
1981.
4. "Carcinogens-Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, August 1977.
5. "OSHA Safety and Health Standards, General Industry," (29CFR1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
6. "Safety in Academic Chemistry Laboratories," American Chemical
Society Publication, Committee on Chemical Safety, 3rd Edition,
1979.
7. R.F. Arrendale, R.F. Severson, and O.T. Chortyk, "Open Split Inter-
face for Capillary Gas Chromatography/Mass Spectrometry," Anal. Chem.
1984, 56, 1533.
8. J.J. Flesch, P.S. Fair, "The Analysis of Cyanogen Chloride in Drink-
ing Water," Proceedings of Water Quality Technology Conference,
American Water Works Association, St. Louis, MO., November 14-16,
1988.
30
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17. TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. MOLECULAR WEIGHTS AND QUANTITATION IONS FOR METHOD ANALYTES
Primary Secondary
Quantitation Quantitation
Compound MWa Ion Ions
Internal standard
Fluorobenzene 96 96 77
Surrogates
4-Bromofl uorobenzene 174 95 174,176
l,2-Dichlorobenzene-d4 150 152 115,150
Target Analvtes
Acetone 58 43 58
Acrylonitrile 53 52 53
Allyl chloride 76 76 49
Benzene 78 78 77
Bromobenzene 156 156 77,158
Bromochloromethane 128 128 49,130
Bromodichloromethane 162 83 85,127
Bromoform 250 173 175,252
Bromomethane 94 94 96
2-Butanone 72 43 57,72
n-Butylbenzene 134 91 134
sec-Butyl benzene 134 105 134
tert-Butylbenzene 134 ' 119 91
Carbon disulfide 76 76
Carbon tetrachloride 152 117 119
Chloroacetonitrile 75 48 75
Chlorobenzene 112 112 77,114
1-Chlorobutane 92 56 49
Chloroethane 64 64 66
Chloroform 118 83 85
Chloromethane 50 50 52
2-Chlorotoluene 126 91 126
4-Chlorotoluene 126 91 126
Dibromochloromethane 206 129 127
l,2-Dibromo-3-Chloropropane 234 75 155,157
1,2-Dibromoethane 186 107 109,188
Dibromomethane 172 93 95,174
1,2-DiChlorobenzene 146 146 111,148
1,3-Dichlorobenzene 146 146 111,148
1,4-DiChlorobenzene 146 146 111,148
31
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TABLE 1. (continued)
Primary
Quantitation
Ion.
Secondary
Quantitation
Ions
trans-1 , 4-Di chl oro-2-butene
Di chl orodi f 1 uoromethane
1,1-Dichloroethane
1,2-Dichloroethane
1,1-Dichloroethene
ci s-1 , 2-Di chl oroethene
trans-1 , 2-Di chl oroethene
1, 2-Di chl oropropane
1 , 3-Di chl oropropane
2 , 2-Di chl oropropane
1 , 1-Di chl oropropene
1 , 1-Di chl oropropanone
ci s-1 , 3-di chl oropropene
trans-1 ,3-di chl oropropene
Di ethyl ether
Ethyl benzene
Ethyl methacrylate
Hexachl orobutadi ene
Hexachloroethane
2-Hexanone
Isopropyl benzene
4-Isopropyl to! uene
Methacrylonitrile
Methyl acrylate
Methyl ene chloride
Methyl iodide
Methyl met hacryl ate
4-Methyl -2-pentanone
Methyl -t-butyl ether
Naphthalene
Nitrobenzene
2-Nitropropane
Pentachl oroethane
Propionitrile
n-Propyl benzene
Styrene
1,1,1, 2-Tetrachl oroethane
1,1,2, 2-Tetrachl oroethane
Tetrachl oroethene
Tetrahydrofuran
Toluene
1,2, 3-Tr i chl orobenzene
1,2, 4-Tri chl orobenzene
1,1, 1-Tri chl oroethane
1,1, 2-Tri chl oroethane
124
120
98
98
96
96
96
112
112
112
110
126
110
110
74
106
114
258
234
100
120
134
67
86
84
142
100
100
88
128
123
89
200
55
120
104
166
166
164
72
92
180
180
132
132
53
85
63
62
96
96
96
63
76
77
75
43
75
75
59
91
69
225
117
43
105
119
67
55
84
142
69
43
73
128
51
46
117
54
91
104
131
83
166
71
92
180
180
97
83
88,75
87
65,83
98
61,63
61,98
61,98
112
78
97
110,77
83
110
110
45,73
106
99
260
119,201
58
120
134,91
52
85
86,49
127
99
58,85
57
—
77
—
119,167
—
120
78
133,119
131,85
168,129
72,42
91
182
182
99,61
97,85
32
-------
TABLE 1. (continued)
Primary Secondary
Quantitation Quantitation
Compound MWa Ion Ions
Trichloroethene
Tri chl orof 1 uoromethane
1,2, 3-Tr i chl oropropane
1 ,2 , 4-Tri methyl benzene
1,3, 5-Tr i methyl benzene
Vinyl Chloride
o-Xylene
m-Xyl ene
p-Xylene
130
136
146
120
120
62
106
106
106
95
101
75
105
105
62
106
106
106
130,132
103
77
120
120
64
91
91
91
aMonoisotopic molecular weight calculated from the atomic masses of the
isotopes with the smallest masses.
33
-------
TABLE 2. CHROMATOGRAPHIC RETENTION TINES FOR METHOD ANALYTES
ON THREE COLUMNS WITH FOUR SETS OF CONDITIONS3
Compound
Retention
Column lb Column
Time (min:sec)
r Column 2C Column 3d Column 4e
Internal standard
Fluorobenzene
Surrogates
4-Bromof1uorobenzene
1,2-Di chlorobenzene-d4
Target Analvtes
Acetone
Acrylonitrile
Ally! chloride
Benzene
Bromobenzene
Bromochloromethane
Bromodichloromethane
Bromoform
Bromomethane
2-Butanone
n-Butylbenzene
sec-Butyl benzene
tert-Butylbenzene
Carbon Disulfide
Carbon Tetrachloride
Chloroaceton i tri1e
Chlorobenzene
1-Chlorobutane
Chloroethane
Chloroform
Chloromethane
2-Chlorotoluene
4-Chlorotoluene
Cyanogen chloride (8)
Dibromochloromethane
1,2-Di bromo-3-Chloropropane
1,2-Di bromoethane
Dibromomethane
1,2-Di chlorobenzene
1,3-Di chlorobenzene
1,4-Di chlorobenzene
t-1,4-Di chloro-2-butene
Di chlorodi f1uoromethane
1,1-01chloroethane
8:49
18:38
22:16
7:37
15:46
2:05
6:24
1:38
19:20
19:30
14:23
24:32
14:44
10:39
22:31
21:13
21:33
1:33
4:51
6:27
15:43
19:08
8:14
18:57
6:44
10:35
17:56
2:01
22:13
20:47
20:17
5:40
15:52
4:23
8:29
14:53
0:58
19:29
18:05
17:34
5:16
13:01
4:48
0:44
16:25
16:43
11:51
21:05
11:50
7:56
19:10
18:08
18:23
0:42
2:56
14:06
23:38
27:25
8:03
22:00
31:21
35:51
13:30
24:00
12:22
15:48
22:46
4:48
27:32
26:08
25:36
13:10
20:40
12:36
3:24
24:32
24:46
19:12
19:24
15:26
27:26
26:22
26:36
3:08
10:48
7:25
16:25
5:38
9:20
15:42
1:17
17:57
17:28
17:19
7:25
14:20
1:27
5:33
0:58
16:44
16:49
1:03
12:48
18:02
13:36
9:05
17:47
17:28
17:38
0:53
4:02
16:14
17:49
16:58
21:32
31:52
20:20
23:36
30:32
12:26
19:41
35:41
34:04
33:26
16:30
21:11
23:51
28:26
21:00
20:27
9:11
32:21
32:38
26:57
38:20
27:19
23:22
35:55
34:31
34:45
31:44
7:16
18:46
34
-------
TABLE 2. (continued)
Compound
1,2-Dichloroethane
1,1-Dichloroethene
ci s-1 , 2-Di chl oroethene
trans-1 , 2-Di chl oroethene
1,2-Dichl oropropane
1,3-Di chl oropropane
2 , 2-Di chl qropropane
1 , 1-Di chl oropropanone
1 , 1-Di chl oropropene
ci s-1 ,3-di chl oropropene
trans-1 , 3-di chl oropropene
Di ethyl ether
Ethyl benzene
Ethyl Methacrylate
Hexachl orobutad i ene
Hexachloroethane
Hexanone
I sopropyl benzene
4-Isopropyl toluene
Methacrylonitrile
Methyl acryl ate
Methyl ene Chloride
Methyl Iodide
Methylmethacryl ate
4-Methyl -2-pentanone
Methyl -t-butyl ether
Naphthalene
Nitrobenzene
2-Nitropropane
Pentachloroethane
Propionitrile
n-Propyl benzene
Styrene
1,1,1, 2-Tetrachl oroethane
1,1,2, 2-Tetrachl oroethane
Tetrachl oroethene
Tetrahydrofuran
Toluene
1,2, 3-Tri chl orobenzene
1,2, 4-Tri chl orobenzene
1,1, 1-Tri chl oroethane
1,1, 2-Tr i chl oroethane
Tri chl oroethene
Tri chl orof 1 uoromethane
1 , 2 , 3-Tri chl oropropane
1,2, 4-Trimethyl benzene
Retention
Column lb Column 2b
8:24
2:53
6:11
3:59
10:05
14:02
6:01
7:49
11.58
13.46
15:59
26:59
18:04
21:12
3:36
27:10
19:04
17:19
15:56
18:43
13:44
12:26
27:47
26:33
7:16
13:25
9:35
2:16
19:01
20:20
5:50
1:34
3:54
2:22
7:40
11:19
3:48
5:17
13:23
23:41
15:28
18:31
2:04
23:31
16:25
14:36
13:20
16:21
11:09
10:00
24:11
23:05
4:50
11:03
7:16
1:11
16:14
17:42
Time (min:sec)
Column 2 Column 3
13:38
7:50
11:56
9:54
1,5:12
18:42
11:52
13:06
16:42
17:54
21:00
32:04
23:18
26:30
9:16
32:12
24:20
22:24
20:52
24:04
18:36
17:24
32:58
31:30
12:50
18:18
14:48
6:12
24:08
31:30
7:00
2:20
5:04
3:32
8:56
12:29
5:19
7:10
14:44
19:14
16:25
17:38
2:40
19:04
16:49
15:47
14:44
15:47
13:12
11:31
19:14
18:50
6:46
11:59
9:01
1:46
16:16
17:19
Column 4e
21:31
16:01
19:53
17:54
23:08
26:23
19:54
24:52
21:08
24:24
25:33
15:31
28:37
25:35
42:03
36:45
26:23
30:52
34:27
20:15
20:02
17:18
16:21
23:08
24:38
17:56
42:29
39:02
23:58
33:33
19:58
32:00
29:57
28:35
31:35
26:27
20:26
25:13
43:31
41:26
20:51
25:59
22:42
14:18
31:47
33:33
35
-------
TABLE 2. (continued)
Compound
1,3, 5-Tri methyl benzene
Vinyl chloride
o-Xyl ene
m-Xyl ene
p-Xyl ene
Retention Time (mi n: sec)
Column 1 Column 2 Column 2C Column 3
19:28
1:43
17:07
16:10
16:07
16:54
0:47
14:31
13:41
13:41
24:50
3:56
22:16
21:22
21:18
16:59
1:02
15:47
15:18
15:18
Column 4e
32:26
10:22
29:56
28:53
28:53
8Columns 1-4 are those given in Sect. 6.3.2.1; retention times were measured
from the beginning of thermal desorption from the trap (columns 1-2, and 4) or
from the beginning of thermal release from the cryogenic interface (column 3).
bGC conditions given in Sect. 11.3.1.
CGC conditions given in Sect. 11.3.2.
dGC conditions given in Sect. 11.3.3.
eGC conditions given in Sect. 11.3.4.
36
-------
TABLE 3. ION ABUNDANCE CRITERIA FOR 4-BROMOFLUOROBENZENE (BFB)
Mass
(M/z) Relative Abundance Criteria .
50 15 to 40% of mass 95
75 30 to 80% of mass 95
95 Base Peak, 100% Relative Abundance
96 5 to 9% of mass 95
173 < 2% of mass 174
174 > 50% of mass 95
175 5 to 9% of mass 174
176 > 95% but < 101% of mass 174
177 5 to 9% of mass 176
37
-------
TABLE 4. ACCURACY AND PRECISION DATA FROM 16-31 DETERMINATIONS OF THE METHOD
ANALYTES IN REAGENT WATER USING WIDE BORE CAPILLARY COLUMN la
Compound
Benzene
Bromobenzene
Bromochl oromethane
Bromodi chl oromethane
Bromoform
Bromomethane
n-Butyl benzene
sec-Butyl benzene
tert-Butyl benzene
Carbon tetrachloride
Chlorobenzene
Chloroethane
Chloroform
Chl oromethane
2-Chlorotoluene
4-Chlorotoluene
D1 bromochl oromethane
1 , 2-Di bromo-3-chl oropropane
1,2-Dibromoethane
Dibromomethane
1 , 2-Di chl orobenzene
1 , 3-Di chl orobenzene
1 , 4-Di chl orobenzene
Di chl orodi f 1 uoromethane
1 , 1-Di chl oroethane
1, 2-Di chloroethane
1, 1-Di chl oroethene
cis-1,2 Dichl oroethene
trans-1 , 2-Di chl oroethene
1 , 2-Di chl oropropane
1, 3-Di chl oropropane
2, 2-Dichl oropropane
1 , 1-Di chl oropropene
ci s-1 , 2-Di chl oropropene
trans-1 , 2-Di chl oropropene
Ethyl benzene
Hexachl orobutadi ene
Isopropyl benzene
4-Isopropyl toluene
Methyl ene chloride
Naphthalene
n-Propyl benzene
Styrene
True
Cone.
Range
(UQ/L)
0.1-10
0.1-10
0.5-10
0.1-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.1-10
0.5-10
0.5-10
0.5-10
0.1-10
0.1-10
0.1-10
0.5-10
0.5-10
0.5-10
0.1-10
0.5-10
0.2-20
0.5-10
0.5-10
0.1-10
0.1-10
0.5-10
0.1-10
0.1-10
0.1-10
0.5-10
0.5-10
0.1-10
0.5-10
0.5-10
0.1-10
0.1-10
0.1-100
0.1-10
0.1-100
Mean
Accuracy
(% of True
Value}
97
100
90
95
101
95
100
100
102
84
98
89
90
93
90
99
92
83
102
100
93
99
103
90
96
95
94
101
93
97
96
86
98
99
100
101
99
95
104
100
102
Rel.
Std.
Dev.
m
5.7
5.5
6.4
6.1
6.3
8.2
7.6
7.6
7.3
8.8
5.9
9.0
6.1
8.9
6.2
8.3
7.0
19.9
3.9
5.6
6.2
6.9
6.4
7.7
5.3
5.4
6.7
6.7
5.6
6.1
6.0
16.9
8.9
8.6
6.8
7.6
6.7
5.3
8.2
5.8
7.2
Method
Det.
Limit"
(U.Q/1)
0.04
0.03
0.04
0.08
0.12
0.11
0.11
0.13
0.14
0.21
0.04
0.10
0.03
0.13
0.04
0.06
0.05
0.26
0.06
0.24
0.03
0.12
0.03
0.10
0.04
0.06
0.12
0.12
0.06
0.04
0.04
0.35
0.10
0.06
0.11
0.15
0.12
0.03
0.04
0.04
0.04
38
-------
TABLE 4. (Continued)
Compound
1,1,1, 2-Tetrachl oroethane
1,1,2, 2-Tetrachl oroethane
Tetrachloroethene
Toluene
1,2, 3-Tri chl orobenzene
1,2, 4-Tri chl orobenzene
1,1, 1-Tri chl oroethane
1,1, 2-Tr i chl oroethane
Trichloroethene
Tr i chl orof 1 uoromethane
1,2, 3-Tri chl oropropane
1,2, 4-Tri methyl benzene
1 , 3 , 5-Tri methyl benzene
Vinyl chloride
o-Xylene
m-Xyl ene
p-Xylene
True
Cone.
Range
0.5-10
0.1-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.5-10
0.1-31
0.1-10
0.5-10
Mean
Accuracy
(% of True
Value)
90
91
89
102
109
108
98
104
90
89
108
99
92
98
103
97
104
Rel.
Std.
Dev.
6.8
6.3
6.8
8.0
8.6
8.3
8.1
7.3
7.3
8.1
14.4
8.1
7.4
6.7
7.2
6.5
7.7
Method
Det.
Limitb
fud/U
0.05
0.04
0.14
0.11
0.03
0.04
0.08
0.10
0.19
0.08
0.32
0.13
0.05
0.17
0.11
0.05
0.13
aData obtained by using column 1 with a jet separator interface and a
quadrupole mass spectrometer (Sect. 11.3.1) with analytes divided among
three solutions.
bReplicate samples at the lowest concentration listed in column 2 of this
table were analyzed. These results were used to calculate MDLs.
39
-------
TABLE 5. ACCURACY AND PRECISION DATA FROM SEVEN DETERMINATIONS OF METHOD
ANALYTES IN REAGENT WATER USING THE CRYOGENIC TRAPPING OPTION
AND A NARROW BORE CAPILLARY COLUMN 3a
Compound
Benzene
Bromobenzene
Bromochl oromethane
Bromodi chl oromethane
Bromoform
Bromomethane
n-Butyl benzene
sec-Butyl benzene
tert-Butyl benzene
Carbon tetrachloride
Chl orobenzene
Chloroethane
Chloroform
Chi oromethane
2-Chlorotoluene
4-Chlorotoluene
Cyanogen chloride
Di bromochl oromethane
1 , 2-Di bromo-3-chl oropropane
1 , 2-Di bromoethane
Dibromomethane
1 , 2-Di chl orobenzene
1 , 3-Di chl orobenzene
1 , 4-Di chl orobenzene
Di chl orodi f 1 uoromethane
1,1-Di chloroethane
1 , 2-Di chl oroethane
1,1-Di chl oroethene
cis-1,2 Di chl oroethene
trans-1 , 2-Di chl oroethene
1 , 2-Di chl oropropane
1 , 3-Di chl oropropane
2 , 2-Di chl oropropane
1,1-Di chl oropropene
ci s-1 , 3-Di chl oropropene
trans-1 , 3-Di chl oropropene
Ethyl benzene
Hexachl orobutadi ene
Isopropyl benzene
4-Isopropyl toluene
Methyl ene chloride
Naphthalene
True
Cone.
(UQ/L)
0.1
0.5
0.5
0.1
0.1
0.1
0.5
0.5
0.5
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.5
0.5
0.5
0.1
Mean
Accuracy
(% of True
Value}
99
97
97
100
99
99
94
90
90
92
91
100
95
99
99
96
92
99
92
97
93
97
99
93
99
98
100
95
100
98
96
99
99
98
99
100
98
87
97
98
Rel.
Std.
Dev.
(%)
6.2
7.4
5.8
4.6
5.4
7.1
6.0
7.1
2.5
6.8
5.8
5.8
3.2
4.7
4.6
7.0
10.6
5.6
10.0
5.6
6.9
3.5
6.0
5.7
8.8
6.2
6.3
9.0
3.7
7.2
6.0
5.8
4.9
7.4
5.2
6.7
6.4
13.0
13.0
7.2
Method
Dect.
Limit
(«q/L)
0.03
0.11
0.07
0.03
0.20
0.06
0.03
0.12
0.33
0.08
0.03
0.02
0.02
0.05
0.05
0.05
0.30
0.07
0.05
0.02
0.03
0.05
0.05
0.04
0.11
0.03
0.02
0.05
0.06
0.03
0.02
0.04
0.05
0.02
0.03
0.04
0.10
0.26
0.09
0.04
4ft
40
-------
TABLE 5. (Continued)
Compound
n-Propyl benzene
Styrene
1,1,1, 2-Tetrachl oroethane
1,1,2, 2-Tetrachl oroethane
Tetrachl oroethene
Toluene
1 , 2 , 3-Tr i chl orobenzene
1 , 2 , 4-Tri chl orobenzene
1,1, 1-Tri chl oroethane
1 , 1 , 2-Tri chl oroethane
Tri chl oroethene
Tri chl orof 1 uoromethane
1 , 2 , 3-Tri chl oropropane
1,2, 4-Tri methyl benzene
1,3, 5-Trimethyl benzene
Vinyl chloride
o-Xylene
m-Xyl ene
p-Xyl ene
True
Cone .
(un/L)
0.1
0.1
0.1
0.5
0.1
0.1
0.1
0.1
-0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Mean
Accuracy
(% of True
Value)
99
96
100
100
96
100
98
91
100
98
96
97
96
96
99
96
94
94
97
Rel.
Std.
Dev.
W
6.6
19.0
4.7
12.0
5.0
5.9
8.9
16.0
4.0
4.9
2.0
4.6
6.5
6.5
4.2
0.2
7.5
4.6
6.1
Method
Dect.
Limit
(ua/l}
0.06
0.06
0.04
0.20
0.05
0.08
0.04
0.20
0.04
0.03
0.02
0.07
0.03
0.04
0.02
0.04
0.06
0.03
0.06
aData obtained by using column 3 with a cryogenic interface and a
quadrupole mass spectrometer (Sect 11.3.3).
Reference 8.
41
-------
TABLE 6. ACCURACY AND PRECISION DATA FROM SEVEN DETERMINATIONS
OF THE METHOD ANALYTES IN REAGENT WATER USING WIDE BORE
CAPILLARY COLUMN 2a
Internal Standard
Compound
Mean Accuracy
(% of True
Value, RSD
No.b 2 UQ/l Cone.) (%)
Mean Accuracy
(% of True
Value,
0.2 ua/l Cone.)
RSD
Fluorobenzene 1
Surrogates
4-Bromof1uorobenzene 2 98
l,2-Dichlorobenzene-d4 3 97
Target Analvtes
Benzene
Bromobenzene
Bromochloromethane
Bromodi chloromethane
Bromoform
Bromomethane
n-Butylbenzene
sec-Butyl benzene
tert-Butylbenzene
Carbon tetrachloride
Chlorobenzene
Chloroethane0
Chloroform
Chloromethane
2-Chlorotoluene
4-Chlorotoluene
Di bromochloromethane
1,2-Di bromo-3-chloropropanec
1,2-Di bromoethanec
Dibromomethane 13 99
1,2-Dichlorobenzene 45 93
1,3-Dichlorobenzene 46 100
1,4-Dichlorobenzene 47 98
Dichlorodifluoromethane 14 38
1,1-Dichloroethane 15 97
1,2-Dichloroethane 16 102
1,1-Dichloroethene 17 90
cis-1,2-Dichloroethene 18 100
trans-1,2-Dichloroethene 19 92
37
38
4
5
6
7
39
40
41
8
42
9
10
43
44
11
97
102
99
96
89
55
89
102
101
84
104
97
110
91
89
95
1.8
3.2
4.4
3.0
5.2
1.8
2.4
27.
4.8
3.5
4.5
3.2
3.1
2.0
5.0
2.4
2.0
2.7
2.1
2.7
4.0
4.1
25.
2.3
3.8
2.2
3.4
2.1
96
95
113
101
102
100
90
52
87
100
100
92
103
95
d
108
108
100
95
94
87
94
d
85
100
87
89
85
1.3
1.7
1.8
1.9
2.9
1.8
2.2
6.7
2.3
2.8
2.9
2.6
1.6
2.1
3.1
4.4
3.0
2.2
5.1
2.3
2.8
3.6
2.1
3.8
2.9
2.3
42
-------
TABLE 6. (Continued)
Compound
1 , 2-Di chl oropropane
1 , 3-Di chl oropropane
2 , 2-Di chl oropropane0
1 , 1-Di chl oropropene0
cis-1, 3-Di chl oropropene0
trans-1 , 3-Di chl oropropene
Ethyl benzene
Hexachl orobutadi ene
Isopropyl benzene
4- Isopropyl toluene
Methylene chloride
Naphthalene
n-Propyl benzene
Styrene
1,1,1, 2-Tetrachl oroethane
1,1,2, 2-Tetrachl oroethane
Tetrachloroethene
Toluene
1,2, 3-Tri chl orobenzene
1,2, 4-Tri chl orobenzene
1,1, 1-Tri chl oroethane
1,1, 2-Tri chl oroethane
Trichloroethene
Tri chl orof 1 uoromethane
1,2, 3-Tri chl oropropane
1,2, 4-Tri methyl benzene
1 , 3 , 5-Trimethyl benzene
Vinyl chloride
o-Xylene
m-Xyl ene
p-Xylene
No.b
20
21
25
48
26
49
50
27
51
52
53
28
29
30
54
55
56
31
32
33
34
35
57
58
36
59
60
61
Mean Accuracy
(% of True
Value,
2 UQ/L Cone.)
102
92
96
96
91
103
95
e
93
102
95
99
101
97
105
90
92
94
107
99
81
97
93
88
104
97
f
98
RSD
2.2
3.7
1.7
9.1
5.3
3.2
3.6
7.6
4.9
4.4
2.7
4.6
4.5
2.8
5.7
5.2
3.9
3.4
2.9
4.6
3.9
3.1
2.4
3.5
1.8
2.3
Mean Accuracy
(% of True
Value,
0.2 UQ/L Cone.
103
93
99
100
88
101
95
e
78
97
104
95
84
92
126
78
83
94
109
106
48
91
106
97
115
98
f
103
RSD
) M
2.9
3.2
2.1
4.0
2.4
2.1
3.1
8.3
2.1
3.1
3.8
3.6
3.3
1.7
2.9
5.9
2.5
2.8
2.5
13.
2.8
2.2
3.2
14.
1.7
1.4
aData obtained using column 2 with the open split interface and an ion
trap mass spectrometer (Sect. 11.3.2) with all method analytes in the same
reagent water solution.
Designation in Figures 1 and 2.
°Not measured; authentic standards were not available.
dNot found at 0.2 /ig/L.
*Not measured; methylene chloride was in the laboratory reagent blank.
m-xylene coelutes with and cannot be distinguished from its isomer p-xylene,
No 61.
43
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TABLE 7. ACCURACY AND PRECISION DATA FROM SEVEN DETERMINATIONS
OF METHOD ANALYTES IN REAGENT WATER USING WIDE BORE
CAPILLARY COLUMN 4
Compound
Acetone
Acrylonitrile
Ally! chloride
2-Butanone
Carbon disulfide
Chloroacetonitrile
1-Chlorobutane
t-1 , 2-Di chl oro-2-butene
1 , 1-Di chl oropropanone
Di ethyl ether
Ethyl methacryl ate
Hexachloroethane
2-Hexanone
Methacryl on itrile
Methyl acryl ate
Methyl iodide
Methyl methacryl ate
4-Methyl -2-pentanone
Methyl -tert-butyl ether
Nitrobenzene
2-Nitropropane
Pentachloroethane
Propionitrile
Tetrahydrofuran
True
Cone.
1.0
1.0
1.0
2.0
0.20
1.0
1.0
1.0
5.0
1.0
0.20
0.20
1.0
1.0
1.0
0.20
1.0
0.40
0.40
2.0
1.0
0.20
1.0
5.0
Mean
Cone.
Detected
(ug/L)
1.6
0.81
0.90
2.7
0.19
0.83
0.87
1.3
4.2
0.92
0.23
0.18
1.1
0.92
1.2
0.19
1.0
0.56
0.52
2.1
0.83
0.23
0.87
3.9
Rel.
Std.
Dev.
5.7%
8.7%
4.7%
5.6%
15%
4.7%
6.6%
8.7%
7.7%
9.5%
3.9%
10%
12%
4.2%
12%
3.1%
13%
9.7%
5.6%
18%
6.2%
20%
5.3%
13%
Method
Det.
Limit
0.28
0.22
0.13
0.48
0.093
0.12
0.18
0.36
1.0
0.28
0.028
0.057
0.39
0.12
0.45
0.019
0.43
0.17
0.090
1.2
0.16
0.14
0.14
1.6
44
-------
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FIGURE 1. PURGING DEVICE
47
-------
PACKING PflOCSKJIE
CONSTRUCTION
GLASS a
•Ott *
ACTIVATE), _
CHAKCOAL.7J
GRADEH
w OV-T
GLASS
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RTTINGNUT
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HUP INLET
FIGURE 2. TRAP PACKINGS AND CONSTRUCTION TO INCLUDE
DESORB CAPABILITY
48
-------
I
i
8
1
a
a
o
2
3
-------
50
-------
METHOD 515.2. DETERMINATION OF CHLORINATED ACIDS IN WATER
USING LIQUID-SOLID EXTRACTION AND GAS
CHROMATOGRAPHY WITH AN ELECTRON CAPTURE DETECTOR
Revision 1.0
August 1992
R.C. Dressman and J.J. Lichtenberg - EPA 600/4-81-053, Revision 1.0 (1981)
J.W. Hodgeson - Method 515, Revision 2.0 (1986)
T. Engels (Battelle Columbus Laboratories) - National Pesticide Survey
Method 3, Revision 3.0 (1987)
R.L. Graves - Method 515.1, Revision 4.0 (1989)
J.W. Hodgeson - Method 515.2, Revision 1.0 (1992)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
51
-------
METHOD 515.2
DETERMINATION OF CHLORINATED ACIDS IN WATER USING
LIQUID-SOLID EXTRACTION AND GAS CHRONATOGRAPHY
WITH AN ELECTRON CAPTURE DETECTOR
1. SCOPE AND APPLICATION
1.1
1.2
1.3
This is a gas chromatographic (GC) method applicable to the determi-
nation of certain chlorinated acids in ground water and finished
drinking water. The following compounds can be determined by this
method:
Chemical Abstract Services
Registry Number
1.4
50594-
25057-
94-
94-
1861-
1918-
51-
120-
88-
7600-
87-
1918-
93-
93-
66-6
89-0
75-7
82-6
32-1
00-9
•36-5
•36-5
•85-7
•50-2
•86-5
•02-1
•76-5
•72-1
Analvte
Acifluorfen
Bentazon
2-,4-D
2,4-DB
Dacthal(a>
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
Pentachlorophenol (PCP)
Picloram
2,4,5-T
2,4,5-TP(Silvex)
(a) Dacthal monoacid and diacid metabolites included in method
scope; Dacthal diacid metabolite used for validation studies.
This method is applicable to the determination of salts and esters
of analyte acids. The form of each acid is not distinguished by
this method. Results are calculated and reported for each listed
analyte as the total free acid.
Single laboratory accuracy and precision data and method detection
limits (MDLs) have been determined for the analytes above (Sect.
13). Observed detection limits may vary among water matrices,
depending upon the nature of interferences in the sample matrix and
the specific instrumentation used.
This method is restricted to use by or under the supervision of
analysts experienced in the use of GC and in the interpretation of
gas chromatograms. Each analyst must demonstrate the ability to
generate acceptable results with this method using the procedure
described in Sect. 9.3.
52
-------
1.5 Analytes that are not separated chromatographically, (i.e., have
very similar retention times) cannot be individually identified and
measured in the same calibration mixture or water sample unless an
alternative technique for identification and quantitation exists
(Sect. 11.6).
1.6 When this method is used to analyze unfamiliar samples for any or
all of the analytes above, analyte identifications shoul-d be con-
firmed by analysis on a second gas chromatographic column or by gas
chromatography/mass spectrometry (GC/MS).
2. SUMMARY OF METHOD
2.1 A 250-mL measured volume of sample is adjusted to pH 12 with 6 N
sodium hydroxide for 1 hr to hydrolyze derivatives. Extraneous
organic material is removed by a solvent wash. The sample is
acidified, and the chlorinated acids are extracted with a 47 mm
resin based extraction disk. The acids are converted to their
methyl esters using diazomethane. Excess derivatizing reagent is
removed, and the esters are determined by capillary column GC using
an electron capture detector (ECO).
3. DEFINITIONS
3.1 INTERNAL STANDARD (IS) — A pure analyte(s) added to a sample,
extract, or standard solution in known amount(s), and used to
measure the relative responses of other method analytes and surro-
gates that are components of the same sample or solution. The IS
must be an analyte that is not a sample component.
3.2 SURROGATE ANALYTE (SA) — A pure analyte(s), which is extremely
unlikely to be found in any sample, and which is added to a sample
aliquot in known amount(s) before extraction or other processing,
and is measured with the same procedures used to measure other
sample components. The purpose of the SA is to monitor method
performance with each sample.
3.3 LABORATORY DUPLICATES (LD1 AND LD2) — Two aliquots of the same
sample taken in the analytical laboratory and analyzed separately
with identical procedures. Analyses of LD1 and LD2 indicate the
precision associated with laboratory procedures, but not with sample
collection, preservation, or storage procedures.
3.4 FIELD DUPLICATES (FD1 AND FD2) -- Two separate samples collected at
the same time and place under identical circumstances and treated
exactly the same throughout field and laboratory procedures.
Analyses of FD1 and FD2 give a measure of the precision associated
with sample collection, preservation and storage, as well as with
laboratory procedures.
3.5 LABORATORY REAGENT BLANK (LRB) ~ An aliquot of reagent water or
other blank matrix that is treated exactly as a sample including
53
-------
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The LRB
is used to determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or the appara-
tus.
3.6 FIELD REAGENT BLANK (FRB) — An aliquot of reagent water or other
blank matrix that is placed in a sample container in the laboratory
and treated as a sample in all respects, including shipment to the
sampling site, exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB
is to determine if method analytes or other interferences are
present in the field environment.
3.7 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) ~ A solution of one or
more method analytes, surrogates, internal standards, or other test
substances used to evaluate the performance of the instrument system
with respect to a defined set of criteria.
3.8 LABORATORY FORTIFIED BLANK (LFB) — An aliquot of reagent water or
other blank matrix to which known quantities of the method analytes
are added in the laboratory. The LFB is analyzed exactly like a
sample, and its purpose is to determine whether the methodology is
in control, and whether the laboratory is capable of making accurate
and precise measurements.
3.9 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) — An aliquot of an envi-
ronmental sample to which known quantities of the method analytes
are added in the laboratory. The LFM is analyzed exactly like a
sample, and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background concen-
trations of the analytes in the sample matrix must be determined in
a separate aliquot, and the measured values in the LFM corrected for
background concentrations.
3.10 STOCK STANDARD SOLUTION (SSS) -- A concentrated solution containing
one or more method analytes prepared in the laboratory using
assayed reference materials or purchased from a reputable commercial
source.
3.11 PRIMARY DILUTION STANDARD SOLUTION (PDS) — A solution of several
analytes prepared in the laboratory from stock standard solutions,
and diluted as needed to prepare calibration solutions and other
needed analyte solutions.
3.12 CALIBRATION STANDARD (CAL) — A solution prepared from the primary
dilution standard solution or stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
54
-------
3.13 QUALITY CONTROL SAMPLE (QCS) ~ A solution of method analytes of
known concentrations which is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally prepared
test materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware and other sample processing apparatus that lead
to discrete artifacts or elevated baselines in gas chromatograms.
All reagents and apparatus must be routinely demonstrated to be free
from interferences under analytical conditions by analyzing labora-
tory reagent blanks as described in Sect. 9.2.
4.1.1 Glassware.must be scrupulously cleaned. (1) Clean all glass-
ware as soon as possible after use by thoroughly rinsing with
the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with dilute acid,
tap and reagent water. Drain dry, and heat in an oven or
muffle furnace at 400°C for 1 hr. Do not heat volumetric
ware. Thermally stable materials such as PCBs might not be
eliminated by this treatment. Thorough rinsing with acetone
may be substituted for the heating. After glassware is dry
and cool, store it in a clean environment to prevent any
accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
4.1.2 The use of high purity reagents and solvents helps to mini-
mize interference problems. Purification of solvents by
distillation in all-glass systems may be required.
WARNING: When a solvent is purified, stabilizers and preser-
vatives added by the manufacturer are removed, thus poten-
tially making the solvent hazardous and reducing the shelf
life.
4.2 The acid forms of the analytes are strong organic acids which react
readily with alkaline substances and can be lost during sample
preparation. Glassware and glass wool must be acid-rinsed with 1 N
hydrochloric acid and the sodium sulfate must be acidified with
sulfuric acid prior to use to avoid analyte losses due to adsorp-
tion.
4.3 Organic acids and phenols, especially chlorinated compounds, cause
the most direct interference with the determination. Alkaline
hydrolysis and subsequent extraction of the basic sample removes
many chlorinated hydrocarbons and phthalate esters that might
otherwise interfere with the electron capture analysis.
4.4 Interferences by phthalate esters can pose a major problem in pesti-
cide analysis when using the ECD. Phthalates generally appear in
55
-------
the chromatogram as large peaks. Common flexible plastics contain
varying amounts of phthalates, that are easily extracted or leached
during laboratory operations. Cross-contamination of clean glass-
ware routinely occurs when plastics are handled during extraction
steps, especially when solvent-wetted surfaces are handled. Inter-
ferences from phthalates can best be minimized by avoiding the use
of plastics in the laboratory. Exhaustive purification of reagents
and glassware may be required to eliminate background phthalate
contamination. (2,3)
4.5 Interfering contamination may occur when a sample containing low
concentrations of analytes is analyzed immediately following a
sample containing relatively high concentrations of analytes.
Between-sample rinsing of the sample syringe and associated equip-
ment with methyl-tert-butyl-ether (MTBE) can minimize sample cross-
contamination. After analysis of a sample containing high concen-
trations of analytes, one or more injections of MTBE should be made
to ensure that accurate values are obtained for the next sample.
4.6 Matrix interferences may be caused by contaminants that are coex-
tracted from the sample. Also, note that all analytes listed in the
Scope and Application Section are not resolved from each other on
any one column, i.e., one analyte of interest may interfere with
another analyte of interest. The extent of matrix interferences
will vary considerably from source to source, depending upon the
water sampled. The procedures in Sect. 11 can be used to overcome
many of these interferences. Tentative identifications should be
confirmed (Sect. 11.6).
4.7 It is important that samples and working standards be contained in
the same solvent. The solvent for working standards must be the
same as the final solvent used in sample preparation. If this is
not the case, chromatographic comparability of standards to sample
extracts may be affected.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound must
be treated as a potential health hazard. Accordingly, exposure to
these chemicals must be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file
of OSHA regulations regarding the safe handling of the chemicals
specified in this method. A reference file of material safety data
sheets should also be made available to all personnel involved in
the chemical analysis. Additional references to laboratory safety
are available and have been identified (5-7) for the information of
the analyst.
5.2 DIAZOMETHANE — A toxic carcinogen which can explode under certain
conditions. The following precautions must be followed:
56
-------
5.2.1 Use the diazomethane generator behind a safety shield in a
well ventilated fume hood. Under no circumstances can the
generator be heated above 90°C, and all grinding surfaces
such as ground glass joints, sleeve bearings, and glass
stirrers must be avoided. Diazomethane solutions must not be
stored. Only generate enough for the immediate needs. The
diazomethane generator apparatus used in the esterification
procedure (Sect. 11.4) produces micromolar amounts of diazo-
methane in solution to minimize safety hazards. If the
procedure is followed exactly, no possibility for explosion
exists.
5.3 METHYL-TERT-BUTYL ETHER — Nanograde, redistilled in glass, if
necessary. Must be free of peroxides as indicated by EM Quant test
strips (available from Scientific Products Co., Cat. No. PI 126-8,
and other suppliers).
5.4 WARNING: When a solvent is purified, stabilizers added by the
manufacturer are removed, thus potentially making the solvent
hazardous.
EQUIPMENT AND SUPPLIES (All specifications are suggested. Catalog
numbers are included for illustration only.)
6.1 KONTES FILTER FUNNELS — Fisher Cat. No. 953755-0000 or equivalent.
6.2 VACUUM FLASKS — 1000 mL with glass side arm
6.3 VACUUM MANIFOLD — The manifold should be capable of holding 6-8
filter flasks in series with house vacuum. Commercial manifolds are
available from a number of suppliers, e.g., Baker, Fisher, and
Varian.
6.4 CULTURE TUBES (25 x 200 mm) WITH TEFLON-LINED SCREW CAPS — Fisher
Cat. No. 14-933-1C, or equivalent.
6.5 PASTEUR PIPETS — Glass disposable (5 mL)
6.6 LARGE VOLUME PIPETS — Disposable, Fisher Cat. No. 13-678-8 or
equivalent.
6.7 BALANCE -- Analytical, capable of weighing to .0001 g.
6.8 pH METER --Wide range capable of accurate measurements in the pH =
1-12 range.
6.9 DIAZOMETHANE GENERATOR -- See Figure 1 for a diagram of an all glass
system custom made for these validation studies. A micromolar
generator is also available from Aldrich Chemical.
6.10 ANALYTICAL CONCENTRATOR — Six or twelve positions, Organomation N-
EVAP Model No. 111-6917 or equivalent.
57
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6.11 GAS CHROMATOGRAPHY — Analytical system complete with gas chromato-
graph equipped with ECD, split/splitless capillary injector, temper-
ature programming, differential flow control and all required acces-
sories. A data system is recommended for measuring peak areas. An
autoinjector is recommended to improve precision of analysis.
6.12 GC COLUMNS AND RECOMMENDED OPERATING CONDITIONS
6.12.1 Primary -- DB-5 or equivalent, 30 m x .32 mm ID, 0.25 fm film
thickness. Injector Temp. = 200°C, Detector Temp. = 280°C,
Helium linear velocity is 30 cm/sec at 200°C and 10 psi, 2 jtL
splitless injection with purge on 3 min. Program: Hold at
60°C 1 min., increase to 260°C at 5°C/min. and hold 5 min.
6.12.2 Confirmation — DB-1701 or equivalent, 30 m x .32 mm ID, 0.25
IOR film thickness. Injector Temp. - 200 °C, Detector Temp. =
280°C, Helium linear velocity is 30 cm/sec at 200°C and 10
psi, 2 fiL splitless injection with purge on 3 min. Program:
Hold at 60°C 1 min., increase to 260°C at 5°C/min. and hold 5
min.
6.13 GLASS WOOL — Acid washed with IN HC1 and heated at 450°C for 4 hr.
6.14 SHORT RANGE pH PAPER (pH=0-3).
6.15 VOLUMETRIC FLASKS -- 50 mL, 100 mL, and 250 mL
6.16 MICROSYRINGES — 25 /iL, 50 /iL, 100 /iL, 250 /iL, 500 fil
6.17 AMBER BOTTLES — 15 mL, with Teflon-lined screw caps
6.18 GRADUATED CYLINDER -- 250 mL
6.19 SEPARATORY FUNNEL — 500 mL
6.20 GRADUATED CENTRIFUGE TUBES — 15 mL or 10 mL Kuderna Danish Concen-
trator tubes
7. REAGENTS AND STANDARDS
7.1 EXTRACTION DISKS, 47 mm — Resin based polystyrenedivinyl benzene
7.2 REAGENT WATER — Reagent water is defined as a water in which an
interference is not observed at the MDL of each analyte of interest.
7.2.1 A Mi 111 pore Super-Q water system or its equivalent may be
used to generate deionized reagent water. Distilled water
that has been passed through granular charcoal may also be
suitable.
7.2.2 Test reagent water each day it is used by analyzing according
to Sect. 11.
58
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7.3 METHANOL — Pesticide quality or equivalent.
7.4 METHYL-TERT-BUTYL ETHER (MTBE) — Nanograde, redistilled in glass if
necessary. Ether must be demonstrated to be free of peroxides. One
test kit (EM Quant Test Strips), is available from EM Science,
Gibbstown, NJ. Procedures for removing peroxides from the ether are
provided with the test strips. Ethers must be periodically tested
(at least monthly) for peroxide formation during use. Any reliable
test kit may be used,
7.5 SODIUM SULFATE — (ACS) GRANULAR, ANHYDROUS — Heat in a shallow
tray at 400°C for a minimum of 4 hr to remove phthalates and other
interfering organic substances. Alternatively, extract with methy-
lene chloride in a Soxhlet apparatus for 48 hr.
7.5.1 Sodium sulfate drying tubes — Plug the bottom of a large
volume disposable pipet with a minimum amount of acidified
glass wool (Supelco Cat. No. 20383 or equivalent). Fill the
pipet halfway (3 g) with acidified sodium sulfate (See Sect.
7.9).
7.6 SULFURIC ACID -- Reagent grade.
7.6.1 Sulfuric acid, 12 N — Slowly add 335 ml concentrated sulfu-
ric acid to 665 ml of reagent water.
7.7 SODIUM HYDROXIDE — ACS reagent grade or equivalent.
7.7.1 Sodium hydroxide IN -- Dissolve 4.0 g reagent grade sodium
hydroxide in reagent water and dilute to 100 ml in volumetric
flasks.
7.7.2 Sodium hydroxide 6N
7.8 ETHYL ETHER, UNPRESERVED ~ Nanograde, redistilled in glass if
necessary. Must be free of peroxides as indicated by EM Quant test
strips (available from Scientific Products Co., Cat. No. PI126-8,
and other suppliers). Procedures recommended for removal of per-
oxides are provided with the test strips.
7.9 ACIDIFIED SODIUM SULFATE — Cover 500 g sodium sulfate (Sect. 7.5)
with ethyl ether (Sect. 7.8). While agitating vigorously, add
dropwise approximately 0.7 mL concentrated sulfuric acid. Remove
the ethyl ether overnight under vacuum and store the sodium sulfate
in a 100°C oven.
7.10 CARBITOL, ACS GRADE — Available from Aldrich Chemical.
7.11 DIAZALD, ACS GRADE -- Available from Aldrich Chemical.
7.12 DIAZALD SOLUTION -- Prepare a solution containing 10 g Diazald in
100 mL of a 50:50 by volume mixture of ethyl ether and carbitol.
59
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This solution is stable for 1-month or longer when stored at 4°C in
an amber bottle with a Teflon-lined screw cap.
7.13 4,4'-DIBROMOOCTAFLUOROBIPHENYL (DBOB) — 99% purity, for use as
internal standard.
7.14 2,4-DICHLOROPHENYLACETIC ACID (DCAA) — 99% purity, for use as
surrogate standard.
7.15 POTASSIUM HYDROXIDE -- ACS reagent grade or equivalent.
7.15.1 Potassium hydroxide solution, 37% — Using extreme caution,
dissolve 37 g reagent grade potassium hydroxide in reagent
water and dilute to 100 ml.
7.16 STOCK STANDARD SOLUTIONS (1.00-2.00 /ig//iL) — Stock standard solu-
tions may be purchased as certified solutions or prepared from pure
standard materials using the following procedure:
7.16.1 Prepare stock standard solutions by accurately weighing
approximately 0.0100-0.0200 g of pure material. Dissolve the
material in methanol and dilute to volume in a 10-mL volu-
metric flask. Larger volumes may be used at the convenience
of the analyst. If compound purity is certified at 96% or
greater, the weight may be used without correction to calcu-
late the concentration of the stock standard. Commercially
prepared stock standards may be used at any concentration if
they are certified by the manufacturer or by an independent
source.
7.16.2 Transfer the stock standard solutions into 15-mL TFE-fluoro-
carbon-sealed screw cap amber vials. Store at 4°C or less
when not in use.
7.16.3 Stock standard solutions should be replaced after 2 months or
sooner if comparison with laboratory fortified blanks, or QC
samples indicate a problem.
7.16.4 Primary Dilution Standards -- Prepare two sets of standards
according to the sets labeled A and B in Table 1. For each
set, add approximately 25 mL of methanol to a 50 mL volumet-
ric flask. Add aliquots of each stock standard in the range
of approximately 20 to 400 ML and dilute to volume with
methanol. Individual analyte concentrations will then be in
the range of 0.4 to 8 /jg/mL (for a 1.0 mg/mL stock). The
minimum concentration would be appropriate for an analyte
with strong electron capture detector (ECD) response, e.g.
pentachlorophenol. The maximum concentration is for an
analyte with weak response, e.g., 2,4-DB. The concentrations
given in Table 2 reflect the relative volumes of stock stan-
dards used for the primary dilution standards used in gener-
ating the method validation data. Use these relative values
60
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to determine the aliquot volumes of individual stock stan-
dards above.
7.17 INTERNAL STANDARD SOLUTION — Prepare a stock internal standard
solution by accurately weighing approximately 0.050 g of pure DBOB.
Dissolve the DBOB in methanol and dilute to volume in a 10-mL
volumetric flask. Transfer the DBOB solution to a TFE-fluorocarbon-
sealed screw cap bottle and store at room temperature. Prepare a
primary dilution standard at approximately 1.00 /ig/mL by the addi-
tion of 20 /iL of the stock standard to 100 mL of methanol. Addition
of 100 /iL of the primary dilution standard solution to the final 5
mL of sample extract (Sect. 11) results in a final internal standard
concentration of 0.020 /ig/mL. Solution should be replaced when
ongoing QC (Sect. 9) indicates a problem. Note that DBOB has been
shown to be an effective internal standard for the method analytes,
but other compounds may be used if the QC requirements in Sect. 9
are met.
7.18 SURROGATE ANALYTE SOLUTION — Prepare a surrogate analyte stock
standard solution by accurately weighing approximately 0.050 g of
pure DCAA. Dissolve the DCAA in methanol and dilute to volume in a
10-mL volumetric flask. Transfer the surrogate analyte solution to
a TFE-fluorocarbon-sealed screw cap bottle and store at room temper-
ature. Prepare a primary dilution standard at approximately 2.0
/ig/mL by addition of 40 til at the stock standard to 100 mL of
methanol. Addition of 250 /iL of the surrogate analyte solution to a
250-mL sample prior to extraction results in a surrogate concentra-
tion in the sample of 2 /ig/L and, assuming quantitative recovery of
DCAA, a surrogate analyte concentration in the final 5 mL extract of
0.1 /ig/mL. The surrogate standard solution should be replaced when
ongoing QC (Sect. 9) indicates a problem. DCAA has been shown to be
an effective surrogate standard for the method analytes, but other
compounds may be used if the QC requirements in Sect. 10 are met.
7.19 INSTRUMENT PERFORMANCE CHECK SOLUTION — Prepare a diluted dinoseb
solution by adding 10 /iL of the 1.0 /ig//iL dinoseb stock solution to
the MTBE and diluting to volume in a 10-mL volumetric flask. To
prepare the check solution, add 40 /iL of the diluted dinoseb solu-
tion, 16 /tL of the 4-nitrophenol stock solution, 6 /iL of the 3,5-
dichlorobenzoic acid stock solution, 50 /iL of the surrogate standard
solution, 25 /iL of the internal standard solution, and 250 /iL of
methanol to a 5-mL volumetric flask and dilute to volume with MTBE.
Methylate sample as described in Sect. 11.4. Dilute the sample to
10 mL in MTBE. Transfer to a TFE-fluorocarbon-sealed screw cap
bottle and store at room temperature. Solution should be replaced
when ongoing QC (Sect. 9) indicates a problem.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 Grab samples should be collected in 1-L amber glass containers.
Conventional sampling practices (7) should be followed; however, the
bottle must not be prerinsed with sample before collection.
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8.2 SAMPLE PRESERVATION AND STORAGE
8.2.1 Add hydrochloric acid (diluted 1:1 in water) to the sample at
the sampling site in amounts to produce a sample pH ^ 2.
Short range (0-3) pH paper (Sect. 6.14) may be used to moni-
tor the pH.
8.2.2 If residual chlorine is present, add 80 mg of sodium thiosul-
fate per liter of sample to the sample bottle prior to col-
lecting the sample.
8.2.3 After the sample is collected in the bottle containing pre-
servative(s), seal the bottle and shake vigorously for 1 min.
8.2.4 The samples must be iced or refrigerated at 4°C away from
light from the time of collection until extraction. Preser-
vation study results indicate that the sample analytes (mea-
sured as total acid), except 5-hydroxy-dicamba, are stable in
water for 14 days when stored under these conditions (Tables
8 and 9). The concentration of 5-hydroxydicamba is seriously
degraded over 14 days in a biologically active matrix. How-
ever, analyte stability will very likely be affected by the
matrix; therefore, the analyst should verify that the preser-
vation technique is applicable to the samples under study.
8.3 EXTRACT STORAGE
8.3.1 Extracts should be stored at 4°C or less away from light.
Preservation study results indicate that most analytes are
stable for 14 days (Tables 8 and 9); however, the analyst
should verify appropriate extract holding times applicable to
the samples under study.
9. QUALITY CONTROL
9.1 Minimum QC requirements are initial demonstration of laboratory
capability, determination of surrogate compound recoveries in each
sample and blank, monitoring internal standard peak area or height
in each sample and blank (when internal standard calibration proce-
dures are being employed), analysis of laboratory reagent blanks,
laboratory fortified samples, laboratory fortified blanks, and QC
samples.
9.2 LABORATORY REAGENT BLANKS (LRB) — Before processing any samples,
the analyst must demonstrate that all glassware and reagent inter-
ferences are under control. Each time a set of samples is extracted
or reagents are changed, a LRB must be analyzed. If within the
retention time window of any analyte the LRB produces a peak that
would prevent the determination of that analyte, determine the
source of contamination and eliminate the interference before
processing samples.
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9.3 INITIAL DEMONSTRATION OF CAPABILITY
9.3.1 Select a representative fortified concentration (about 10 to
20 times MDL) for each analyte. Prepare a sample concen-
trate (in rnethanol) containing each analyte at 1000 times
selected concentration. With a syringe, add 250 /iL of the
concentrate to each of at least four 250 mL aliquots of
reagent water, and analyze each aliquot according to proce-
dures beginning in Sect. 11.
9.3.2 For each analyte the recovery value for all four of these
samples must fall in the range of ± 40% of the fortified
concentration. For those compounds that meet the acceptance
criteria, performance is considered acceptable and sample
analysis may begin. For compounds failing this criteria,
this procedure must be repeated using five fresh samples
until satisfactory performance has been demonstrated for all
analytes.
9.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples via a
new, unfamiliar method prior to obtaining some experience
with it. As laboratory personnel gain experience with this
method the quality of data should improve beyond those
required here.
9.4 The analyst is permitted to modify GC columns, GC conditions,
detectors, concentration techniques (i.e., evaporation techniques),
internal standard or surrogate compounds. Each time such method
modifications are made, the analyst must repeat the procedures in
Sect. 9.3.
9.5 ASSESSING SURROGATE RECOVERY
9.5.1 When surrogate recovery from a sample or a blank is <60% or
> 140%, check (1) calculations to locate possible errors,
(2) fortifying solutions for degradation, (3) contamination,
and (4) instrument performance. If those steps do not
reveal the cause of the problem, reanalyze the extract.
9.5.2 If a blank extract reanalysis fails the 60-140% recovery
criteria, the problem must be identified and corrected
before continuing.
9.5.3 If sample extract reanalysis meets the surrogate recovery
criteria, report only data for the reanalyzed extract. If
sample extract continues to fail the recovery criteria,
report all data for that sample as suspect.
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9.6 ASSESSING THE INTERNAL STANDARD
9.6.1 When using the internal standard (IS) calibration procedure,
the analyst is expected to monitor the IS response (peak
area or peak height) of all samples during each analysis
day. The IS response for any sample chromatogram should not
deviate from the daily calibration check standard's IS
response by more than 30%.
9.6.2 If >30% deviation occurs with an individual extract, opti-
mize instrument performance and inject a second aliquot of
that extract.
9.6.2.1 If the reinjected aliquot produces an acceptable
internal standard response, report results for
that aliquot.
9.6.2.2 If a deviation of greater than 30% is obtained for
the reinjected extract, analysis of the samples
should be repeated beginning with Sect. 11, pro-
vided the sample is still available. Otherwise,
report results obtained from the reinjected ex-
tract, but annotate as suspect.
9.6.3 If consecutive samples fail the IS response acceptance
criteria, immediately analyze a medium calibration standard.
9.6.3.1 If the standard provides a response factor (RF)
(Sect. 10.2.2) within 20% of the predicted value,
then follow procedures itemized in Sect. 9.6.2 for
each sample failing the IS response criterion.
9.6.3.2 If the check standard provides a response factor
which deviates more than 20% of the predicted
value, then the analyst must recalibrate as spec-
ified in Sect. 10.
9.7 ASSESSING LABORATORY PERFORMANCE — LABORATORY FORTIFIED BLANK
9.7-.1 The laboratory must analyze at least one laboratory forti-
fied blank (LFB) sample with every 20 samples or one per
sample set (all samples extracted within a 24-hr period)
whichever is greater. The concentration of each analyte in
the LFB should be 10 times the MDL. Calculate percent
recovery (X,-). If the recovery of any analyte falls outside
the control limits (See Sect. 9.7.2), that analyte is judged
out of control, and the source of the problem should be
identified and resolved before continuing analyses.
9.7.2 Until sufficient data become available, usually a minimum of
results from 20 to 30 analyses, each laboratory should
64
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assess laboratory performance against the control limits in
Sect. 9.3.2 that are derived from the data in Table 2. When
sufficient internal performance data become available,
develop control limits from the mean percent recovery (X)
and standard deviation (S) of the percent recovery. These
data are used to establish upper and lower control limits as
fol1ows:
UPPER CONTROL LIMIT = X + 3S
LOWER CONTROL LIMIT = X - 3S
After each five to ten new recovery measurements, new con-
trol limits should be calculated using only the most recent
20-30 data points. These calculated control limits should
never exceed those established in Sect. 9.3.2.
9.7.3 Method detection limits (MDL) must be determined using the
procedure given in reference (8). The MDLs must be suffi-
cient to detect analytes at the required levels according to
SDWA regulations.
9.7.4 At least quarterly, analyze a QCS (Sect. 3.13) from an
outside source.
9.7.5 Laboratories are encouraged to participate in external
performance evaluation studies such as the laboratory cert-
ification programs offered by many states or the studies
conducted by USEPA.
9.8 ASSESSING ANALYTE RECOVERY - LABORATORY FORTIFIED SAMPLE MATRIX
9.8.1 Each laboratory must analyze a LFM for 10% of the samples or
one sample concentration per set, whichever is greater. The
concentration should not be less then the background concen-
tration of the sample selected for fortification. Ideally,
the concentration should be the same as that used for the
laboratory fortified blank (Sect. 9.7). Over time, samples
from all routine sample sources should be fortified.
9.8.2 Calculate the percent recovery, P of the concentration for
each analyte, after correcting the measured concentration,
X, from the fortified sample for the background concentra-
tion, b, measured in the unfortified sample.
P = 100 (X - b) / fortified concentration,
and compare these values to control limits appropriate for
reagent water data collected in the same fashion. If the
analyzed unfortified sample is found to contain NO back-
ground concentrations and the added concentrations are those
specified in Sect. 9.7, then the appropriate control limits
would be the acceptance limits in Sect. 9.7. If, on the
other hand, the analyzed unfortified sample is found to
65
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contain background concentration, b, estimate the standard
deviation at the background concentration, s^, using regres-
sions or comparable background data and, similarly, estimate
the mean, X and standard deviation, sa, of analytical
results at the total concentration after fortifying. Then
the appropriate percentage control limits would be P ± 3sp ,
where:
P = 100 X / (b + fortifying concentration)
1/2
2 2
and s - 100 (s + s ) /fortifying concentration
P a b
For example, if the background concentration for Analyte A
was found to be 1 jug/L and the added amount was also 1 pg/L,
and upon analysis the laboratory fortified sample measured
1.6 /jg/L, then the calculated P for this sample would be
(1.6 /xg/L minus 1.0 /ig/L) /I pg/L or 60%. This calculated P
is compared to control limits derived from prior reagent
water data. Assume that analysis of an interference free
sample at 1 /zg/L yields_an s of 6.12 /zg/L and similar analy-
sis at 2.0 jjg/L yields X and s of 2.01 /zg/L and 0.20 /zg/L,
respectively. The appropriate limits to judge the reason-
ableness of the percent recovery, 60%, obtained on the
fortified matrix sample is computed as follows:
[100 (2.01 iig/L) / 2.0 /zg/L]
2 2 1/2
± 3 (100) [(0.12 0g/L)2 + (0.20 zzg/L)2] / 1.0 /zg/L =
100.5% ± 300 (0.233) =
100.5% ± 70% or 30% to 170% recovery of the added analyte.
9.8.3 If the recovery of any such analyte falls outside the desig-
nated range, and the laboratory performance for that analyte
is shown to be in control (Sect. 9.7), the recovery problem
encountered with the fortified sample is judged to be matrix
related, not system related. The result for that analyte in
the unfortified sample is labeled suspect/matrix to inform
the data user that the results are suspect due to matrix
effects.
9.9 ASSESSING INSTRUMENT SYSTEM/INSTRUMENT PERFORMANCE CHECK (IPC)
SAMPLE — Instrument performance should be monitored on a daily
basis by analysis of the IPC sample. The IPC sample contains
compounds designed to indicate appropriate instrument sensitivity,
column performance (primary column) and chromatographic perfor-
mance. IPC sample components and performance criteria are listed
in Table 11. Inability to demonstrate acceptable instrument
66
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performance indicates the need for reevaluation of the instrument
system. The sensitivity requirements are set based on the MDLs
published in this method. MDLs will vary from laboratory to
laboratory.
9.10 The laboratory may adopt additional QC practices for use with this
method. The specific practices that are most productive depend
upon the needs of the laboratory and the nature of the samples.
For example, field or laboratory duplicates may be analyzed to
assess the precision of the environmental measurements or field
reagent blanks may be used to assess contamination of samples under
site conditions, transportation, and storage.
10. CALIBRATION AND STANDARDIZATION
10.1 Establish GC operating parameters equivalent to those indicated in
Sect. 6.12. This calibration procedure employs procedural stan-
dards, i.e., fortified aqueous standards which are processed
through most of the method (Sect. 11). The GC system is calibrated
by means of the internal standard technique (Sect. 10.2). NOTE:
Calibration standard solutions must be prepared such that no
unresolved analytes are mixed together (See Table 1).
10.2 INTERNAL STANDARD CALIBRATION PROCEDURE — To use this approach,
the analyst must select one or more internal standards compatible
in analytical behavior to the compounds of interest. The analyst
must further demonstrate that the measurement of the internal
standard is not affected by method or matrix interferences. DBOB
(Sect. 7.13) has been identified as a suitable internal standard.
10.2.1 Prepare aqueous calibration standards at a minimum of three
(five are recommended) concentration levels for each method
analyte as follows: for each concentration, fill a 250-mL
volumetric flask with 240 mL of reagent water at pH 1 and
containing 20% by weight of dissolved sodium sulfate. Add
an appropriate aliquot of the primary dilution standard
(Sect. 7.16.4) and dilute to 250 mL with the same reagent
water. Process each aqueous calibration sample through the
analytical procedure beginning with Sect. 11.2, i.e., omit
the hydrolysis and cleanup step (Sect. 11.1). The lowest
calibration standard should represent analyte concentrations
near, but above, the respective MDLs. The remaining stan-
dards should bracket the analyte concentrations expected in
the sample extracts, or should define the working range of
the detector. The internal standard is added to the final 5
mL extract as specified in Sect. 11.
10.2.2 Analyze each calibration standard according to the procedure
beginning in Sect. 11.2. Tabulate response (peak height or
area) against concentration for each compound and internal
standard. Calculate the response factor (RF) for each anal-
yte and surrogate using Equation 1.
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(AS) (c,8)
RF = _ Equation 1
(A,.) (C8)
where :
As = Response for the analyte to be measured.
Ais = Response for the internal standard.
Cis = Concentration of the internal standard (ng/l).
Cs ~ Concentration of the analyte to be measured
10.2.3 If the RF value over the working range is constant (30% RSD
or less) the average RF can be used for calculations.
Alternatively, the results can be used to plot a calibration
curve of response ratios (As/Ais) vs. Cs. A data station may
be used to collect the chromatographic data, calculate
response factors and generate linear or second order regres-
sion curves.
10.2.4 The working calibration curve or RF must be verified on each
working shift by the measurement of one or more calibration
standards. A new calibration standard need not be deriva-
tized each day. The same standard extract can be used up to
14 days. If the response for any analyte varies from the
predicted response by more than +30%, the test must be re-
peated using a fresh calibration standard. If the repeti-
tion also fails, a new calibration curve must be generated
for that analyte using freshly prepared standards.
10.2.5 Verify calibration standards periodically, at least quarter-
ly is recommended, by analyzing a standard prepared from
reference material obtained from an independent source. Re-
sults from these analyses must be within the limits used to
routinely check calibration.
11. PROCEDURE
11.1 MANUAL HYDROLYSIS AND CLEAN-UP
11.1.1 Remove the sample bottles from cold storage and allow them
to equilibrate to room temperature. Acidify and add sodium
thiosulfate to blanks and QC check standards as specified
in Sect. 8.
11.1.2 Measure a 250-mL aliquot of each sample with a 250-mL
graduated cylinder and pour into a 500-mL separatory fun-
nel . Add 250 /tL of the surrogate primary dilution standard
(Sect. 7.18) to each 250-mL sample. The surrogate will be
at a concentration of 2 /tg/L. Dissolve 50 g sodium sulfate
in the sample.
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11.1.3 Add 4 ml of 6 N NaOH to each sample, seal, and shake.
Check the pH of the sample with pH paper or a pH meter; if
the sample does not have a pH greater than or equal to 12,
adjust the pH by adding more 6 N NaOH. Let the sample sit
at room temperature for 1 hr, shaking the separatory funnel
and contents periodically.
11.1.4 Add 1.5 ml methylene chloride to the graduated cylinder to
rinse the walls, transfer the methylene chloride to the
separatory funnel and extract the sample by vigorously
shaking the funnel for 2 min with periodic venting to
release excess pressure. Allow the organic layer to sepa-
rate from the water phase for a minimum of 10 min. If the
emulsion interface between layers is more than one-third
the volume of the solvent layer, the analyst must employ
mechanical techniques to complete the phase separation.
The optimum technique depends upon the sample, but may
include stirring, filtration through glass wool, centrifu-
gation, or other physical methods. Discard the methylene
chloride phase.
11.1.5 Add a second 15-mL volume of methylene chloride to the
separatory funnel and repeat the extraction procedure a
second time, discarding the methylene chloride layer.
Perform a third extraction in the same manner.
11.1.6 Drain the contents of the separatory funnel into a 500-mL
beaker. Adjust the pH to 1.0 ± 0.1 by the dropwise addi-
tion of concentrated sulfuric acid with constant stirring.
Monitor the pH with a pH meter (Sect. 6.8) or short range
(0-3) pH paper (Sect. 6.14).
11.2 SAMPLE EXTRACTION
11.2.1 Vacuum Manifold -- Assemble a manifold (Sect. 6.3) consist-
ing of 6-8 vacuum flasks with filter funnels (Sect.
6.1,6;2). Individual vacuum control, on-off and vacuum
release valves and vacuum gauges are desirable. Place the
47 mm extraction disks (Sect. 7.1) on the filter frits.
11.2.2 Add 20 mL of 10% by volume of methanol in MTBE to the top
of each disk without vacuum and allow the solvent to remain
for 2 min. Turn on full vacuum and pull the solvent
through the disks, followed by room air for 5 min.
11.2.3 Adjust the vacuum to approximately 5 in. (mercury) and add
the following in series to the filter funnel (a) 20 mL
methanol (b) 20 mL reagent water (c) sample. Do not allow
the disk to dry between steps and maintain the vacuum at 5
in.
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11.2.4 After the sample is extracted completely, apply maximum
vacuum and draw room air through the disks for 20 min.
11.2.5 Place the culture tubes (Sect. 6.4) in the vacuum tubes to
collect the eluates. Elute the disks with two each 2-mL
aliquots of 10% methanol in MTBE. Allow each aliquot to
remain on the disk for one min before applying vacuum.
11.2.6 Rinse each 500-mL beaker (Sect.11.1.6) with 4 ml of pure
MTBE and elute the disk with this solvent as in Sect.
11.2.5.
11.2.7 Remove the culture tubes and cap.
11.3 EXTRACT PREPARATION
11.3.1 Pre-rinse the drying tubes (Sect. 7.5.1) with 2 ml of MTBE.
IK 3.2 Remove the entire extract with a 5-mL pi pet and drain the
lower aqueous layer back into the culture tube. Add the
organic layer to the sodium sulfate drying tube (Sect.
7.5.1). Maintain liquid in the drying tube between this
and subsequent steps. Collect the dried extract in a 15-mL
graduated centrifuge tube or a 10-mL Kuderna-Danish tube.
11.3.3 Rinse the culture tube with an additional 1 ml of MTBE and
repeat Sect. 11.3.2.
11.3.4 Repeat step Sect. 11.3.3 and finally add a 1-mL aliquot of
MTBE to the drying tube before it empties. The final
volume should be 6-9 ml. In this form the extract is
esterified as described below.
11.4 EXTRACT ESTERIFICATION
11.4.1 Assemble the diazomethane generator (Figure 1) in a hood.
11.4.2 Add 5 ml of ethyl ether to Tube 1. Add 4 ml of Diazald
solution (Sect. 7.12) and 3 ml of 37% KOH solution (Sect.
7.15.1) to the reaction tube 2. Immediately place the exit
tube into the collection tube containing the sample
extract. Apply nitrogen flow (10 mL/min) to bubble diazo-
methane through the extract. Each charge of the generator
should be sufficient to esterify four samples. The
appearance of a persistent yellow color is an indication
that esterification is complete. The first sample should
require 30 sec to 1 min and each subsequent sample somewhat
longer. The final sample may require 2-3 min.
11.4.3 Cap each collection tube and allow to remain stored at room
temperature in a hood for 30 min. No significant fading of
the yellow color should occur during this period. Fortify
70
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each sample with 100 /tL of the internal standard primary
dilution solution (Sect. 7.17) and reduce the volume to 5.0
ml with the analytical concentrator (Sect. 6.10), a stream
of dry nitrogen, or an equivalent concentration technique.
NOTE: The excess diazomethane is volatilized from the
extract during the concentration procedure.
11.4.4 Cap the tubes and store in a refrigerator if further pro-
cessing will not be performed immediately. Analyze by
GC-ECD.
11.5 GAS CHROMATOGRAPHY
11.5.1 Sect. 6.12 summarizes the recommended GC operating
conditions. Included in Table 1 are retention times ob-
served using this method. Figures 2A and 2B illustrate the
chromatographic performance of the primary column (Sect.
6.12.1) for groups A and B of the method analytes. Other
GC columns, chromatographic conditions, or detectors may be
used if the requirements of Sect. 9.3 are met.
11.5.2 Calibrate the system daily as described in Sect. 10.
11.5.3 Inject 2 /iL of the sample extract. Record the resulting
peak size in area units.
11.5.4 If the response for any sample peak exceeds the working
range of the detector, dilute the extract and reanalyze.
11.6 IDENTIFICATION OF ANALYTES
11.6.1 Identify a sample component by comparison of its retention
time to the retention time of a reference chromatogram. If
the retention time of an unknown compound corresponds,
within limits, to the retention time of a standard com-
pound, then an analyte is considered to be identified.
11.6.2 The width of the retention time window used to make identi-
fications should be based upon measurements of actual
retention time variations of standards over the course of a
day. Three times the standard deviation of a retention
time can be used to calculate a suggested window size for a
compound. However, the experience of the analyst should
weigh heavily in interpretation of chromatograms.
11.6.3 Identification requires expert judgment when sample compo-
nents are not resolved chromatographically. When GC peaks
obviously represent more than one sample component (i.e.,
broadened peak with shoulder(s) or valley between two or
more maxima, or any time doubt exists over the identifica-
tion of a peak in a chromatogram, appropriate alternative
techniques to help confirm peak identification need to be
71
-------
employed. For example, more positive identification may be
made by the use of an alternative detector which operates
on a chemical /physical principle different from that origi-
nally used, e.g., mass spectrometry, or the use of a second
chromatography column. A suggested alternative column is
described in Sect. 6.12.2.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Calculate analyte concentrations in the sample from the response
for the analyte using the calibration procedure described in Sect.
10.
12.2 Calculate the concentration (C) in the sample using the response
factor (RF) determined in Sect. 10.2.2 and Equation 2, or determine
sample concentration from the calibration curve (Sect. 10.2.3).
(A.) (I.)
C (/ig/L) = _ Equation 2.
(A,8)(RF)(V0)
As = Response for the analyte to be measured.
Ais = Response for the internal standard.
Is = Amount of internal standard added to each
where:
extract
V0 = Volume of water extracted (L).
13. METHOD PERFORMANCE
13.1 In a single laboratory, analyte recoveries from reagent water were
determined at three concentration levels. Tables 2-4. Results were
used to determine the analyte MDLs (8) listed in Table 2.
13.2 In a single laboratory, analyte recoveries from dechlorinated tap
water were determined at two concentrations, Tables 5 and 6. In
addition, analyte recoveries were determined at two concentrations
from an ozonated surface (river) water, Tables 7 and 8, and at one
level from a high humectant surface (reservoir) water, Table 10.
Finally, a holding study was conducted on the preserved, ozonated
surface water and recovery data are presented for day 1 and day 14
of this study, Tables 8 and 9. The ozonated surface water was
chosen as the matrix in which to study analyte stability during a
14-day holding time because it was very biologically active.
14. POLLUTION PREVENTION
14.1 This method utilizes the new liquid-solid extraction technology
which requires the use of very small quantities of organic sol-
vents. This feature eliminates the hazards involved with the use
of large volumes of potentially harmful organic solvents needed for
72
-------
conventional liquid-liquid extractions. Also, mercuric chloride, a
highly toxic and environmentally hazardous chemical, has been
replaced with hydrochloric acid as the sample preservative. These
features make this method much safer and a great deal less harmful
to the environment. Some of the phenolic herbicides on the analyte
list are very difficult to methyl ate and diazomethane is still
required to derivatize these compounds.
14.2 For information about pollution prevention that may be applicable
to laboratory operations, consult "Less is Better: Laboratory
Chemical Management for Waste Reduction" available from the
American Chemical Society's Department of Government Relations and
Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.
15. WASTE HANAGEHENT
15.1 Due to the nature of this method, there is little need for waste
management. No large volumes of solvents or hazardous chemicals
are used. The matrices of concern are finished drinking water or
source water. However, the Agency requires that laboratory waste
management practices be conducted consistent with all applicable
rules and regulations, and that laboratories protect the air,
water, and land by minimizing and controlling all releases from
fume hoods and bench operations. Also, compliance is required with
any sewage discharge permits and regulations, particularly the
hazardous waste identification rules and land disposal restric-
tions. For further information on waste management, consult "The
Waste Management Manual for Laboratory Personnel," also available
from the American Chemical Society at the address in Sect. 14.2.
16. REFERENCES
1. ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82,
"Standard Practice for Preparation of Sample Containers and for
Preservation," American Society for Testing and Materials,
Philadelphia, PA, p. 86, 1986.
2. Giam, C.S., H.S. Chan, and G.S. Nef. "Sensitive Method for Deter-
mination of Phthalate Ester Plasticizers in Open-Ocean Biota
Samples," Analytical Chemistry. 47. 2225 (1975).
3. Giam, C.S., and H.S. Chan. "Control of Blanks in the Analysis of
Phthalates in Air and Ocean Biota Samples," U.S. National Bureau of
Standards, Special Publication 442, pp. 701-708, 1976.
4. "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
73
-------
5. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910), ™
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
6. "Safety in Academic Chemistry Laboratories," American Chemical
Society Publication, Committee on Chemical Safety, 3rd Edition,
1979.
7. ASTM Annual Book of Standards, Part 11, Volume 11.01, D3370-82,
"Standard Practice for Sampling Water," American Society for Testing
and Materials, Philadelphia, PA, p. 130, 1986.
8. Glaser, J.A., Foerst, D.L., McKee, G.D., Quave, S.A., and Budde,
W.L., "Trace Analyses for Wastewaters," Environ. Sci. Techno!. 1981,
15, 1426-1435.
9. 40 CFR, Part 136, Appendix B.
74
-------
17. TABLES. DIAGRAMS. FLOWCHARTS AND VALIDATION DATA
TABLE 1. RETENTION DATA
Analvte
3,5-Dichlorobenzoic acid
2, 4-Dichlorophenyl acetic acid
Dicamba
Dichlorprop
2,4-D
4 , 4 ' -Di bromooctaf 1 uorobi phenyl
Pentachl orophenol
Silvex
5-Hydroxydi camba
2,4,5-T
2,4-DB
Dinoseb
Bentazon
Picloram
Dacthal
Acifluorfen
Grouo3
A
(SA) A,B
B
A
B
(IS) A,B
A
B
B
A
B
A
B
B
A
B
Retention Time,
Primary
16.72
19.78
20.18
22.53
23.13
24.26
25.03
25.82
26.28
26.57
27.95
28.03
28.70
29.93
31.02
35.62
min."
Confirmation
18.98
22.83
23.42
25.90
27.01
26.57
27.23
29.08
30.18
30.33
31.47
33.02
33.58
35.90
34.32
40.58
a Analytes were divided into two groups during method development to
avoid chromatographic overlap.
b Columns and chromatographic conditions are described in Sect. 6.12.
75
-------
TABLE 2. SINGLE LABORATORY RECOVERY, PRECISION DATA
AND METHOD DETECTION LIMIT WITH FORTIFIED
REAGENT WATER - LEVEL 1
Analvte
Acifluorfen
Bentazon
2,4-D
2,4-DB
Dacthalb
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
Pentachlorophenol
Picloram
2,4,5-T
2,4,5-TP
Fortified
Cone.
ua/L
0.50
2.50
0.25
2.50
0.25
0.75
1.25
0.25
0.50
0.75
0.25
0.75
0.25
0.25
Mean9
Recovery
%
70
70
96
79
96
109
126
106
87
90
103
95
116
98
Relative
Std. Dev.
%
21
11
38
12
16
11
24
15
22
12
18
15
18
9
MDL
ua/L
0.25
0.63
0.28
0.72
0.13
0.28
1.23
0.13
0.28
0.25
0.16
0.35
0.16
0.06
a Based on the analyses of seven replicates.
b Measurement includes the mono- and diacid metabolites.
76
-------
TABLE 3. SINGLE LABORATORY RECOVERY AND PRECISION DATA
FOR FORTIFIED REAGENT WATER - LEVEL 2
Analvte
Acifluorfen
Bentazon
2,4-D
2,4-DB
Dacthal"
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
Pentachl orophenol
Picloram
2,4,5-T
2,4,5-TP
Fortified
Cone.
ua/L
0.80
4.0
0.40
4.0
0.40
1.20
2.00
0.40
0.80
1.20
0.40
1.20
0.40
0.40
Mean3
Recovery
%
61
81
96
90
96
109
126
76
87
90
66
68
116
105
Relative
Std. Dev.
%
27
8
38
13
16
11
24
21
22
12
26
21
18
7
a Based on the analyses of six-seven replicates.
b Measurement includes the mono- and diacid metabolites.
77
-------
TABLE 4. SINGLE LABORATORY RECOVERY AND PRECISION DATA
FOR FORTIFIED REAGENT WATER - LEVEL 3
Analvte
Aclfluorfen
Bentazon
2,4-D
2,4-DB
Dactha1b
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
Pentachlorophenol
Picloram
2,4,5-T
2,4,5-TP
Fortified
Cone.
ua/L
2.0
10.0
1.0
10.0
1.0
3.0
5.0
1.0
2.0
3.0
1.0
3.0
1.0
1.0
Mean8
Recovery
%
59
68
90
74
60
75
62
97
63
77
69
66
64
68
Relative
Std. Dev.
%
13
8
20
6
10
9
18
17
10
8
11
9
15
8
a Based on the analyses of six-seven replicates.
b Measurement includes the mono- and diacid metabolites.
78
-------
TABLE 5. SINGLE LABORATORY RECOVERY AND PRECISION DATA
FOR FORTIFIED, DECHLORINATED TAP WATER - LEVEL 1
Anal vte
Acifluorfen
Bentazon
2,4-D
2,4-DB
Dactha1b
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
Pentachl orophenol
Picloram
2,4,5-T
2,4,5-TP
Fortified
Cone.
ua/l
0.50
2.50
0.25
2.50
0.25
0.75
1.25
0.25
0.50
0.75
0.25
0.75
0.25
0.25
Mean8
Recovery
%
117
96
59c
112
101
91
103
218d
134
90
91
76
118
99
Relative
Std. Dev.
%
21
12
55
15
10
14
15
37
10
14
8
28
16
10
a Based on the analyses of six-seven replicates.
b Measurement includes the mono- and diacid metabolites.
c 2,4-D background value was 0.29 pg/L.
d Probable interference.
7.9
-------
TABLE 6. SINGLE LABORATORY RECOVERY AND PRECISION DATA
FOR FORTIFIED, DECHLORINATED TAP WATER - LEVEL 2
Analvte
Acifluorfen
Bentazon
2,4-D
2,4-DB
Dacthalb
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
Pentachlorophenol
Picloram
2,4,5-T
2,4,5-TP
2, 4-Dichlorophenyl acetic acidc
Fortified
Cone.
ua/L
2.0
10.0
1.0
10.0
1.0
3.0
5.0
1.0
2.0
3.0
1.0
3.0
1.0
1.0
1.0
Mean3
Recovery
% .
150
112
90
111
118
86
111
88
121
96
96
132
108
115
120
Relative
Std. Dev.
%
7
9
16
10
8
10
5
30
6
6
6
12 :
10
7
19
a Based on the analyses of six-seven replicates.
b Measurement includes the mono- and diacid metabolites.
c Surrogate analyte.
80
-------
TABLE 7. SINGLE LABORATORY RECOVERY AND PRECISION DATA
FOR FORTIFIED, OZONATED SURFACE WATER - LEVEL 1
Analvte
Acifluorfen
Bentazon
2,4-D
2,4-DB
Dacthalb
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
Pentachlorophenol
Picloram
2,4,5-T
2,4,5-TP
2, 4-Dichlorophenyl acetic acidc
Fortified
Cone.
UQ/L
0.50
2.50
0.25
2.50
0.25
0.75
1.25
0.25
0.50
0.75
0.25
0.75
0.25
0.25
0.25
Mean3
Recovery
%
172
92
127
154
113
107
100
115
134
89
110
109
102
127
72
Relative
Std. Dev.
%
14
22
13
19
17
13
17
20
28
13
22
27
19
8
31
a Based on the analyses of six-seven replicates.
b Measurement includes the mono- and diacid metabolites.
c Surrogate analyte.
81
-------
TABLE 8. SINGLE LABORATORY RECOVERY AND PRECISION DATA FOR FORTIFIED,
OZONATED SURFACE WATER - LEVEL 2, STABILITY STUDY DAY lc
Anal vte
Acifluorfen
Bentazon
2,4-D
2,4-DB
Dacthalb
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
Pentachlorophenol
Picloram
2,4,5-T
2,4,5-TP
2, 4-Dichlorophenyl acetic acidd
Fortified
Cone.
ua/L
2.0
10.0
1.0
10.0
1.0
3.0
5.0
1.0
2.0
3.0
1.0
3.0
1.0
1.0
1.0
Mean*
Recovery
%
173
122
126
130
116
109
115
116
116
121
118
182
112
122
110
Relative
Std. Dev.
%
11
7
10
7
11
9
11
11
9
9
10
14
9
10
26
a Based on the analyses of six-seven replicates.
b Measurement includes the mono- and diacid metabolites.
c Samples preserved at pH - 2.0.
d Surrogate analyte.
82
-------
TABLE 9. SINGLE LABORATORY RECOVERY AND PRECISION DATA FOR FORTIFIED,
OZONATED SURFACE MATER - LEVEL 2, STABILITY STUDY DAY 14C
Analvte
Acifluorfen
Bentazon
2,4-D
2,4-DB
Dactha1b
Dicamba
3,5-Dichlorobenzoic acid
Dlchlorprop
Dinoseb
5-Hydroxydicamba
Pentachlorophenol
Picloram
2,4, 5-T
2,4,5-TP
2, 4-Dichlorophenyl acetic acidd
Fortified
Cone.
ua/L
2.0
10.0
1.0
10.0
1.0
3.0
5.0
1.0
2.0
3.0
1.0
3.0
1.0
1.0
1.0
Mean3
Recovery
%
151
97
84
128
116
103
81
107
118
20
94
110
113
113
87
Relative
Std. Dev.
%
18
9
11
10
7
9
12
11
7
14
7
32
8
11
6
a Based on the analyses of six-seven replicates.
b Measurement includes the mono- and diacid metabolites.
c Samples preserved at pH =2.0.
d Surrogate analyte.
83
-------
TABLE 10. SINGLE LABORATORY RECOVERY AND PRECISION DATA FOR
FORTIFIED, HIGH HUH1C CONTENT SURFACE WATER
Analvte
Acifluorfen
Bentazon
2,4-D
2,4-DB
Dacthalb
Dlcamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydi camba
Pentachl orophenol
Picloram
2,4,5-T
2,4,5-TP
Fortified
Cone.
ua/L
2.0
10.0
1.0
10.0
1.0
3.0
5.0
1.0
2.0
3.0
1.0
3.0
1.0
1.0
Mean8
Recovery
%
120
87
59
80
100
76
87
110
97
82
70
124
101
80
Relative
Std. Dev.
%
13
11
7
14
6
9
4
22
6
9
5
9
4
6
a Based on the analyses of six-seven replicates.
b Measurement includes the mono- and diacid metabolites.
84
-------
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FIGURE 1.DIAZOMETHANE GENERATOR
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METHOD 548.1 DETERMINATION OF ENDOTHALL IN DRINKING WATER BY
ION-EXCHANGE EXTRACTION, ACIDIC METHANOL HETHYLATION
AND GAS CHROMATOGRAPHY/MASS SPECTROMETRY
Revision 1.0
August 1992
Jimmie W. Hodgeson
Jeffrey Collins (Technology Applications, Incorporated)
W. J. Bashe (Technology Applications, Incorporated)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
89
-------
METHOD 548.1
DETERMINATION OF ENDOTHALL IN DRINKING WATER BY
ION EXCHANGE EXTRACTION, ACIDIC METHANOL METHYLATION AND
GAS CHROMATOGRAPHY/MASS SPECTROMETRY
1. SCOPE AND APPLICATION
1.1 This method is for the identification and simultaneous measurement
of endothall in drinking water sources and finished drinking
water. The following analyte can be determined by this method:
Chemical Abstract Services
Analvte Registry Number
Endothall 145-73-3
1.2 This is a gas chromatographic/mass spectrometric (GC/MS) method.
However, a flame ionization detector (FID) may be utilized for the
determination, but must be supported by an additional analysis
using a confirmatory gas chromatographic column.
1.3 The method detection limit (1) (MDL, defined in Sect. 13) for
endothall is listed in Table 1 for both GC/MS and FID. The MDL
may differ from the listed value depending upon the nature of
interferences in the sample matrix. In particular, water sources
containing high levels of dissolved calcium, magnesium and sulfate
may require sample dilution before extraction to obtain adequate
endothall recovery. Guidelines (Sect. 4.2 and Sect. 11.2.1) are
provided on levels of these ions above which dilution is recom-
mended, as well as appropriate dilution factors.
1.4 In this ion exchange liquid-solid extraction procedure, endothall
may be esterified directly in the elution solvent, acidic metha-
nol.
1.5 The method performance data provided in this method were obtained
using both a GC/MS system .and a gas chromatograph with a flame
ionization detector (FID). Modern GC/MS instruments have sensi-
tivities at least equivalent to the FID. If the analyst has
access to a GC/MS system meeting the specifications described in
Sect. 6.10, it should be as the primary means of identification
and measurement.
2. SUMMARY OF METHOD
2.1 Liquid-solid extraction (LSE) cartridges containing an intermedi-
ate strength, primarily tertiary amine anion exchanger are mounted
on a vacuum manifold and conditioned with appropriate solvents.
LSE disks may be used instead of cartridges of all quality control
90
-------
criteria specified in Sect. 9 are met. A 100-mL sample is ex-
tracted and the analyte is eluted with 8-mL of acidic methanol.
After addition of a small volume of methylene chloride as a co-
solvent, the dimethyl ester of endothall is formed within 30 min
with modest heating (50°C). After addition of salted reagent
water, the ester is partitioned into 8-10 ml of methylene chlo-
ride. The extract volume is reduced to 1 ml with nitrogen purge
for a concentration factor of 100. The extract is analyzed by
GC/MS or GC/FID with a megabore capillary column.
3. DEFINITIONS
3.1 INTERNAL STANDARD (IS) -- A pure analyte(s) added to a sample,
extract, or standard solution in known amount(s) and used to
measure the relative responses of other method analytes and
surrogates that are components of the same sample or solution.
The internal standard must be an analyte that is not a sample
component.
3.2 SURROGATE ANALYTE (SA) -- A pure analyte(s), which is extremely
unlikely to be found in any sample, and which is added to a sample
aliquot in known amount(s) before extraction or other processing
and is measured with the same procedures used to measure other
sample components. The purpose of the SA is to monitor method
performance with each sample.
3.3 LABORATORY DUPLICATES (LD1 and LD2) — Two aliquots of the same
sample taken in the laboratory and analyzed separately with
identical procedures. Analyses of LD1 and LD2 indicate the
precision associated with laboratory procedures, but not with
sample collection, preservation, or storage procedures.
3.4 FIELD DUPLICATES (FD1 and FD2) — Two separate samples collected
at the same time and place under identical circumstances and
treated exactly the same throughout field and laboratory proce-
dures. Analyses of FD1 and FD2 give a measure of the precision
associated with sample collection, preservation and storage, as
well as with laboratory procedures.
3.5 LABORATORY REAGENT BLANK (LRB) — An aliquot of reagent water or
other blank matrix that is treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The
LRB is used to determine if method analytes or other interferences
are present in the laboratory environment, the reagents, or the
apparatus.
3.6 FIELD REAGENT BLANK (FRB) — An aliquot of reagent water or other
blank matrix that is placed in a sample container in the labora-
tory and treated as a sample in all respects, including shipment
to the sampling site, exposure to sampling site conditions,
storage, preservation, and all analytical procedures. The purpose
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of the FRB is to determine if method analytes or other interfer-
ences are present in the field environment.
3.7 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) — A solution of one
or more method analytes, surrogates, internal standards, or other
test substances used to evaluate the performance of the instrument
system with respect to a defined set of method criteria.
3.8 LABORATORY FORTIFIED BLANK (LFB) -- An aliquot of reagent water or
other blank matrix to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed exactly
like a sample, and its purpose is to determine .whether the method-
ology is in control, and whether the laboratory is capable of
making accurate and precise measurements.
3.9 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) — An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the
LFM corrected for background concentrations.
3.10 STOCK STANDARD SOLUTION (SSS) — A concentrated solution contain-
ing one or more method analytes prepared in the laboratory using
assayed reference materials or purchased from a reputable commer-
cial source.
3.11 PRIMARY DILUTION STANDARD SOLUTION (PDS) — A solution of several
analytes prepared in the laboratory from stock standard solutions
and diluted as needed to prepare calibration solutions and other
needed analyte solutions.
3.12 CALIBRATION STANDARD (CAL) — A solution prepared from the primary
dilution standard solution or stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.13 QUALITY CONTROL SAMPLE (QCS) — A solution of method analytes in
known concentrations which is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally pre-
pared test materials.
4. INTERFERENCES
4.1 Method interference may be caused by contaminants in solvents,
reagents, glassware, and other sample processing hardware that
lead to discrete artifacts and/or elevated baselines in the
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chromatograms. All of these materials must be routinely demon-
strated to be free from interferences under the analytical condi-
tions by analyzing laboratory reagent blanks as described in Sect.
9.2.
4.1.1 Glassware must be scrupulously cleaned (2) as soon as
possible after use by rinsing with the last solvent used in
it. This should be followed by detergent washing with hot
water, and rinses with tap water and distilled water. It
should then be drained dry, and heated in a laboratory oven
at 400°C for several hours before use. Solvent rinses with
methanol may be substituted for the oven heating. After
drying and cooling, glassware should be stored in a clean
environment to prevent any accumulation of dust or other
contaminants.
4.1.2 The use of high purity reagents and solvents is absolutely
necessary to minimize interference problems. Purification
of solvents by distillation in all-glass systems immediate-
ly prior to use may be necessary.
4.2 The major potential interferences in this ion-exchange procedure
are other naturally occurring ions in water sources, namely,
dissolved calcium, magnesium and sulfate. These are the only ions
thus far demonstrated to be interferences when present at concen-
trations possibly occurring in drinking water sources. For
example, the sources identified in Tables 3 and 4 contained
elevated concentrations of these ions and reduced recoveries were
observed. Sulfate is an effective counter ion, and displaces
endothall from the column when present at high concentrations. On
the other hand, both calcium and magnesium complex the endothall
anion, which then is no longer available in ionic form for ion-
exchange extraction. Table 4 illustrates that sample dilution or
the addition of ethylenediamine tetraacetic acid for complexing
the cations, or a combination of the two, may be used. Figure 1
illustrates quantitatively the separate effects of these ions on
recovery.
4.3 The extent of interferences that may be encountered using this
method has not been fully assessed. Although the GC conditions
described allow for a unique resolution of endothall, other matrix
components may interfere. Matrix interferences may be caused by
contaminants that are coextracted from the sample. Matrix inter-
ferences will vary considerably from source to source, depending
on the nature of the matrix being sampled. A distinct advantage
of this method is that the anion exchange cartridge provides an
effective clean-up mechanism for many potential organic matrix
interferences. Many neutral and basic organics retained by the
column are removed by the methanol wash step of Sect. 11.2.3. The
most probable matrix interferences are other organic acids or
phenols retained by the column. For the cartridge to effectively
serve for both sample clean-up and analyte extraction, it is
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critical that the conditioning steps described in Sect. 11.2.1 be
followed exactly.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this
method has not been precisely defined; however, each chemical
compound should be treated as a potential health hazard. From
this viewpoint, exposure to these chemicals must be minimized.
The laboratory is responsible for maintaining a current awareness
file of OSHA regulations regarding the safe handling of the
chemical specified in this method. A reference file of material
data handling sheets should also be made available to all person-
nel involved in the chemical analysis. Additionally references to
laboratory safety are available (3-5).
6. EQUIPMENT AND SUPPLIES
6.1 SAMPLING EQUIPMENT (for discrete or composite sampling). Amber
glass bottles (250 mL or larger) fitted with screw caps lined with
Teflon. If amber bottles are not available, protect samples from
light. The container must be washed, rinsed with methanol, and
dried before use to minimize contamination.
6.2 SEPARATOR FUNNELS — 125 mL, with Teflon stopcocks, ground glass
or Teflon stoppers.
6.3 SCREW CAP — 125 x 13 mm, culture tubes. Screw caps should have
Teflon liners.
6.4 Graduated 15 mL centrifuge tubes with #13 ground glass stoppers
6.5 PASTEUR PIPETS — Glass, disposable 5-3/4" length
6.6 BALANCE — Analytical, capable of weighing to .0001 g.
6.7 Six or twelve position analytical concentrator (Organomation, N-
EVAP model # 111/6917 or equivalent).
6.8 pH METER
6.9 GAS CHROMATOGRAPH — Analytical system complete with GC suitable
for flame ionization detection, split/splitless capillary injec-
tion temperature programming, and all required accessories includ-
ing syringes, analytical columns, gases and strip chart recorder.
A data system is recommended for measuring peak areas. An auto
injector is recommended for improved precision of analysis.
6.10 GAS CHROMATOGRAPH/MASS SPECTROMETER/DATA SYSTEM (GC/MS/DS) —
6.10.1 The GC must be capable of temperature programming and be
equipped for split/splitless or on-column capillary injec-
tion. The injection tube liner should be quartz and about
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3 mm in diameter. The injection system must not allow the
analytes to contact hot stainless steel or other metal
surfaces that promote decomposition.
6.10.2 The GC/MS interface should allow the capillary column or
transfer line exit to be placed within a few mm of the ion
source. Other interfaces, for example, the open split
interface, are acceptable as long as the system has ade-
quate sensitivity (See Sect. 10 for calibration require-
ments).
6.10.3 The mass spectrometer must be capable of electron ioniza-
tion at a nominal electron energy of 70 eV and of scanning
from 45 to 450 amu with a complete scan cycle time (includ-
ing scan overhead) of 1.5 sec or less. (Scan cycle time =
Total MS data acquisition time in sec divided by total
number of scans in the chromatogram). The spectrometer
must produce a mass spectrum that meets all criteria in
Table 5 when 5 to 10 ng of DFTPP is introduced into the GC.
An average spectrum across the DFTPP GC peak may be used to
test instrument performance.
6.10.4 An interfaced data system is required to acquire, store,
reduce, and output mass spectral data. The computer soft-
ware must have the capability of processing stored data by
recognizing a GC peak within any given retention time
window, comparing the mass spectra from the GC peak with
spectral data in a user-created data base, and generating a
list of tentatively identified compounds with their reten-
tion times and scan numbers. The software must also allow
integration of the ion abundance of any specific ion be-
tween specified time or scan number limits, calculation of
response factors as defined in Sect. 10.3.6 (or construc-
tion of a second or third order regression calibration
curve), calculation of response factor statistics (mean and
standard deviation), and calculation of concentrations of
analytes as described in Sect. 12.
6.11 GC COLUMNS
6.11.1 GC/MS — DB5, 30 m x 0.25 mm, 0.25 /im film thickness
6.11.2 FID Primary -- RTX Volatiles, 30 m x 0.53 mm. ID, 2.0 urn
film thickness, Restek Catalog No. 10902.
6.11.3 FID Confirmation — DBS, 30 m x 0.32 mm ID, 0.25 urn film
thickness
6.12 LIQUID-SOLID EXTRACTION VACUUM SYSTEM — May be used.
6.13 8 mL LIQUID-SOLID EXTRACTION CARTRIDGES WITH FRITS — Also avail-
able from a number of commercial suppliers. Appropriate liquid-
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solid extraction disks may also be used in this method if equiva- ^^
lent or better quality assurance data can be demonstrated (See
Sect. 9).
6.14 LIQUID-SOLID EXTRACTION 70 mL RESERVOIRS AND ADAPTERS — Baxter
Catalog # 9442 (adapter catalog # 9430) or equivalent.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 REAGENT WATER — Reagent water is defined as water in which an
interference is not observed at the endothall method detection.
7.1.1 A Mi Hi pore Super-Q Water System or its equivalent may be
used to generate deionized reagent water. Distilled water
that has been charcoal filtered may also be suitable.
7.2 METHANOL — Pesticide quality.
7.3 METHYLENE CHLORIDE — Pesticide quality or equivalent.
7.4 SODIUM SULFATE-ACS GRANULAR -- Heat in a shallow tray for 4 hrs at
400°C to remove phthlates and other interfering organic substances
or extract with methylene chloride in a Soxhlet apparatus for 48
hrs.
7.5 10% SULFURIC ACID IN METHANOL — Using extreme caution, slowly
dissolve reagent grade sulfuric (10% v/v) acid in methanol.
7.6 SODIUM HYDROXIDE (NAOH) 1 N — Dissolve 4 g ACS grade in reagent
water and dilute up to 100 mL in a 100 mL volumetric flask.
7.7 10% SODIUM SULFATE IN REAGENT WATER — Dissolve 100 g sodium
sulfate in reagent water and dilute to volume in a 1-L volumetric
flask.
7.8 BIOREX 5 ANION EXCHANGE RESIN — BioRad Laboratories Catalog
No. 140-7841.
7.9 DISODIUM ETHYLENEDIAMINE TETRAACETATE (EDTA) — Certified ACS
Fisher or equivalent.
7.10 ENDOTHALL, MONOHYDRATE -- Available as neat material from Ultra
Scientific, North Kingston, RI or as a concentrated solution from
NSI Environmental Solutions, Research Triangle Park, NC.
7.11 ACENAPTHENE-dlO — Available from MSD Isotopes or Cambridge
Chemicals.
7.12 STOCK STANDARD SOLUTIONS
7.12.1 Endothall — 50 /ig/mL in methanol
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7.12.2 Acenaphthene-dlO — 500 /ag/mL in methanol. Dissolve 25 mg
(approximately 32.2 #L) Acenapthnene-dlO in 50 ml methanol.
Prepare a working standard at 10 ng/mL by a 1:50 dilution
of the stock standard.
7.12.3 Decafluorotriphenylphosphine (DFTPP) — 5 /jg/mL.
7.12.4 Stock standard solutions must be replaced after 6 months,
or sooner if comparison with check standards indicates a
problem.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 Grab samples must be collected in glass containers. Conventional
sampling practices should be followed, except that the bottle must
not be prewashed with sample before collection. Composite samples
should be collected in refrigerated glass containers. Automatic
sampling equipment must be as free as possible of plastic tubing
and other potential sources of contamination.
8.2 SAMPLE PRESERVATION
8.2.1 If residual chlorine is present, add 80 mg of sodium thio-
sulfate per liter of sample to the sample bottle prior to
collecting the sample.
8.2.2 After adding the sample to the bottle containing the sodium
thiosulfate, seal the bottle and shake vigorously for 1
min.
8.2.3 The samples must be iced or refrigerated at 4°C from the
time of collection until extraction and analysis. Endo-
thall is not known to be light sensitive, but excessive
exposure to light,and heat should be avoided.
8.2.4 A graphical representation of the results of a 14-day
holding stability study on endothall in three different
water matrices is presented in Figure 2. These matrices
were a dechlorinated tap water sample, a filtered river
water sample containing considerable biological activity
and the same river water biologically preserved at pH 2.
These data indicate that the samples may be held for 7 days
before extraction under the conditions of Sect. 8.2.3.
Endothall appears to be biologically stable over 7 days.
However, the chemical and biological stability of endothall
may be matrix dependent. The analyst may verify analyte
stability in the matrix of interest by conducting appropri-
ate holding studies. Samples with unusually high biologi-
cal activity should be acidified to pH 1.5 to 2.0 with 1:1
HC1:H20.
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8.3 EXTRACT STORAGE — Sample extracts should be stored in the dark at
4°C or less. A maximum extract holding time of 14 days is recom-
mended.
9. QUALITY CONTROL
9.1 Each laboratory that uses this method is required to operate a
formal quality control (QC) program. The minimum QC requirements
are initial demonstration of laboratory capability, analysis of
laboratory reagent blanks, laboratory fortified blanks, laboratory
fortified matrix samples and QC check standards.
9.2 LABORATORY REAGENT BLANKS — Before processing any samples, the
analyst must demonstrate that all glassware and reagent interfer-
ences are under control. Each time a set of samples is analyzed
or reagents are changed, a laboratory reagent blank must be
analyzed. For this method, the blank matrix is filtered reagent
water. If within the retention time window of endothall, the
reagent blank produces a peak which prevents the measurement of
endothall, determine the source of contamination and eliminate the
interference before processing samples.
9.3 INITIAL DEMONSTRATION OF CAPABILITY
9.3.1 Select a representative fortified concentration for
endothall. Prepare a methanol solution containing
endothall at 1000 times the selected concentration. The
concentrate must be prepared independently from the stan-
dards used to prepare the calibration curve (Sect. 10.2).
With a syringe, add 100 fil of the concentrate to each of
four to seven 100-mL aliquots of reagent water and analyze
each aliquot according to procedures in Sect. 11.
9.3.2 Calculate the mean percent recovery (R), the relative
standard deviation of the recovery (RSD in Table 2), and
the MDL (1). The mean recovery must fall in the range of R
± 20% using the values for R (Recovery) for reagent water
(Table 2). The standard deviation should be less than 30%.
If these acceptance criteria are met, performance is
acceptable and sample analysis may begin. If either of
these criteria fails, initial demonstration of capability
should be repeated until satisfactory performance has been
demonstrated.
9.3.3 The initial demonstration of capability is used primarily
to preclude a laboratory from analyzing unknown samples by
a new, unfamiliar method prior to demonstrating a basic
level of skill at performing the technique. As laboratory
personnel gain experience with this method the quality of
the data should improve beyond the requirements stated in
Sect. 9.3.2.
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9.4 The analyst is permitted to modify GC columns or GC conditions to
improve separations or lower analytical costs. Each time such
method modifications are made, the analyst must repeat the proce-
dures in Sect. 9.3.
9.5 ASSESSING THE INTERNAL STANDARD — In using the IS calibration
procedure, the analyst is expected to monitor the IS response
(peak area) of all samples during each analysis day. The IS
response for any sample chromatogram should not deviate from the
most recent calibration check standard IS response by more than
30%.
9.5.1 If a deviation of greater than 30% is encountered for a
sample, reinject the extract.
9.5.1.1 If acceptable IS response is achieved for the
reinjected extract, then report the results for
that sample.
9.5.1.2 If a deviation of greater than 30% is obtained
for the reinjected extract, analysis of the
sample should be repeated beginning with Sect.
11, provided the sample is still available.
Otherwise, report results obtained from the
reinjected extract, but annotate as suspect.
9.5.2 If consecutive samples fail the IS response acceptance
criterion, immediately analyze a medium calibration check
standard.
9.5.2.1 If the check standard provides a response fac-
tor (RF) within 20% of the predicted value,
then follow procedures itemized in Sect. 9.5.1
for each sample failing the IS response crite-
rion.
9.5.2.2 If the check standard provides a response fac-
tor (RF) which deviates more than 20% from the
predicted value, then the analyst must recali-
brate, as specified in Sect. 10.2.
9.6 ASSESSING LABORATORY PERFORMANCE
9.6.1 The laboratory must analyze at least one laboratory forti-
fied blank (LFB) per sample set (all samples extracted
within a 24-hr period). The fortifying concentration in
the LFB should be 10 to 20 times the MDL. Calculate accu-
racy as percent recovery (R,-). If the recovery falls
outside the control limits (See Sect. 9.6.2), the system is
judged out of control, and the source of the problem must
be identified and resolved before continuing analyses.
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9.6.2 Until sufficient LFB data become available, usually a
minimum of results from 20 to 30 analyses, the laboratory
should assess its performance against the control limits
described in Sect. 9.3.2. When sufficient laboratory
performance data become available, develop control limits
from the mean percent recovery (R) and standard deviation
(S) of the percent recovery. These data are used to estab-
lish upper and lower control limits as follows:
Upper Control Limit = R + 3S
Lower Control Limit = R - 3S
After each group of five to ten new recovery measurements,
control limits should be recalculated using only the most
recent 20 to 30 data points.
9.6.3 Each laboratory should periodically determine and document
its detection limit capabilities for endothall.
9.6.4 Each quarter the laboratory should analyze quality control
samples (if available). If criteria provided with the QCS
are not met, corrective action should be taken and docu-
mented.
9.7 ASSESSING ANALYTE RECOVERY
9.7.1 The laboratory must add a known fortified concentration to
a minimum of 10% of samples or one fortified matrix sample
per set, whichever is greater. The fortified concentration
should not be less than the background concentration of the
sample selected for fortification. The fortified concen-
tration should be the same as that used for the LFB (Sect.
9.6). Over time, samples from all routine sample sources
should be fortified.
9.7.2 Calculate the percent recovery for endothall, corrected for
background concentrations measured in the unfortified
sample, and compare these values to the control limits
established in Sect. 9.6.2 for the analyses of LFBs.
9.7.3 If the recovery falls outside the designated range and the
laboratory performance for that sample set is shown to be
in control (Sect. 9.6), the recovery problem encountered
with the fortified sample is judged to be matrix related,
not system related. The result in the unfortified sample
must be labelled suspect/matrix to inform the data user
that the results are suspect due to matrix effects.
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10. CALIBRATION AND STANDARDIZATION
10.1 PREPARATION OF CALIBRATION STANDARDS
10. 1.1 Calibration standards as dimethyl esters are prepared by
addition of aliquots of the eridothall stock standard (Sect.
7.12.1) to the esterification reaction mixture, consisting
of 8 mL of 10% H2S04/methanol and 6 ml of methylene chlo-
ride in the screw cap culture tubes (Sect. 6.3). The
standards are then esterified and partitioned into the
organic phase according to Sect. 11.4. Prepare endothall
acid standards equivalent to aqueous standards at 100, 50,
25 and 5 itg/l by addition of the following aliquots of the
stock standard solution (Sect. 7.12) to the esterification
reaction mixture - 200 #L, 100 nl, 50 /tL and 10 /iL. By way
of illustration, 200 /iL of the 50 jtg/mL stock contains 10
/ig of endothall . When dissolved in 100 ml of water, the
aqueous concentration is 100
10.1.2 Process each standard as described in Sect. 11.4.1 and
Sect. 11.4.2. The internal standard is added as described
in Sect. 11.4.3. Triplicate samples should be prepared at
each concentration level.
10.2 Demonstration and documentation of acceptable initial calibration
are required before any samples are analyzed and intermittently
throughout sample analyses as dictated by results of continuing
calibration checks. After initial calibration is successful, a
continuing calibration check is required at the beginning of each
8-hr period during which analyses are performed. Additional
periodic calibration checks are good laboratory practice.
10.3 INITIAL CALIBRATION
10.3.1 Calibrate the mass spectrometer with calibration compounds
and procedures prescribed by the manufacturer with any
modifications necessary to meet the requirements in Sect.
10.3.2.
10.3.2 Inject into the GC a 1-or 2-/tL aliquot of the 5 ng//iL DFTPP
solution and acquire a mass spectrum that includes data for
m/z = 45-450. Use GC conditions that produce a narrow (at
least five scans per peak) symmetrical peak. If the spec-
trum does not meet all criteria (Table 5), the MS must be
retuned to meet all criteria before proceeding with cali-
bration. An average spectrum across the GC peak may be
used to evaluate the performance of the system.
10.3.3 Inject a l-fil aliquot of a medium concentration calibration
solution, for example 50 /ig/L, and acquire and store data
from m/z 45-450 with a total cycle time (including scan
overhead time) of 1.5 sec or less. Cycle time should be
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adjusted to measure at least five or more spectra during
the elution of the GG peak. Figure 3 illustrates a total
ion chromatogram and mass spectrum of endothall and the
internal standard, acenaphthene-dlO, using the prescribed
conditions.
10.3.4 If all performance criteria are met, inject a 1-/JL aliquot
of each of the other calibration solutions using the same
GC/MS conditions.
10.3.5 Calculate a response factor (RF) for endothall for each
calibration solution by use of the internal standard re-
sponse as expressed below. This calculation is supported
in acceptable GC/MS data system software (Sect. 6.10.4),
and many other software programs. The RF is a unitless
number, but units used to express quantities of analyte and
internal standard must be equivalent.
RF =
-(Ax) (Q,-s>
Ais=
where: Ax = integrated abundance of the quantitation
ion of the analyte (m/z 183).
integrated abundance of the quantitation
ion internal standard (m/z 164).
Qx - quantity of analyte injected in ng or
concentration units.
Qjs= quantity of internal standard injected
in ng or concentration units.
10.3.5.1 Calculate the mean RF from the analyses of the
calibration solutions. Calculate the standard
deviation (SD) and the relative standard devi-
ation (RSD) from each mean: RSD = 100 (SD/M).
If the RSD of any analyte or surrogate mean RF
exceeds 30%, either analyze additional aliquots
of appropriate calibration solutions to obtain
an acceptable RSD of RFs over the entire con-
centration range or take action to improve
GC/MS performance. See Sect. 10.4.5 for possi-
ble remedial actions.
10.3.6 As an alternative to calculating mean response factors and
applying the RSD test, use the GC/MS data system software
or other available software to generate a linear or second
order regression calibration curve.
10.4 Continuing calibration check. Verify the MS tune and initial
calibration at the beginning of each 8-hr work shift during which
analyses are performed using the following procedure.
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10.4.1 Inject a 1-0L aliquot of the 5 ng//*L DFTPP solution and
acquire a mass spectrum that includes data for m/z 45-450.
If the spectrum does not meet all criteria (Table 5), the
MS must be retuned to meet all criteria before proceeding
with the continuing calibration check.
10.4.2 Inject a 1-/JL aliquot of a medium concentration calibration
solution and analyze with the same conditions used during
the initial calibration.
10.4.3 Determine that the absolute area of the quantitation ion of
the internal standard has not decreased by more than 30%
from the area measured in the most recent continuing cali-
bration check, or by more than 50% from the area measured
during initial calibration. If the area has decreased by
more than these amounts, adjustments must be made to re-
store system sensitivity. These adjustments may require
cleaning of the MS ion source, or other maintenance as
indicated in Sect. 10.4.5, and recalioration. Control
charts are useful aids in documenting system sensitivity
.changes.
10.4.4 Calculate the RF for endothall from the data measured in
the continuing calibration check. The RF must be within
30% of the mean value measured in the initial calibration.
Alternatively, if a linear or second order regression is
used, the concentration measured using the calibration
curve must be within 30% of the true value of the concen-
tration in the medium calibration solution. If these
conditions do not exist, remedial action must be taken
which may require repeating the initial calibration.
10.4.5 Some possible remedial actions: major maintenance such as
cleaning an ion source, cleaning quadrupole rods, etc.
require returning to the initial calibration step.
10.4.5.1 Check and adjust GC and/or MS operating condi-
tions; check the MS resolution, and calibrate
the mass scale.
10.4.5.2 Clean or replace the splitless injection liner;
silanize a new injection liner.
10.4.5.3 Flush the GC column with solvent according to
manufacturer's instructions.
10.4.5.4 Break off a short portion (about 1 meter) of
the column from the end near the injector; or
replace GC column. This action will cause a
slight change in retention times.
10.4.5.5 Prepare fresh CAL solutions* and repeat the
initial calibration step.
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10.4.5.6 Clean the MS ion source and rods (if a quad-
rupole).
10.4.5.7 Replace any components that allow analytes to
come into contact with hot metal surfaces.
10.4.5.8 Replace the MS electron multiplier or any other
faulty components.
11. PROCEDURE
11.1 PREPARATION OF ANION EXCHANGE CARTRIDGES
11.1.1 Prepare a 50% (v/v) slurry of Bio-Rex 5 resin and reagent
water.
11.1.2 Attach the required number of 8-mL extraction cartridges
(Sect. 6.13) to the vacuum manifold (Sect. 6.12), and
insert bottom fritted disks into each cartridge.
11.1.3 Fill the cartridges completely with Bio-Rex 5 slurry. Draw
off excess water with vacuum. The final wet resin bed
height should be 3.5 ±0.1 cm. Adjust the height by adding
more slurry and repeating procedure, or add more reagent
water to reservoir and remove excess resin slurry.
11.1.4 After the bed heights are adjusted to 3.5 cm and with
excess water removed under vacuum, insert a fritted disk on
top of the resin bed. The fritted disk should press firmly
into the resin and be horizontal to the reservoir to pre-
vent sample channeling around the disk. Fill the cart-
ridges with reagent water and draw half of the water into
the resin. Maintain the resin cartridges in this condition
until ready for use.
NOTE: The use of liquid-solid extraction disks instead of cart-
ridges is permissible as long as all the quality control
criteria specified in Sect. 9 of this method are met.
11.2 SAMPLE PREPARATION
11.2.1 As discussed above (Sect. 1.3 and Sect. 4.2), reduced
recoveries will be observed if the sample contains elevated
levels of Call, Mgll or sulfate. If facilities are avail-
able, measure the concentrations of these ions. Figure 1
graphically presents analyte recovery versus individual ion
concentration. Reduced recoveries may be anticipated when
the combined Call + Mgll exceeds approximately 100 mg/L or
sulfate exceeds approximately 250 mg/L. If measurement of
ion concentration is not feasible, determine the actual
recovery for a laboratory fortified sample matrix as de-
scribed in Sect. 9.7. In the event of anticipated or mea-
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sured low recoveries, treat the sample as described in
Sect, 11.2.2.
11.2.2 For samples containing moderately high levels of these
ions, add 186 mg of EDTA (Sect. 7.9) per 100-mL sample
(0.005 M). The treated ground water characterized in Table
3 is an example of a matrix successfully treated this way.
For samples containing very high levels of sulfate, sample
dilution may be required in addition to the EDTA. The
western surface water characterized in Table 3 (ca. 2000
mg/L sulfate) was successfully analyzed after dilution by a
factor of 10 and the addition of 75 mg EDTA per 100 ml of
the diluted sample (0.002 M). Samples containing interme-
diate levels of sulfate can be analyzed with smaller dilu-
tion factors. Guidelines on dilution factors and EDTA
addition are given below.
SULFATE. mo/L DILUTION FACTOR ADDED EDTA. mg/100 ml
< 250 1:1 186
250 - 500 1:2 125
500 - 1250 1:5 75
> 1250 1:10 75
NOTE: Dilution should not be employed if adequate recovery
is attained by the addition of EDTA alone.
11.2.3 The addition of EDTA results in a large reagent peak near
the end of the temperature program. Therefore, complete
the entire program described in Table 1.
11.2.4 If the ionic nature of the samples being processed is
completely unknown, the analyst as an option may routinely
dilute all samples by a factor of 10 and add EDTA as above.
However, the analyst should be able to demonstrate reagent
water MDLs of 2 jttg/L or lower. In this event the MDL will
be 20 jug/L or less for the diluted sample, still a factor
of 5 below the regulated maximum contaminant level.
11.3 SAMPLE EXTRACTION
11.3.1 Attach the 70-mL reservoir to the resin cartridge with the
adapter (Sect. 6.14).
11.3.2 Condition the resin cartridge by drawing the following
reagents through the cartridge in the following order:
1. 10 ml methanol
2. 10 ml reagent water
3. 10 ml 10% H2S04 in methanol
4. 10 ml reagent water
5. 20 ml 1 N NaOH
6. 20 ml reagent water
105
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Do not allow the cartridge to become dry between steps.
Draw each reagent through the cartridge at a rate of 10
mL/min. Leave a 1-cm layer of reagent water over the resin
bed.
11.3.3 Fill the 70-mL reservoir with 60 ml of the sample. Adjust
sample flow rate to 3 mL/min. Add the balance of sample
when needed to prevent the reservoir from going dry.
11.3.4 After the sample passes through the cartridge, remove the
70-mL reservoir and the adapter. Draw 10 mL of methanol
through the resin cartridge. Make sure that any visible
water inside the cartridge dissolves in methanol. Next
draw room air through the cartridge for 5 min under a
vacuum of 10-20 in. Hg. Position the culture tube (Sect.
6.3) inside the manifold to collect the eluent.
11.3.5 Elute the cartridge with 8 mL of 10% H2S04 in methanol,
followed by 6 mL of methylene chloride under vacuum over a
1 min period.
11.4 SAMPLE DERIVATIZATION, PARTITION AND ANALYSIS
11.4.1 Cap the culture tube and hold at 50°C for 1 hr in a heating
block or water bath. Remove from heat and allow the tube
to cool for 10 min. ;
11.4.2 Pour the contents of the culture tube into a 125-mL separa-
tory funnel. Rinse the tube with two x 0.5 mL aliquots of
methylene chloride and add the rinsings to the separatory
funnel. Add 20 mL of 10% sodium sulfate in reagent water
to the separatory funnel. Shake the funnel three times
vigorously, venting with the stopcock, and then shake
vigorously for an additional 15 sec. After the phases have
separated, drain the lower organic layer into a 15-mL
graduated centrifuge tube (Sect. 6.4). Repeat the extrac-
tion procedure above with two additional 2-mL aliquots of
methylene chloride, adding the organic phase to the centri-
fuge tube each time.
11.4.3 Fortify the extract with 250 p.1 of the internal standard
working solution (Sect. 7.12.2) and concentrate to a final
volume of 1.0 mL, using the N-EVAP (Sect. 6.7) and dry
nitrogen.
11.4.4 Inject 2 nl of the concentrated extract (Sect. 11.4.3) and
analyze by GC/MS using the conditions described in Table 1.
This table includes the retention time and MDL that were
obtained under these conditions. A sample total ion chro-
matogram of endothall and d-10 acenaphthene illustrating
retention times, and the mass spectrum of the dimethylated
endothall are shown in Figure 3. Other columns, chromato-
106
-------
graphic conditions, or detectors may be used if the re-
quirements of Sect. 9.3 are met.
11.4.5 If the peak area exceeds the linear range of the calibra-
tion curve, a smaller sample volume should be used.
11.5 IDENTIFICATION OF THE ANALYTE
11.5.1 Identify endothall by comparison of its mass spectrum
(after background subtraction) to a reference spectrum in a
user created spectral library. The GC retention time of
the sample component should be within 10 sec of the reten-
tion time of endothall in the latest calibration standard.
If a FID is used, identifications should be confirmed by
retention time comparisons on the second GC column (Table
1).
11.5.2 In general, all ions present above 10% relative abundance
in the mass spectrum of the standard should be present in
the mass spectrum of the sample component and should agree
within absolute 20%. For example, if an ion has a relative
abundance of 30% in the standard spectrum, its abundance in
the sample spectrum should be in the range of 10-50%.
However, the experience of the analyst should weigh heavily
in the interpretation of spectra and chromatograms.
11.5.3 Identification requires expert judgement when sample compo-
nents are not resolved chromatographically, that is, when
GC peaks from interferences are present. When endothall
coelutes with an interference, indicated by a broad peak or
a shoulder on the peak, the identification criteria can
usually be met, but the endothall spectrum will contain
extraneous ions contributed by the coeluting interfering
compound.
12. DATA ANALYSIS AND CALCULATIONS
12.1 When using GC/MS, complete chromatographic resolution is not
necessary for accurate and precise measurements of analyte concen-
trations if unique ions with adequate intensities are available
for quantitation. However, when using FID, complete resolution is
essential.
12.1.1 Calculate endothall concentration.
(Ax)(Qis)
(Ais) RF V
where:
C« = (
lig/L in the water sample.
107
Cx = concentration of endothall in
-------
Ax = integrated abundance of the quantitation ^^
ion of endothall (m/z 183) in the
sample.
Ais = integrated abundance of the quantitation
ion of the internal standard (m/z 164)
in the sample.
Qis = total quantity (in micrograms) of
internal standard added to the water
sample.
V = original water sample volume in liters.
RF = mean response factor endothall from the
initial calibration.
12.1.2 Alternatively, use the GC/MS data system software or other
available proven software to compute the concentration of
the endothall from the linear calibration or the second
order regression curves.
12.1.3 Calculations should utilize all available digits of preci-
sion, but final reported concentrations should be rounded
to an appropriate number of significant figures (one digit
of uncertainty). Experience indicates that three signifi-
cant figures may be used for concentrations above 99 jtg/L,
two significant figures for concentrations between 1-99
/jg/L, and one significant figure for lower concentrations.
13. METHOD PERFORMANCE
13.1 METHOD DETECTION LIMITS — The MDL is defined as the minimum
concentration of a substance that can be measured and reported
with 99% confidence that the value is above the background level
(1). The MDLs listed in Table 1 were obtained using reagent water
for detection by GC/MS and FID.
13.2 In a single laboratory study on fortified reagent water and ground
water matrices, the mean recoveries and relative standard devia-
tions presented in Table 2 were obtained. Table 3 provides the
concentrations of Call, Mgll and sulfate for two high ionic
strength drinking water sources studied. Table 4 presents mean
recovery data for these fortified sources with and without the
addition of EDTA and/or sample dilution.
14. POLLUTION PREVENTION
14.1 This method utilizes the new liquid-solid extraction technology
which requires the use of very little organic solvent thereby
eliminating the hazards involved with the use of large volumes of
organic solvents in conventional liquid-liquid extractions. It
also uses acidic methanol as the derivatizing reagent in place of
the highly toxic and explosive diazomethane. These features make
this method much safer for the analyst to employ and a great deal
less harmful to the environment..
108
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15. HASTE MANAGEMENT
15.1 Due to the nature of this method, there is very little need for
waste management. No large volumes of solvents or hazardous
chemicals are used. The matrices are drinking water or source
water, and can be discarded down the sink.
16. REFERENCES
1. 40 CFR Part 136, Appendix B.
2. ASTM Annual Book of Standards, Part 31, D3694-78. "Standard
Practices for Preparation of Sample Containers and for Preserva-
tion of Organic Constituents," American Society for Testing and
Materials, Philadelphia, PA.
3. "Carcinogens-Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, August 1977.
4. "OSHA Safety and Health Standards, General Industry," (29CFR1910),
Occupational Safety and Health Administration, OSHA 2206, (Re-
vised, January 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical
Society Publication, Committee on Chemical Safety, 3rd Edition,
1979.
109
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17. TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA
TABLE 1. RETENTION TIMES AND METHOD DETECTION LIMITS
RETENTION TIME, min. METHOD DETECTION LIMIT
COMPOUND Column A Column B Column C GC/MS Jtg/Lci) FID
Endothall 16.02 19.85 18.32 1.79 0.7
dlO-Acenaphthene 14.69
<1> Based on 7 replicate analyses of a reagent water fortified at 2
Column A: DB-5 fused silica capillary for GC/MS, 30 m x 0.25 mm, 0.25 micron
film
MS inlet temperature = 200°C
Injector temperature « 200°C
Temperature Program: Hold 5 min at 80°C, increase to 260°C at
10°/min, hold 10 min.
Column B: FID primary column, RTX Volatiles, 30 m x 0.53 mm I.D., 2 micron
film thickness.
Detector temperature = 280°C
Injector Temperature = 200°C
Carrier gas velocity = 50 cm/sec.
Temperature program: Same as Column A
Column C: FID confirmation column, DB-5, 30 m x 0.32 mm ID, 0.25 micron film
Carrier Gas velocity = 27 cm/sec
Same injector, detector and temperature program as Column A.
110
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TABLE 2. ENDOTHALL METHOD DEVELOPMENT DATA
Matrix
Reagent Water
Reagent Water
Reagent Water
Ground Water3
Ground Water
Ground Water
Cone.
M9/L
2
10
100
2
10
100
Recovery1
%
101
86
95
91
82
88
RSD2
%
10
10
3
25
14
6
1 Based on analysis of 7 replicates.
2 Relative Standard Deviation.
3 High Humic Content Florida Ground Water.
Ill
-------
TABLE 3. MATRIX ANALYSES1
Western Surface,
Major Ion mg/L
Ca 330
Mg 132
i
Na 400
Sulfate 1850
Eastern Ground
mg/L
122
33
23
102
1 Determination by inductively coupled plasma - mass spectrometry for
cations and ion chromatography for sulfate.
112
-------
TABLE 4. ENDOTHALL METHOD VALIDATION DATA
Matrix
WS3
WS - 1/104
WS - 1/10
EG5
EG
EG - 1/5
Cone.
/*9/L
25
50
50
25
25
25
EDTA1
Mole/L
0
0
0.002
0
0.005
0
Recovery2
%
9
66
88
43
97
97
RSD
%
19
13
5
17
6
5
1
Ethylenediamine Tetraacetic Acid
2 Based on 7 Replicates
3 WS - Treated Western Surface Water
4 Dilution Factor in Reagent Water
5 WG - Eastern Ground Water
113
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TABLE 5. ION ABUNDANCE CRITERIA FOR BIS(PERFLUOROPHENYL)PHENYL
PHOSPHINE (DECAFLUOROTRIPHENYLPHOSPHINE, DFTPP)
Mass
Relative Abundance
Criteria _
Purpose of Checkpoint1
51 10-80% of the base peak
68 <2% of mass 69
70 <2% of mass 69
127 10-80% of the base peak
197 <2% of mass 198
198 base peak or >50% of 442
199 5-9% of mass 198
275 10-60% of the base peak
365 >1% of the base peak
441 Present and < mass 443
442 base peak or >50% of 198
443 15-24% of mass 442
low mass sensitivity
low mass resolution
low mass resolution
low-mid mass sensitivity
mid-mass resolution
mid-mass resolution and sensitivity
mid-mass resolution and isotope ratio
mid-high mass sensitivity
baseline threshold
high mass resolution
high mass resolution and sensitivity
high mass resolution and isotope ratio
1A11 ions are used primarily to check the mass measuring accuracy of the mass
spectrometer and data system, and this is the most important part of the perfor-
mance test. The three resolution checks, which include natural abundance isotope
ratios, constitute the next most important part of the performance test. The
correct setting of the baseline threshold, as indicated by the presence of low
intensity ions, is the next most important part of the performance test. Finally,
the ion abundance ranges are designed to encourage some standardization to
fragmentation patterns.
114
-------
h-
8
1"
^
S <
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CM
= O
o
o
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o
o
O
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o
o
en
o o o o o o
O 01 00 N 10 IO
O O O
-t cn CM
o o
o
o
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w
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H
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w
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115
-------
CO
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5
o
X
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O
ill
1
LLI
o
X
co
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H
O
AU3AOO3U
116
-------
sundance
250000
200000
150000
100000 •
50000 -
TIC: 716ML10.D
O
T3
til
y^\J A^r/v^vAj^J W^ys/\
rime •>
' ' I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' ' ' 1 ' ' ' ' I ' r
13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50
tounggnge
3000
2500
2000
1500-
1000-
500
0
K/Z ->
Scan
6
59
i
III
III!
bill
[I HI
7
1
i •
60
8
Illi
Lit
I8
1737 (16.024 min) : 716ML10.D (-)
5
80
i;
113
9
1
H
5
till li i! i i
'in ii H i.i
hi
i !i> i
100 120
3
155
L27
1-
ll
1 1
,5
,J|L
\l\\
183
II I I ilk
nil
140 160 180
FIGURE 3. ENDOTHALL GC/MS
UPPER: TOTAL ION CHROMATOGRAPHY ENDOTHALL: 16.02 MIN., 10 NG
LOWER: RELATIVE ION ABUNDANCE
117
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-------
METHOD 549.1 DETERMINATION OF DIQUAT AND PARAQUAT IN DRINKING WATER BY
LIQUID-SOLID EXTRACTION AND HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY WITH ULTRAVIOLET DETECTION
Revision 1.0
August 1992
J. W. Hodgeson
W. J. Bashe (Technology Applications Inc.)
James W. Eichelberger
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
119
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METHOD 549.1
DETERMINATION OF DIQUAT AND PARAQUAT IN DRINKING MATER
BY LIQUID-SOLID EXTRACTION AND HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY WITH ULTRAVIOLET DETECTION
1. SCOPE AND APPLICATION
1.1 This is a high performance liquid chromatography (HPLC) method for
the determination of diquat (I,r-ethylene-2j2'-bipyridilium
dibromide salt) and paraquat (l,r-dimethyl-4,4'- bipyridilium
dichloride salt) in drinking water sources and finished drinking
water (1,2).
Chemistry Abstract Services
Analvtes Registry Number
Diquat 85-00-7
Paraquat 1910-42-5
1.2 When this method is used to analyze unfamiliar samples, compound
identification should be supported by at least one additional
qualitative technique. The use of a photodiode array detector
provides ultraviolet spectra that can be used for the qualitative
confirmation.
1.3 The method detection limits (MDL, defined in Sect. 13) (3) for
diquat and paraquat are listed in Table 1.
1.4 This method is restricted to use by or under the supervision of
analysts experienced in the use of HPLC. Each analyst must
demonstrate the ability to generate acceptable results with this
method using the procedure described in Sect. 9.3.
2. SUMMARY OF METHOD
2.1 A measured volume of liquid sample, approximately 250 mL, is
adjusted to pH 10.5. The sample is extracted using a C8 solid
sorbent cartridge or a C-8 disk which has been specially prepared
for the reversed-phase, ion-pair mode. The disk or cartridge is
eluted with 4.5 mL of an acidic aqueous solvent. After the ion-
pair reagent is added to the eluate, the final volume is adjusted
to 5.0 mL. Liquid chromatographic conditions are described which
permit the separation and measurement of diquat and paraquat in
the extract by absorbance detection at 308 nm and 257 nm,
respectively. A photodiode array detector is utilized to provide
simultaneous detection and confirmation of the method analytes
(1,2).
120
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2.2 Analysis of diquat and paraquat is complicated by their ionic
nature. Glassware should be deactivated to prevent loss of
analytes. The substitution of polyvinylchloride (PVC) for glass
is recommended.
3. DEFINITIONS
3.1 LABORATORY REAGENT BLANK (LRB) — An aliquot of reagent water or
other blank matrix that is treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The
LRB is used to determine if method analytes or other interferences
are present in the laboratory environment, the reagents, or the
apparatus.
3.2 FIELD REAGENT BLANK (FRB) — An aliquot of reagent water or other
blank matrix that is placed in a sample container in the
laboratory and treated as a sample in all respects, including
shipment to the sampling site, exposure to sampling site
conditions, storage, preservation and all analytical procedures.
The purpose of the FRB is to determine if method analytes or other
interferences are present in the field environment.
3.3 LABORATORY FORTIFIED BLANK (LFB) — An aliquot of reagent water or
other blank matrix to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed exactly
like a sample, and its purpose is to determine whether the
methodology is in control, and whether the laboratory is capable
of making accurate and precise measurements.
3.4 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) — An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the
LFM corrected for background concentrations.
3.5 STOCK STANDARD SOLUTION (SSS) — A concentrated solution
containing one or more method analytes prepared in the laboratory
using, assayed reference materials or purchased from a reputable
commercial source.
3.6 PRIMARY DILUTION STANDARD SOLUTION (PDS) ~ A solution of several
analytes prepared in the laboratory from stock standard solutions
and diluted as needed to prepare calibration solutions and other
needed analyte solutions.
3.7 CALIBRATION STANDARD (CAL) ~ A solution prepared from the primary
dilution standard solution and stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
121
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used to calibrate the instrument response with respect to analyte
concentration.
3.8 QUALITY CONTROL SAMPLE (QCS) — A solution of method analytes of
known concentration which is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally
prepared test materials.
3.9 EXTERNAL STANDARD (ES) -- A pure analyte(s) that is measured in an
experiment separate from the experiment used to measure the
analyte(s) in the sample. The signal observed for a known
quantity of the pure external standard(s) .is used to calibrate the
instrument response for the corresponding analyte(s). The
instrument response is used to calculate the concentrations of the
analyte(s) in the sample.
4. INTERFERENCES
4.1 Method interference may be caused by contaminants in solvents,
reagents, glassware, and other sample processing hardware that
lead to discrete artifacts and/or elevated baselines in the
chromatogram. All of these materials must be routinely
demonstrated to be free from interferences under the conditions of
the analysis by analyzing laboratory reagent blanks as described
in Sect. 9.2.
4.1.1 Glassware must be scrupulously cleaned (4). Clean all
glassware as soon as possible after use by rinsing with
the last solvent used in it. This should be followed by
detergent washing with hot water and rinses with tap water
and distilled water. It should then be drained dry and
heated in a laboratory oven at 130°C for several hours
before use. Solvent rinses with methanol may be
substituted for the oven heating. After drying and
cooling, glassware should be stored in a clean environment
to prevent any accumulation of dust or other contaminants.
4.1.2 Before the initial use of all glassware, the procedure
described in Sect. 4.1.1 should be followed. Silanization
of all glassware which will come in contact with the
method analytes is necessary to prevent adsorption of the
diquat and paraquat cations onto glass surfaces (7.13).
4.1.3 Plasticware should be washed with detergent and rinsed in
tap water and distilled water. It should be drained dry
before use.
4.1.4 The use of high purity reagents and solvents helps to
minimize interference problems. Purification of solvents
by distillation in all-glass systems may be required.
122
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4.2 Interferences may be caused by contaminants that are coextracted
from the sample. The extent of matrix interferences will vary
considerably from source to source. Because of the selectivity of
the detection system used here, no interferences have been
observed in the matrices studied. If interferences occur, some
additional cleanup may be necessary.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this
method has not been precisely defined. Each chemical compound
should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be minimized. The
laboratory is responsible for maintaining a current awareness file
of OSHA regulations regarding the safe handling of the chemicals
specified in this method. A reference file of material data
handling sheets should also be made available to all personnel
involved in the chemical analysis.
EQUIPMENT AND SUPPLIES
6.1 SAMPLING EQUIPMENT, discrete or composite sampling.
6.1.1 Grab sample bottle — Amber polyvinylchloride (PVC) high
density, 1-L, fitted with screw caps. If amber bottles
are not available, protect samples from light. The
container must be washed, rinsed with deionized water, and
dried before use to minimize contamination.
6.2 GLASSWARE
6.2.1 Volumetric flask — 5 mL, silanized
6.2.2 Autosampler vials — 4 mL, silanized
6.3 BALANCE — analytical, capable of accurately weighing 0.0001 g
6.4 pH METER -- capable of measuring pH to 0.1 units
6.5 HPLC APPARATUS
6.5.1 Isocratic pumping system, constant flow (Waters M6000A
HPLC pump or equivalent).
6.5.2 Manual injector or automatic injector, capable of
delivering 200 juL.
6.5.3 Analytical column -- any column which produces results
equal to or better than those listed below may be used.
123
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6.5.3.1 Hamilton PRP-1, (5 urn, 150 mm x 4.1 mm), or
equivalent.
6.5.3.2 Guard column, C8 packing
6.5.4 Column Oven (Matron, Model CH-30 and controller, Model
TC-50, or equivalent).
6.5.5 Photodiode array detector (LKB 2140 Rapid Spectral
Detector or equivalent). Any detector which has the
capability to switch between 257 nm and 308 nm may be
used.
6.5.6 Data system — Use of a data system to report retention
times and peak areas is recommended but not required.
6.6 EXTRACTION APPARATUS
6.6.1 Liquid solid extraction cartridges, C8, 500 mg or
equivalent.
6.6.2 Liquid solid extraction system (Baker - 10 SPE, or
equivalent).
6.6.3 Liquid solid extraction disks (C-8 Empore, 47 mm, or
equivalent).
6.6.4 Liquid solid extraction system, Empore, 47 mm, 6 position
manifold (Varian Associates or equivalent). If a disk
extraction manifold is not used, all glassware used
instead should be silanized or substituted with
polypropylene ware.
6.6.5 Vacuum pump, 100 VAC, capable of maintaining a vacuum of
8-10 mm of Hg.
6.6.6 Membrane Filters, 0.45 /zm pore-size, 47 mm diameter,
Nyl on.
7. REAGENTS AND STANDARDS
7.1 DEIONIZED WATER — Water which has been processed through a series
of commercially available filters including a particulate filter,
carbon bed, ion exchange resin and finally a bacterial filter to
produce deionized, reagent grade water. Any other source of
reagent water may be used provided the requirements of Sect. 9 are
met.
7.2 METHANOL — HPLC grade or higher purity
7.3 ORTHOPHOSPHORIC ACID, 85% (w/v) — Reagent grade
124
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7.4 DIETHYLAMINE -- Reagent grade
7.5 CONCENTRATED SULFURIC ACID — ACS reagent grade
7.6 SODIUM HYDROXIDE — Reagent grade
7.7 CONCENTRATED HYDROCHLORIC ACID, 12 N ~ Reagent grade
7.8 CETYL TRIMETHYL AMMONIUM BROMIDE, 95% — Aldrich Chemical
7.9 SODIUM THIOSULFATE — Reagent grade
7.10 1-HEXANESULFONIC ACID, sodium salt, 98%, Aldrich Chemical
7.11 1-HEPTANESULFONIC ACID, sodium salt, 98%, Aldrich Chemical
7.12 AMMONIUM HYDROXIDE, ACS, Concentrated
7.13 'SYLON CT — Silanization solution, Supelco
7.14 REAGENT SOLUTIONS
7.14.1 Conditioning solution A — Dissolve 0.500 g of cetyl
trimethyl ammonium bromide and 5 mL of concentrated
ammonium hydroxide in 500 mL of deionized water and dilute
to 1000 mL in volumetric flask.
7.14.2 Conditioning solution B — Dissolve 10.0 g of 1-hexanesul-
fonic acid, sodium salt and 10 mL of concentrated ammonium
hydroxide in 250 mL of deionized water and dilute to 500
mL in volumetric flask.
7.14.3 Sodium hydroxide solution, 10% w/v — Dissolve 50 g of
sodium hydroxide into 400 mL of deionized water and dilute
to 50.0 mL in a volumetric flask.
7.14.4 Hydrochloric acid, 10% v/v — Add 50 mL of concentrated
hydrochloric acid to 400 mL of deionized water and dilute
to 500 mL in a volumetric flask.
7.14.5 Disk or cartridge eluting solution — Add 13.5 mL of
orthophosphoric acid and 10.3 mL of diethylamine to 500 mL
of deionized water and dilute to 1000 mL in a volumetric
flask.
7.14.6 Ion-pair concentrate — Dissolve 3.75 g of 1-hexanesul-
fonic acid in 15 mL of the disk or cartridge eluting
solution and dilute to 25 mL in a volumetric flask with
the disk eluting solution.
125
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7.15 STOCK STANDARD SOLUTIONS
7.15.1 Diquat dibromide and Paraquat dichloride.
7.15.2 Stock dlquat and paraquat solutions (1000 tng/L). Dry
diquat and paraquat salts in an oven at 110°C for 3 hr.
Cool in a desiccator. Repeat process to a constant
weight. Weigh 0.1968 g of dried diquat salt and 0.1770 g
of dried paraquat salt and place into a silanized glass or
polypropylene 100-mL volumetric flask. Dissolve with
approximately 50 ml of deionized water. Dilute to the
mark with deionized water.
7.15.3 The salts used in preparing the stock standards (Sect.
7.15.2) were taken to be diquat dibromide monohydrate and
paraquat dichloride tetrahydrate (5). The drying
procedure described in Sect. 7.15.2 will provide these
hydration levels.
7.16 MOBILE PHASE -- Make mobile phase by adding the following to 500
mL of deionized water: 13.5 mL of orthophosphoric acid; 10.3 mL of
diethylamine; 3.0 g of 1-hexanesulfonic acid, sodium salt. Mix
and dilute with deionized water to a final volume of 1 L.
8. SAHPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Grab samples must be collected in either amber PVC high density
bottles or silanized amber glass bottles. Conventional sampling
procedures should be followed (6). Automatic sampling equipment
must be free as possible of adsorption sites which might extract
the sample.
8.2 The samples must be iced or refrigerated at approximately 4°C from
the time of collection until extraction. The analytes are light-
sensitive, particularly diquat.
8.3 Samples which are known or suspected to contain residual chlorine
must be preserved with sodium thiosulfate (100 mg/L). Samples
which are biologically active must be preserved by adding sulfuric
acid to pH 2 to prevent adsorption of method analytes by the
humectant material.
8.4 Analyte stability over time may depend on the matrix tested.
Analyte stability in representative drinking water matrices is
listed in Table 3. All samples must be extracted within 7 days of
collection. Extracts must be analyzed within 21 days of
extraction (1).
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9. QUALITY CONTROL
9.1 Minimum quality control (QC) requirements are initial
demonstration of laboratory capability, analysis of laboratory
reagent blanks, laboratory fortified matrix samples, and
laboratory fortified blanks. The laboratory must maintain records
to document the quality of the data generated. Additional quality
control practices are recommended.
9.2 LABORATORY REAGENT BLANKS (LRB) — Before processing any samples,
the analyst must analyze a LRB to demonstrate that all deactivated
glassware or plasticware, and reagent interferences are reasonably
free of contamination. In addition, each time a set of samples is
extracted or reagents are changed, a LRB must be analyzed. If
within the retention time window (Sect. 11.3.2) of the analyte of
interest, the LRB produces a peak that would prevent the
determination of that analyte, determine the source of
contamination and eliminate the interference before processing
samples.
9.3 INITIAL DEMONSTRATION OF CAPABILITY
9.3.1 Prepare laboratory fortified blanks (LFBs) at analyte
concentrations of 100 tig/I. With a syringe, add 25 nl of
the stock standard (Sect. 7.14.2) to at least four 250 mL
aliquots of reagent water and analyze each aliquot
according to procedures beginning in Sect. 11.2.
9.3.2 Calculate the recoveries, relative standard deviation
(RSD), arid the MDL (3). The recovery (R) values should be
within ± 30% of the R values listed in Table 2 for at
least three of four consecutive samples. The RSD of the
mean recovery should be less than 30%. The MDL must be
sufficient to meet the requirements of the SDWA
regulations. For analytes that fail this critera, initial
demonstration procedures should be repeated.
9.3.3 The initial demonstration of capability is used primarily
to preclude a laboratory from analyzing unknown samples
via a new, unfamiliar method prior to obtaining some
experience with it. As laboratory personnel gain
experience with this method the quality of the data should
improve beyond the requirements stated in Sect. 9.3.2.
9.4 The analyst is permitted to use other HPLC columns, HPLC
conditions, or detectors to improve separations or lower
analytical costs. Each time such method modifications are made,
the analyst must repeat the procedures in Sect. 9.3.
127
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9.5 LABORATORY FORTIFIED BLANKS
9.5.1 The laboratory must analyze at least one laboratory
fortified blank (LFB) sample per sample set (all samples
extracted within a 24-hr period). The fortified
concentration of each analyte in the LFB should be 10
times the MDL. If the recovery of either analyte falls
outside the control limits (Sect. 9.5.2), that analyte is
judged out of control, and the source of the problem must
be identified and resolved before continuing analyses.
9.5.2 Until sufficient data become available, usually a minimum
of results from 20 to 30 analyses, the laboratory should
assess laboratory performance against the control limits
in Sect. 9.3.2. When sufficient internal performance data
become available, develop control limits from the mean
percent recovery (R) and standard deviation (S ) of the
percent recovery. These data are used to establish upper
and lower control limits as follows:
UPPER CONTROL LIMIT = R + 3S_
LOWER CONTROL LIMIT = R - 3S
r
After each five to ten new recovery measurements, new
control limits should be calculated using only the most
recent 20-30 data points.
9.6 LABORATORY FORTIFIED SAMPLE MATRIX
9.6.1 The laboratory must add a known fortified concentration to
a minimum of 10% of the samples or one fortified sample
per set, whichever is greater. The fortified
concentration should not be less than the background
concentration of the original sample. Ideally, the
fortified concentration should be the same as that used
for the laboratory fortified blank (Sect. 9.5). Over
time, samples from all routine samples sources should be
fortified.
9.6.2 Calculate the accuracy as percent recovery (R) for each
analyte, corrected for background concentrations measured
in the original sample, and compare these values to the
control limits established in Sect. 9.5.2 from the
analyses of LFBs.
9.6.3 If the recovery of any such analyte falls outside the
designated range, and the laboratory performance for that
analyte is shown to be in control (Sect. 9.5), the
recovery problem encountered with the dosed sample is
judged to be matrix related, not system related. The
result for that analyte in the original sample is labeled
128
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suspect/matrix to inform the data user that the results
are suspect due to matrix effects.
9.7 QUALITY CONTROL SAMPLES (QCS) — Each quarter the laboratory
should analyze one or more QCS. If criteria provided with the QCS
are not met, corrective action should be taken and documented.
9.8 The laboratory may adopt additional quality control practices for
use with this method. The specific practices that are most
productive depend upon the needs of the laboratory and the nature
of the samples. For example, field or laboratory duplicates may
be analyzed to assess the precision of the environmental
measurements or field reagent blanks may be used to assess
contamination of samples under site conditions, transportation and
storage.
10. CALIBRATION AND STANDARDIZATION
10.1 Establish HPLC operating conditions indicated in Table 1. The
chromatographic system can be calibrated using the external
standard technique.
10.2 In order to closely match calibration standards to samples,
process standards by the following method: Using C-8 disks or C.
cartridges conditioned according to Sect. 11.2.1, pass 250 mL of
reagent water through the disk or cartridge and discard the water.
Dry the disk or cartridge by passing 5 mL of methanol through it.
Discard the methanol. Pass 4.0 mL of the eluting solution through
the disk or cartridge and catch in a 5 mL silanized volumetric
flask. Fortify the eluted solution with 100 p,L of the ion-pair
concentrate and with 500 /iL of the stock standard and dilute to
the mark with eluting solution. This provides a 10:1 dilution of
the stock. Use serial dilution of the calibration standard by the
same method to achieve lower concentration standards.
10.3 Analyze a minimum of three calibration standards prepared by the
procedure described in Sect. 10.2 utilizing the HPLC conditions
given in Table 1. From full spectral data obtained, extract the
308 nm chromatographic trace for diquat and the 257 nm trace for
paraquat. Integrate and record the analyte peak areas. Any
mathematical manipulations performed to aid in data reduction must
be recorded and performed on all sample chromatograms. Tabulate
the peak area against quantity injected. The results may be used
to prepare calibration curves for diquat and paraquat.
10.4 The working calibration curve must be verified on each working day
by measurement of a calibration check standard, at the beginning
of the analysis day. These check standards should be at two
different concentration levels to verify the calibration curve.
For extended periods of analysis (greater than 8 hr), it is
strongly recommended that check standards be interspersed with
129
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samples at regular intervals. If the response for any analyte
varies from the predicted response by more than ±20%, the test
must be repeated using a fresh calibration standard. If the
results still do not agree, generate a new calibration curve.
11. PROCEDURE
11.1 SAMPLE CLEANUP -- Cleanup procedures
relatively clean sample matrix. The
recommended in this method have been
various sample types. If particular
of an alternative cleanup procedure,
that the recovery of the analytes is
by the method.
may not be necessary for a
cleanup procedures
used for the analysis of
circumstances demand the use
the analyst must demonstrate
within the limits specified
11.1.1 If the sample contains particulates, or the complexity is
unknown, the entire sample should be passed through a
0.45 jum Nylon membrane filter into a silanized glass or
plastic container.
11.1.2 Store all samples at 4°C unless extraction is to be
performed immediately.
11.2 CARTRIDGE EXTRACTION
11.2.1 Before sample extraction, the C-8 extraction cartridges
must be conditioned by the following procedure.
11.2.1.1 Place a C8 cartridge on the cartridge
extraction system manifold.
11.2.1.2 Elute the following solutions through the
cartridge in the stated order. Take special
care not to let the column go dry. The flow
rate through the cartridge should be
approximately 10 mL/min.
11.2.1.2.1 Cartridge Conditioning Sequence
a. Deionized water, 5 mL
b. Methanol, 5 mL
c. Deionized water, 5 mL
d. Conditioning Solution A, 5 mL
e. Deionized water, 5 mL
f. Methanol, 10 mL
g. Deionized water, 5 mL
h. Conditioning Solution B, 10 mL
11.2.1.2.2 Retain conditioning solution B in
the C8 cartridge to keep it
activated.
130
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11.2.2 The C8 cartridges should not be prepared more than 48 hr
prior to use. After conditioning, the cartridge should be
capped and stored at 4°C.
11.2.3 Measure a 250-mL aliquot of the sample processed through
Sect. 11.1 in a silanized, volumetric flask.
11.2.4 Immediately before extraction, adjust the pH of sample to
10.5+0.2 with 10% w/v NaOH (aq) or 10% v/v HC1 (aq).
11.2.5 Place a conditioned C? cartridge on the solid phase
extraction vacuum manifold. Attach a 60-mL reservoir to
the C8 cartridge with the appropriate adapter. Put a 250-
mL beaker inside the extraction manifold to catch waste
solutions and sample. Transfer the measured volume in
aliquots to the reservoir. Turn on the vacuum pump or
house vacuum and adjust the flow rate to 3 to 6 mL/min.
Filter the sample through the C8 cartridge, and wash the
column with 5 ml of HPLC grade methanol. Continue to draw
the vacuum through the cartridge for one additional minute
to dry the cartridge. Release the vacuum and discard the
sample waste and methanol.
11.2.6 Place a silanized 5-mL volumetric flask beneath the
collection stem in the vacuum manifold. Add 4.5 ml of the
eluting solution to the sample cartridge. Turn on the
vacuum and adjust the flow rate to 1 to 2 mL/min.
11.2.7 Remove the 5-mL volumetric flask with the extract.
Fortify the extract with 100 nl of the ion-pair
concentrate. Adjust the volume to the mark with cartridge
eluting solution, mix thoroughly, and seal tightly until
analyzed.
11.2.8 Analyze sample by HPLC using conditions described in
Table.1. Integration and data reduction must be
consistent with that performed in Sect. 10.3. Figure 1
presents a representative, sample chromatogram.
11.3 DISK EXTRACTION — The top surface of the disk matrix must remain
covered with liquid at all times. If the disk is exposed to air
at any step in the disk cleanup procedure, the elution,procedure
should be restarted. Eluants applied to the disk should be
allowed to soak into the disk before drawing them through. Vacuum
should then be applied to draw most of the eluant through the
disk, leaving a thin layer of solution on the top of the disk.
Flow rate through the disk is not critical.
11.3.1 Assemble the 47 mm disk in the disk holder or a filter
apparatus. Be sure that the surfaces of the holder are
either glass or Teflon coated to avoid adsorption or
decomposition of the analytes.
131
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11.3.2 Adjust the pH of the sample to 10.5 ± 0.2 with 10% w/v
aqueous sodium hydroxide or 10% v/v aqueous hydrochloric
acid solution. Once the pH has been adjusted, the steps
below must be performed immediately.
11.3.3 Apply 10 ml of methanol to the disk. Apply vacuum to
begin elution, then immediately vent the vacuum when drops
of liquid appear at the drip tip. Allow the methanol to
soak into the disk for a minimum of 1 min, then reapply
the vacuum to bring the methanol to just above the top
surface of the disk.
11.3.4 Draw 2 10-mL aliquots of reagent water through to just
above the top surface of the disk to remove the methanol.
11.3.5 Apply 10 ml of Conditioning Solution A to the disk. As
with the methanol, draw a few drops through, then allow
the disk to soak for at least 1 min. Draw the Conditioning
Solution A through the disk to just above its top surface.
11.3.6 Draw 2 10-mL aliquots of reagent water through to just
above the top surface of the disk.
11.3.7 Apply 10 ml of Conditioning Solution B to the disk. Draw
a few drops through using vacuum and allow the disk to
soak for at least 1 min. Draw the remaining Conditioning
Solution B through to just above the top surface of the
disk.
11.3.8 Measure 250 ml of the pH adjusted sample using a
polypropylene graduated cylinder. Pour the sample aliquot
into the filtration apparatus reservoir and apply vacuum
to draw the sample through the disk. Pass the entire
sample through the disk, leaving no liquid on the top of
the disk, then vent the vacuum.
11.3.9 Assemble a graduated collection tube under the drip tip
with the tip descending into the tube slightly to prevent
losses of eluants. Be sure the tube will hold at least 10
ml of eluate.
,11.3.10 With the vacuum vented, drip enough methanol onto the disk
to cover it completely (0.5-1.0 mL). Allow the methanol
to soak into the disk for 1 min. Add more methanol as
needed to keep the disk covered as it soaks.
11.3.11 Pipet 4 ml of Disk Eluting Solvent onto the disk. Apply
vacuum until drops appear at the drip tip. Vent the
vacuum and allow the disk to soak for 1 min.
11.3.12 Draw the Disk Eluting Solution through to just above the
top surface of the disk. Add 4 ml of Disk Eluting
132
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Solution and draw it completely through the disk. Tap the
disk holder assembly gently to loosen adhering drops into
the collection tube.
11.3.13 Vent the vacuum, disassemble the disk extraction device,
and remove the collection tube. Add disk elution solution
to the tube to a known volume.
11.3.14 Analyze samples by HPLC. Some suggested conditions, which
were used in developing this method, are listed in Table
1. This table includes the retention times and MDLs that
were obtained using the suggested conditions. Figure 1
displays a representative sample chromatogram.
11.4 IDENTIFICATION OF ANALYTES
11.4.1 Identify a sample component by comparison of its retention
time to the retention time of a reference chromatogram.
If the retention time of an unknown compound corresponds,
within limits (Sect. 11.4.2), to the retention time of a
standard compound, then identification is considered
positive.
11.4.2 The width of the retention time window used to make
identification should be based upon measurements of actual
retention time variations of standards over the course of
a day. Three times the standard deviation of a retention
time can be used to calculate a suggested window size for
a compound. However, the experience of the analyst should
weigh heavily in the interpretation of chromatograms.
11.4.3 Identification requires expert judgment when sample
components are not resolved chromatographically. When
peaks obviously represent more than one sample component
(i.e., broadened peak with shoulder(s) or valley between
two or more maxima), or any time doubt exists over the
identification of a peak in a chromatogram, a confirmatory
technique must be employed. Through the use of the
photodiode array detector, full spectra of the analyte
peaks are obtained (Figure 2). When a peak of an unknown
sample falls within the retention time windows of method
analytes, confirm the peak identification by spectral
comparison with analyte standards.
If additional confirmation is required, replace the 1-
hexanesulfonic acid salt with 1-heptanesulfonic acid,
sodium salt in the mobile phase and reanalyze the samples.
Comparison of the ratio of retention times in the samples
by the two mobile phases with that of the standards will
provide additional confirmation.
133
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11.4.4 If the peak area exceeds the linear range of the
calibration curve, a smaller sample volume should be used.
Alternatively, the final solution may be diluted with
mobile phase and reanalyzed.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Determine the concentration of the analytes in the sample.
12.1.1 Calculate the concentration of each analyte injected from
the peak area using the calibration curves in Sect. 10.3
and the following equation.
Concentration, ng/L = (A) x (VF)
(VS)
where: A = Peak area of analyte in sample extract
VF * Final volume of sample extract, in ml
VS = Sample volume, in ml
12.2 Report results as micrograms per liter without correction for
recovery data. When duplicate and fortified samples are analyzed,
report all data obtained with sample results.
13. METHOD PERFORMANCE
13.1 METHOD DETECTION LIMITS — The method detection limit (MDL) is
defined as the minimum concentration of a substance that can be
measured and reported with 99% confidence that the value is above
the background level (3). The MDL data listed in Table 1 were
obtained using both cartridges and disks with reagent water as the
matrix.
13.2 This method has been tested for linearity of recovery from
fortified reagent water and has been demonstrated to be applicable
over the range from 4 x MDL to 1000 x MDL.
13.3 Single-laboratory precision and accuracy results at several
concentration levels in drinking water matrices using cartridges
are presented in Table 2A. Single laboratory accuracy and
precision data at a low, a medium, and a fairly high concentration
of each compound in several matrices are listed in Table 2B.
14. POLLUTION PREVENTION
14.1 Only an extremely small volume of an organic solvent is used in
this method. A maximum of 15 ml of methanol is used per sample to
condition each cartridge or disk. Methanol is not considered to
be a toxic or hazardous solvent. All other chemicals used in this
method are not considered toxic when used in the prescribed
amounts.
134
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14.2 For information about pollution prevention that may be applicable
to laboratory operations, consult "Less is Better: Laboratory
Chemical Management for Waste Reduction" available from the
American Chemical Society's Department of Government Relations and
Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.
15. WASTE MANAGEMENT
15.1 There are generally no waste management problems involved with
discarding spent or left over samples in this method since most
often the sample matrix is drinking water. If a sample is
analyzed which appears to be highly contaminated with chemicals,
analyses should be carried out to assess the type and degree of
contamination so that the samples may be discarded properly. The
Agency requires that laboratory waste management practices be
conducted consistent with all applicable rules and regulations,
and that laboratories protect the air, water, and land by
minimizing and controlling all releases from fume hoods and bench
operations. Also, compliance is required with any sewage
discharge restrictions. For further information on waste
management, consult "The Waste Management Manual for Laboratory
Personnel" also available from the American Chemical Society at
the address in Sect. 14.2.
16. REFERENCES
1. Bashe, W. J., "Determination of Diquat and Paraquat in Drinking
Waters by High Performance Liquid Chromatography with Ultraviolet
Detection," final report, U.S. Environmental Protection Agency,
March, 1989.
2. Lagman, L. H. and J. R. Hale, "Analytical Method for the
Determination of Diquat in Aquatic Weed Infested Lakes and Rivers in
South Carolina," Technology Conference Proceedings, WQTC-15,
American Water Works Association, November 15-20, 1987.
3. Glaser, J. A., D. L. Foerst, G. M. McKee, S. A. Quave, and W. L.
Budde, "Trace Analyses for Wastewaters," Environ. Sci. Techno!.. 15,
1426, 1981.
4. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice
for Preparation of Sample Container and for Preservation," American
Society for Testing and Materials, Philadelphia, PA, p. 679, 1980.
5. Worobey, B. L., "Analytical Method for the Simultaneous
Determination of Diquat and Paraquat Residues in Potatoes by High
Pressure Liquid Chromatography," Pestic. Sci 18(4). 245, 1987.
6. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice
for Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
135
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7. "Handbook of Quality Control in Water and Wastewater Laboratories,"
EPA-600/4-79-019, U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio
45268, March 1979.
136
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17. TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
CONDITIONS AND METHOD DETECTION LIMITS
Analyte
Diquat
Paraquat
Retention Time (min)
2.1
2.3
Method Detection Limits3
(fj.g/1)
(cartridges)
0.44
0.80
Method Detection Limits6
(/ig/L)
(disks)
0.51
0.59
HPLC Conditions:
Column: Hamilton PRP-1, 5^, 4.1 mm x 150 mm
Column Temperature: 35.0 C
Flow Rate: 2.0 mL/min., Ion-Pair Mobile Phase
(Sect. 7.16)
Injection Volume: 200 jui
Photodiode Array Detector Settings:
Wavelength Range: 210 - 370 nm
Sample Rate: 1 scan/sec.
Wavelength Step: 1 nm
Integration Time: 1 sec.
Run Time: 5.0 min.
Quantitation
Wavelengths: Diquat - 308 nm
Paraquat - 257 nm
0 MDL data were obtained from six samples fortified at 2 jug/L diquat and 2.3 jug/L
paraquat.
b MDL data were obtained from eight samples fortified at 1 /jg/L diquat and 1 /tg/L
paraquat.
137
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TABLE 2A. SINGLE OPERATOR ACCURACY AND PRECISION
USING CARTRIDGES
Analyte
Dlquat
Paraquat
Matrix
Type
Reagent
Water
Ground
Water
Tap8
Water
Reagent
Water
Ground
Water
Tap8
Water
Number
of
Analyses
6
6
7
7
6
6
6
7
7
6
6
Fortified
Concentration
M9/U
2.0
10
100
1000
100
100
2.3
11
113
113
113
Relative
Accuracy
(Recovery)
%
85.6
92.1
96.2
90.0
102.
91.3
87.6
99.7
94.4
92.1
74.2
Relative
Standard
Deviation
%
5.1
7.3
5.6
9.8
3.7
4.7
9.1
6.9
12.
3.4
1.8
Dechlorinated with Na2S203 (100 mg/L)
138
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TABLE 2B. SINGLE OPERATOR ACCURACY AND PRECISION
USING DISK (N = 5 FOR EACH TYPE OF WATER)
Type of
Water
RW
DW
GW
SW
WW
Type of
Water
RW
OW
GW
SW
WW
Fortified 2.
Mean % Rec.
99.2
93.5
87.4
86.5
90.5
Fortified 2.
Mean % Rec.
94.7
100.1
86.4
77.4
85.9
62 pg/L
% RSD
7.0
5.4
5.8
4.2
7.1
47 iig/l
% RSD
9.9
6.1
6.4
8.9
6.4
DIQUAT
Fortified
Mean % Rec
88.0
82.2
90.3
82.4
78.0
PARAQUAT
Fortified
Mean % Rec
92.6
85.2
81.4
71.8
81.6
10.5 iig/L
. % RSD
2.5
4.1
4.6
5.6
5.2
9.9 jig/L
. % RSD
4.7
4.1
3.2
5.3
6.1
Fortified 52.
Mean % Rec.
83.8
84.1
90.5
87.4
86.1
Fortified 49.
Mean % Rec.
90.1
85.7
86.1
81.0
87.2
5 pg/L
% RSD
5.9
2.2
1.4
6.5
3.4
5 pg/L
% RSD
1.4
1.2
4.0
2.6
2.3
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TABLE 3. 14-DAY SAMPLE HOLDING/PRESERVATION DATA3
Analvte Matrix
Dav 0
Percent Recovery
Dav 7
Dav 14
E
R
Diquat
Paraquat
RWb
TWC
GWd
RW
TW
GW
98.8 + 8.6
84.1 + 1.0
84.9 ± 6.6
90.8 + 4.4
72,1 + 0.8
98.1 + 1.4
93.2 + 1.4
94.1 + 5.8
87.5 ± 3.1
86.8 + 4.4
86.7 + 4.7
72.5 + 4.8
102. + 2.9
94.4 + 12.0
72.4 ± 4.5
89.2 + 3.9
84.7 + 2.9
66.4 + 7.9
a Average of four samples for each matrix. All matrices were
preserved with H2SO, (pH = 2). Concentration of each analyte was
100 /itg/L.
b RW = Reagent Water
c TW » Tap Water - Dechlorinated with Na2S203 (100 mg/L)
d GW * Groundwater
140
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£
M
i
8
I
I
I?
3
S
i
Figure 1. HPLC sample chroraatograms of dlquat
(A* 308 nm) and paraquat (X* 257 nn). Retention
time of dlquat (C * 10 ug/l) 1s 2.03 sin.; retention
time of paraquat (C *> 11 ug/L) 1s 2.2S mln.
141
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si-
OIQUAT
l.t »«•.« as«.i aM.«
.t »•••
Figure 2.
UV spectra of dlquat at 10 ug/L
and paraquat at 11 ug/L.
9
t
1
9
N
S
1
142
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METHOD 552.1 DETERMINATION OF HALOACETIC ACIDS AND DALAPON IN
DRINKING MATER BY ION-EXCHANGE LIQUID-SOLID
EXTRACTION AND GAS CHROMATOGRAPHY WITH AN
ELECTRON CAPTURE DETECTOR
Revision 1.0
August 1992
Jimmie W. Hodgeson
David Becker (Technology Applications Inc.)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
143
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METHOD 552.1
DETERMINATION OF HALOACETIC ACIDS AND DALAPON
IN DRINKING WATER BY ION-EXCHANGE LIQUID-SOLID EXTRACTION
AND GAS CHROMATOGRAPHY WITH ELECTRON CAPTURE DETECTION
1. SCOPE AND APPLICATION
1.1 This is a gas chromatographic (GC) method (1) applicable to the
determination of the listed halogenated acetic acids in drinking
water, ground water, raw water and water at any intermediate
treatment stage. In addition, the chlorinated herbicide, Dalapon,
is determined using this method.
Chemical Abstract Services
Analvte Registry Number
Monochloroacetic Acid 79-11-8
Dichloroacetic Acid 79-43-6
Trichloroacetic Acid 76-03-9
Monobromoacetic Acid 79-08-3
Bromochloroacetic Acid 5589-96-3
Dibromoacetic Acid 631-64-1
Dalapon 75-99-0
1.2 This is a liquid-solid extraction method and is designed as a
simplified alternative to the liquid-liquid extraction approach of
Method 552 for the haloacetic acids. This method also provides a
much superior technique for the determination of the herbicide,
dalapon, compared to the complex herbicide procedure described in
Method 515.1. The procedure also represents a major step in the
incorporation of pollution prevention in methods development, in
that the use of large volumes of organic solvents is eliminated.
1.3 This method is applicable to the determination of the target
analytes over the concentration ranges typically found in drinking
water (2, 3), subject to the method detection limits (MDL) listed in
Table 2. The MDLs observed may vary according to the particular
matrix analyzed and the specific instrumentation employed. The
haloacetic acids are observed ubiquitously in chlorinated supplies
at concentrations ranging from < 1 to > 50 /itg/L.
1.4 Reduced analyte recoveries may be observed in high ionic strength
matrices, particularly waters containing elevated sulfate concentra-
tions. Improved recoveries may be obtained by sample dilution at
the expense of higher MDLs. This effect is discussed more exten-
sively in Sect. 4.2.
1.5 Tribromoacetic acid has not been included because of problems
associated with stability and chromatography with this method.
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Mixed bromochloroacetic acids have recently been synthesized.
Bromochloroacetic acid is present in chlorinated supplies and method
validation data are provided here. Commercial standards are now
available for this compound. The mixed trihalogenated acids may
also be present. These are not included because of current problems
with purity and the chromatography for these compounds.
1.6 This method is designed for analysts skilled in extract concentra-
tion techniques, derivatization procedures and the use of GC and
interpretation of gas chromatograms.
1.7 When this method is used for the analyses of waters from unfamiliar
sources, analyte identifications must be confirmed by at least one
additional qualitative technique, such as gas chromatography/mass
spectrometry (GC/MS) or by GC using dissimilar columns.
2. SUMMARY OF METHOD
2.1 A 100-mL volume of sample is adjusted to pH 5.0 and extracted with a
preconditioned miniature anion exchange column. NOTE: The use of
liquid-solid extraction disks is certainly permissible as long as
all the quality control criteria specified in Sect. 9 of this method
are met. The analytes are eluted with small aliquots of acidic
methanol and esterified directly in this medium after the addition
of a small volume of methyl-tert-butyl ether (MTBE) as co-solvent.
The methyl esters are partitioned into the MTBE phase and identified
and measured by capillary column gas chromatography using an elec-
tron capture detector (GC/ECD).
3. DEFINITIONS
3.1 INTERNAL STANDARD (IS) — A pure analyte(s) added to a sample,
extract, or standard solution in known amount(s) and used to measure
the relative responses of other method analytes and surrogates that
are components of the same sample or solution. The internal stan-
dard must be an analyte that is not a sample component.
3.2 SURROGATE ANALYTE (SA)— A pure analyte(s), which is extremely
unlikely to be found in any sample, and which is added to a sample
aliquot in known amount(s) before extraction or other processing and
is measured with the same procedures used to measure other sample
components. The purpose of the SA is to monitor method performance
with each sample.
3.3 LABORATORY DUPLICATES (LD1 AND LD2) — Two aliquots of the same
sample taken in the laboratory and analyzed separately with identi-
cal procedures. Analyses of LD1 and LD2 indicate the precision
associated with laboratory procedures, but not with sample collec-
tion, preservation, or storage procedures.
3.4 FIELD DUPLICATES (FD1 AND FD2) — Two separate samples collected at
the same time and place under identical circumstances and treated
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exactly the same throughout field and laboratory procedures.
Analyses of FD1 and FD2 give a measure of the precision associated
with sample collection, preservation and storage, as well as with
laboratory procedures.
3.5 LABORATORY REAGENT BLANK (LRB) — An aliquot of reagent water or
other blank matrix that are treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The LRB
is used to determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or the appara-
tus.
3.6 FIELD REAGENT BLANK (FRB) — An aliquot of reagent water or other
blank matrix that is placed in a sample container in the laboratory
and treated as a sample in all respects, including shipment to the
sampling site, exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB
is to determine if method analytes or other interferences are
present in the field environment.
3.7 LABORATORY FORTIFIED BLANK (LFB) -- An aliquot of reagent water or
other blank matrix to which known quantities of the method analytes
are added in the laboratory. The LFB is analyzed exactly like a
sample, and its purpose is to determine whether the methodology is
in control, and whether the laboratory is capable of making accurate
and precise measurements.
3.8 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) — An aliquot of an envi-
ronmental sample to which known quantities of the method analytes
are added in the laboratory. The LFM is analyzed exactly like a
sample, and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background concen-
trations of the analytes in the sample matrix must be determined in
a separate aliquot and the measured values in the LFM corrected for
background concentrations.
3.9 STOCK STANDARD SOLUTION (SSS) — A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed
reference materials or purchased from a reputable commercial source.
3.10 PRIMARY DILUTION STANDARD SOLUTION (PDS) — A solution of several
analytes prepared in the laboratory from stock standard solutions
and diluted as needed to prepare calibration solutions and other
needed analyte solutions.
3.11 CALIBRATION STANDARD (CAL) ~ A solution prepared from the primary
dilution standard solution and stock standard solutions of the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
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3.12 QUALITY CONTROL SAMPLE (QCS) -- A solution of method analytes of
known concentration which is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally prepared
test materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware and other sample processing apparatus that lead
to discrete artifacts or elevated baselines in gas chromatograms.
All reagents and apparatus must be routinely demonstrated to be free
from significant interferences under the conditions of the analysis
by analyzing laboratory reagent blanks as described in Sect. 9.2.
4.1.1 For each set of samples analyzed, the reagent blank concen-
tration values exceeding 0.1 /zg/L should be subtracted from
the sample concentrations. A persistent reagent blank of
approximately 1 /zg/L was observed for bromochloroacetic acid
(BCAA) on the primary DB-1701 column. The background was
clean on the DB-210 confirmation column and the MDL for BCAA
in Table 2 was determined using this column.
4.1.2 Glassware must be scrupulously cleaned (4). Clean all
glassware as soon as possible after use by thoroughly rins-
ing with the last solvent used in it. Follow by washing
with hot water and detergent and thorough rinsing with tap
water, dilute acid, and reagent water. Drain and heat in an
oven or muffle furnace at 400 C for 1 hr. Do not heat
volumetric ware. Thermally stable materials such as PCBs
may not be eliminated by this treatment. Thorough rinsing
with reagent grade acetone may be substituted for the heat-
ing. After drying and cooling, store glassware in a clean
environment to prevent any accumulation of dust or other
contaminants. Store inverted or capped with aluminum foil.
4.1.3 The use of high purity reagents and solvents helps to mini-
mize interference problems. Purification of solvents by
distillation in all-glass systems may be required. The
extraction solvent, MTBE, may need to be redistilled.
4.2 The major potential interferences in this ion-exchange procedure are
other naturally occurring ions in water sources, principally sul-
fate. This is the only ion thus far demonstrated as an interfer-
ence, when present at concentrations possibly occurring in drinking
water sources. Sulfate as an effective counter ion displaces the
haloacids from the column when present at concentrations above 200
mg/L. Table 4 illustrates this effect for fortified reagent water
containing 500 mg/L and 400 mg/L of Na2S04 and NaCl respectively
(approximately 3.7 millimole (mM) in both cases). Markedly reduced
recoveries are observed for all analytes in the presence of high
147
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concentrations of sulfate. Reduced recoveries may be observed for
the monohaloacetic acids in very high ionic strength waters, as
illustrated for the sample with 400 mg/L NaCl. However, normal
recoveries were observed from a water sample containing the same
molar concentration of CaCl2. The only preventive measure currently
available for high ionic strength waters is sample dilution.
Dilution by a factor of 5 will suffice in the vast majority of
cases, although a factor of 10 may be required in a few extreme
sites (e.g. western waters with sulfate > 1000 mg/L). The MDLs will
still be approximately 1 pg/L for a dilution factor of 5. However,
for many chlorinated supplies the monohaloacetic acids may occur at
concentrations near 1 ng/l. In any event, this is the recommended
method to determine dalapon.
4.3 The acid forms of the analytes are strong organic acids which react
readily with alkaline substances, and can be lost during sample
preparation. Glassware must be acid rinsed with 1:9 hydrochloric
acid: water prior to use to avoid analyte losses due to adsorption.
4.4 Organic acids and phenols, especially chlorinated compounds, are the
most direct potential interferences with the determination. The
procedure includes a methanol wash step after the acid analytes are
adsorbed on the column. This step eliminates the potential for
interferences from neutral or basic, polar organic compounds present
in the sample.
4.5 Interfering contamination may occur when a sample containing low
concentrations of analytes is analyzed immediately following a
sample containing relatively high concentrations of analytes.
Routine between-sample rinsing of the sample syringe and associated
equipment with MTBE can minimize sample cross-contamination. After
analysis of a sample containing high concentrations of analytes, one
or more injections of MTBE should be made to ensure that accurate
values are obtained for the next sample.
4.6 Matrix interferences may be caused by contaminants that are coex-
tracted from the sample. The extent of matrix interferences will
vary considerably from source to source, depending upon the water
sampled. Tentative identifications should be confirmed using the
confirmation column specified in Table 1 or by the use of gas
chromatography with mass spectrometric detection.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound must
be treated as a potential health hazard. From this viewpoint,
exposure to these chemicals must be minimized. The laboratory is
responsible for maintaining a current awareness file of OSHA regula-
tions regarding the safe handling of the chemicals specified in this
method. A reference file of material data handling sheets should
also be made available to all personnel involved in the chemical
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analysis. Additional references to laboratory safety are available
and have been identified (5-7) for the information of the analyst.
5.2 The toxicity of the extraction solvent, MTBE, has not been well
defined. Susceptible individuals may experience adverse affects
upon skin contact or inhalation of vapors. For such individuals a
mask may be required. Protective clothing and gloves should be used
and MTBE should be used only in a chemical fume hood or glove box.
6. EQUIPMENT AND SUPPLIES
6.1 SAMPLE CONTAINERS — Amber glass bottles, approximately 250 ml,
fitted with Teflon-lined screw caps. At least 200 ml of sample
should be collected.
6.2 GAS CHROMATOGRAPH (GC) — Analytical system complete with GC
equipped for electron capture detection, split/splitless capillary
injection, temperature programming, differential flow control, and
with all required accessories including syringes, analytical col-
umns, gases and strip-chart recorder. A data system is recommended
for measuring peak areas. The gases flowing through the electron
capture detector should be vented through the laboratory fume hood
system.
6.3 PRIMARY GC COLUMN — DB-1701 or equivalent bonded, fused silica
column, 30 m x 0.32 mm ID, 0.25 pi film thickness. Another type of
column may be used if equivalent or better separation of analytes
can be demonstrated.
6.4 CONFIRMATORY GC COLUMN — DB-210 or equivalent bonded, fused silica
column, 30 m x 0.32 mm ID, 0.50 fim film thickness. Another type of
column may be used if equivalent or better separation of analytes
can be demonstrated.
6.5 PASTEUR PIPETS, GLASS DISPOSABLE
6.6 pH METER — Wide range with the capability of accurate pH measure-
ments at pH 5 ± 0.5.
6.7 15-mL amber colored bottles with Teflon-lined screw caps.
6.8 LIQUID-SOLID EXTRACTION VACUUM MANIFOLD ~ Available from a number
of suppliers.
6.9 LSE CARTRIDGES (1 mL) AND FRITS -- Also available from a number of
suppliers. The use of LSE disks instead of cartridges is permissi-
ble as long as all the quality control criteria in Sect. 9 of this
method are met.
6.10 75-mL RESERVOIRS PLUS ADAPTERS — Available from J. T. Baker, Cat
No. 7120-03 and Cat. No. 7122-00.
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6.11 GRADUATED CONICAL CENTRIFUGE TUBES WITH TEFLON-LINED SCREW CAPS (15
mL).
6.12 SCREW CAP CULTURE TUBES — Suggested size 13 x 100 mm.
6.13 BLOCK HEATER — Capable of holding screw cap culture tubes in Sect.
6.12.
6.14 VORTEX MIXER
7. REAGENTS AND STANDARDS
7.1 REAGENT WATER — Reagent water is defined as a water in which an
interference is not observed at the MDL of each analyte of interest.
7.1.1 A Mi 11ipore Super-Q water system or its equivalent may be
used to generate deionized reagent water. Distilled water
that has been passed through granular charcoal may also be
suitable.
7.1.2 Test reagent water each day it is used by analyzing accord-
ing to Sect. 11.
7.2 METHANOL — Pesticide quality or equivalent.
7.3 METHYL-TERT-BUTYL ETHER -- Nanograde, redistilled in glass if
necessary. Ethers must be demonstrated to be free of peroxides.
One test kit (EM Quant Test Strips), is available from EM Science,
Gibbstown, NJ. Procedures for removing peroxides from the ether are
provided with the test strips. Ethers must be periodically tested
(at least monthly) for peroxide formation during use. Any reliable
test kit may be used.
7.4 SODIUM SULFATE -- (ACS) granular, anhydrous. Heat in a shallow tray
at 400°C for a minimum of 4 hr to remove phthalates and other
interfering organic substances. Alternatively, extract with methy-
lene chloride in a Soxhlet apparatus for 48 hr.
7.5 SODIUM HYDROXIDE (NaOH), IN -- Dissolve 4 g ACS grade in reagent
water in a 100-mL volumetric flask and dilute to the line.
7.6 1,2,3-TRICHLOROPROPANE, 99+% — For use as the internal standard.
7.7 2-BROMOPROPIONIC ACID — For use as a surrogate compound.
7.8 10% Na?S04/H20 (BY WEIGHT) SOLUTION — Dissolve lOg Na2S04 in 90 g
reagent water.
7.9 10% H2S04/MeOH SOLUTION — Prepare a solution containing 10 mL H2S04
in 90 mL methanol.
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7.10 1M HCl/MeOH — Prepare a solution containing 8.25 mL HC1 (ACS grade)
with 91,75 ml methanol.
7.11 AG-1-X8 ANION EXCHANGE RESIN — Rinse resin with three consecutive
500-mL aliquots of deionized water and store in deionized water.
Available from Biorad, Richmond, CA.
7.12 ACETONE -- ACS reagent grade or equivalent.
7.13 AMMONIUM CHLORIDE ~ ACS reagent grade or equivalent.
7.14 SODIUM SULFITE.— ACS reagent grade or equivalent.
7.15 STOCK STANDARD SOLUTIONS
7.15.1 Analytes and Surrogates (Table 1) — Prepare at 1 to 5 mg/mL
in MTBE.
7.15.2 Internal Standard Fortifying Solution — Prepare a solution
of 1,2,3-trichloropropane at 1 mg/mL by adding 36 /tL of the
neat material (Sect. 7.6) to 50 mL of MTBE. From this stock
standard solution, prepare a primary dilution standard at 10
mg/L by the addition of 1 mL to 100 mL MTBE.
7.15.3 Surrogate Standard Fortifying Solution -- Prepare a surro-
gate stock standard solution of 2-bromopropionic acid at a
concentration of 1 mg/mL by accurately weighing approximate-
ly 10 mg of 2-bromopropionic acid, transferring it to a 10-
mL volumetric, and diluting to the mark with MTBE. Prepare
a primary dilution standard at a concentration of 2.5 /jg/mL
by diluting 250 (il of the stock standard to 100 mL with
methanol.
8. SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Grab samples must be collected in accordance with conventional
sampling practices (9) using amber glass containers with TFE-lined
screw-caps and capacities in excess of 100 mL.
8.1.1 Prior to shipment to the field, to combine residual chlo-
rine, add crystalline ammonium chloride (NH4C1) to the
sample container in an amount to produce a concentration of
100 mg/L in the sample. Alternatively, add 1.0 mL of a
10 mg/mL aqueous solution of NH4C1 to the sample bottle for
each 100 mL of sample bottle capacity immediately prior to
sample collection. Granular ammonium chloride may also be
added directly to the sample bottle.
8.1.2 After collecting the sample in the bottle containing the
dechlorination reagent, seal the bottle and agitate for 1
min.
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8.1.3 Samples must be iced or refrigerated at 4°C and maintained
at these conditions away from light until extraction.
Holding studies performed to date have suggested that, in
samples dechlorinated with NH,C1, the analytes are stable
for up to 28 days. Since stability may be matrix dependent,
the analyst should verify that the prescribed preservation
technique is suitable for the samples under study.
8.1.4 Extract concentrates (Sect. 11.3.6) should be stored at 4°C
or less away from light in glass vials with Teflon-lined
caps. Extracts should be analyzed within 48 hrs following
preparation.
9. QUALITY CONTROL
9.1 Minimum quality control (QC) requirements are initial demonstration
of laboratory capability, determination of surrogate compound
recoveries in each sample and blank, monitoring internal standard
peak area or height in each sample and blank, analysis of laboratory
reagent blanks, laboratory fortified blanks, laboratory fortified
sample matrices, and QC samples. Additional QC practices are
recommended.
9.2 LABORATORY REAGENT BLANKS (LRB) — Before processing any samples,
the analyst must analyze at least one LRB to demonstrate that all
glassware and reagent interferences are under control. In addition,
each time a set of samples is extracted or reagents are changed, a
LRB must be analyzed. If within the retention time window (Sect.
11.4.4) of any analyte, the LRB produces an interference signifi-
cantly in excess of that anticipated (Sect. 4.1.1), determine the
source of contamination and eliminate the interference before
processing samples.
9.3 INITIAL DEMONSTRATION OF CAPABILITY
9.3.1 Select a representative fortified concentration for each of
the target analytes. Concentrations near level 2 (Table 4)
are recommended. Prepare 4 to 7 replicate laboratory forti-
fied blanks by adding an appropriate aliquot of the primary
dilution standard or another certified quality control
sample. Be sure to add the internal standard, 1,2,3-tri-
chloropropane, and the surrogate compound, 2 bromopropionic
acid, to these samples (See Sect. 11). Analyze the LFBs
according to the method beginning in Sect. 11 and calculate
mean recoveries and standard deviation for each analyte.
9.3.2 Calculate the mean percent recovery, the standard deviation
of the recoveries, and the MDL (10); For each analyte, the
mean recovery value, expressed as a percentage of the true
value, must fall in the range of 70-130% and the standard
deviation should be less than 30%. For those compounds that
meet these criteria, performance is considered acceptable
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and sample analysis may begin. For those compounds that
fail these criteria, this procedure must be repeated using a
minimum of four fresh samples until satisfactory performance
has been demonstrated. Maintain this data on file to demon-
strate initial capabilities.
9.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples via a
new, unfamiliar method prior to obtaining some experience
with it. As laboratory personnel gain experience with this
method, the quality of data should improve beyond those re-
quired here.
9.3.4 The analyst is permitted to modify GC columns, GC condi-
tions, detectors, extraction techniques, concentration
techniques (i.e., evaporation techniques), internal standard
or surrogate compounds. Each time such method modifications
are made, the analyst must repeat the procedures in Sect.
9.3.1 and also analyze a laboratory fortified matrix sample.
9.4 ASSESSING SURROGATE RECOVERY
9.4.1 When surrogate recovery from a sample or blank is < 70% or
> 130%, check (1) calculations to locate possible errors,
(2) standard solutions for degradation, (3) contamination,
and (4) instrument performance. If those steps do not
reveal the cause of the problem, reanalyze the extract.
9.4.2 If the extract reanalysis fails the 70-130% recovery crite-
rion, the problem must be identified and corrected before
continuing. It may be necessary to extract another aliquot
of sample.
9.4.3 If the extract reanalysis meets the surrogate recovery
criterion, report only data for the reanalyzed extract. If
sample extract continues to fail the recovery criterion,
report all data for that sample as suspect.
9.4.4 Develop and maintain control charts on surrogate recovery as
described in Sect. 9.6.2. Charting of surrogate recoveries
Is an especially valuable activity, since these are present
in every sample and the analytical results will form a
significant record of data quality.
9.5 ASSESSING THE INTERNAL STANDARD
9.5.1 When using the internal standard calibration procedure
prescribed in this method, the analyst is expected to moni-
tor the IS response (peak area or peak height) of all sam-
ples during each analysis day. The IS response for any
sample chromatogram should not deviate from the daily cali-
bration standard IS response by more than 30%.
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9.5.2 If > 30% deviation occurs with an individual extract, opti-
mize instrument performance and inject a second aliquot of
that extract.
9.5.2.1 If the reinjected aliquot produces an acceptable
internal standard response, report results for
that aliquot.
9.5.2.2 If a deviation of greater than 30% is obtained for
the reinjected extract, analysis of the samples
should be repeated beginning with Sect. 11, pro-
vided the sample is still available. Otherwise,
report results obtained from the reinjected ex-
tract, but annotate as suspect.
9.5.3 If consecutive samples fail the IS response acceptance
criteria, immediately analyze a medium calibration standard.
9.5.3.1 If the calibration standard provides a response
factor (RF) within 20% of the predicted value,
then follow procedures itemized in Sect. 9.5.2 for
each sample failing the IS response criterion.
9.5.3.2 If the check standard provides a response factor
which deviates more than 20% of the predicted
value, then the analyst must recalibrate (Sect.
10).
9.6 LABORATORY FORTIFIED BLANK
9.6.1 The laboratory must analyze at least one laboratory forti-
fied blank (LFB) sample with every 20 samples or one per
sample set (all samples extracted within a 24-hr period),
whichever is greater. Fortified concentrations near level 2
(Table 4) are recommended. Calculate percent recovery (R).
If the recovery of any analyte falls outside the control
limits (see Sect. 9.6.2), that analyte is judged out of
control, and the source of the problem should be identified
and resolved before continuing analyses.
9.6.2 Prepare control charts based on mean upper and lower control
limits, R ± 3 SR. The initial demonstration of capability
(Sect. 9.3) establishes the initial limits. After each 4-6
new recovery measurements, recalculate R and SR using all
the data, and construct new control limits. When the total
number of data points reach 20, update the control limits by
calculating R and SR using only the most recent 20 data
points. At least quarterly, replicates of LFBs should be
analyzed to determine the precision of the laboratory mea-
surements. Add these results to the ongoing control charts
to document data quality.
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9.7 LABORATORY FORTIFIED SAMPLE MATRIX
9.7.1 Chlorinated water supplies will usually contain significant
background concentrations of several method analytes, espe-
cially dichloroacetic acid (DCAA) and trichloroacetic acid
(TCAA). The concentrations of these acids may be equal to
or greater than the fortified concentrations. Table 6
illustrates the relatively poor accuracy and precision which
may be anticipated when a large background must be subtract-
ed. The water supply used in the development of this method
contained only moderate concentrations of DCAA and TCAA.
For many supplies, the concentrations may be so high that
fortification may lead to a final extract with instrumental
responses exceeding the linear range of the electron capture
detector. If this occurs, the extract must be diluted. In
spite of these problems, sample sources should be fortified
and analyzed as described below. Poor accuracies and high
precisions across all analytes likely indicate the presence
of interfering ions, especially sulfate, and the requirement
for sample dilution.
9.7.2 the laboratory must add known concentrations of analytes to
a minimum of 10% of samples or one sample per sample set,
whichever is greater. The concentrations should be equal to
or greater than the background concentrations in the sample
selected for fortification. Ideally, the concentration
should be the same as that used for the laboratory fortified
blank (Sect. 9.6). Over time, samples from all routine
sample sources should be fortified.
9.7.3 Calculate the mean percent recovery, R, of the concentration
for each analyte, after correcting the total mean measured
concentration, A, from the fortified sample for the back-
ground concentration, B, measured in the unfortified sample,
i.e.:
R = 100 (A - B) / C,
where C is the fortifying concentration. Compare these
values to control limits appropriate for reagent water data
collected in the same fashion (Sect. 9.6).
9.7.4 If the analysis of the unfortified sample reveals the ab-
sence of measurable background concentrations, and the added
concentrations are those specified in Sect. 9.6, then the
appropriate control limits would be the acceptance limits in
Sect. 9.6.
9.7.5 If the sample contains measurable background concentrations
of analytes, calculate mean recovery of the fortified con-
centration, R, for each such analyte after correcting for
the background concentration (Sect. 9.7.3). Compare these
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values to reagent water recovery data, R*, at comparable
fortified concentrations from Tables 2, 4, and 5. Results
are considered comparable if the measured recoveries fall
within the range,
R ± 3SC,
where S is the estimated percent relative standard devia-
tion in°the measurement of the fortified concentration. By
contrast to the measurement of recoveries in reagent water
(Sect. 9.6.2) or matrix samples without background (Sect.
9.7.3), the relative standard deviation, Sc, must be ex-
pressed as the statistical sum of variation from two
sources, the measurement of the total concentration as well
as the measurement of background concentration. In this
case, variances, defined as S2, are additive and Se can be
expressed as,
or
- (Sa + Sb)
21'2
where S and Sb are the percent relative standard deviations
of the lotal measured concentration and the background
concentration respectively. The value of S may be estimat-
ed from the mean measurement of A above or from data at
comparable concentrations from Tables 2, 4, and 5. Like-
wise, Sb can be measured from repetitive measurements of the
background concentration or estimated from comparable con-
centration data from Tables 2, 4, and 5.
9.7.6 If the recovery of any such analyte falls outside the desig-
nated range, and the laboratory performance for that analyte
is shown to be in control (Sect. 9.6), the recovery problem
encountered with the fortified sample is judged to be matrix
related, not system related. The result for that analyte in
the unfortified sample is labeled suspect/matrix to inform
the data user that the results are suspect due to matrix
effects.
9.8 QUALITY CONTROL SAMPLE (QCS) — At least quarterly, analyze a QCS
from an external source. If measured analyte concentrations are not
of acceptable accuracy, check the entire analytical procedure to
locate and correct the problem source.
9.9 The laboratory may adapt additional QC practices for use with this
method. The specific practices that are most productive depend upon
the needs of the laboratory and the nature of the samples. For
example, field or laboratory duplicates may be analyzed to assess
the precision of the environmental measurements or field reagent
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blanks may be used to assess contamination of samples under site
conditions, transportation and storage.
10. CALIBRATION AND STANDARDIZATION
10.1 Establish GC operating parameters equivalent to the suggested
specifications in Table 1. The GC system must be .calibrated using
the internal standard (IS) technique. Other columns or conditions
may be used if equivalent or better performance can be demonstrated.
10.2 INTERNAL STANDARD CALIBRATION PROCEDURE — This approach requires
the analyst to select one or more internal standards which are
compatible in analytical behavior with the method analytes. For the
single laboratory precision and accuracy data reported in Tables
2-9, one internal standard, 1,2,3-trichloropropane, was used as a
concentration of 0.4 /ig/mL in the final 5.0-mL concentrate.
10.2.1 Prepare separate stock standard solutions for each analyte
of interest at a concentration of 1-5 mg/mL in MTBE. Method
analytes may be obtained as neat materials or ampulized
solutions (> 99% purity) from a number of commercial suppli-
ers.
10.2.2 Prepare primary dilution standard solutions by combining and
diluting stock standard solutions with methanol. As a
guideline to the analyst, the primary dilution standard
solution used in the validation of this method is described
here. Stock standard solutions were prepared in the 1-2
mg/mL range for all analytes and the surrogate. Aliquots of
each stock standard solution (approximately 50-250 /iL) were
added to 100-mL methanol to yield a primary dilution stan-
dard containing the following approximate concentrations of
analytes:
Concentration. ua/mL
Monochloroacetic acid 3
Monobromoacetic acid 2
Dalapon 2
Dichloroacetic acid 3
2-Bromopropionic acid 1
Trichloroacetic acid 1
Bromochloroacetic acid 2
Dibromoacetic acid 1
The primary dilution standards are used to prepare calibra-
tion standards, which comprise at least three concentration
levels (optimally five) of each analyte with the lowest
standard being at or near the MDL of each analyte. The
concentrations of the other standards should define a range
containing the expected sample concentrations or the working
range of the detector.
157
-------
10.2.2.1 Calibration standards — Calibration is performed
by extracting procedural standards, i.e.; forti-
fied reagent water. A five-point calibration
curve may be prepared by fortifying a 100- ml re-
agent water samples at pH 5 with 20, 50, 100, 250,
and 500 /tl_ of the primary dilution standard pre-
pared above. Alternatively, three levels of cali-
bration solutions may be prepared. Analyze each
calibration standard in triplicate according to
the procedure outlined in Sect. 11. In addition,
a reagent water blank must be analyzed in tripli-
cate.
10.2.3 Include the surrogate analyte., 2-bromopropionic acid,
within the calibration standards prepared in Sect. 10.2.2.
10.2.4 Inject 2 pi of each standard and calculate the relative
response for each analyte (RRa) using the equation:
RR8 = Aa/Ais
where Aa is the peak area of the analyte.
Ais the peak area of the internal standard.
10.2.5 Generate a calibration curve of RRa versus analyte concen-
tration of the standards expressed in equivalent /jg/L in the
original aqueous sample. The working calibration curve must
be verified daily by measurement of one or more calibration
standards. If the response for any analyte falls outside
the predicted response by more than 15%, the calibration
check must be repeated using a freshly prepared calibration
standard. Should the retest fail, a new calibration curve
must be generated.
10.2.6 A data system may be used to collect the chromatographic
data, calculate response factors, and calculate linear or
second order calibration curves.
11. PROCEDURE
11.1 PREPARATION AND CONDITIONING OF EXTRACTION COLUMNS
11.1.1 Preparation — Place 1 ml liquid-solid extraction cartridges
(Sect. 6.9) onto the vacuum manifold. Place frits into the
tubes and push down to place them flat on the bottom. Add
the AG-1-X8 resin solution dropwise to the tubes with a
Pasteur pipet until there is a solid layer of resin 10 mm in
height. Add reagent water and apply vacuum to settle out
the suspended resin particles. Do not allow the resin to go
dry. At this point extraction of samples can begin or the
columns can be stored for later use by maintaining the resin
under water and sealing the top with aluminum foil.
158
-------
11.1.2 Conditioning — Attach adapters and 75-mL reservoirs to the
liquid-solid extraction cartridges. To condition the col-
umns, add to the reservoirs and pass the following series of
solvents in 10-mL aliquots through the resin under vacuum:
methanol, reagent water, 1 M HCl/MeOH, reagent water, 1 M
NaOH, and reagent water. The conditioning solvents should
pass through the resin at the rate of « 2 mL/min. without
allowing the resin bed to dry and the sample should be added
(Sect. 11.2.3) immediately after the last reagent water
aliquot.
11.2 SAMPLE EXTRACTION AND ELUTION
11.2.1 Remove the samples from storage (Sect. 8.1.3) and allow them
to equilibrate to room temperature.
11.2.2 Adjust the pH of a 100-mL sample to 5 ± 0.5 using 1:2 H2S04
water and check the pH with a pH meter or narrow range pH
paper.
11.2.3 Add 250 /nL of the surrogate primary dilution standard (Sect.
7.15.3) to each sample
11.2.4 Transfer the 100-mL sample to the reservoir and apply a
vacuum to extract the sample at the rate of « 2 mL/min.
11.2.5 Once the sample has completely passed through the column add
10 mL MeOH to dry the resin.
11.2.6 Remove the reservoirs and adapters, disassemble the vacuum
manifold and position screw cap culture tubes (Sect. 6.12)
under the columns to be eluted. Reassemble the vacuum
manifold, add 4 mL 10% H2S04/methanol to the column and
elute at the rate of approximately 1.5 mL/min. Turn off the
vacuum and remove the culture tubes containing the eluants.
11.3 SOLVENT PARTITION
11.3.1 Add 2.5 mL MTBE to each eluant and agitate in the vortex
mixer at a low setting for about 5 sec.
11.3.2 Place the capped culture tubes in the heating block (Sect.
6.13) at 50°C and maintain for 1 hr. At this stage, quanti-
tative methyl ati on of all method analytes is attained.
11.3.3 Remove the culture tubes from the heating block and add to
each tube 10 mL of 10% by weight of sodium sulfate in re-
agent water (Sect. 7.8). Agitate each solution for 5-10 sec
in the vortex mixer at a high setting.
11.3.4 Allow the phases to separate for approximately 5 min. Trans-
fer the upper MTBE layer to a 15-mL graduated conical cen-
159
-------
„...„;,,. ____ x _____ ____ , ______ - r ______ r ,---- — r---
extraction two more times with approximately 1 ml MTBE each
time. Combine the MTBE sample extracts in the graduated
centrifuge tube.
11.3.5 Add 200 /tL of the internal standard fortifying solution
(Sect. 7.15.2) to each extract and add MTBE to each to a
final volume of 5 ml.
11.3.6 Transfer a portion of each extract to a vial and analyze
using 6C-ECD. A duplicate vial should be filled from excess
extract. Analyze the samples as soon as possible. The
sample extract may be stored up to 48 hr if kept at 4°C or
less away from light in glass vials with Teflon-lined caps.
11.4 GAS CHROMATOGRAPHY
11.4.1 Table 1 summarizes recommended GC operating conditions and
retention times observed using this method. Figure 1 illus-
trates the performance of the recommended column with the
method analytes. Other GC columns, chromatographic condi-
tions, or detectors may be used if the requirements of Sect.
9.3 are met.
11.4.2 Calibrate the system daily as described in Sect. 10. The
standards and extracts must be in MTBE.
11.4.3 Inject 2 [il of the sample extract. Record the resulting
peak size in area units.
11.4.4 The width of the retention time window used to make identi-
fications should be based upon measurements of actual reten-
tion time variations of standards over the course of a day.
Three times the standard deviation of a retention time can
be used to calculate a suggested window size for a compound.
However, the experience of the analyst should weigh heavily
in the interpretation of chromatograms.
11.4.5 If the response for the peak exceeds the working range of
the system, dilute the extract and reanalyze.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Calculate analyte concentrations in the sample and reagent blanks
from the response for the analyte relative to the internal standard
(RRa) using the equation in Sect. 10.2.4.
12.2 For samples processed as part of a set where recoveries falls
outside of the control limits established in Sect. 9, results for
the affected analytes must be labeled as suspect.
160
-------
13. METHOD PERFORMANCE
13.1 In a single laboratory (EMSL-Cincinnati), recovery and precision
data were obtained at three concentrations in reagent water (Tables
2, 4, and 5). In addition, recovery and precision data were ob-
tained at a medium concentration for high ionic strength reagent
water (Table 3), dechlorinated tap water, high humectant ground
water, and an ozonated surface water (Tables 6-9). The MDL (10)
data are given in Table 2.
14. POLLUTION PREVENTION
14.1 This method utilizes the new LSE technology which requires the use
of very small quantities of organic solvents. This feature elimi-
nates the hazards involved with the use of large volumes of poten-
tially harmful organic solvents needed for conventional liquid-
liquid extractions. This method also uses acidic methanol as the
derivatizing reagent in place of the highly toxic diazomethane.
These features make this method much safer for use by the analyst in
the laboratory and much less harmful to the environment.
14.2 For information about pollution prevention that may be applicable to
laboratory operations consult "Less is Better: Laboratory Chemical
Management for Waste Reduction" available from the American Chemical
Society's Department of Government Relations and Science Policy,
1155 16th Street N.W., Washington, D.C. 20036.
15. HASTE MANAGEMENT
15.1 Due to the nature of this method there is little need for waste
management. No large volumes of solvents or hazardous chemicals are
used. The matrices of concern are finished drinking water or source
water. However, the Agency requires that laboratory waste manage-
ment practices be conducted consistent with all applicable rules and
regulations, and that laboratories protect the air, water, and land
by minimizing and controlling all releases from fume hoods and bench
operations. Also compliance is required with any sewage discharge
permits and regulations, particularly the hazardous waste identifi-
cation rules and land disposal restrictions. For further informa-
tion on waste management, consult "The Waste Management Manual for
Laboratory Personnel" also available from the American Chemical
Society at the address in Sect. 14.2.
16. REFERENCES
1. Hodgeson, J. W., Collins, J. D., and Becker, D, A., "Advanced Tech-
niques for the Measurement of Acidic Herbicides and Disinfection
Byproducts in Aqueous Samples," Proceedings of the 14th Annual EPA
Conference on Analysis of Pollutants in the Environment, Norfolk, VA.,
May 8-9, 1991. Office of Water Publication No. 821-R-92-001, U.S.
Environmental Protection Agency, Washington, DC, pp 165-194.
161
-------
2. Uden, P.C. and Miller, J.W., J. Am. Water Works Assoc. 75, 1983, pp.
524-527.
3. Fair. P.S., Earth, R.C., Flesch, J. J. and Brass, H. "Measurement of
Disinfection By-products in Chlorinated Drinking Water," Proceedings
Water Quality Technology Conference (WQTC-15), Baltimore, Maryland,
November 15-20, 1987, American Water Works Association, Denver, CO,
pp. 339-353.
4. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for
Preparation of Sample Containers and for Preservation," American
Society for Testing and Materials, Philadelphia, PA, p. 679, 1980.
5. "Carcinogens-Working with Carcinogens," Publication No. 77-206,
Department of Health, Education, and Welfare, Public Health Service,
Center for Disease Control, National Institute of Occupational Safety
and Health, Atlanta, GA, August 1977.
6. "OSHA Safety and Health Standards, General Industry," (29CFR1910),
OSHA 2206, Occupational Safety and Health Administration, Washington,
D.C. Revised January 1976.
7. "Safety In Academic Chemistry Laboratories," 3rd Edition, American
Chemical Society Publication, Committee on Chemical Safety, Washing-
ton, D.C., 1979.
8. Hodgeson, J.W. and Cohen, A.L. and Collins, J.D., "Analytical Methods
for Measuring Organic Chiorination By-products," Proceedings Water
Quality Technology Conference (WQTC-16), St. Louis, MO, Nov. 13-17,
1988, American Water Works Association, Denver, CO, pp. 981-1001.
9. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials, Philadel-
phia, PA, p. 76, 1980.
10. Glaser, J. A., Foerst, D. L., McKee, G. D., Quave, S. A. and Budde, W.
L., Environ. Sci. Technol. 15, 1981, pp. 1426-1435.
162
-------
17. TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA
TABLE 1. RETENTION DATA AND CHROMATOGRAPHIC CONDITIONS
Analyte
Monochloroacetic Acid (MCAA)
Monobromoacetic Acid (MBAA)
Oalapon
Dichloroacetic Acid (DCAA)
2-Bromopropionic acid (b)
Trichloroacetic Acid (TCAA)
1,2,3-Trichloropropane (a)
Bromochloroacetic Acid (BCAA)
Dibromoacetic Acid (DBAA)
Retention Time,
Column A
5.16
7.77
8.15
8.37
8.80
11.43
12.62
12.92
15.50
min.
Column B
9.44
11.97
11.97
11.61
12.60
13.34
12.91
14.20
16.03
Column A: DB-1701, 30 m x 0.32 mm i.d., 0.25 fim film thickness,
Injector Temp. = 200°C, Detector Temp. = 260°C, Helium
Linear Velocity = 27 cm/sec, Splitless injection with 30 s
delay
Program: Hold at 50°C for 10 min, to 200°C at 10°C/min. and hold 5
min., to 230°C at 10°C/min. and hold 5 min.
Column B: DB-210, 30 m x 0.32 mm i.d., 0.50 /zm film thickness,
Injector Temp. = 200°C, Detector Temp. = 260°C, Linear
Helium Flow = 25 cm/sec, splitless injection with 30 s
delay.
Program: Hold at 50°C for 10 min., to 200°< at 10°C/min and hold 5
min., to 230° at 10°C/min. and hold 5 min.
(a) Internal Standard
(b) Surrogate Compound
163
-------
TABLE 2. ANALYTE RECOVERY AND PRECISION DATA
AND METHOD DETECTION LIMITS3
LEVEL 1 IN REAGENT WATER
Fortified
Cone.
Analyte pg/L
Monochloroacetic Acid
Monobromoacetic Acid
Dichloroacetic Acid
2-Bromopropionic Acidb
Trichloroacetic Acid
Bromochloroacetic acid
Dibromoacetic Acid
Dal apon
1.5
1.0
1.5
0.05
0.50
1.0
0.50
1.0
Mean
Meas.
Cone.
/ig/L
1.47
0.73
1.65
0.47
0.30
0.75
0.29
0.81
Std.
Dev.
/*g/L
0.07
0.08
0.14
0.03
0.02
0.03
0.03
0.10
Rel.
Std.
Dev., %
4.6
7.9
7.7
5.6
4.0
3.4
6.4
12
Mean
Recovery
%
98
73
110
94
60
75
58
81
Method
Detection
Limit
Mg/L
0.21
0.24
0.45
0.08
0.07
0.10
0.09
0.32
8 Produced by analysis of seven aliquots of fortified reagent water (Reference 10).
b Surrogate Compound
164
-------
TABLE 3. RECOVERY AND PRECISION DATA IN HIGH
IONIC STRENGTH WATERS
MEAN RECOVERY ± RSDa
Fortified
Cone.
Analyte /uj/L
Monochloroacetic Acid
Monobromoacetic Acid
Dichloroacetic Acid
2-Bromoprop ionic Acid
Trichloroacetic Acid
Bromochloroacetic Acid
Dibromoacetic Acid
Dalapon
7.5
5.0
7.5
2.5
2.5
5.0
2.5
5.0
Reagent Reagent Water +
Water (RW) 500 mg/L Na2S04b
109 ±
83 ±
107 ±
108 ±
101 ±
101 ±
93 ±
93 ±
1.5
18 5.0 ± 10
3.6 59 + 2.4
1.8 32 ± 0.3
0.4 8 ± 3.0
2.6 85 ± 0.7
1.9 40 ± 22
1.9 57 ± 5.3
Reagent Water +
400 mg/L NaClb.
46 ±
50 ±
114 ±
137 ±
64 ±
107 ±
89 ±
99 ±
10
13
0.1
2'.1
11
3.5
5.0
1.7
a Based on the analysis of three replicate samples.
b Molar concentration of added salt is 3.7 mM in both cases.
165
-------
TABLE 4. ANALYTE RECOVERY AND PRECISION DATA8
LEVEL 2 IN REAGENT WATER
Analyte
Honochloroacetic Acid
Monobromoacetic Acid
Dichloroacetic Acid
2-Bromopropionic Acidb
Trichloroacetic Acid
Bromochloroacetic Acid
Dibromoacetic Acid
Dal apon
Fortified
Cone.
P9/L
7.5
5.0
7.5
2.5
2.5
5.0
2.5
5.0
Mean
Meas.
Cone.
09/L
7.73
3.95
8.06
2.57
2.32
5.22
2.41
4.03
Std.
Dev.
M9/L
0.18
0.65
0.16
0.06
0.14
0.12
0.09
0.36
Rel.
Std.
Dev., %
2.3
16
2.0
2.4
5.8
2.2
3.4
7.5
Mean
Recovery
%
103
79
108
103
93
104
96
97
8 Produced by the analysis of seven aliquots of fortified reagent water.
b Surrogate Compound
166
-------
TABLE 5. AMALYTE RECOVERY AND PRECISION DATA8
LEVEL 3 IN REAGENT WATER
Analyte
Monochloroacetic Acid
Monobromoacetic Acid
Dichloroacetic Acid
2-Bromoprop ionic Acid
Trichloroacetic Acid
Bromochloroacetic Acid
Dibromo acetic Acid
Dalapon
Fortified
Cone .
M9/L
15.0
10.0
15.0
5.0
5.0
10.0
5.0
10.0
Mean
Meas.
Cone.
M9/L
14.5
7.82
15.1
4.98
4.89
10.3
4.85
9.02
Std.
Dev.
M9/L
0.15
0.68
6.09
0.08
0.07
0.25
0.04
0.16
Rel.
Std.
Dev., %
1.0
8.4
0.6
1.5
1.4
2.4
0.7
1.8
Mean
Recovery
%
99
78
101
100
98
103
97
90
a Produced by the analysis of seven aliquots of fortified reagent water.
167
-------
TABLE 6. ANALYTE RECOVERY AND PRECISION DATA8
DECHLORINATED TAP WATER
Fortified
Cone.
Analyte
Monochloroacetic Acid
Monobromoacetic Acid
Dichloroacetic Acid
2-Bromopropionic Acid0
Trichloroacetic Acid
Bromochloroacetic Acid
Dibromoacetic Acid
Dal apon
W/L
7.5
5.0
7.5
7.5
2.5
5.0
2.5
5.0
Meanb
Meas.
Cone.
mil
5.70
4.57
5.62
2.22
1.48
5.70
2.42
4.69
Std.
Dev.
mil
0.63
0.45
0.76
0.16
0.42
0.92
0.13
0.21
Rel.
Std.
Dev., %
11
9.8
14
7.2
28
16
5.4
4.5
Mean
Recovery
%
76
91
75
89
59
114
97
94
a Produced by the analysis of seven aliquots of fortified dechlorinated tap water.
b Significant background concentrations (> 5-15 ng/l) have been subtracted from these
values for dichloroacetic acid, trichloroacetic acid, bromochloroacetic acid, and
dibromoacetic acid.
c Surrogate Compound
168
-------
TABLE 7. ANALYTE RECOVERY AND PRECISION DATA8
HIGH HUMIC CONTENT GROUND WATER
Analyte
Monochloroacetic Acid
Monobromoacetic Acid
Dichloroacetic Acid
2-Bromopropionic Acidb
Trichloroacetic Acid
Bromochloroacetic Acid
Dibromoacetic Acid
Dalapon
Fortified
Cone.
Wf/L
7.5
5.0
7.5
2.5
2.5
5.0
2.5
5.0
Mean
Meas.
Cone.
mli
3.55
2.21
7.60
1.83
2.37
5.53
2.58
4.92
Std.
Dev.
W/L
0.32
0.21
0.08
0.09
0.12
0.16
0.13
0.29
Rel.
Std.
Dev., %
8.9
11
1.1
4.9
5.1
2.9
5.0
6.0
Mean
Recovery
%
47
44
101
73
95
111
103
90
Produced by the analysis of seven aliquots of fortified high humic content ground
water.
Surrogate Compound
169
-------
TABLE 8. ANALYTE RECOVERY AND PRECISION DATA8
HIGH HUMIC CONTENT GROUND WATER DILUTED 1:5
Analyte
Monochloroacetic Acid
Honobromoacetic Acid
Dichloroacetic Acid
2-Bromopropionic Acidb
Trichloroacetic Acid
Bromochloroacetic Acid
Dibromoacetic Acid
Dal apon
Fortified
Cone.
M9/L
1.5
1.0
1.5
0.5
0.5
1.0
0.5
1.0
Mean
Meas.
Cone.
pg/L
1.50
0.97
1.89
0.49
0.28
0.43
0.30
0.88
Std.
Dev.
/*g/L
0.17
0.06
0.16
0.01
0.03
0.07
0.02
0.12
Rel.
Std.
Dev., %
11
6.2
8.5
2.0
11
16
6.7
14
Mean
Recovery
%
100
97
126
98
56
43
60
88
8 Produced by the analysis of seven aliquots of fortified high humic content ground
water diluted 1:5.
b
Surrogate Compound
170
-------
TABLE 9. ANALYTE RECOVERY AND PRECISION DATA3
OZONATED RIVER WATER
Analyte
Monochloroacetic Acid
Monobromoacetic Acid
Dichlorpacetic Acid
2-Bromopropionic Acidb
Trichloroacetic Acid
Bromochloroacetic Acid
Dibromoacetic Acid
Dalapon
Fortified
Cone.
W/L
7.5
5.0
7.5
2.5
2.5
5.0
2.5
5.0
Mean
Meas.
Cone.
M9/L
6.22
4.28
7.09
2.31
2.65
5.20
2.36
5.08
Std.
Dev.
M9/L
0.91
0.34
0.22
0.09
0.13
0.18
0.09
0.17
Rel.
Std.
Dev.,.%
15
7.9
3.1
3.7
4.9
3.5
3.8
3.4
Mean
Recovery
%
83
86
94
92
106
104
94
102
a Produced by the analysis of seven aliquots of fortified ozonated river water.
b Surrogate Compound
171
-------
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172
-------
METHOD 553. DETERMINATION OF BENZIDINES AND NITROGEN-CONTAINING
PESTICIDES IN WATER BY LIQUID-LIQUID EXTRACTION
OR LIQUID-SOLID EXTRACTION AND REVERSE PHASE
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/
PARTICLE BEAM/MASS SPECTROMETRY
Revision 1.1
August 1992
Thomas D. Behymer
Thomas A. Bellar
James S. Ho
William L. Budde
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
173
-------
METHOD 553
DETERMINATION OF BENZIDINES AND NITROGEN-CONTAINING PESTICIDES IN WATER BY
LIQUID-LIQUID EXTRACTION OR LIQUID-SOLID EXTRACTION AND REVERSE PHASE
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/PARTICLE BEAM/MASS SPECTROMETRY
1. SCOPE AND APPLICATION
1.1 This is a general purpose method that provides procedures for
determination of benzidines and nitrogen-containing pesticides in
water and wastewater. The method is applicable to a wide range of
compounds that are efficiently partitioned from a water sample into
methylene chloride or onto a liquid-solid extraction device. The
compounds must also be amenable to separation on a reverse phase
liquid chromatography column and transferable to the mass spectrome-
ter with a particle beam interface. Particulate bound organic
matter will not be partitioned onto the liquid-solid extraction
system, and more than trace levels of particulates in the water may
disrupt the partitioning process. The compounds listed below are
potential method analytes and single-laboratory accuracy and preci-
sion data have been determined for the compounds as described in
Sect. 13. The specific analytical conditions given in the method
are applicable to those compounds for which accuracy and precision
data are given. Other analytes (Sect. 1.2) may require slight
adjustments of analytical conditions. A laboratory may use this
method to identify and measure additional analytes after the labora-
tory obtains acceptable (defined in Sect. 9) accuracy and precision
data for each added analyte.
Compound
benzidine
benzoylprop ethyl
caffeine
carbaryl
o-chlorophenyl thiourea
3,3'-dichlorobenzidine
S.S'-dimethoxybenzidine
3,3'-dimethylbenzidine
diuron
ethylene thiourea
linuron (Lorox)
monuron
rotenone
siduron
1Monoisotopic molecular weight calculated from the atomic masses of
the isotopes with the smallest masses.
Abbre-
viation
BZ
BP
CF
CL
PT
DB
MB
LB
DI
ET
LI
MO
RO
SI
MW1
184
365
194
201
186
252
244
212
232
102
248
198
394
232
Chemical Abstracts Service
Reaistrv Number fCASRN)
92-87-5
33878-50-1
58-08-2
63-25-2
5344-82-1
91-94-1
119-90-4
612-82-8
330-54-1
96-45-7
330-55-2
150-68-5
83-79-4
1982-49-6
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1.2 Preliminary investigation indicates that the following compounds may
be amenable to this method: Aldicarb sulfone, Carbofuran, Methio-
carb, Methomyl (Larmate), Mexacarbate (Zectran), and N-(l-Naphthyl)
thiourea. Caffeine, Ethylene thiourea and o-Chlorophenyl thiourea
have been successfully analyzed by HPLC/PB/MS, but have not been
successfully extracted from a water matrix.
1.3 Method detection limit (MDL) is defined as the statistically calcu-
lated minimum amount that can be measured with 99% confidence that
the reported value is greater than zero (1). The MDL is compound
dependent and is particularly dependent on extraction efficiency and
sample matrix. For the analytes listed in Tables 3-5, the estimated
MDLs range from 2 to 30 ng/L.
2. SUMMARY OF METHOD
2.1 Organic compound, analytes and surrogates are extracted from 1 L of
water sample by liquid-liquid extraction (LLE) with methylene
chloride or by passing 1 L of sample water through a cartridge or
disk containing a solid inorganic matrix coated with a chemically
bonded C^ organic phase or a neutral polystyrene/divinyl benzene
polymer (liquid-solid extraction, LSE). If LLE is used, the
analytes are concentrated in methanol by evaporation of the methy-
lene chloride and addition of methanol (solvent exchange). If LSE
is used, the analytes are eluted from the LSE cartridge or disk with
a small quantity of methanol and concentrated further by evaporation
of some of the solvent. The sample components are separated,
identified, and measured by injecting an aliquot of the concentrated
methanol solution into a high performance liquid chromatograph
(HPLC) containing a reverse phase HPLC column and Interfaced to a
mass spectrometer (MS) with a particle beam (PB) interface. Com-
pounds eluting from the HPLC column are identified by comparing
their measured mass spectra and retention times to reference spectra
and retention times in a data base. Reference spectra and retention
times for analytes are obtained by measurement of calibration
standards under the same conditions used for samples. The concen-
tration of each identified component is measured by relating the MS
response of the quantitation ion produced by that compound to the MS
response of the quantitation ion produced by the same compound in a
calibration standard (external standard). Surrogate analytes, whose
concentrations are known in every sample, are measured with the same
external standard calibration procedure. An optional isotope
dilution procedure is included for samples which contain interfering
matrix or coeluting compounds.
3. DEFINITIONS
3.1 EXTERNAL STANDARD (ES) — A pure analyte(s) that is measured in an
experiment separate from the experiment used to measure the
analyte(s) in the sample. The signal observed for a known quantity
of the pure external standard(s) is used to calibrate the instrument
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response for the corresponding analyte(s). The instrument response
is used to calculate the concentrations of the analyte(s) in the
sample.
3.2 SURROGATE ANALYTE (SA) — A pure analyte(s), which is extremely
unlikely to be found in any sample, and which is added to a sample
aliquot in known amount(s) before extraction and is measured with
the same procedures used to measure other sample components. The
purpose of a surrogate analyte is to monitor method performance with
each sample.
3.3 LABORATORY DUPLICATES (LD1 and LD2) — Two aliquots of the same
sample taken in the laboratory and analyzed separately with identi-
cal procedures. Analyses of LD1 and LD2 indicate the precision
associated with laboratory procedures, but not with sample collec-
tion, preservation, or storage procedures.
3.4 FIELD DUPLICATES (FD1 and FD2) — Two separate samples collected at
the same time and place under identical circumstances and treated
exactly the same throughout field and laboratory procedures.
Analyses of FD1 and FD2 give a measure of the precision associated
with sample collection, preservation and storage, as well as with
laboratory procedures.
3.5 LABORATORY REAGENT BLANK (LRB) — An aliquot of reagent water or
other blank matrix that is treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, and
surrogates that are used with other samples. The LRB .is used to
determine if method analytes or other interferences are present in
the laboratory environment, the reagents, or the apparatus.
3.6 FIELD REAGENT BLANK (FRB) — An aliquot of reagent water or other
blank matrix that is placed in a sample container in the laboratory
and treated as a sample in all respects, including shipment to the
sampling site, exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB
is to determine if method analytes or other interferences are
present in the field environment.
3.7 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) ~ A solution of one or
more method analytes, surrogates, internal standards, or other test
substances used to evaluate the performance of the instrument system
with respect to a defined set of method criteria.
3.8 LABORATORY FORTIFIED BLANK (LFB) — An aliquot of reagent water or
other blank matrix to which known quantities of the method analytes
are added in the laboratory. The LFB is analyzed exactly like a
sample, and its purpose is to determine whether the methodology is
in control, and whether the laboratory is capable of making accurate
and precise measurements.
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3.9 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) — An aliquot of an envi-
ronmental sample to which known quantities of the method analytes
are added in the laboratory. The LFM is analyzed exactly like a
sample, and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background concen-
trations of the analytes in the sample matrix must be determined in
a separate aliquot and the measured values in the LFM corrected for
background concentrations.
3.10 STOCK STANDARD SOLUTION (SSS) — A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed
reference materials or purchased from a reputable commercial source.
3.11 PRIMARY DILUTION STANDARD SOLUTION (PDS)— A solution of several
analytes prepared in the laboratory from stock standard solutions
and diluted as needed to prepare calibration solutions and other
needed analyte solutions.
3.12 CALIBRATION STANDARD (CAL) — A solution prepared from the primary
dilution standard solution or stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.13 QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of
known concentrations which is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally prepared
test materials.
3.14 INSTRUMENT DETECTION LIMIT (IDL) - The minimum quantity of analyte
or the concentration equivalent which gives an analyte signal equal
to three times the standard deviation of the background signal at
the selected wavelength, mass, retention time, absorbance line, etc.
4. INTERFERENCES
4.1 When two compounds coelute, the transport efficiency of both com-
pounds through the particle beam interface generally improves and
enhanced ion abundances are observed in the mass spectrometer (2).
The degree of signal enhancement by coelution is compound dependent.
This coelution effect invalidates the external calibration curve
and, if not recognized, will result in incorrect concentration
measurements. Procedures given in this method to check for co-
el ut ing compounds must be followed to preclude inaccurate measure-
ments (Sect. 10.2.6.5 and Sect. 12.1). An optional isotope dilution
calibration procedure has been included for use when interfering
matrix or coeluting compounds are present.
4.2 During analysis, major contaminant sources are reagents, chromatog-
raphy columns, and liquid-solid extraction columns or disks.
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Analyses of field and laboratory reagent blanks provide information
about the presence of contaminants.
4.3 Interfering contamination may occur when a sample containing low
concentrations of compounds is analyzed immediately after a sample
containing relatively high concentrations of compounds. Syringes,
injectors, and other equipment must be cleaned carefully or replaced
as needed. After analysis of a sample containing high concentra-
tions of compounds, a laboratory reagent blank should be analyzed to
ensure that accurate values are obtained for the next sample.
5. SAFETY
5.1 The toxicity or carcinogenicity of chemicals used in this method has
not been precisely defined; each chemical should be treated as a
potential health hazard, and exposure to these chemicals should be
minimized. Each laboratory is responsible for maintaining awareness
of procedures and regulations for safe handling of chemicals used in
this method (3-5).
5.2 Some method analytes have been tentatively classified as known or
suspected human or mammalian carcinogens. Pure standard materials
and stock standard solutions of all analytes should be handled with
suitable protection to skin, eyes, etc.
6. EQUIPMENT AND SUPPLIES
6.1 All glassware must be meticulously cleaned. This may be accom-
plished by washing with detergent and water, rinsing with water,
distilled water, or solvents, air-drying, and heating (where appro-
priate) in an oven. Volumetric glassware is never heated.
6.2 SAMPLE CONTAINERS -- 1-L or 1-qt amber glass bottles fitted with a
Teflon-lined screw cap. (Bottles in which high purity solvents were
received can be used as sample containers without additional clean-
ing if they have been handled carefully to avoid contamination
during use and after use of original contents.)
6.3 SEPARATORY FUNNELS — 2-L and 100-mL with a Teflon stopcock.
6.4 LIQUID CHROMATOGRAPHY COLUMN RESERVOIRS ~ Pear-shaped 100- or 125-
mL vessels without a stopcock but with a ground glass outlet sized
to fit the liquid-solid extraction column. (Lab Glass, Inc., Part
No. ML-700-706S, with a 24/40 top outer joint and a 14/35 bottom
inner joint, or equivalent.) A 14/35 outlet joint fits some commer-
cial cartridges.
6.5 SYRINGE NEEDLES — No. 18 or 20 stainless steel.
6.6 VACUUM FLASKS — 1 or 2 L with solid rubber stoppers.
6.7 VOLUMETRIC FLASKS — Various sizes.
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6.8 LABORATORY OR ASPIRATOR VACUUM SYSTEM — Sufficient capacity to
maintain a slight vacuum of 13 cm (5 in) of mercury in the vacuum
flask.
6.9 MICRO SYRINGES — Various sizes.
6.10 VIALS — Various sizes of amber vials with Teflon-lined screw caps.
6.11 DRYING COLUMN — 0.6 cm x 40 cm with 10 mL graduated collection
vial.
6.12 CONCENTRATOR TUBE — Kuderna-Danish (K-D) 10 mL graduated with
ground glass stoppers.
6.13 ANALYTICAL BALANCE — Capable of weighing 0.0001 g accurately.
6.14 LIQUID CHROMATOGRAPHY COLUMN - A 15-25 cm x 2 mm (i.d.) stainless
steel tube (e.g., Waters C-18 Novapak or equivalent) packed with
silica particles (4-10 /Ltm) with octadecyldimethylsilyl (C-18)
groups chemically bonded to the silica surface. Residual acidic
sites should be blocked (endcapped) with methyl or other non-polar
groups and the stationary phase must be bonded to the solid support
to minimize column bleed. Column selection for minimum bleeding is
strongly recommended. The column must be conditioned over night
before each use by pumping a 75-100% v/v acetonitrile: water
solution through it at a rate of about 0.05 mL/min. Other packings
and column sizes may be used if equivalent or better performance can
be achieved.
6.15 Guard column of similar packing used in the analytical column is
recommended.
6.16 LIQUID CHROMATOGRAPH/MASS SPECTROMETER/DATA SYSTEM (LC/MS/DS)
6.16.1 The LC-must accurately maintain flow rates between 0.20-
0.40 mL/min while performing a gradient elution from 100%
solvent A to 100% solvent B. Pulse dampening is recommend-
ed but not required. An autoinjector is highly desirable
and should be capable of accurately delivering 1-10 fj,L
injections without affecting the chromatography.
6.16.2 The system should include a post-column mixing tee and an
additional 1C pump for post-column addition of acetonitrile
at a constant rate of 0.1 - 0.7 mL/min.
6.16.3 The particle beam LC/MS interface must reduce the system
pressure to a level fully compatible with the generation of
classical electron ionization (El) mass spectra, i.e.,
about 1 X 10"6 to 1 X 10"4 Torr, while delivering sufficient
quantities of analytes to the conventional El source to
meet sensitivity, accuracy, and precision requirements.
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All significant background components with mass greater
than 62 Dal tons should be removed to a level that does not
produce ions greater than a relative abundance of 10% in
the mass spectra of the analytes.
6.16.4 The mass spectrometer must be capable of electron ioniza-
tion at a nominal electron energy of 70 eV. The spectrome-
ter must be capable of scanning from 45 to 500 amu with a
complete scan cycle time (including scan overhead) of 1.5
sec or less. (Scan cycle time = Total MS data acquisition
time in seconds divided by number of scans in the chromato-
gram). The spectrometer must produce a mass spectrum that
meets all criteria in Table 1 when 500 ng or less of DFTPPO
(Sect. 7.11) is introduced into the LC. An average spec-
trum across the DFTPPO LC peak may be used to test instru-
ment performance.
6.16.5 An interfaced data system is required to acquire, store,
reduce, and output mass spectral data. The computer soft-
ware should have the capability of processing stored LC/MS
data by integration of the ion abundance of any specific
ion between specified time or scan number limits, construc-
tion of a first or second order regression calibration
curves, calculation of response factors as defined in Sect.
10.2.9, calculation of response factor statistics (mean and
standard deviation), and calculation of concentrations of
analytes from the calibration curve or the equation in
Sect. 12.
6.17 MILLIPORE STANDARD FILTER APPARATUS, ALL GLASS — This will be used
if the disks are to be used to carry out the extraction instead of
cartridges.
7. REAGENTS AND STANDARDS
7.1 Helium nebulizer/carrier gas as contaminant free as possible.
7.2 LIQUID-SOLID EXTRACTION (LSE) MATERIALS
7.2.1 Cartridges are inert non-leaching plastic, for example
polypropylene, or glass and must not contain contaminants
that leach into methanol. The cartridges are packed with
various amounts of sorbents such as C18 or a neutral poly-
styrene/divinyl benzene polymer. The packing must have a
narrow size distribution and must not leach organic com-
pounds into methanol. One liter of water should pass
through the cartridge in about 2 hr with the assistance of
a slight vacuum of about 13 cm (5 in) of mercury. Faster
flow rates are acceptable if equivalent accuracy and preci-
sion are obtained. Robotic systems typically pump the
sample through a cartridge in less than 2 hr. These sys-
tems are also acceptable if equivalent accuracy and preci-
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sion are obtained. Sect. 9 and Tables 4 and 5 provide
criteria for acceptable LSE cartridges which are available
from several commercial suppliers.
7.2.2 Extraction disks (Empore) are thin filter-shaped materials
with C18 modified silica, or neutral polystyrene/divinyl-
benzene polymer, impregnated in a Teflon or other inert
matrix. As with cartridges, the disks should not contain
any organic compounds, either from the Teflon or the bonded
silica, which will leach into the methanol eluant. One
liter of reagent water should pass through the disks in 5-
20 min using a vacuum of about 66 cm (26 in) of mercury.
Sect. 9 provides criteria for acceptable LSE disks which
are available commercially.
7.3 SOLVENTS
7.3.1 Acetonitrile, methylene chloride, and methanol — HPLC
grade and pesticide quality or equivalent.
7.3.2 Reagent water — Water in which an interferant is not
observed at the MDL of the compound of interest. Prepare
reagent water by passing tap water through a filter bed
containing about 0.5 kg of activated carbon or by using a
water purification system. Store in clean, narrow-mouth
bottles with Teflon-lined septa and screw caps.
7.4 Hydrochloric acid, concentrated.
7.5 Sodium sulfate, anhydrous.
7.6 REDUCING AGENTS FOR CHLORINATED WATER — Sodium sulfite, sodium
thiosulfate or sodium arsenite.
7.7 AMMONIUM ACETATE, SODIUM CHLORIDE, AND SODIUM HYDROXIDE (IN) ~ ACS
reagent grade.
7.8 STOCK STANDARD SOLUTIONS (SSS) -- Individual solutions of analytes,
surrogates, and isotopically labelled analogues of the analytes may
be purchased as certified solutions or prepared from pure materials.
To prepare, add 10 mg (weighed on an analytical balance to 0.1 mg)
of the pure material to 1.9 mL of methanol or acetonitrile in a 2-mL
volumetric flask, dilute to the mark, and transfer the solution to
an amber glass vial. Certain analytes, such as 3,3'-dimethoxy-
benzidine, may require dilution in 50% v/v acetonitrile or methanol:
water solution. If the analytical standard is available only in
quantities smaller than 10 mg, reduce the volume of solvent accord-
ingly. If compound purity is certified by the supplier at >96%, the
weighed amount can be used without correction to calculate the
concentration of the solution (5 jug/juL). Store the amber vials in
a freezer at < 0°C.
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7.8.1 Benzidines as the free base or as acid chlorides may be
used for calibration purposes. However, the concentration
of the standard must be calculated as the free base.
7.9 PRIMARY DILUTION STANDARD SOLUTION (PDS) — The stock standard
solutions are used to prepare a primary dilution standard solution
that contains multiple analytes. The recommended solvent for this
dilution is a 50% v/v acetonitrile:water mixture. Aliquots of each
of the stock standard solutions are combined to produce the primary
dilution in which the concentration of the analytes is at least
equal to the concentration of the most concentrated calibration
solution. Store the primary dilution standard solution in an amber
vial in a freezer at < 0°C, and check frequently for signs of
deterioration or evaporation, especially just before preparing
calibration solutions.
7.10 FORTIFICATION SOLUTION OF SURROGATES ~ The analyst should monitor
the performance of the extraction, cleanup (when used), and analyti-
cal system and the effectiveness of the method in dealing with each
sample matrix by spiking each sample, standard, and blank with 1 or
2 surrogates recommended to encompass the range of the gradient
elution program used in this method. The compounds recommended as
surrogates for the analysis of benzidines and nitrogen-containing
pesticides are benzidine-D8 (DBZ), caffeine- 5N2 (NCF), 3,3'-di-
chlorobenzidine-D6 (DCB), and bis-(perfluorophenyl)-phenylphosphine
oxide (OD) unless their unlabelled counterpart is being analyzed or
they will be used for isotope dilution calibration (Abbreviations in
parentheses are used in Figure 4). Prepare a solution of the
surrogates in methanol or acetonitrile at a concentration of 5 mg/mL
of each. Other surrogates may be included in this solution as
needed. (A 10-0L aliquot of this solution added to 1 L of water
gives a concentration of 50 /ug/L of each surrogate). Store the
surrogate fortifying solution in an amber vial in .a freezer at
< 0°C.
7.11 MS PERFORMANCE CHECK SOLUTION — Prepare a 100 ng//iL solution of
bis-(perfluorophenyl)-phenylphosphine oxide (DFTPPO) in acetoni-o
trile. Store this solution in an amber vial in a freezer at < 0 C.
DFTPPO is not currently commercially available. For this method
development work, DFTPPO was synthesized from bis-(perfluorophenyl)
phenyl phosphine (DFTPP) in solution by adding a slight excess of
hydrogen peroxide (DFTPP + H202 •+ DFTPPO + H20). The solvent was
removed and the resulting crystals were thoroughly washed with water
to remove any residual hydrogen peroxide. It is critical to remove
all residual hydrogen peroxide before adding the DFTPPO to the CAL
solution. Any residual hydrogen peroxide will degrade some
analytes.
7.12 CALIBRATION SOLUTIONS (CAL1 - CAL6) -- Prepare a series of six
concentration calibration solutions in acetonitrile which contain
all analytes at concentrations of 2, 5, 10, 25, 50 and 100 times the
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instrument detection limit of each compound with a constant concen-
tration of each surrogate in each CAL solution. This calibration
range may be optimized by the operator, but each analyte must be
bracketed by at least 2 calibration points. CAL1 through CAL6 are
prepared by combining appropriate aliquots of the primary dilution
standard solution (Sect. 7.9) and the fortification solution of
surrogates (Sect. 7.10). DFTPPO may be added to one or more CAL
solutions to verify MS tune (See Sect. 10.3.1.). Store these
solutions in amber vials in a freezer at < 0°C. Check these solu-
tions quarterly for signs of deterioration.
7.12.1 For isotope dilution calibration, prepare the calibration
solutions as described above with the addition of one
coeluting isotopically labelled analog for each analyte of
interest. The concentration for each coeluting labelled
standard should be approximately 25 to 50 times the instru-
ment detection limit of the analyte of interest and must be
constant in all calibration solutions (CAL1 through CAL6).
These solutions permit the relative response (unlabelled to
labelled) to be measured as a function of the amount of
analyte injected. If more than one labelled compound is
used, one spiking solution containing all labelled com-
pounds should be prepared.
7.13 MOBILE PHASE — Solvent A is a 75:25 v/v water:acetonitrile solution
containing ammonium acetate at a concentration of O.Oi M. This
composition is used to eliminate biological activity in the A Phase.
Solvent B is acetonitrile. Both solvents are degassed in an ultra-
sonic bath under reduced pressure and maintained by purging with a
low flow of helium.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 SAMPLE COLLECTION — When sampling from a water tap, open the tap
and allow the system to flush until the water temperature has
stabilized (usually about 2-5 min). Adjust the flow to about 500
mL/min and collect samples from the flowing stream. Keep samples
sealed from collection time until analysis. When sampling from an
open body of water, fill the sample container with water from a
representative area. Sampling equipment, including automatic
samplers, must be free of plastic tubing^ gaskets, and other parts
that may leach analytes into water. Automatic samplers that compos-
ite samples over time must use refrigerated glass sample containers.
8.2 SAMPLE DECHLORINATION AND PRESERVATION — All samples should be iced
or refrigerated at 4°C from the time of collection until extraction.
Residual chlorine should be reduced at the sampling site by addition
of a reducing agent. Add 40-50 mg of sodium sulfite or sodium
thiosulfate (these may be added as solids with stirring until
dissolved) to each liter of water.
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8.3 HOLDING TIME — Samples must be extracted within 7 days and the IP
extracts analyzed within 30 days of sample collection. Extracts
should be stored in an amber vial in a freezer at < 0°C.
8.4 FIELD BLANKS
8.4.1 Processing of a field reagent blank (FRB) is recommended
along with each sample set, which is composed of the sam-
ples collected from the same general sample site at approx-
imately the same time. At the laboratory, fill a sample
container with reagent water, seal, and ship to the sam-
pling site along with the empty sample containers. Return
the FRB to the laboratory with filled sample bottles.
9. QUALITY CONTROL
9.1 Quality control (QC) requirements are the initial demonstration of
laboratory capability followed by regular analyses of LRBs, LFBs,
and laboratory fortified matrix samples. The laboratory must
maintain records to document the quality of the data generated.
Additional QC practices- are recommended.
9.2 Initial demonstration of low system background and acceptable
particle size and packing. Before any samples are analyzed, or any
time a new supply of LSE cartridges or disks is received from a
supplier, or a new column is installed, it must be demonstrated that
a LRB is reasonably free of contamination that would prevent the
determination of any analyte of concern. In this same experiment it
must be demonstrated that the particle size and packing of the LSE
cartridge are acceptable. Consistent flow rate is an indication of
acceptable particle size distribution and packing.
9.2.1 A source of potential contamination may be, the liquid-
solid extraction (LSE) cartridges and disks and columns
which may contain silicon compounds and other contaminants
that could prevent the determination of method analytes.
Generally, contaminants will be leached from the cart-
ridges, disks, or columns into the solvent and produce a
variable background. If the background contamination is
sufficient to prevent accurate and precise analyses, this
condition must be corrected before proceeding with the '
initial demonstration. Figure 1 shows unacceptable back-
ground contamination from a column with stationary phase
bleed.
9.2.2 Other sources of background contamination are solvents,
reagents, and glassware. Background contamination must be
reduced to an acceptable level before proceeding with the
next section. In general, background for method analytes
should be below the MDL.
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9.2.3 One liter of water should pass through the cartridge in
about 2 hr (faster flow rates are acceptable if precision
and accuracy are acceptable) with a partial vacuum of about
13 cm (5 in) of mercury. The extraction time should not
vary unreasonably among LSE cartridges. Robotic systems
typically pump the sample through a cartridge in less than
2 hr. These systems are also acceptable if equivalent
accuracy and precision are obtained. Extraction disks may
be used at a faster flow rate (See Sect. 7.2.2).
9.3 INITIAL DEMONSTRATION OF LABORATORY ACCURACY AND PRECISION ~
Analyze 5-7 replicates of a LFB containing each analyte of concern
at a concentration in the range of 10-50 times the instrument
detection limits (see regulations and maximum contaminant levels for
guidance on appropriate concentrations).
9.3.1 Prepare each replicate by adding an appropriate aliquot of
the PDS, or another certified quality control sample, to
reagent water. Analyze each replicate according to the
procedures described in Sect. 11 and on a schedule that
results in the analyses of all replicates with 48 hr.
9.3.2 Calculate the measured concentration of each analyte in
each replicate, the mean concentration of each analyte in
all replicates, and mean accuracy (as mean percentage of
true value) for each analyte, and the precision (as rela-
tive standard deviation, RSD) of the measurements for each
analyte. Calculate the MDL of each analyte using the
referenced procedures (1).
9.3.3 For each analyte and surrogate, the mean accuracy,
expressed as a percentage of the true value, should be
70-130% and the RSD should be < 30%, The MDLs should be
sufficient to detect analytes at the regulatory levels. If
these criteria are not met for an analyte, take remedial
action and repeat the measurements for that analyte to
demonstrate acceptable performance before samples are
analyzed.
9.3.4 Develop and maintain a system of control charts to plot the
precision and accuracy of analyte and surrogate measure-
ments as a function of time. Charting of surrogate recov-
eries is an especially valuable activity since these are
present in every sample and the analytical results will
form a significant record of data quality.
9.4 LABORATORY REAGENT BLANKS (LRBs) — With each batch of samples
processed as a group within a work shift, analyze a laboratory
reagent blank to determine the background system contamination. Any
time a new batch of LSE cartridges or disks is used, or new supplies
of other reagents are used, repeat the demonstration of low back-
ground described in Sect. 9.2.
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9.5 With each batch of samples processed as a group within a work shift,
analyze a single LFB containing each analyte of concern at a concen-
tration as determined in Sect. 9.3. Evaluate the accuracy of the
measurements (Sect. 9.3.3), and estimate whether acceptable MDLs can
be obtained. If acceptable accuracy and MDLs cannot be achieved,
the problem must be located and corrected before further samples are
analyzed. Add these results to the ongoing control charts to
document data quality.
9.6 Determine that the sample matrix does not contain materials that
adversely affect method performance. This is accomplished by
analyzing replicates of laboratory fortified matrix samples and
ascertaining that the precision, accuracy, and MDLs of analytes are
in the same range as obtained with LFBs. If a variety of different
sample matrices are analyzed regularly, for example, drinking water
from groundwater and surface water sources, matrix independence
should be established for each.
9.7 With each set of field samples a FRB should be analyzed. The
results of these analyses will help define contamination resulting
from field sampling and transportation activities.
9.8 At least quarterly, replicates of LFBs should be analyzed to deter-
mine the precision of the laboratory measurements. Add these
results to the ongoing control charts to document data quality.
9.9 At least quarterly, analyze a QCS from an external source. If
measured analyte concentrations are not of acceptable accuracy
(Sect. 9.3.3), check the entire analytical procedure to locate and
correct the problem source.
9.10 Numerous other specific QC measures are incorporated into other
parts of this procedure, and serve to alert the analyst to potential
problems.
10. CALIBRATION AND STANDARDIZATION
10.1 Demonstration and documentation of acceptable initial calibration
and system optimization are required before any samples are analyzed
and is required intermittently during sample analysis as indicated
by results of continuing calibration checks. After initial calibra-
tion is successful, a continuing calibration check is required at
the beginning of each 8-hr period during which analyses are per-
formed. Additional periodic calibration checks are good laboratory
practice.
10.2 INITIAL CALIBRATION
10.2.1 Optimize the interface according to the manufacturer's
instructions. This usually is accomplished on initial
installation by flow injection with caffeine or benzidine
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and should utilize a mobile phase of 50% v/v acetonitrile:
water. Major maintenance may require reoptimization.
10.2.2 Calibrate the MS mass and abundance scales using the cali-
bration compound and manual (not automated) ion source
tuning procedures specified by the manufacturer. Calibra-
tion must be accomplished while a 50% v/v acetonitrile:
water mixture is pumped through the 1C column and the
optimized particle beam interface. For optimum long-term
stability and precision, interface and ion source
parameters should be set near the center of a broad signal
plateau rather than at the peak of a sharp maximum (sharp
maxima vary short term with particle beam interfaces and
gradient elution conditions).
10.2.3 Fine tune the interface by making a series of injections
into the LC column of a medium level CAL standard and
adjusting the operating parameters until optimum sensitivi-
ty and precision are obtained for the maximum number of
target compounds (6). Suggested additional operating
conditions are:
mobile phase purge: helium at 30 mL/min continuous,
mobile phase flow rate: 0.3 mL/min through the column,
gradient elution: Hold for 1 min at 25% acetonitrile,
then linearly program to « 70% acetonitrile in 29 min,
start data acquisition immediately,
post-column addition: acetonitrile at 0.1-0.7 mL/min,
depending on the interface requirements. Maintain a
minimum of 30% acetonitrile in the interface to improve
system precision and possibly sensitivity,
desolvation chamber temperature: 45°-80°C,
ion source temperature: 250° - 290°C,
electron energy: 70 eV, and
scan range: 62-465 amu at 1-2 sec/scan.
10.2.4 The medium level standard (CAL) used in Sect. 10.2.3 should
contain DFTPPO, or separately inject into the LC a 5-/iL
aliquot of the 100 ng//zL DFTPPO solution and acquire a
mass spectrum that includes data for m/z 62-465. Use LC
conditions that produce a narrow (at least ten scans per
peak) symmetrical peak. If the spectrum does not meet all
criteria (Table 1), the MS ion source must be retuned and
adjusted to meet all criteria before proceeding with cali-
187
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in
bration. An average spectrum across the LC peak may be ^^
used to evaluate the performance of the system. Figure 2
represents the average composite spectrum obtained for
DFTPPO from a multilaboratory study involving 5 different
particle beam interfaces from 13 laboratories.
10.2.5 Inject a 5-/iL aliquot of a medium concentration calibra-
tion solution, for example 50 ng//iL, and acquire and store
data from m/z 62-465 with a total cycle time (including
scan overhead time) of 1.5 sees or less. Cycle time should
be adjusted to measure at least ten spectra during the
elution of each LC peak.
10.2.6 Performance criteria for the medium calibration. Examine
the stored LC/MS data with the data system software. Fig-
ure 3 shows an acceptable total ion chromatogram.
10.2.6.1 LC performance. 3,3'-dimethyl- and 3,3'- dimeth-
oxybenzidine should be separated by a valley whose
height is less than 25% of the average peak height
of these two compounds. If the valley between
them exceeds 25%, modify the gradient. If this
fails, the LC column requires maintenance. (See
Sect. 10.3.6)
10.2.6.2 Peak tailing — Examine a total ion chromatogram
and determine the degree of peak tailing. Severe
tailing indicates a major problem and system main-
tenance is required to correct the problem. (See
Sect. 10.3.6)
10.2.6.3 MS sensitivity — Signal/noise in any analyte mass
spectrum should be at least 3:1.
10.2.6.4 Column bleed — Figure 1 shows an unacceptable
chromatogram with column bleed. Figure 3 is the
mass spectrum of dimethyloctadecylsilanol, a com-
mon stationary phase bleed product. If unaccept-
able column bleed is present, the column must be
changed or conditioned to produce an acceptable
background (Figure 4).
10.2.6.5 Coeluting compounds — Compounds which coelute
cannot be measured accurately because of carrier
effects in the particle beam interface. Peaks
must be examined carefully for coeluting substanc-
es and if coeluting compounds are present at
greater than 10% the concentration of the target
compound, conditions must be adjusted to resolve
the components, the target compound must be
188
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flagged as positively biased, or isotope dilution
calibration should be used.
10.2.7 If all performance criteria are met, inject a 5-p.l aliquot
of each of the other CAL solutions using the same LC/MS
conditions.
10.2.8 The general method of calibration (external) is a second
order regression of integrated ion abundances of the
quantitation ions (Table 2) as a function of amount inject-
ed. For second order regression, a sufficient number of
calibration points must be obtained to accurately determine
the- equation of the curve. For some individual analytes
over a short concentration range, reasonable linearity may
be observed and response factors may be used. Calculate
response factors using the equation below. Second order
regressions and response factor calculations are supported
in acceptable LC/MS data system software (Sect. 6.16.5),
and many other software programs.
(Ax)
RF = ——
where: Ax = integrated abundance of the quantitation ion
of the analyte.
(|x = quantity of analyte injected in ng or
concentration units.
10.2.9 If response factors are used (i.e., linear calibration with
the line going through the origin), calculate the mean RF
from the analyses of the six CAL solutions for each analyte
and surrogate. Calculate the standard deviation (SD) and
the relative standard deviation (RSD) from each mean (M):
RSD = 100 (SD/M). If the RSD of any analyte or surrogate
mean RF exceeds 20%, either analyze additional aliquots of
appropriate CAL solutions to obtain an acceptable RSD of
RFs over the entire concentration range, take action to
improve LC/MS performance, or use the second order regres-
sion calibration. (See Sect. 10.2.8)
10.3 CONTINUING CALIBRATION CHECK — Verify the MS tune and initial
calibration at the beginning of each 8-hr work shift during which
analyses are performed using the following procedure:
10.3.1 Inject a 5-jiL aliquot of the 100 ng//iL DFTPPO solution
(this may be contained in the medium level CAL solution
used in Sect. 10.3.2) and acquire a mass spectrum that
includes data for m/z 62-465. If the spectrum does not
meet all criteria (Table 1), the MS must be retuned and
189
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adjusted to meet all criteria before proceeding with the
continuing calibration check.
10.3.2 Inject a 5-/uL aliquot of a medium level CAL solution and
analyze with the same conditions used during the initial
calibration. One or more additional CAL solutions should
be analyzed.
10.3.3 Demonstrate acceptable performance for the criteria shown
in Sect. 10.2.6.
10.3.4 Determine that the absolute areas of the quantitation ions
of the external standards and surrogate(s) have not changed
by more than 20% from the areas measured during initial
calibration. If these areas have changed by more than 20%,
recalibration and other adjustments are necessary. These
adjustments may require cleaning of the MS ion source, or
other maintenance as indicated in Sect. 10.3.6, and
recalibration. Control charts are useful aids in document-
ing system sensitivity changes.
10.3.5 Using the previously generated second order regression
curve, calculate the concentrations in the medium level CAL
solution and compare the results to the known values in the
CAL solution. If calculated concentrations deviate by more
than 20% from known values, recalibration of the system
with the 6 CAL solutions is required. If response factors
were used, calculate the RF for each analyte and surrogate
from the data measured in the continuing calibration check.
The RF for each analyte and surrogate must be within 20% of
the mean value measured in the initial calibration.
10.3.6 Some possible remedial actions — Major maintenance such as
cleaning an ion source, cleaning quadrupole rods, etc.
require returning to the initial calibration step.
10.3.6.1 Check and adjust LC and/or MS operating condi-
tions; check the MS resolution, and calibrate
the mass scale.
10.3.6.2 Replace the mobile phases with fresh solvents.
Verify that the combined flow rate from the LC
and post-column addition pumps is constant.
10.3.6.3 Flush the LC column with acetonitrile.
10.3.6.4 Replace LC column; this action will cause a
change in retention times.
10.3.6.5 Prepare fresh CAL solutions and repeat the
initial calibration step. dlk
190
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10.3.6.6 Clean the MS ion source, entrance lens, and
rods (if a quadrupole).
10.3.6.7 Replace any components that leak.
10.3.6.8 Replace the MS electron multiplier or any other
faulty components.
10.3.6.9 Clean the interface to eliminate plugged compo-
nents and/or replace skimmers according to the
manufacturer's instructions.
10.3.6.10 If automated peak areas are being used, verify
values by manual integration.
10.3.6.11 Increasing ion source temperature can reduce
peak tailing, but excessive ion source tempera-
ture can affect the quality of the spectra for
some compounds'.
10.3.6.12 Air leaks into the interface may affect the
quality of the spectra (e.g. DFTPPO), especial-
ly when ion source temperatures are operated in
excess of 280°.
10.4 CALIBRATION WITH ISOTOPE DILUTION (OPTIONAL) — For samples with
interfering matrix or coeluting peaks, the most reliable method for
quantitation is the use of coeluting isotope labelled internal
standards (7). Isotope dilution calibration will be limited by the
availability and cost of the labelled species and the requirement
that each analyte must coelute with the labelled internal standard.
Because the labelled internal standard must coelute with the
analyte, the quantitation ion for the internal standard must be
larger than that of the analyte and not present in the analyte's
mass spectrum. 'In addition, it must be verified that the labelled
internal standard is not contaminated by its unlabelled counterpart.
10.4.1 A calibration curve encompassing the concentration range is
prepared for each compound to be determined. The relative
response (analyte integrated ion abundances to labelled
integrated ion abundance) vs. amount of analyte injected is
plotted using linear regression. A minimum of five data
points are employed for this type of calibration.
10.4.2 To calibrate, inject a 5.0 ill aliquot of each of the cali-
bration standards (Sect. 7.12.1) and compute the relative
response (analyte integrated ion abundances to labelled
compound integrated ion abundance). Plot this versus the
amount of analyte injected by linear regression. This
plotted line or the equation of this line should be used '
for quantitative calculations. Unless this line goes
through the origin, the response factors at each point will
• 191
-------
.
cannot be used. These calculations are supported in ac-
ceptable LC/MS data system software (Sect. 6.15.5), and in
many other software programs.
10.4.3 Follow Sect. 10.3 to verify calibration at the beginning of
each 8-hr work shift by injecting a 5.0 /iL aliquot of a
medium CAL solution and analyze it with the same conditions
used during the initial calibration. Using the previously
generated first order regression line (relative response
versus amount of analyte injected), calculate the concen-
trations in the medium level CAL solution and compare the
results to the known values in the CAL solution. If calcu-
lated concentrations deviate by more than 20% from known
values, recalibration of the system with the CAL solutions,
containing the isotopically labelled analogues, is re-
quired.
11. PROCEDURE
11.1 The extraction procedure depends on the analytes selected and the
nature of the sample. LSE (cartridge or disk) is limited to partic-
ulate-free water, e.g., drinking water. Consult Tables 3-5 to
determine which analytes are amenable to liquid-solid and
liquid-liquid extractions. Sect. 11.2 provides the LSE procedure
using cartridges and Sect. 11.3 provides the LSE procedure using
disks. Sect. 11.4 provides the procedure for LLE. After the
extraction is complete, proceed to Sect. 11.5 to continue with the
method.
11.2 LIQUID-SOLID EXTRACTION (LSE) PROCEDURE USING CARTRIDGES (This
procedure may be manual or automated).
11.2.1 Set up the extraction apparatus shown in Figure 5. The
reservoir is not required but recommended for convenient
operation. Water drains from the reservoir through the LSE
cartridge and into a syringe needle which is inserted
through a rubber stopper into the suction flask. A slight ,
vacuum of 13 cm (5 in) of mercury is used during all opera-
tions with the apparatus. The pressure used is critical as
a vacuum greater than 13 cm may result in poor precision.
About 2 hr is required to draw a liter of water through the
cartridge, but faster flow rates are acceptable if preci-
sion and accuracy are acceptable. The use of robotic
extraction systems is acceptable if equivalent MDLs, preci-
sion and accuracy are obtained.
11.2.2 Mark the water meniscus on the side of the sample bottle
for later determination of the sample volume. A 1-L sample.
is recommended. Pour the water sample into the 2-L separa-
tory funnel with the stopcock closed. Adjust the pH to 7.0
by the dropwise addition of hydrochloric acid or 1 N sodium
192
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hydroxide. Residual chlorine must not be present, as a
reducing agent should have been added at the time of sam-
pling. For extractions using C.8 cartridges, add 0.01 M
ammonium acetate (0.77 g in 1 L) to the water sample and
mix until homogeneous. Add a 10-juL aliquot of the forti-
fication solution for surrogates, and mix until homoge-
neous. The concentration of surrogates in the water should
be 10-50 times the instrument detection limit.
11.2.3 Flush each cartridge with two 10-mL aliquots of methanol,
letting the cartridge drain dry after each flush. This
solvent flush may be accomplished by adding methanol di-
rectly to the solvent reservoir in Figure 5. Add 10 ml of
reagent water to the solvent reservoir, but before the
reagent water level drops below the top edge of the packing
in the LSE cartridge, open the stopcock of the separatory
funnel and begin adding sample water to the solvent reser-
voir. Close the stopcock when an adequate amount of sample
is in the reservoir.
11.2.4 Periodically open the stopcock and drain a portion of the
sample water into the solvent reservoir. The water sample
will drain into the cartridge, and from the exit into the
suction flask. Maintain the packing material in the car-
tridge immersed in water at all times. Wash the separatory
funnel and cartridge with 10 ml of reagent water, and draw
air through the cartridge for 10 min.
11.2.5 Transfer the LSE cartridge to the elution apparatus shown
in Figure 5B. Wash the 2-L separatory funnel with 15 ml of
methanol, close the stopcock of the 100-mL separatory
funnel of the elution apparatus, and elute the cartridge
with two 7.5-mL aliquots of the methanol washings. Concen-
trate the extract to the desired volume under a gentle
stream of nitrogen. Record the exact volume of the ex-
tract.
11.2.5.1 If isotope dilution calibration is used, spike the
extract with the isotopically labelled standards
prior to solvent evaporation. The concentration
of these isotopically labelled compounds after the
desired extract volume is reached should be the
same as the concentration in each CAL solution.
11.2.6 Determine the original sample volume by refilling the
sample bottle to the mark and transferring the liquid to a
1000-mL graduated cylinder. Record the sample volume to
the nearest 5 rat.
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11.3 LIQUID-SOLID EXTRACTION (LSE) PROCEDURE USING DISKS (This procedure
may be manual or automated).
11.3.1 Mark the water meniscus on the side of the sample bottle
for later determination of the sample volume. A 1-L sample
is recommended. Pour the water sample into the 2-L separa-
tory funnel with the stopcock closed. Adjust the pH to 7.0
by the dropwise addition of hydrochloric acid or 1 N sodium
hydroxide. Residual chlorine must not be present because
a reducing agent should have been added at the time of sam-
pling. For extractions using C18 disks, add 0.01 M ammoni-
um acetate (0.77 g in 1 L) to the water sample and mix
until homogeneous. Add a 10-juL aliquot of the fortifica-
tion solution for surrogates, and mix until homogeneous.
The concentration of surrogates in the water should be 10-
50 times the instrument detection limit.
11.3.2 Preparation of Disks
11.3.2.1 Insert the disk into the 47 mm filter apparatus
(See Figure 6). Wash and pre-wet the disk with 10
mL methanol (MeOH) by adding the MeOH to the disk
and allowing it to soak for about a min, then
pulling most of the remaining MeOH through. A
layer of MeOH must be left on the surface of the
disk, which should not be allowed to go dry from
this point until the end of the sample extraction.
THIS IS A CRITICAL STEP FOR A UNIFORM FLOW AND
GOOD RECOVERY.
11.3.2.2 Rinse the disk with 10 mL reagent water by adding
the water to the disk and pulling most through,
again leaving a layer of water on the surface of
the disk.
11.3.3 Add the water sample to the reservoir and turn on the
vacuum to begin the extraction. Full aspirator vacuum may
be used. Particulate-free water may pass through the disk
in as little as 10 min or less. Extract the entire sample,
draining as much water from the sample container as possi-
ble.
11.3.4 Remove the filtration top from the flask, but do not disas-
semble the reservoir and fritted base. Empty the water
from the flask, and insert a suitable sample tube to con-
tain the eluant. The only constraint on the sample tube is
that it must fit around the drip tip of the fritted base.
Reassemble the apparatus.
11.3.5 Add 5 mL MeOH to the sample bottle, and rinse the inside
walls thoroughly. Allow the MeOH to settle to the bottom
194
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of the bottle, and transfer to the disk with a disposable
pipet, rinsing the sides of the glass filtration reservoir
in the process. Pull about half of the MeOH through the
disk, release the vacuum, and allow the disk to soak for a
minute. Pull the remaining MeOH through the disk.
11.3.6 Repeat the above step twice. Concentrate the combined
extracts to the desired volume under a gentle stream of
nitrogen. Record the exact volume of the extract. (Prelim-
inary investigation indicates that acetonitrile is a better
extraction solvent for rotenone when extracting water,
containing high levels of particulate matter, with LSE
disks.)
11.3.6.1 If isotope dilution calibration is used, spike the
extract with the isotopically labelled standards
prior to solvent evaporation. The concentration
of these isotopically labelled compounds after the
desired extract volume is reached should be the
same as the concentration in each CAL solution.
11.3.7 Determine the original sample volume by refilling the
sample bottle to the mark and transferring the liquid to a
1000-mL graduated cylinder. Record the sample volume to
the nearest 5 ml.
11.4 LIQUID-LIQUID EXTRACTION (LLE) PROCEDURE
11.4.1 Mark the water meniscus on the side of the sample bottle
for later determination of the sample volume. A 1-L sample
is recommended. Pour the water sample into a 2-L separa-
tory funnel with the stopcock closed. Residual chlorine
should not be present as a reducing agent should have been
added at the time of sampling. Add a 10-juL aliquot of the
fortification solution for surrogates, and mix until homo-
geneous. The concentration of surrogates in the water
should be 10-50 times the instrument detection limit.
11.4.2 Adjust the pH of the sample to 7.0 by dropwise addition of
hydrochloric acid or 1 N sodium hydroxide. Add 100 g of
sodium chloride to the sample and shake to dissolve the
salt.
11.4.3 Add 60 mL of methylene chloride to the sample bottle,
shake, and transfer the solvent to the separatory funnel
and extract the sample by vigorously shaking the funnel for
2 min with periodic venting to release excess pressure.
Allow the organic layer to separate from the water phase
for a minimum of 10 min. If the emulsion interface between
layers is more than onerthird the volume of the solvent
layer, mechanical techniques must be employed to complete
195
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the phase separation. The optimum technique depends on the
sample, but may include stirring, filtration of the emul-
sion through glass wool, centrifuging, etc. Collect the
methylene chloride extract in a 500-mL Erlenmeyer flask.
11.4.4 Add a second 60-mL volume of methylene chloride and repeat
the extraction a second time, combining the extracts in the
Erlenmeyer flask. Perform a third extraction in the same
manner.
11.4.5 Assemble a K-D concentrator by attaching a 10-mL concentra-
tor tube to a 500-mL evaporative flask. Dry the extract by
pouring it through a solvent-rinsed drying column contain-
ing about 10 cm of anhydrous sodium sulfate. Collect the
extract in the K-D concentrator, and rinse the column with
20-30 mL of methylene chloride.
11.4.6 Add 1 or 2 clean boiling stones to the evaporative flask
and attach a macro Snyder column. Pre-wet the Snyder
column by adding about 1 ml of methylene chloride to the
top. Place the K-D apparatus on a hot water bath, 65-70°C,
so that the concentrator tube is partially immersed in the
hot water, and the entire lower rounded surface of the
flask is bathed with hot vapor. Adjust the vertical posi-
tion of the apparatus and the water temperature as required
to complete the concentration in 15-20 min. At the proper
rate of distillation, the balls of the column will actively
chatter, but the chambers will not flood. When the appar-
ent volume of the liquid reaches 2 ml, add 20 ml of metha-
nol through the Snyder column using a syringe and needle.
Raise the temperature of the hot water bath to 90°C, and
concentrate the sample to about 2 ml. Concentrate the
extract to the desired volume under a gentle stream of
nitrogen. Record the exact volume of the concentrated
extract.
11.4.6.1 If isotope dilution calibration is used, spike the
extract with the isotopically labelled standards
prior to solvent evaporation. The concentration
of these isotopically labelled compounds after the
desired extract volume is reached should be the
same as the concentration in each CAL solution.
11.4.7 Determine the original sample volume by refilling the
sample bottle to the mark and transferring the liquid to a
1000-mL graduated cylinder. Record the sample volume to
the nearest 5 ml.
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11.5 LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY (LC/MS)
11.5.1 Analyze a 5-/LtL aliquot with the LC/MS system under the
same conditions used for the initial and continuing cali-
brations (Sect. 10.2).
11.6 IDENTIFICATION OF ANALYTES
11.6.1 At the conclusion of data acquisition, use the system
software to display the chromatogram, mass spectra and
retention times of the peaks in the chromatogram.
11.6.2 Identify a sample component by comparison of its mass
spectrum (after background subtraction) to a reference
spectrum in the user-created data base. The LC retention
time of the sample component should be within 10 sec of the
time observed for that same compound when a calibration
solution was analyzed. In general, all ions that are
present above 10% relative abundance in the mass spectrum
of the standard should be present in the mass spectrum of
the sample component and should agree within absolute 20%.
For example, if an ion has a relative abundance of 30% in
the standard spectrum, its abundance in the sample spectrum
should be in the range of 10 to 50%. Some ions, particu-
larly the molecular ion, are of special importance, and
should be evaluated even If they are below 10% relative
abundance.
11.6.3 Use the data system software to examine the ion abundances
of components of the chromatogram. If any ion abundance
exceeds the system working range, dilute the sample aliquot
and analyze the diluted aliquot.
11.6.4 Identification is hampered when sample components are not
resolved chromatographically and produce mass spectra
containing ions contributed by more than one analyte. When
LC peaks obviously represent more than one sample component
(i.e., broadened peak with shoulder(s) or vallies between
two or more maxima), appropriate analyte spectra and back-
ground spectra can be selected by examining plots of char-
acteristic ions for tentatively identified components.
When analytes coelute (i.e., only one LC peak is apparent),
the identification criteria can be met but each analyte
spectrum will contain extraneous ions contributed by the
coeluting compound.
11.6.5 Structural isomers that produce very similar mass spectra
can be explicitly identified only if they have sufficiently
different LC retention times. (See Sect. 10.2.6.1) Ac-
ceptable resolution is achieved if the height of the valley
between two isomer peaks is less than 25% of the average
197
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height of the two peak heights. Otherwise, structural
isomers are identified as isomeric pairs.
11.6.6 Background components appear in variable quantities in
laboratory and field reagent blanks, and generally subtrac-
tion of the concentration in the blank from the concentra-
tion in the sample is not recommended because the
concentration of the background in the blank is highly
variable. If method analytes appear in the blank, then
resample.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Complete chromatographic resolution is necessary for accurate and
precise measurements of analyte concentrations. Compounds which
coelute cannot be measured accurately because of carrier effects in
the particle beam interface (2). Peaks must be examined carefully
for coeluting substances and if coeluting compounds are present at
greater than 10% the.concentration of the target compound, either
conditions must be adjusted to resolve the components, or the target
compound must be removed from the list of quantitative analytes.
12.2 Use the LC/MS system software or other available proven software to
compute the concentrations of the analytes and surrogates from the
second order regression curves. Manual verification of automated
integration is recommended.
12.2.1 For isotope dilution calculations, use the first order plot
of relative response (analyte integrated ion abundances to
labelled integrated ion abundance) vs. amount of analyte
injected or the equation of the line to compute concentra-
tions. If the plotted line does not go through the origin,
response factors will not be constant at each calibration
point; therefore, average response factors cannot be used,.
12.3 If appropriate, calculate analyte and surrogate concentrations from
response factors and the following equation.
(Ax) Ve
C
x RF V V,-
where: Cx = Concentration of analyte or surrogate in
IJ.g/1 in the water sample.
Ax = integrated abundance of the quantitation
ion of the analyte in the sample.
V = original water sample volume in liters.
RF - mean response factor of analyte from the
initial calibration.
Ve = volume of final extract in juL
V.- = injection volume in pi
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13. METHOD PERFORMANCE
13.1 Single laboratory accuracy and precision data (Tables 3-5) for each
listed analyte were obtained. Five to seven 1-L aliquots of reagent
water containing approximately 5 times the MDL of each analyte were
analyzed with this procedure. (For these experiments, the final
extract volume was 0.5 ml.)
13.1.2 With these data, MDLs were calculated using the formula:
MDL = S t(n-l,l-alpha = 0.99)
where:
t(n-l,l-alpha = 0.99) =
Student's t value for the 99% confidence level
with n-1 degrees of freedom, where n = number of
replicates, and S = standard deviation of repli-
cate analyses.
13.2 A multilaboratory (12 laboratories) validation of the determinative
step was done for four of the analytes (benzidine - BZ, 3,3'-di-
methoxybenzidine - MB, 3,3'-dimethylbenzidine - LB, 3,3'-dichloro-
benzidine - DB). Table 6 gives the results from this study for
single laboratory precision, overall laboratory precision, and over-
all laboratory accuracy. The two concentration levels shown repre-
sent the two extremes of the concentration range studied.
14. POLLUTION PREVENTION
14.1 Although this method allows the use of either LLE or LSE, LSE is
highly recommended whenever possible. Only small amounts of metha-
nol are used with this procedure as compared to much larger amounts
of methylene chloride used for LLE. All other compounds used are
neat materials used to prepare standards and sample preservatives.
All compounds are used in small amounts and pose minimal threat to
the environment if properly disposed.
14.2 For information about pollution prevention that may be applicable to
laboratory operations, consult "Less Is Better: Laboratory Chemical
Management for Waste Reduction" available from the American Chemical
Society's Department of Government Relations and Science Policy,
1155 16th Street N.W., Washington, D.C., 20036.
15. WASTE MANAGEMENT
15.1 There are generally no waste management problems involved with
discarding spent or left over samples in this method since most
often the sample matrix is drinking water. If a sample is analyzed
which appears to be highly contaminated with chemicals, analyses
should be carried out to assess the type and degree of contamination
so that the samples may be discarded properly. All other expired
199
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standards should be discarded properly. It is the laboratory's
responsibility to comply with all applicable regulations for waste
. disposal. The Agency requires that laboratory waste management
practices be conducted consistent with all applicable rules and
regulations, and that laboratories protect the air, water, and land
by minimizing and controlling all releases from fume hoods and bench
operations. Also, compliance is required with any sewage discharge
permits and regulations, particularly the hazardous waste identifi-
cation rules and land disposal restrictions. For further informa-
tion on waste management, consult "The Waste Management Manual for
Laboratory Personnel" also available from the American Chemical
Society at the address in Sect. 14.2.
16. REFERENCES
1. Glaser, J.A., D.L. Foerst, 6.D. McKee^ S.A. Quave, and W.L. Budde,
"Trace Analyses for Wastewaters," Environ. Sci. Techno!. 1981 15,
1426-1435.
2. Bellar, T.A., T.D. Behymer, and W.L. Budde, "Investigation of
Enhanced Ion Abundances from a Carrier Process in High-Performance
Liquid Chromatography Particle Beam Mass Spectrpmetry," J. Am. Soc.
Mass Soectrom.. 1990, 1, 92-98.
3. "Carcinogens - Working With Carcinogens," Department of Health, .
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-20.6, Aug. 1977.
4. "OSHA Safety and Health Standards, General Industry," (29CFR1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical
Society Publication, Committee on-Chemical Safety, 3rd Edition,
1979.
6. Behymer, T.D., T.A. Bellar, and W.L. Budde, "Liquid
Chromatography/Particle Beam/Mass Spectrometry of Polar Compounds of
Environmental Interest," Anal. Chem.. 1990, 62, 1686-1690.
7. Ho, J.S., T.D. Behymer, W.L. Budde, and T.A. Bellar, "Mass Transport
and Calibration in Liquid Chromatography/Particle Beam/Mass Spec-
trometry," J. Am. Soc. Mass Soectrom.. 1992, 3, 662-671.
200
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17. TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA
TABLE I. ION ABUNDANCE CRITERIA FOR BIS(PERFLUOROPHENYL)PHENYLPHOSPHINE OXIDE
(DECAFLUOROTRIPHENYLPHOSPHINE OXIDE, DFTPPO)
Mass Relative Abundance
(M/z) Criteria
Purpose of Checkpoint1
77 present, major ion
168 present, major ion
169 4-10% of 168
271 present, major ion
365 5-10% of base peak
438 present
458 present
459 15-24% of mass 458
low mass sensitivity
mid-mass sensitivity
mid-mass resolution and isotope ratio
base peak
baseline threshold check
important high mass fragment
molecular ion
high mass resolution and isotope ratio
All ions are used primarily to check the mass measuring accuracy of the mass
spectrometer and data system, and this is the most important part of the
performance test. The resolution checks, which include natural abundance
isotope ratios, constitute the next most important part of the performance
test. The correct setting of the baseline threshold, as indicated by the
presence of low intensity ions, is the next most important part of the
performance test. Finally, the ion abundance ranges are designed to encourage
some standardization of fragmentation patterns.
201
-------
TABLE 2. RETENTION TIME DATA AND QUANTITATION IONS FOR METHOD ANALYTES
Retention
Time(min:sec) Quantitation
Compound Aa Bb Ion (m/z)
benzidine
benzoylprop ethyl
caffeine
carbaryl
o-chlorophenyl thiourea
3,3'-dich1orobenzidine
3,3'-dimethoxybenzidine
3, 3 '-dimethyl benzidine
diuron
ethyl ene thiourea
linuron
rotenone
siduron
Surrogates:0
benzidine-D»
caffeine-N2
3 , 3 ' -di chl orobenzi di ne-D6
bi s (perf 1 uorophenyl ) -
phenylphosphine oxide
4.3
24.8
1.4
10.1
2.7
16.6
8.1
8.5
11.0
1.2
16.0
21.1
14.8
4.2
1.3
16.5
22.0
4.9
31.3
1.6
14.7
3.0
22.7
11.5
12.4
16.1
1.4
21.9
27.4
20.6
4.8
1.6
22.6
28.9
184
105
194
144
151
252
244
212
72
102
161
192
93
192
196
258
271
"These retention times were obtained on a Hewlett-Packard 1090 liquid
chromatograph with a Waters C18 Novapak 15 cm x 2 mm column using gradient
conditions given in Sect. 10.2.3.
hrhese retention times were obtained on a Waters 600 MS liquid
chromatograph with a Waters C18 Novapak 15 cm x 2 mm column using gradient
conditions given in Sect. 10.2.3.
°These compounds cannot be used if unlabelled compounds are present
(See Sect. 4.1).
202
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METHOD 554. DETERMINATION OF CARBONYL COMPOUNDS IN DRINKING WATER
BY DINITROPHENYLHYDRAZINE DERIVATIZATION AND HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY
Revision 1.0
August 1992
James W. Eichelberger
W. J. Bashe (Technology Applications, Incorporated)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
213
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METHOD 554
DETERMINATION OF CARBONYL COMPOUNDS IN DRINKING WATER BY
DINITROPHENYLHYDRAZINE DERIVATIZATION AND HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY (HPLC)
1. SCOPE AND APPLICATION
1.1 This is a high performance liquid chromatographic (HPLC) method
optimized for the determination of selected carbonyl compounds in
finished drinking water and raw source water. The analytes applica-
ble to this method are partitioned from the water onto a reverse
phase C18 bonded silica packed cartridge, then eluted with ethanol.
Liquid-solid extraction disks may also be used for this purpose.
Single-laboratory accuracy and precision data have been generated
for the following compounds:
Chemical Abstract Services
Analvte Registry Number
Formaldehyde 50-00-0
Acetaldehyde 75-07-0
Propanal - 123-38-6
Butanal 123-72-8
Pentanal 110-62-3
Hexanal 66-25-1
Heptanal 111-71-7
Octanal 124-13-0
Nonanal 124-19-6
Decanal 112-31-2
Cyclohexanone 108-94-1
Crotonaldehyde 123-73-9
1.2 The method detection limits (MDLs) for the analytes are listed in
Tables 1 and 2. The MDL is defined as the statistically calculated
minimum amount that can be measured with 99% confidence that the
reported value is greater than zero (1). The MDLs for a specific
sample may differ from that of the standard matrix and by the volume
of sample used in the procedure. For the listed analytes, MDLs
range from 3.0 to 69.0 /jg/L.
1.3 This method is restricted to use by or under the supervision of
analysts experienced in the use of HPLC and in the interpretation of
chromatograms. Each analyst must demonstrate the ability to gener-
ate acceptable results with this method, using the procedure de-
scribed in Sect. 11.
214
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2. SUMMARY OF HETHQD
2.1 A measured volume of aqueous sample, approximately 100 ml, is
buffered to pH 3 and the analytes are derivatized at 40°C for 1 hr
with 2,4-dinitrophenylhydrazine (DNPH). The derivatives are ex-
tracted from the water by passing the sample through a series of 3
cartridges each of which contains 500 mg of a chemically bonded C,.,
organic phase (liquid-solid extraction, LSE). The solid sorbent
cartridges are then eluted with 10 ml of ethanol. LSE disks may
also be used as long as all the (QC) criteria specified in Sect. 9
of this method are met. Liquid chromatographic conditions are
described which permit the separation and measurement of the car-
bony! compounds in the extract by absorbance detection at 360 nm.
3. DEFINITIONS
3.1 LABORATORY REAGENT BLANK (LRB) — An aliquot of reagent water or
other blank matrix that is treated exactly as a sample including
exposure to all glassware, equipment, solvents and reagents, inter-
nal standards, and surrogates that are used with other samples. The
LRB is used to determine if method analytes or other interferences
are present in the laboratory environment, the reagents, or the
apparatus.
3.2 FIELD REAGENT BLANK (FRB) — An aliquot of reagent water or other
blank matrix that is placed in a sample container in the laboratory
and treated as a sample in all respects, including shipment to the
sampling site, exposure to sampling site conditions, storage,
preservation, and all analytical procedures. The purpose of the FRB
is to determine if method analytes or other interferences are
present in the field environment.
3.3 LABORATORY FORTIFIED BLANK (LFB) — An aliquot of reagent water or
other blank matrix to which known quantities of the method analytes
are added in the laboratory. The LFB is analyzed exactly like a
sample, and its purpose is to determine whether the methodology is
in control, and whether the laboratory is capable of making accurate
and precise measurements.
3.4 LABORATORY FORTIFIED MATRIX SAMPLE (LFM) ~ An aliquot of an envi-
ronmental sample to which known quantities of the method analytes
are added in the laboratory. The LFM is analyzed exactly like a
sample, and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background concen-
trations of the analytes in the sample matrix must be determined in
a separate aliquot and the measured values in the LFM corrected for
background concentrations.
3.5 STOCK STANDARD SOLUTION (SSS) — A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed
reference materials or purchased from a reputable commercial source.
215
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3.6 PRIMARY DILUTION STANDARD SOLUTION (PDS) — A solution of several
analytes prepared in the laboratory from stock standard solutions
and diluted as needed to prepare calibration solutions and other
needed analyte solutions.
3.7 CALIBRATION STANDARD (CAL) — A solution prepared from the primary
dilution standard solution and stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.8 QUALITY CONTROL SAMPLE (QCS) — A solution of method analytes of
known concentrations which is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally prepared
test materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware, and other sample processing hardware that lead
to discrete artifacts and/or elevated baselines in the chromato-
grams. All of these materials must be routinely demonstrated to be
free from interferences under the conditions of the analysis by
analyzing laboratory reagent blanks as described in Sect. 9.2.
4.1.1 Glassware must be scrupulously cleaned as soon as possible
after use by rinsing with the last solvent used. This
should be followed by detergent washing with hot water, and
rinses with tap water and distilled water. Glassware should
then be drained, dried, and heated in a laboratory oven at
130°C for several hours before use. Solvent rinses with
methanol may be substituted for the oven heating. After
drying and cooling, glassware should be stored in a clean
environment to prevent any accumulation of dust or other
contaminants.
4.1.2 The use of high purity reagents and solvents helps to mini-
mize interference problems. Purification of solvents by
distillation in all-glass systems may be required.
4.2 Matrix interferences may be caused by contaminants that are coex-
tracted from the sample. The extent of matrix interferences will
vary considerably from source to source, depending upon the nature
and diversity of the matrix being sampled. If interferences occur,
cleanup may be necessary.
4.3 The extent of interferences that may be encountered using liquid
chromatographic techniques has not been fully assessed. Although
the HPLC conditions described allow for resolution of the specific
216
-------
compounds covered by this method, other matrix components may
interfere.
4.4 Acetaldehyde is generated during the derivatization step due to the
use of ethanol as the solvent for the DNPH. This background will
impair the measurement of acetaldehyde at levels below 250 /zg/L.
Accordingly, if acetaldehyde is a compound of interest, use of
another solvent, such as acetonitrile, for the DNPH solution is
suggested.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
should be treated as a potential health hazard. From this view-
point, exposure to these chemicals must be reduced to the lowest
possible level by whatever means available. The laboratory is
responsible for maintaining a current awareness file of OSHA regula-
tions regarding the safe handling of the chemicals specified in this
method (2). A reference file of material safety data sheets should
also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available.
5.2 Formaldehyde and acetaldehyde have been tentatively classified as
known or suspected human or mammalian carcinogens.
6. EQUIPMENT AND SUPPLIES
6.1 SAMPLE CONTAINERS
6.1.1 Grab Sample Bottle (aqueous samples) — 8 oz. (237 mL) amber
glass, screw cap bottles and caps equipped with Teflon-faced
silicone septa. Prior to use, wash bottles and septa.
6.1.2 Grab Sample Bottle (solids) -- 8 oz., amber glass, wide
mouth, screw cap bottles and caps equipped with Teflon-lined
closures. Prior to use wash bottles and septa.
6.2 REACTION VESSEL ~ 250 mL Florence flask.
6.3 VIALS ~ 10 and 25 mL, glass with Teflon-lined screw-caps.
6.4 VOLUMETRIC FLASKS — 10 mL, with ground glass stopper.
6.5 BALANCE — Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.6 pH METER — Capable of measuring to the nearest 0.01 units.
6.7 HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC APPARATUS (MODULAR)
6.7.1 Pumping System — Gradient, with constant flow control cap-
able of 1.50 mL/min.
217
-------
6.7.2 High pressure injection valve with 20 nl loop.
6.7.3 Column — 250 mm x 4.6 mm i.d., 5 jLtm particle size, C18
(Zorbax or equivalent).
6.7.4 Absorbance detector — 360 run.
6.7.5 Strip-chart recorder compatible with detector ~ Use of a
data system for measuring peak area and retention times is
recommended.
6 8 LSE CARTRIDGES — Packed with about 500 mg silica whose surface is
modified by chemically bonded octadecyl (C-18) groups. These car-
tridges are available from several commercial suppliers. LSE disks
may also be used as long as all the QC criteria specified in Sect. 9
of this method are met.
6.9 VACUUM MANIFOLD — Capable of simultaneous extraction of 10 samples
6.10 SAMPLE RESERVOIRS -- 60-mL capacity.
6.11 PIPET — Capable of accurately delivering 0.10 mL solution.
6.12 SYRINGES -- Luer-Lok, 5 mL, 500 til and 100 /iL.
6.13 ENVIRONMENTAL SHAKER — Controlled temperature incubator (± 2°C)
with orbital shaking (Lab-Line Orbit Environ-Shaker Model 3527 or
equivalent).
7. REAGENTS AND STANDARDS
7 1 Reagent grade chemicals must be used in all tests. Unless otherwise
indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the
American Chemical Society, where such specifications are available.
Other grades may be used, provided it is first ascertained that the
reagent is of sufficiently high purity to permit its use without
lessening the accuracy of the determination.
7.2 REAGENT WATER — All references to reagent water in this method
refer to water in which an interference is not observed at the MDL
of the compound of interest. Reagent water can be generated by
passing tap water through a carbon filter bed containing about 1 Ib.
of activated carbon. Subsequently, while maintaining the tempera-
ture at 90°C, bubble a contaminant-free inert gas through the water
for 1 hr. A water purification system may be used to generate
organic-free deionized water.
7.3 METHANOL — HPLC grade or equivalent.
7.4 ETHANOL ~ Reagent grade
7.5 2,4- DINITROPHENYLHYDRAZINE (DNPH) (70% (W/W)) in water.
218
-------
7.6 CITRIC ACID, C6H807
7.7 SODIUM CITRATE — Trisodium salt dihydrate.
7.8 SODIUM HYDROXIDE — Concentrated
7.9 SODIUM CHLORIDE
7.10 SODIUM SULFITE, Na2S03.
7.11 SODIUM SULFATE, Na2S04 — Granular, anhydrous.
7.12 HYDROCHLORIC ACID, 0.1 N.
7.13 ACETIC ACID — Glacial.
7.14 AMMONIUM CHLORIDE, NH4C1.
7.15 EXTRACTION FLUID — Dilute 64.3 mL of 1.0 N NaOH and 5.7 mL of
glacial acetic acid to 900 mL with water. The pH should be
4.93 ± 0.02. Dilute to 1-L with water.
7.16 STOCK STANDARD SOLUTIONS
7.16.1 Stock standard formaldehyde solution approximately 1 mg/mL
— Prepare by diluting 265 /iL of formalin to 100 mL with
water.
7.16.2 Standardization of formaldehyde stock solution — Transfer a
25-mL aliquot of a 0.1 M Na2S03 solution to a beaker and
record the pH. Add a 25.0 mL aliquot of the formaldehyde
stock solution (Sect. 7.16.1) and record the pH. Titrate
this mixture back to the original pH using 0.1 N HC1. The
formaldehyde concentration is calculated using the following
equation:
Concentration (mg/mL) = 30.03 x (N HC1) x
(mL HC1) / 25.0
Where: N HC1 = Normality of HC1 solution used mL HC1 = mL
of standardized HC1 solution used, and
30.03 = MW of formaldehyde
Note: The pH value of the 0.1 Na2S03 should be 10.5 ± 0.2.
When the stock formaldehyde solution and the 0.1 M
Na2S03 solution are mixed together as in Sect.
7.16.2, the pH should be 11.5 ± 0.2. It is recom-
mended that new solutions be prepared if the pH
deviates from this value.
7.16.3 Stock aldehyde(s) and ketone(s) — Prepare by adding an
appropriate amount of the analyte to 90 mL of methanol, then
219
-------
dilute to 100 mL to give a final concentration of 1.0 mg/mL.
7.16.4 Stock standard solutions must be replaced after 6 weeks, or
sooner, if comparison with check standards indicates a
problem.
7.17 REACTION SOLUTIONS
7.17.1 DNPH (3.00 g/L) — Dissolve 428.7 mg of 70% (w/w) reagent in
100 ml absolute ethanol. Slight heating or sonication may
be necessary to effect dissolution.
Note: If the DHPH does not complete by dissolve, filter the
solution to remove the undissolved compound.
7.17.2 pH 3 Citrate buffer (1M) — Prepare by adding 80 ml of 1 M
citric acid solution to 20 ml 1 M sodium citrate solution.
Mix thoroughly. Adjust pH with 6N NaOH or 6N HC1 as needed.
7.17.3 Sodium chloride solution (saturated) — Prepare by mixing an
excess of the reagent grade solid with water.
7.17.4 Reducing Agent, Ammonium chloride (100 mg/L) — Added to all
samples containing residual chlorine. Sodium thiosulfate is
not recommended because it may produce a residue of elemen-
tal sulfur that can interfere with some analytes. The
ammonium chloride may be added as a solid with stirring
until dissolved, to each volume of water.
7.18 SYRINGE FILTERS -- 0.45 /im filtration disks (Gelman Acrodisc No.
4438, or equivalent).
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 SAMPLE COLLECTION — When sampling from a water tap, open the tap
and allow the system to flush until the water temperature has
stabilized (usually about 2-5 min). Adjust the flow to about 500
mL/min and collect samples from the flowing stream. Keep samples
sealed from collection time until analysis. When sampling from an
open body of water, fill the sample container with water from a
representative area. Sampling equipment must be free of plastic and
other parts that may leach analytes into water. Follow other
conventional sampling procedures (3) as needed.
8.2 SAMPLE DECHLORINATION AND PRESERVATION — All samples should be iced
or refrigerated at 4°C from the time of collection until extraction.
Residual chlorine should be reduced at the sampling site by addition
of a reducing agent (Sect. 7.17.4).
8.2.1 The use of HCL as a sample preservative has not been imple-
mented in this method. Method 554 has been designed to
measure "free" aldehydes and cyclohexanone by employing mild
reaction conditions. The evolution of aldehydes can occur
220
-------
in samples whose pH is low (less than or equal to 2 (4),
(5).
8.3 HOLDING TIME — Samples must be derivatized and extracted within 3
days of sample collection. In reagent water, the analyte concentra-
tions remained constant over a 7-day period. In ground water,
hexanal, octanal and decanal experienced losses after the first day.
The other analytes degraded after the third day. Matrices, such as
groundwater, which are biologically active, should be extracted upon
receipt. All samples should be extracted within three days of
collection.
8.4 FIELD REAGENT BLANKS — Processing of a field reagent blank (FRB) is
recommended along with each sample set, which is composed of the
samples collected from the same general sampling site at approxi-
mately the same time. At the laboratory, fill a sample container
with reagent water, seal and ship to the sampling site along with
the empty sample containers. Return the FRB to the laboratory with
filled sample bottles.
9. QUALITY CONTROL
9.1 Each laboratory that uses this method is required to operate a
formal QC program. Minimum QC requirements are initial demonstra-
tion of laboratory capability, analysis of laboratory reagent
blanks, laboratory fortified blanks, laboratory fortified sample
matrices, and QC samples. Additional QC practices are encouraged.
9.2 LABORATORY REAGENTS BLANKS (LRB) — Before processing any samples,
the analyst must analyze at least one LRB to demonstrate the absence
of contaminants that would prevent the determination of any method
analyte. In addition, each time a set of samples is extracted or
reagents are changed, a LRB must be analyzed. If within the reten-
tion time window of any analyte, the LRB produces a peak that would
prevent the determination of that analyte, determine the source of
contamination and eliminate the interference before processing
samples.
9.3 INITIAL DEMONSTRATION OF CAPABILITY
9.3.1 Select a representative fortified concentration for each of
the target analytes at approximately 250 /zg/L. Prepare a
primary dilution standard (PDS) in methanol 1000 times more
concentrated than the selected concentration. This primary
dilution standard must be prepared independently from the
standards used to prepare the calibration curve. With a
syringe, add 100 fiL of the PDS to each of four to seven 100
mL aliquots of reagent water. Analyze the aliquots accord-
ing to the method beginning in Sect. 11.
9.3.2 Calculate the measured concentration of each analyte in each
replicate, the mean concentration of each analyte in all
replicates, the mean accuracy (as mean percentage of true
221
-------
value), the precision (RSD), and the MDL (1). Determine ,
accuracy based upon extracted standards as called for in
Sect. 10. For each analyte, the mean accuracy must fall in
the range of R ± 30% using the values for reagent water
listed in Table 3 at the lower concentration level. The
calculated standard deviation should be less than ± 30% or 3
Sr (value listed in Table 3), whichever is larger. For
those compounds that meet these criteria, performance is
considered acceptable and sample analysis may begin. For
those compounds that fail these criteria, the procedures in
Sect. 9.3.1 must be repeated using a minimum of four fresh
samples until satisfactory performance has been demonstrat-
ed.
9.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples using a
new, unfamiliar method prior to obtaining some experience
with it. As laboratory personnel gain experience with this
method, the quality of data should improve beyond those re-
quired here.
9.3.4 The analyst is permitted to modify HPLC conditions. Each
time a method modification is made, the analyst must repeat
the procedures in Sect. 9.3.1.
9.4 LABORATORY FORTIFIED BLANK
9.4.1 The laboratory must analyze at least one laboratory forti-
fied blank (LFB) sample with every 20 samples or one per
sample set (all samples extracted within a 24-hr period),
whichever frequency is greater. A fortified concentration
near the lower value in Table 3 is recommended. The LFB
sample must be prepared from a primary dilution standard
which is prepared separately and independently from the
standards used to prepare the calibration curve. Calculate
the mean recovery (R). If the accuracy for any analyte
falls outside the control limits (See Sect. 9.4.2), that
analyte is judged out of control, and the source of the
problem should be identified and resolved before continuing
analyses.
9.4.2 Prepare control charts based on mean upper and lower
control limits R ± 3 Sr. The initial demonstration of
capability (Sect. 9.3) establishes the initial limits.
After each 4-6 new accuracy measurements, recalculate R
and S using all the data, and construct new control limits.
When the total number of data points reach 20, update the
control limits by calculating R and Sr using only the most
recent 20 data points. At least quarterly, replicates of
LFBs should be analyzed to determine the precision of the
laboratory measurements. Add these results to the ongoing
control charts to document data quality.
222
-------
9.5 LABORATORY FORTIFIED SAMPLE MATRIX
9.5.1 The analyst must add known concentrations of analytes to a
minimum of 10% of the routine samples or one concentration
per sample set, whichever frequency is greater. The concen-
trations should be equal to or greater than the background
concentrations in the sample selected for fortification.
Ideally, the concentration should be the same as that used
for the laboratory fortified blank (Sect. 9.4). Over time,
samples from all routine sample sources should be fortified.
9.5.2 Calculate the mean percent recovery (R) for each analyte,
after correcting the total mean measured concentration, A,
from the fortified sample for the background concentration,
B, measured in the unfortified sample, i.e.,
R = 100 (A - B) / C,
where C is the fortified concentration. Compare these
values to control limits appropriate for reagent water data
collected in the same fashion (Sect. 9.4).
9.5.3 If the accuracy of any analyte falls outside the designated
range, and the laboratory performance for that analyte is
shown to be in control (Sect. 9.4), the accuracy problem
encountered with the fortified sample is judged to be matrix
related, not system related. The result for that analyte in
the unfortified sample is labeled suspect/matrix to inform
the data user that the results are suspect due to matrix
effects.
9.6 QUALITY CONTROL SAMPLE (QCS) — At least quarterly, analyze a QCS
from an external source. If measured analyte concentrations are not
of acceptable accuracy, check the entire analytical procedure to
locate and correct the problem source.
9.7 The laboratory may adopt additional QC practices for use with this
method. The specific practices that are most productive depend upon
the needs of the laboratory and the nature of the samples. For
example, field or laboratory duplicates may be analyzed to assess
the precision of the environmental measurements, or field reagent
blanks may be used to assess contamination of samples under site,
transportation and storage conditions.
10. CALIBRATION AND STANDARDIZATION
10.1 Establish HPLC operating parameters to completely separate the
peaks. Table 1 lists some retention times produced using the
following conditions:
HPLC Column: C18, 250 mm x 4.6 mm i.d., 5 [im particle size
223
-------
Mobile Phase: 70%/30% methanol/water (v/v) for 20 min, up to 100%
methanol in 15 min, hold at 100% methanol for 10 min
Flow Rate: 1.5 mL/min
UV Detector: 360 nm
Injection Size: 20 /*L
10.2 Prepare procedural calibration standards according to the
procedure in Sect. 10.2.1. Calibrate the chromatographic system
using the external standard technique (Sect. 10.2.2).
10.2.1 Preparation of Calibration Standards
10.2.1.1 Prepare calibration solutions at a minimum of
five concentration levels for each analyte of
interest by adding volumes of stock standard
solutions (Sect. 7.16) to reagent water and
diluting to 100 ml. The lowest concentration
level of each analyte should be near to, but
above, the MDLs listed in Tables 1 or 2, while
the other concentration levels should corre-
spond to the expected range of concentrations
found in real samples.
10.2.1.2 Process each calibration standard solution
through derivatization and extraction using the
same extraction option employed for sample pro-
cessing (Sect. 11.1.3).
10.2.2 External standard calibration procedure
10.2.2.1 Analyze each processed calibration standard
(some suggested chromatographic conditions are
listed in Sect. 10.1) and tabulate peak area
(y axis) versus calibration solution concentra-
tion (x axis) in /jg/L. The results may be used
to prepare calibration curves for the analytes.
By linear regression, determine the slope, m, of
the calibration curve.
10.2.2.2 The working calibration curve must be verified
on each working day by the measurement of one or
more fresh calibration standards. If the re-
sponse for any analyte varies from the previous-
ly established responses by more than 10%, the
test must be repeated after it is verified that
the analytical system is in control. Alterna-
tively, a new calibration curve may be prepared
for that compound. If an autosampler is avail-
able, it may be convenient to prepare a calibra-
224
-------
tion curve daily by analyzing standards along
with test samples.
11. PROCEDURE
11.1 DERIVATIZATION AND EXTRACTION
11.1.1 Measure a 100-mL aliquot of the sample. Other sample vol-
umes, from 50 ml to 100 ml, may be used to accommodate the
anticipated analyte concentration range. Quantitatively
transfer the sample aliquot to a 250 ml Florence flask.
Note: In cases where the selected sample or extract volume
is less than 100 ml, the total volume of the aqueous
phase should be adjusted to 100 ml with reagent
water.
11.1.2 Derivatization and .extraction of the derivatives can be
accomplished using "the liquid-solid extraction (Sect.
11.1.3).
11.1.3 Liquid-Solid Extraction (Either LSE cartridges or disks may
be used.)
11.1.3.1 Add 4 ml of citrate buffer to the sample and
adjust the pH to 3.0 ± 0.1 with 6 M HC1 or 6 M
NaOH. Add 6 ml of DNPH reagent, seal the con-
tainer, and place in a heated, orbital shaker
(Sect. 6.13), set at 40"C, for 1 hr. Adjust the
agitation to gently swirl the reaction solution.
11.1.3.2 Assemble the vacuum manifold and connect it to a
water aspirator or vacuum pump. Entrain three
solid sorbent cartridges and attach the nested
cartridges to the vacuum manifold. Condition
the cartridges by passing 10 ml of dilute ci-
trate buffer (10 mL of 1 M citrate buffer dis-
solved in 250 ml of water) through the cartridge
train.
11.1.3.3 Remove the reaction vessel from the shaker and
add 10 ml of saturated NaCl solution to the
vessel.
11.1.3.4 Add the reaction solution to the cartridge train
and apply a vacuum to draw the solution through
the cartridges at a rate of 3 to 5 mL/min.
Continue applying the vacuum for about 10 min
after the liquid sample has passed through the
cartridges.
225
-------
11.1.3.5 While maintaining vacuum conditions, elute the
cartridges with approximately 9 ml of absolute
ethanol, directly into a 10-mL volumetric flask.
Dilute the eluate to volume with absolute etha-
nol, mix thoroughly, and place in a tightly
sealed vial until analysis.
NOTE: Because this method uses an excess of
DNPH, the cartridges will retain a yellow
color after this step. This color is not
indicative of incomplete recovery of the
analyte derivatives from the cartridges.
11 2 Analyze samples by HPLC, using recommended conditions provided in
Sect. 10.1. Tables 1 and 2 list the retention times and MDLs that
were obtained under these conditions. Other HPLC columns, chromato-
graphic conditions, or detectors may be used provided the require-
ments of Sect. 9.3 are met.
11.3 The width of the retention time window used to make identifications
should be based upon measurements of actual retention time varia-
tions of standards over the course of a day. Three times the
standard deviation of a retention time for a compound can be used to
calculate a suggested window size; however, the experience of the
analyst should weigh heavily in the interpretation of the chromato-
grams.
11.4 If an analyte peak area exceeds the linear range of the calibration
curve, a smaller sample volume should be used. Alternatively, the
final solution may be diluted with ethanol and reanalyzed.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Identify the method analytes in the sample chromatogram by comparing
the retention time of the suspect peak to the retention time of an
analyte peak in a calibration standard or the laboratory fortified
blank. Computerized linear regression analysis is recommended for
assessment of the linearity of the calibration and for slope calcu-
lation. Linearity is achieved when the coefficient of correlation
(R) for the linear regression is > 0.99. Alternatively, the slope,
m, may be determined by calculating the average peak area to concen-
tration ratio for each calibration standard. The percent relative
standard deviation (% RSD) of the slope must be less than 10% if the
latter procedure is to be employed.
12.2 Calculate the analyte concentrations as follows:
C - A x Iflfi (mL)
m Vs
where:
226
-------
m = slope of calibration curve, L//jg.
Cs = sample concentration in fig/I',
A = area of signal; and
Vs = volume of sample in ml.
13. METHOD PERFORMANCE
13.1 The MDL concentrations listed in Tables 1 and 2 were obtained in
reagent water and dechlorinated tap water using LSE. Results
reported in both tables were achieved using fortified 100-mL sam-
ples.
13.2 This method has been tested for linearity of recovery from fortified
reagent water and has been demonstrated to be applicable over the
range from 10 x MDL to 1000 x MDL.
13.3 Single operator precision and accuracy data are provided in Tables
3, 4, 5 and 6. The tables report data at two fortification levels
for reagent water, ground water and dechlorinated tap water. Data
for ozone treated water are reported at a single fortification level
(500/ig/L).
13.4 To generate the MDL and precision and accuracy data reported in this
section, analytes were segregated into two fortification groups (A
and B) and analyzed separately. Representative chromatograms using
LSE for both Groups A and B are presented in Figures 1 and 2,
respectively.
14. POLLUTION PREVENTION
14.1 This method utilizes the new liquid-solid extraction (LSE) techno-
logy to remove the analytes from water. It requires the use of very
little organic solvent, thereby eliminating the hazards involved
with the use of large volumes of organic solvents in conventional
liquid-liquid extractions. A 10 mL aliquot of ethanol, a nontoxic
solvent, is used per sample to elute the derivatized aldehydes and
ketone from the LSE cartridge. This method is safe for the labora-
tory analyst to use and will not harm the environment.
15. HASTE MANAGEMENT
15.1 Due to the nature of this method and the reagents employed, there is
no need for waste management in disposing of the used or unused
samples. No toxic solvents or hazardous chemicals are used. The
matrices are drinking water or source water and can be discarded in
the sink drain.
16. REFERENCES
1. Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, S.A., and W.L.
Budde, "Trace Analyses for Wastewaters," Environ. Sci. Techno!.
1981, 15, 1426-1435.
227
-------
2. "OSHA Safety and Health Standards, General Industry," (29CRF1910).
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
3. ASTM Annual Book of Standards, Part II, Volume 11.01, D3370-82,
"Standard Practice for Sampling Water," American Society for Testing
and Materials, Philadelphia, PA, 1986.
4. "Aldehydes - Photometric Analysis," Sawicki, E and C.R. Sawicki,
Volume 5; Academic Press, London: 1.975.
5. Bicking, M.K.L., W.M. Cooke, F.K. Kawahara, and J.E. Longbottom,
"Effect of pH on the Reaction of 2,4-Dinitrophenylhydrazine with
Formaldehyde and Acetaldehyde," J. of Chromatographv. 1988, 455,
310-315.
228
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17..TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. METHOD DETECTION LIMITS3 USING LIQUID-SOLID
EXTRACTION IN REAGENT WATER
Analyte
Formaldehyde
Acetaldehyde
Propanal
Butanal
Pentanal
Hexanal
Heptanal
Octanal
Nonanal
Decanal
Cyclohexanone
Crotonaldehyde
Retention Time (min)
5.3
7.5
11.7
18.1
26.9
32.5
36.6
40.4
43.0
45.5
27.9
16.7
MDL (/ig/L)
6.2
43. 7b
11.0
6.3
15.3
10.7
10.0
6.9
13.6
4.4
5.8
7.7
MDL was computed as follows: MDL = t(N-l, 0.01) x S.D.
where t(n-l, 0.01) is the upper first percentile point of the t-distri-
bution with n-1 degrees of freedom and S.D. is the standard deviation in
/*g/L. With the exception of acetaldehyde all reported MD.Ls are based
upon analyses of 6 to 8 replicate, fortified blanks (25 #g/L).
(See Reference 1)
Reported MDL based upon analyses of 3 replicate, fortified blanks at 250
229
-------
TABLE 2. METHOD DETECTION LIMITS3 USING LIQUID-SOLID
EXTRACTION IN DECHLORINATED TAP WATER
Analyte
Formaldehyde
Acetal dehyde
Propanal
Butanal
Pentanal
Hexanal
Heptanal
Octanal
Nonanal
Decanal
Cycl ohexanone
Crotonal dehyde
Retention Time (min)
5.3
7.5
11.7
18.1
26.9
32.5
36.6
40.4
43.0
45.5
27.9
16.7
MDL (u.g/1)
8.1
69. Ob
3.4
8.6
3.3
9.6
7.3
6.0
24.3
12.9
9.5
6.3
HDL was computed as follows: MDL = t(N-l, 0.01) x S.D.
where t(n-l, 0.01) is the upper first percentile point of the t-distri-
bution with n-1 degrees of freedom and S.D. is the standard deviation in
/ig/L. With the exception of acetaldehyde all reported MDLs are based
upon analyses of 6 to 8 replicate, fortified blanks (25 ng/L).
(See Reference 1)
Reported MDL based upon analyses of 3 replicate, fortified blanks at 250
230
-------
TABLE 3. SINGLE OPERATOR ACCURACY AND PRECISION USING
LIQUID-SOLID EXTRACTION IN REAGENT WATER
Analyte
Formaldehyde
Acetaldehyde
Propanal
Butanal
Pentanal
Hexanal
Heptanal
Octanal
Nonanal
Decanal
Crotonaldehyde
Cyclohexanone
FLa
250
2500
250
2500
250
2500,
250
2500
250
2500
250
2500
250
2500
250
2500
250
2500
250
2500
250
2500
250
2500
Rb
96.3
109.8
40.2
112.2
93.8
110.8
91.1
108.2
91.6
100.5
87.0
94.6
90.1
104.9
89.2
97.1
90.2
105.3
85.0
98.9
87.6
104.3
94.8
116.7
Src
7.6
1.5
1.0
21.3
2.3
2.4
2.9
2.6
.20
2.0
4.7
5.4
2.4
1.7
.09
1.0
3.0
2.2
1.1
1.6
7.3
1.5
4.1
4.7
Number of
Analyses
7
3
3
3
7
3
7
3
7
3
7
3
7
3
7
3
7
3
7
3
7
3
7
3
a FL = Fortification Level in fj.g/1
b R = Average Percent Recovery
231
-------
TABLE 4. SINGLE OPERATOR ACCURACY AND PRECISION USING
LIQUID-SOLID EXTRACTION IN GROUND WATER
Analyte
Formaldehyde
Acetaldehyde
Propanal
Butanal
Pentanal
Hexanal
Heptanal
Octanal
Nonanal
Decanal
Crotonaldehyde
Cyclohexanone
FLa
250
2500
250
2500
250
2500
250
2500
250
2500
250
2500
250 -
2500
250
2500
250
2500
250
2500
250
2500
250
2500
Rb
103.2
118.4
50.2
109.2
99.1
105.4
94.7
95.9
90.0
96.7
89.0
95.9
96.4
98.0
94.1
96.9
93.1
97.9
86.0
98.5
93.6
100.2
107.6
111.1
Src
10.3
9.2
3.9
6.5
1.3
9.4
3.9
8.7
12.7
1.6
4.7
1.9
6.3
9.6
1.8
1.5
5.1
11.6
4.3
2.2
3.0
2.6
4.0
10.8
Number of
Analyses
7
3
3
3
7
3
7
3
3
3
6
3
7
3
7
3
7
3
7
3
7
3
7
3
0 FL = Fortification Level in /tg/L
b R » Average Percent Recovery
Sr = Standard Deviation of Percent Recovery
232
-------
TABLE 5. SINGLE OPERATOR ACCURACY AND PRECISION USING
LIQUID-SOLID EXTRACTION IN DECHLORINATED TAP WATER
Analyte
Formaldehyde
Acetaldehyde
Propanal
Butanal
Pentanal
Hexanal
Heptanal
Octanal
Nonanal
Decanal
Crotonaldehyde
Cyclohexanone
FLa
25
250
250d
25
250
25
250
25
250
25
250
25
250
25
250
25
250
25
250
25
250
25
250
Rb
90.0
90.8
52.0
120.2
83.4
91.6
79.4
106.1
72.3
99.2
70.4
97.2
79.2
60.5
75.6
120.6
69.9
109.8
91.5
86.8
97.7
94.8
104.5
Src
1.1
11.6
9.7
17.9
6.3
11.4
.81
1.1
3.9
3.2
4.0
9.8
5.4
2.0
14.8
32.4
12.7
23.9
34.2
2.1
5.5
12.7
10.8
Number of
Analyses
3
8
8
8
8
8
3
8
3
8
3
8
8
8
3
8
3
8
3
8
8
8
8
8 FL = Fortification Level in /tg/L
b R = Average Percent Recovery
c Sr = Standard Deviation of Percent Recovery
d Background levels of this analyte will impair measurement if the
fortification level is below 250 /ig/L.
233
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TABLE 6. SINGLE OPERATOR ACCURACY AND PRECISION USING
LIQUID-SOLID EXTRACTION IN OZONE TREATED WATER
Analyte
Formaldehyde
Acetaldehyde
Propanal
Butanal
Pentanal
Hexanal
Heptanal
Octanal
Nonanal
Decanal
Crotonaldehyde
Cyclohexanone
FLa
500
500
500
500
500
500
500
500
500
500
500
500
Rb
78.8
99.4
93.7
97.6
89.6
91.8
99.0
93.9
100.0
93.4
89.5
97.2
Src
1.1
2.3
1.8
1.2
1.7
1.7
3.2
2.5
3.8
6.9
2.5
.83
Number of
Analyses
8
8
8
8
8
8
8
8
8
8
8
8
8 FL = Fortification Level in /jg/L
b R = Average Percent Recovery
Sr - Standard Deviation of Percent Recovery
234
-------
-0.80
-1.00
.-1.20
£-1.40-
-1.60-
-1.80-
-2.00-
JL
i
1.00
».0§ 3.00
» 10* •inutec
4.00
RT (Bin)
5.33
11.68
18.13
27.93
36.60
42.99
Analvte
Formaldehyde
Propanal
Butanal
Cyclohexanone
Heptanal
Nonanal
Figure 1 Liquid-solid Procedural Standard of Group A
Analytes at 625 jtg/L.
235
-------
-0.60-
-o.eo
-t.oo-
-1.40-
-1.60-
LL
—T r
i.OO
2.00 3.00
x iO* Minutes
4.00
RT (nln)
7.50
16.68
26.83
32.53
40.36
45.49
Analvte
Acetaldehyde
Crotonaldehyde
Pentanal
Hexanal
Octanal
Decanal
Figure 2 Liquid-solid Procedural Standard of Group B
Analytes at 625 0g/L.
236
-------
METHOD 555. DETERMINATION OF CHLORINATED ACIDS IN WATER BY
HIGH PERFORMANCE LIQUID CHROMAT06RAPHY WITH A
PHOTODIODE ARRAY ULTRAVIOLET DETECTOR
Revision 1.0
August 1992
.James W. Eichelberger
Winslow J. Bashe (Technology Applications, Inc.)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
237
-------
METHOD 555
DETERMINATION OF CHLORINATED ACIDS IN WATER BY HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY WITH A PHOTODIODE ARRAY
ULTRAVIOLET DETECTOR
1. SCOPE AND APPLICATION
1.1 This is a high performance liquid chromatographic (HPLC) method
for the determination of certain chlorinated acids in ground water
and finished drinking water. The following compounds can be
determined by this method:
Chemical Abstract Services
Analvte Registry Number
Acifluorfen 50594-66-6
Bentazon 25057-89-0
Chloramben* 133-90-4
2,4-D 94-75-7
2,4-DB 94-82-6
Dicamba 1918-00-9
3,5-Dichlorobenzoic acid 51-36-5
Dichlorprop 120-36-5
Dinoseb 88-85-7
5-Hydroxydicambaa 7600-50-2
MCPA
MCPP
4-Nitrophendla 100-02-7
Pentachlorophenor (PCP) 87-86-5
Picloram8 1918-02-1
2,4,5-T 93-76-5
2,4,5-TP . 93-72-1
8 Analytes measurable from 20 ml sample volume only.
b Use a 100 ml sample for pentachlorophenol in order to attain a
HDL of 0.3 pg/L. The MLC for this compound is 1.0 /tg/L.
1.2 This method is applicable to the determination of salts and esters
of analyte acids. The form of each analyte is not distinguished
by this method. Results are calculated and reported for each
listed analyte as the total free acid.
1.3 This method has been validated in a single laboratory and method
detection limits (MDLs) (1) have been determined from a 20-mL
sample for the analytes above. Observed MDLs may vary among
ground waters, depending on the nature of interferences in the
sample matrix and the specific instrumentation used.
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1.4 This method is restricted to use by or under the supervision of
analysts experienced in the use of HPLC and in the interpretation
of chromatograms. Each analyst must demonstrate the ability to
generate acceptable results with this method using the procedure
described in Sect. 9.3.
1.5 Analytes that are not separated chromatographically cannot be
individually identified and measured in the same calibration
mixture or water sample unless an alternative technique for
identification and quantitation exists (Sect. 11.3).
1.6 When this method is used to analyze unfamiliar samples, analyte
identifications must be confirmed by at least one additional
qualitative technique.
2. SUMMARY OF METHOD
2.1 _A measured sample volume of approximately 100 ml is adjusted to pH
12 with 6 N sodium hydroxide, shaken, and allowed to set for 1 hr
to hydrolyze chlorinated esters. The sample is acidified with
HjPO,, filtered, and the chlorinated acids are extracted from a
20-ml aliquot. The 20-mL aliquot is pumped through an HPLC
cartridge (containing C^-silica), trapping the chlorinated acids.
The concentrator cartridge is valved in-line with the C18 analyti-
cal column following extraction. The analytes are separated and
measured by photodiode array - ultraviolet detection (PDA-UV).
NOTE: A liquid-solid extraction disk is perfectly acceptable for
use in the in-line extraction of the analytes providing all
quality control (QC) criteria in Sect. 9 are met or exceeded.
.2.2 The method measures the analytes from 20-mL volumes. Volumes of
up to 100 mL may be analyzed by this procedure for certain
analytes. The analytes which may not be determined in a larger
volume are indicated in Sect. 1.1.
3. DEFINITIONS
3.1 LABORATORY DUPLICATES (LD1 AND LD2) — Two aliquots of the same
sample taken in the laboratory and analyzed separately with
identical procedures. Analyses of LD1 and LD2 indicate the
precision associated with laboratory procedures, but not with
sample collection, preservation, or storage procedures.
3.2 FIELD DUPLICATES (FD1 AND FD2) -- Two separate samples collected
at the same time and place under identical circumstances and
treated exactly the same throughout field and laboratory proce-
dures. Analyses of FD1 and FD2 give a measure of the precision
associated with sample collection, preservation and storage, as
well as with laboratory procedures.
3.3 LABORATORY REAGENT BLANK (LRB) — An aliquot of reagent water or
other blank matrix that is treated exactly as a sample including
exposure to all glassware, equipment, solvents, and reagents that
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are used with other samples. The LRB is used to determine if
method analytes or other interferences are present in the labora-
tory environment, the reagents, or the apparatus.
3.4 FIELD REAGENT BLANK (FRB) — An aliquot of reagent water or other
blank matrix that is placed in a sample container in the labora-
tory and treated as a sample in all respects, including shipment
to the sampling site, exposure to sampling site conditions,
storage, preservation, and all analytical procedures. The purpose
of the FRB is to determine if method analytes or other interfer-
ences are present in the field environment.
3.5 LABORATORY FORTIFIED BLANK (LFB) — An aliquot of reagent water or
other blank matrix to which known quantities of the method
analytes are added in the laboratory. The LFB is analyzed exactly
like a sample, and its purpose is to determine whether the method-
ology is in control, and whether the laboratory is capable of
making accurate and precise measurements.
3.6 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) — An aliquot of an
environmental sample to which know quantities of the method
analytes are added in the laboratory. The LFM is analyzed
exactly like a sample, and its purpose is to determine whether the
sample matrix contributes bias to the analytical results. The
background concentrations of the analytes in the sample matrix
must be determined in a separate aliquot and the measured values
in the LFM corrected for background concentrations.
1 ^
3.7 STOCK STANDARD SOLUTION (SSS) — A concentrated solution contain-
ing one or more method analytes prepared in the laboratory using
assayed reference materials or purchased from a reputable commer-
cial supplier.
3.8 PRIMARY DILUTION STANDARD SOLUTION (PDS) -- A solution of several
analytes prepared in the laboratory from stock standard solutions
and diluted as needed to prepare calibration solutions and other
needed analyte solutions.
3.9 CALIBRATION STANDARD (CAL) — A solution prepared from the primary
dilution standard solution or stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.10 QUALITY CONTROL SAMPLE (QCS) — A solution of method analytes of
known concentrations which is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards,
It is used to check laboratory performance with externally pre-
pared test materials.
3.11 METHOD DETECTION LIMIT (MDL) — The minimum concentration of an
analyte that can be identified, measured and reported with 99%
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confidence that the analyte concentration is greater than zero.
3.12 EXTERNAL STANDARD (ES) — A pure analyte(s) that is measured in
an experiment separate from the experiment used to measure the
analyte(s) in the sample. The signal observed for a known quanti-
ty of the external standard(s) is used to calibrate the instrument
response for the corresponding analytes(s). The instrument
response is used to calculate the concentrations of the analyte(s)
in the sample.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware, and other sample processing apparatus that
lead to discrete artifacts or elevated baselines in liquid chroma-
tograms. All reagents and apparatus must be routinely demonstrat-
ed to be free from interferences under the conditions of the
analysis by analyzing laboratory reagent blanks as described in
Sect. 9.2.
4.1.1 Glassware must be scrupulously cleaned (3). Clean all
glassware as soon as possible after use by thoroughly rins-
ing with the last solvent used in it. Follow by washing
with hot water and detergent and thorough rinsing with
dilute acid, tap and reagent water. Drain dry, and heat in
an oven or muffle furnace at 400°C for 1 hr. Do not heat
volumetric ware. Thermally stable materials such as PCBs
might not be eliminated by this treatment. Thorough rinsing
with acetone may be substituted for the heating. After
drying and cooling, seal and store glassware in a clean
environment to prevent any accumulation of dust or other
contaminants. Store inverted or capped with aluminum foil.
4.1.2 The use of high purity reagents and solvents helps to mini-
mize interference problems. Purification of solvents by
distillation in all-glass systems may be required.
WARNING: When a solvent is purified, stabilizers added by
the manufacturer are removed, thus potentially making the
solvent hazardous. Removal of preservatives by distillation
may also reduce the shelf-life of the solvent.
4.2 The acid forms of the analytes are strong organic acids which
react readily with alkaline substances and can be lost during
sample preparation. Glassware must be acid-rinsed with 1 N hydro-
chloric acid prior to use to avoid analyte losses due to adsorp-
tion.
4.3 Matrix interferences may be caused by contaminants that are
coextracted from the sample. Also, note that all method analytes
are not resolved from each other on a single column, i.e., one
analyte of interest may interfere with another analyte of inter-
est. The extent of matrix interferences will vary considerably
from source to source, depending upon the water sampled. The
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procedures in Sect. 11 can be used to overcome many of these
interferences. Tentative identifications should always be con-
firmed (Sect. 11.3).
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this
method has not been precisely defined; however, each chemical
compound must be treated as a potential health hazard. According-
ly, exposure to these chemicals must be reduced to the lowest
possible level. The laboratory is responsible for maintaining a
current awareness file of OSHA regulations regarding the safe
handling of the chemicals specified in this method. A reference
file of material safety data sheets should also be made available
to all personnel involved in the chemical analysis.
5.2 WARNING: When a solvent is purified, stabilizers added by the
manufacturer are removed, thus potentially making the solvent
hazardous. Therefore, storage of large volumes of purified sol-
vents may be hazardous. Therefore, only small volumes of solvents
should be purified just before use.
6. EQUIPMENT AND SUPPLIES
6.1 SAMPLE BOTTLE — Borosilicate, 125-mL volume, graduated, fitted
with Teflon-lined screw cap. Protect samples from light. The
container must be washed and dried as described in Sect. 4.1.1
before use to minimize contamination. Cap liners may be cut to
fit from Teflon sheets and extracted with methanol overnight prior
to use.
6.2 GLASSWARE
6.2.1 Volumetric flask, Class A — 100 mL, with ground glass
stoppers.
6.2.2 Graduated cylinder — 100 mL
6.2.3 Disposable pipets, Transfer — borosilicate glass
6.2.4 Glass syringe — 50 mL, with Luer-Lok fitting
6.2.5 Volumetric pipette, Class A — 20 mL
6.3 BALANCE — Analytical, capable of accurately weighing to the
nearest 0.0001 g.
6.4 LIQUID CHROMATOGRAPH — Analytical system complete with gradient
programmable HPLC suitable for use with analytical HPLC columns
and all required accessories including an injector, analytical
column, semi-prep guard column, and photodiode array UV detector.
A data system is necessary for measuring the peak areas and for
assessing the confirmation of the peak identification. A personal
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computer (PC) of at least the AT-class is generally needed to
control and collect data from the photodiode array UV detector.
Table 1 lists the retention times observed for the method analytes
using the column and analytical conditions described below.
Figure 1 is a schematic drawing of the analytical system including
the sample concentrator column (semi-prep guard column).
6.4.1 Primary Column —- 250 mm x 4.6 mm I.D.- ODS-AQ, 5 fim spheri-
cal (YMC Ltd.). Any column may be used if equivalent or
better performance (better peak shape, better analyte effi-
ciency, or more complete separation of analytes) can be
demonstrated. Mobile phase flow rate is established at 1.0
mL/min (linear velocity of 6.0 cm/min). Two mobile phase
components are used: A — 0.025 M H3P04; B — Acetonitrile.
A gradient solvent program is used to separate the analytes:
90:10 A:B to 10:90 A:B in 30 min, linear ramp / hold at
10:90 for 10 min. Reverse the gradient and establish ini-
tial conditions: 10:90 A:B to 90:10 A:B in 10 min, linear
ramp. Allow column backpressure to restablize for 5 to 10
min before beginning the next analysis. Total restabiliza-
tion time will be determined by each analyst.
6.4.2 Confirmation Column ~ 300 mm x 3.9 mm I.D. Nova-Pak C18,
4 ion spherical (Waters Chromatography Division, Millipore).
Any column may be used if equivalent or better performance
(better peak shape, better analyte efficiency, or more
complete separation of analytes) can be demonstrated.
Mobile phase and conditions same as primary column.
6.4.3 Sample Concentrator Column — 30 mm x 10 mm I.D. ODS-AQ,
5 urn spherical (YMC Ltd). An alternative concentrator
column may be used if all QC criteria in Sect. 9 can be
equalled or improved. Also, a liquid-solid extraction disk
may be used if all QC criteria in Sect. 9 can be equalled or
improved.
6.4.4 6-port Switching Valve — Rheodyne Model 7000 (Rheodyne
Corp).
6.4.5 Sample Delivery Pump -- A piston-driven pump capable of
delivering aqueous sample at a flow rate of 5.0 mL/min. An
analytical HPLC pump may serve as the sample delivery pump.
A Waters Model 6000A was used to generate the data presented
in this method.
6.4.6 Detector — Photodiode Array - Ultraviolet (PDA-UV), LKB-
Bromma Model 2140 Rapid Spectral Detector or equivalent.
Detector parameters: Scan Range - 210 to 310 nm at 1
scan/sec, detector integration - 1 sec.
6.4.7 Data Handling System — DOS-based Personal Computer,
AT-class machine or machine of greater capability with 640 K
RAM or more, an 80 Mb hard disk or larger, VGA monitor or
equivalent.
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REAGENTS AND STANDARDS
7.1 ACETONITRILE -- HPLC Grade or equivalent.
7.2 SODIUM SULFITE, GRANULAR, ANHYDROUS — ACS Grade.
7.3 SODIUM HYDROXIDE (NAOH), PELLETS — ACS Grade.
7.3.1 NaOH, 6 N — Dissolve 216 g NaOH in 900 mL reagent water.
7.4 PHOSPHORIC ACID, 85% AR, — ACS grade.
7.4.1 0.025 M — Mix 2.0 mL of H3P04 in 998 mL of reagent water.
7.5 STOCK STANDARD SOLUTIONS (1.00 Jtg/ML) — Stock standard solutions
may be purchased as certified solutions or prepared from pure
standard materials using the following procedure:
7.5.1 Prepare stock standard solutions by accurately weighing
approximately 0.0100 g of pure material. Dissolve the
material in acetonitrile and dilute to volume in a 10-mL
volumetric flask. Larger volumes may be used at the conve-
nience of the analyst. If compound purity is certified at
96% or greater, the weight may be used without correction to
calculate the concentration of the stock standard. Commer-
cially prepared stock standards may be used at any concen-
tration if they are certified by the manufacturer or by an
independent source.
7.5.2 Transfer the stock standard solutions into Teflon-lined
sealed screw cap amber vials. Store at room temperature and
protect from light.
7.5.3 Stock standard solutions should be replaced after two months
or sooner if comparison with laboratory fortified blanks, or
QC samples indicate a problem.
7.6 HYDROCHLORIC ACID — ACS grade.
7.6.1 HC1, 1 N — Dilute 50 mL in 600 mL of reagent water.
7.7 Filters, 0.45 /tm, Nylon, 25 mm i.d. (Gelman Sciences)
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 Grab samples must be collected in glass containers. Conventional
sampling practices (2) should be followed; however, the bottle
must not be prerinsed with sample before collection.
8.2 SAMPLE PRESERVATION AND STORAGE
8.2.1 Add hydrochloric acid (1:1) to the sample to produce a pH of
2. The pH may be measured in the field using pH indicator
strips.
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8.2.2 Residual chlorine should be reduced at the sampling site by
the addition of a reducing agent. Add 4-5 mg of sodium
sulfite (this may be added as a solid with shaking until
dissolved) to each 100 ml of water.
8.2.3 The samples must be iced or refrigerated at 4°C away from
light from the time of collection until extraction. The
samples must be analyzed within 14 days of collection.
However, analyte stability may be affected by the matrix.
Therefore, the analyst should verify that the preservation
technique is applicable to the samples under study. If the
14-day holding time is exceeded, the data should be flagged
so that the data user is aware of possible analyte degrada-
tion.
8.2.4 Field reagent blanks (FRB) — Processing a (FRB) is recom-
mended along with each set, which is composed of the samples
collected from the same general sample site at approximately
the same time. At the laboratory, fill a sample container
with reagent water, seal, and ship to the sampling site
along with the empty sample containers. During sample
collection, open the FRB and add H6 (Sect. 8.2.1) and sodium
sulfite (Sect. 8.2.2) Return the FRB to the laboratory with
filled sample bottles.
9. QUALITY CONTROL
9.1 Minimum QC requirements are initial demonstration of laboratory
capability, analysis of laboratory reagent blanks, laboratory
fortified samples, laboratory fortified blanks, and QC samples.
9.2 LABORATORY REAGENT BLANKS (LRB) — Before processing any samples,
the analyst must demonstrate that all glassware and reagent
interferences are under control. Each time a set of samples is
extracted or reagents are changed, a LRB must be analyzed. If
within the retention time window of any analyte the LRB produces a
peak that would prevent the determination of that analyte, deter-
mine the source of contamination and eliminate the interference
before processing samples.
9.3 INITIAL DEMONSTRATION OF CAPABILITY
9.3.1 Select a representative fortified concentration for each
analyte. Prepare a sample concentrate (in acetonitrile)
containing each analyte at 1000 times the selected concen-
tration. With a syringe, add 100 /tL of the concentrate to
each of at least four 100-mL aliquots of reagent water, and
analyze each aliquot according to procedures beginning in
Sect. 11.
9.3,2 Calculate the recoveries, the relative standard deviation,
and the MDLs (5). For each analyte the recovery value for
all four of these samples must fall in the range of R ± 30%,
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using the value for R for reagent water in Table 2. As the
calibration procedure employs a fortified reagent water
blank for the determination of the calibration curves or
factors, the recovery values for the analytes should, by
definition, be within this range. If the mean recovery of
any analyte fails this demonstration, repeat the measurement
of that analyte to demonstrate acceptable performance.
9.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples using a
new, unfamiliar method prior to obtaining some experience
with it. As laboratory personnel gain experience with this
method the quality of data should improve beyond what is
required here.
9.4 The analyst is permitted to modify LC columns, LC conditions, and
detectors. Each time such method modifications are made, the
analyst must repeat the procedures in Sect. 9.3. NOTE: The LC
column and guard cartridge used to generate the data in this
method were found to be, unique CJ8-silica columns. Before substi-
tuting other C18 columns, a careful review of the literature is
recommended.
9.5 ASSESSING LABORATORY PERFORMANCE — Laboratory fortified blank
9.5.1 The laboratory must analyze at least one laboratory forti-
fied blank (LFB) sample with every 20 samples or one per
sample set (all samples analyzed within a 24-hr period)
whichever is greater. The concentration of each analyte in
the LFB should be 10 times the MDL or the MCL, whichever is
less. Calculate accuracy as percent recovery (X,). If the
recovery of any analyte falls outside the control limits
(See Sect. 9.5.2), that analyte is judged out of control,
and the source of the problem should be identified and
resolved before continuing analyses.
9.5.2 Until sufficient data become available from within the
laboratory, usually a minimum of results from 20 to 30
analyses, the laboratory should assess laboratory perfor-
mance against the control limits in Sect. 9.3.2 that are
derived from the data in Table 2. When sufficient internal
performance data become available, develop control limits
from the mean percent recovery (X) and standard deviation
(S) of the percent recovery. These data are used to estab-
lish upper and lower control limits as follows:
UPPER CONTROL LIMIT = X + 3S
LOWER CONTROL LIMIT = X - 3S
After each five to ten new recovery measurements, new con-
trol limits should be calculated using only the most recent
20-30 data points. These calculated control limits should
never exceed those established in Sect. 9.3.2.
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9.5.3 It is recommended that the laboratory periodically determine
and document its detection limit capabilities for the
analytes of interest.
9.5.4 At least quarterly, analyze a QC sample from an outside
source.
9.5.5 Laboratories are encouraged to participate in external
performance evaluation studies such as the laboratory cer-
tification programs offered by many states or the studies
conducted by USEPA. Performance evaluation studies serve as
independent checks on the analyst's performance.
9.6 ASSESSING ANALYTE RECOVERY -- Laboratory fortified sample matrix
9.6.1 The laboratory should add a known concentration to a minimum
of 10% of the routine samples or one sample per set, which-
ever is greater. The concentration should not be less than
the background concentration of the sample selected for
fortification. Ideally, the concentration should be the
same as that used for the laboratory fortified blank (Sect.
9.5). Over time, samples from all routine sample sources
should be fortified.
9.6.2 Calculate the percent recovery, P of the concentration for
each analyte, after correcting the analytical result, X,
from the fortified sample for the background concentration,
b, measured in the unfortified sample,
P - 100 (X - b) / fortifying concentration,
and compare these values to control limits appropriate for
reagent water data collected in the same fashion. If the
analyzed unfortified sample is found to contain NO back-
ground concentrations, and the added concentrations are
those specified in Sect. 9.5, the appropriate control limits
would be the acceptance limits in Sect. 9.5. If, on the
other hand, the analyzed unfortified sample is found to
contain background concentration, b, estimate the standard
deviation at the background data, s^, using regressions or
comparable background data and similarly, estimate the mean,
Xa, and standard deviation, s , of analytical results or the
total concentration after fortifying. Then the appropriate
percentage control limits would be P + 3sp, where:
P = 100 X / (b + fortifying concentration)
and sp = 100 (sa2 + sb2)172 /fortifying concentration
For example, if the background concentration for Analyte A
was found to be 1 pg/L and the added amount was also 1 /tg/L,
and upon analysis the laboratory fortified sample measured
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1.6 fig/I, then the calculated P for this sample would (1.6
/jg/L minus 1.0 /xg/L)/ 1.0 /t'g/L or 60%. This calculated P is
compared to control limits derived from prior reagent water
data. Assume it is known that analysis of an interference
free sample at 1.0 /ig/L yields an s of 0.12 #g/L and similar
analysis at 2.0 /zg/L yields X and S of 2.01 /tg/L and 0.20
jug/L, respectively. The appropriate limits to judge the
reasonableness of the percent recovery, 60%, obtained on the
fortified matrix sample is computed as follows:
[100 (2.01 pg/L) / 2.0 ug/L]
± 3 (100) [(0.12 /ag/L)2 + (0.20 /ig/L)2]1/2 / 1.0 /tg/L =
100.5% ± 300 (0.233) =
100.5% ± 70 or 30% to 170% recovery of the added analyte.
9.6.3 If the recovery of any such analyte falls outside the desig-
nated range, and the laboratory performance for that analyte
is shown to be in control (Sect. 9.5), the recovery problem
encountered with the fortified sample is judged to be matrix
related, not system related. The result for that analyte in
the unfortified sample is labeled suspect/matrix to inform
the data user that the results are suspect due to matrix
effects.
9.7 The laboratory may adopt additional QC practices for use with this
method. The specific practices that are most productive depend
upon the needs of the laboratory and the nature of the samples.
For example, field or laboratory duplicates may be analyzed to
assess the precision of the environmental measurements. The field
reagent blanks may be used to assess contamination of samples
under site conditions, transportation and storage.
10. CALIBRATION AND STANDARDIZATION
10.1 Establish HPLC operating parameters equivalent to those indicated
in Sect. 6.4.1. The HPLC system should be calibrated using the
external standard technique (Sect. 10.2). NOTE: Calibration
standard solutions must be prepared such that no unresolved
analytes are mixed together. The method analytes have been
separated into two calibration solutions (See Table 1 for Groups A
and B). The analytes in these solutions have been found to be
resolved under the LC conditions listed. Mixtures of these
analytes at concentration levels of 100 /ig/mL (in acetonitrile)
are suggested as a possible secondary dilution standard. Figures
2 and 3 are typical chromatograms of Groups A and B as separated
on the primary HPLC column.
10.2 EXTERNAL STANDARD .CALIBRATION PROCEDURE
10.2.1 Prepare calibration standards (CAL) at a minimum of three
(five are recommended) concentration levels for each
analyte of interest by adding volumes of one or more stock
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standards to volumetric flasks. Alternatively, add vari-
ous volumes of a primary dilution standard solution of
Group A or B (Sect. 10.1) to a volumetric flask. Dilute
to volume with the aqueous mobile phase (0.025 M HjPOJ.
The lowest standard should contain analyte concentrations
near, but above, the respective MDL. The remaining stan-
dards should bracket the analyte concentrations expected
in the sample extracts, or should define the working range
of the detector.
10.2.2 Starting with the standard of the lowest concentration,
process each calibration standard according to Sect. 11.1
and tabulate response (peak area) versus injected quantity
in the standard. The results can be used to prepare a
calibration curve for each compound. Alternatively, if
the ratio of response to concentration (response factor)
is a constant over the working range (20% RSD or less),
linearity through the origin can be assumed and the aver-
age ratio or response factor can be used in place of a
calibration curve.
10.2.3 The working calibration curve or response factor must be
verified on each working day by the measurement of a CAL,
analyzed at the beginning of the analysis day. It is
highly recommended that an additional check standard be
analyzed at the end of the analysis day. For extended
periods of analysis (greater than 8 hr), it is strongly
recommended that check standards be interspersed with
samples at regular intervals during analyses. If the
response for any analyte varies from the predicted re-
sponse by more than ± 25%, the test must be repeated using
a fresh calibration standard. If the results still do not
agree, generate a new calibration curve.
10.2.4 Verify calibration standards periodically, recommend at
least quarterly, by analyzing a standard prepared from
reference material obtained from an independent source.
Results from these analyses must be within the limits used
to routinely check calibration.
11. PROCEDURE
11.1 HYDROLYSIS, PREPARATION, AND EXTRACTION.
11.1.1 Add preservative to blanks and QC check standards. Mark
the water meniscus on the side of the sample bottle for
later determination of sample volume (Sect. 11.1.5).
11.1.2 Add 1.7 ml of 6 N NaOH to the sample, seal, and shake.
Check the pH of the sample with pH paper; if the sample
does not have a pH greater than or equal to 12, adjust the
pH by adding more 6 N NaOH. Let the sample sit at room
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temperature for 1 hr, shaking the sample bottle and con- |||
tents periodically. ^U
11.1.3 Add 2 ml of concentrated H,P04 to the sample, seal, and
shake to mix. Check the pH of the sample with pH paper;
if the sample does not have a pH less than or equal to
two, adjust the pH by adding more H3P04.
11.1.4 From the homogeneous sample, remove a 20-mL aliquot for
analysis. Filter the aliquot through a 0.45 /wn filter
into a graduated cylinder or other convenient graduated
container. Using an HPLC pump (or HPLC reagent delivery
pump), pump the 20-mL aliquot through the on-line concen-
trator column at a flowrate of 5.0 mL/min (See Figure 1).
The use of a liquid-solid extraction disk is perfectly
acceptable providing all QC criteria in Sect. 9 are met or
exceeded. After passing the sample through the concentra-
tor column, follow with an additional 10-mL of the aqueous
mobile phase (0.025 M H3P04).
11.1.5 After analysis is completed, determine the original sample
volume by refilling the sample bottle to the mark and
transferring the water to a 100-mL graduated cylinder.
Record the sample volume to the nearest 1 ml.
11.2 HIGH PERFORMANCE LIQUID CHROMAT06RAPHY
11.2.1 Sect. 6.4.1 summarizes the recommended operating condi-
tions for the HPLC. Included in Table 1 are retention
times observed using this method. Other HPLC columns,
chromatographic conditions, or detectors may be used if
the requirements of Sect. 9.3 are met.
11.2.2 Calibrate the system daily as described in Sect. 10.
11.2.3 After loading the sample (or calibration standard) onto
the concentrator column, valve the sample into the analyt-
ical stream, backflushing the concentrator column. The
photodiode array detector (PDA-UV) is set to scan and
record from 210 to 310 nm, 1 scan per second during the
entire chromatographic run (40 min). Extract the 230 nm
trace from the stored data and record the resulting peak
size in area units for all analytically significant peaks.
11.2.4 If the responses for the peaks exceed the working range of
the system, dilute an additional 20-mL aliquot of the
sample with reagent water, adjust the pH to 12 with NaOH,
and reanalyze according to Sect. 11.1.2.
11.3 IDENTIFICATION OF ANALYTES
11.3.1 Identify a sample component by comparison of its retention
time to the retention time of a reference chromatogram.
250
-------
If the retention time of an unknown compound corresponds,
within limits, to the retention time of a standard com-
pound, then identification is considered positive.
11.3.2 The width of the retention time window used to make iden-
tifications should be based upon measurements of actual
retention time variations of standards over the course of
a day. Three times the standard deviation of a retention
time can be used to calculate a suggested window size for
a compound. However, the experience of the analyst should
weigh heavily in the interpretation of chromatograms.
11.3.3 Identification requires expert judgment when sample compo-
nents are not resolved chromatographically. When peaks
obviously represent more that one sample component (i.e.,
broadened peak with shoulder(s) or vallies between two or
more maxima, or any time doubt exists over the identifica-
tion or a peak on a chromatogram, appropriate alternative
techniques, to help confirm peak identification, should be
used. For this method, the use of the PDA-UV detector
affords the analyst the option of using a secondary wave-
length for the analysis of the questionable identifica-
tion. The response ratio for a compound of interest at
two wavelengths may be determined from standards of known
purity. If the wavelength response ratio and the reten-
tion time matches a given unknown to a method analyte,
more certainty may be assigned to the identification of
the unknown. If this method of compound confirmation is
employed, each analyst will need to determine the wave-
length response ratio for each analyte. Table 3 lists
suggested alternative wavelengths for each analyte in the
scope of the method. An alternative LC column may be used
to separate and confirm the identification of unknown
peaks. A suggested alternative column is described in
Sect.. 6.4.2.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Calculate analyte concentrations in the sample from the response
for the analyte using the calibration procedure described in Sect.
10.
12.2 Calculate the amount of sample analyte injected from the peak
response using the calibration curve or calibration response
factor determined in Sect. 10.2. The concentration (C) in the
sample can be calculated from Equation 1.
= __CAKVJ Equation 1.
where:
A = Amount of standard injected (ng)
251
-------
V,- = Volume of standard injected (ml)
Vt = Volume of sample injected (ml)
Vs - Volume of water sample (ml)
13. METHOD PERFORMANCE
13.1 In a single laboratory, analyte recoveries from reagent water were
determined at two concentration levels. Results were used to
determine analyte MDLs (5) and demonstrated method range. Analyte
MDLs and analyte recoveries and standard deviations about the
percent recoveries at one concentration are given in Table 2.
13.2 In a single laboratory, analyte recoveries from dechlorinated tap
water and ground waters were determined at one concentration
level, 10 ug/L. Results were used to demonstrate applicability of
the method to different tap and ground water matrices. Analyte
recoveries from tap water and ground water are given in Table 4.
MDLs calculated from results of analyses of six 100 ml reagent
water samples at 0.5 /jg/L concentrations for each analyte are
listed in Table 5.
14. POLLUTION PREVENTION
14.1 This method utilizes the new in-line liquid-solid extraction
technology which requires the use of very small quantities of ^^
organic solvents. This feature eliminates the hazards involved |M
with the use of large volumes of potentially harmful organic
solvents needed for conventional liquid-liquid extractions. Also,
this method uses no derivatizing reagents, which are toxic or
explosive, to form gas chromatographable derivatives. These
features make this method much safer for use by the analyst in the
laboratory and a great deal less harmful to the environment.
14.2 For information about pollution prevention that may be applicable
to laboratory operations, consult "Less is Better: Laboratory
Chemical Management for Waste Reduction," available from the
American Chemical Society's Department of Government Relations and
Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.
15. WASTE MANAGEMENT
15.1 Due to the nature of this method, there is little need for waste
management. No large volumes of solvents or hazardous chemicals
are used. The matrices of concern are finished drinking water or
source water. However, the Agency requires that laboratory waste
management practices be consistent with all applicable rules and
regulations, and that laboratories protect the air, water, and
land by minimizing and controlling all releases from fume hoods
and bench operations. Also, compliance is required with any
sewage discharge permits and regulations, particularly the hazard-
ous waste identification rules and land disposal restrictions.
For further information on waste management, consult "The Waste
252
-------
Management Manual for Laboratory Personnel," also available from
the American Chemical Society at the address in Sect. 14.2.
16. REFERENCES
1. Glazer, J.A., Foerst, D.L., McKee, 6.D., Quave, S.A., and Budde,
W.L., Environ. Sci. Techno!. 15, 1981, pp. 1426-1435.
2. "Pesticide Methods Evaluation," Letter Report #33 for EPA Contract
No. 68-03-2697. Available from U.S. Environmental Protection
Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio 45268.
3. ASTM Annual Book of Standards, Part 11, Volume 11.02, D3694-82,
"Standard Practice for Preparation of Sample Containers and for
Preservation," American Society for Testing and Materials,
Philadelphia, PA, p. 86,1986.
4. Giam, C.S., H.S. Chan, and G.S. Nef. "Sensitive Method for
Determination of Phthalate Ester Plasticizers in Open-Ocean Biota
Samples," Analytical Chemistry. 47, 2225 (1975).
5. 40 CFR, Part 136, Appendix B.
253
-------
17. TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. RETENTION TIMES FOR METHOD ANALYTES
Analvte
Picloram
5-Hydroxydi camba
Chloramben
4-Nitrophenol
Di camba
Bentazon
HCPA
2,4-D
3,5-Dichloro-
benzoic acid
MCPP
Dichloroprop
2,4,5-T
2,4-DB
2,4,5-TP
Acifluorfen
Dinoseb
Pentach1 orophenol
Group
(A)
(A)
(A)
(B)
(A)
(A)
(B)
(A)
(B)
(B)
(A)
(B)
(B)
(A)
(A)
(B)
(B)
Retention Times8
(minutes)
Primary Column
19.0
19.7
21.1
21.6
24.0
25.2
25.5
25.6
26.7
27.2
27.3
27.5
28.0
29.2
30.7
32.8
33.4
Confirmation Column
12.8
13.5
14.8
5.0
18.2
19.5
20.1
20.1
21.3
21.8
21.8
*
22.4
22.8
23.9
25.5
27.7
28.3
8 Columns and analytical conditions are described in Sect. 6.4.1 and Sect.
6.4.2.
254
-------
TABLE 2. SINGLE LABORATORY ACCURACY, PRECISION AND METHOD DETECTION LIMITS
(MDLS) FOR ANALYTES FROM REAGENT WATER (a>
Analvte
Acifluorfen
Bentazon
Chloramben
2,4-D
2,4-DB
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
MCPA
MCPP
4-Nitrophenol
Pentachlorophenol (PCP)
Plcloram
2,4,5-T
2,4,5-TP
MDL
«q/Lb
1.7
4.6
3.1
1.3
1.9
2.1
2.1
1.7
1.5
2.2
0.8
1.7
1.2
1.6
0.5
1.3
1.8
Concentration
ua/L
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
Reagent
Rc
104
126
83
112
92
104
94
108
97
132
93
95
95
99
104
93
90
Water
S d
K
1.7
14.6
10.0
4.2
5.9
6.6
6.7
5.4
4.8
7.0
2.5
5.5
4.0
5.2
1.7
4.1
5.8
Data represent the average of 6-7 samples. Sample volume = 20 ml.
MDL = method detection limit; defined in Appendix B to 40 CFR Part 136
Definition and Procedure for the Determination of the Method Detection
Limit - Revision 1.11.
R = average percent recovery.
Sr = standard deviation of the percent recovery
255
-------
TABLE 3. CONFIRMATION WAVELENGTHS AND AREA RESPONSE RATIOS FOR METHOD III
ANALYTES.
Analvte
Acifluorfen
Bentazon
Chloramben
2,4-D
2,4-DB
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxydicamba
MCPA
MCPP
4-Nitrophenol
Pentachlorophenol (PCP)
Plclorara
2,4,5-T
2,4,5-TP
Confirmation
Wavelenqth (nm)
293
240
214
285
285
220
285
285
268
293
285
285
310
290
223
290
293
Area Response
Ratio8
1.72
1.08
0.61
4.02
5.93
0.66
5.15
4.07
0.48
1.89
6.66
6.49
0.56
5.65
0.82
4.00
3.84
a - Area Response Ratio = Peak Area for 230 nm / Peak Area for Conf.
Wavelength
4ft
4ft
256
-------
TABLE 4. SINGLE LABORATORY PRECISION AND ACCURACY DATA FROM TAP WATER AND
GROUND WATER8
Anal vte
Acifluorfen
Bentazon
Chloramben
2,4-D
2,4-DB
Dicamba
3,5-Dichlorobenzoic acid
Dlchlorprop
Dinoseb
5-Hydroxydi camba
MCPA
MCPP
4-Nitrophenol
Pentachlorophenol (PCP)
Picloram
2,4,5-T
2,4,5-TP
Tap
Rb
65.7
86.1
100
117
91.2
94.3
90.2
92.9
94.1
110
92.7
91.4
89.2
102
99.0
88.2
90.3
Dechlorinated
Water Ground Water
SRC Rb S.c
±27.
± 6.0
± 5.5
± 9.6
± 7.0
± 6.1
± 7.2
± 6.1
± 6.2
± 5.5
± 5.0
± 7.7
± 10.
± 4.2
± 4.9
± 7.8
± 5.9
87.3
90.1
88.2
105
97.2
86.0
92.1
98.3
91.2
108
85.2
84.3
103
92.6
84.3
90.0
77.8
±17.
± 9.0
± 5.9
± 8.1
± 6.1
± 7.7
± 5.5
±10.
± 4.1
± 7.0
± 5.7
± 5.9
± 3.4
± 9.1
± 5.8
± 6.2
± 8.9
a - 'Average of six samples fortified at 10
b
- Mean percent recovery, corrected for background levels
c - Standard deviation of the mean percent recovery
257
-------
TABLE 5. SINGLE LABORATORY RECOVERY AND PRECISION DATA AND METHOD
DETECTION LIMITS (MDLS) FOR ANALYTES FROM REAGENT WATER
Analvte
Acifluorfen
Bentazon
Chloramben
2,4-D
*• J * •*•
2,4-DB
Dicamba
3,5-Dichlorobenzoic acid
Dichlorprop
Dinoseb
5-Hydroxdicamba
MCPA
MCPP
4-Nitrophenol
Pentachlorophenol (PCP)
Picloram
2,4,5-T
2,4,5-TP
MDL
ua/Lb
0.40
0.12
N.R.
0.34
0.31
0.24
0.38
0.33
0.26
N.R.
0.35
0.19
N.R.
0.15
N.R.
0.21
0.37
Concentration
ua/L
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Reagent
Rc
114
91
N.R.
121
99
80
105
110
99
N.R.
124
125
N.R.
93
N.R.
80
77
Water J
Sr
23.7
7.3
N.R.
20.2
18.5
14.1
22.5
19.4
15.5
N.R.
21.0
11.1
N.R.
8.6
Nn
.R.
12.7
21.7
a Data represent the average of six samples. Sample Volume = 100 ml
b HDL - Method detection limit; defined in Appendix B to 40 CFR 136 - Definition
and Procedure for the Determination of the Method Detection Limit - Revision
1.11.
c R » Average percent recovery.
d Sr - Standard deviation of the percent recovery.
N.R. - Not Recovered.
258
-------
FIGURE 1. SCHEMATIC DIAGRAM OF SAMPLE CONCENTRATION AND ANALYTICAL HPLC
HARDWARE
Precolumn Extraction Hardware
Analysis Mode
Sanple Pump
Prtcoluen
aste
Rheodyne 7000 Valve
(6-Port)
Extraction Mode
Sample Pump
Analytical Pump (s)
I
Detector
Prtcoluin
aste
y
n
Analytical Pump (s)
L
Detector
259
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