&EPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/600/4-90/020
July 1990
Methods for the
Determination of
Organic Compounds in
Drinking Water
Supplement I
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EPA-600/4-90/020
July 1990
METHODS FOR THE DETERMINATION
OF ORGANIC COMPOUNDS
IN DRINKING WATER
SUPPLEMENT I
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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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
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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.
This publication of the Environmental Monitoring Systems Laboratory -
Cincinnati titled, "Determination of Organic Compounds in Drinking Water
Supplement I" was prepared to gather together under a single cover a set of 9
laboratory analytical methods for organic compounds in drinking water. We are
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 Clark, Director
Environmental Monitoring Systems
Laboratory - Cincinnati
iii
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ABSTRACT
Nine analytical methods covering 54 organic contaminants which may be
present in drinking water or drinking water sources are described in detail.
Seven of these methods cover compounds designated for regulation under the Safe
Drinking Water Act Amendments of 1986. Regulations for this group are in the
proposal stages with promulgation scheduled for June 1992. The other two methods
are for chlorination disinfection byproducts and may be regulated as part of
EPA's disinfectants and disinfectant byproducts rule scheduled for proposal early
in 1992. Most of the analytes may be classified as non-volatile and three of the
methods entail separations by high performance liquid chromatography. The
remainder employ capillary column gas chromatography. One of these requires
detection of a potentially very toxic contaminant, 2,3,7,8-tetrachlorodibenzo-p-
dioxin, at the low parts per trillion level. Labeled isotopes of this analyte are
employed as tracers and high resolution mass spectrometry is required for
detection and unambiguous identification. Three of the methods herein offer new
and simplified liquid-solid extraction procedures, a trend which is likely to
become even more pronounced in the future.
IV
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TABLE OF CONTENTS
Method
Number Title Page
Foreword . i i i
Abstract ............... iv
Acknowledgment vi
Analyte - Method Cross Reference vii
Introduction 1
506 Determination of Phthalate and Adi pate Esters in
Drinking Water by Liquid-Liquid Extraction or
Liquid-Solid Extraction and Gas Chromatography
with Photoionization Detection 5
513 Determination of 2,3,7,8-Tetrachloro-dibenzo-p-dioxin
in Drinking Water by Liquid-Liquid Extraction and Gas
Chromatography with High-Resolution Mass Spectrometry . . 33
547 Determination of Glyphosate in Drinking Water by Direct-
Aqueous-Injection HPLC, Post-Column Derivatization,
and Fluorescence Detection 63
548 Determination of Endothall in Drinking Water by Aqueous
Derivatization, Liquid-Solid Extraction, and Gas
Chromatography with Electron-Capture Detection 81
549 Determination of Diquat and Paraquat in Drinking Water
by Liquid-Solid Extraction and HPLC with Ultraviolet
Detection 101
550 Determination of Polycyclic Aromatic Hydrocarbons in
Drinking Water by Liquid-Liquid Extraction and HPLC
with Coupled Ultraviolet and Fluorescence Detection ... 121
550.1 Determination of Polycyclic Aromatic Hydrocarbons in
Drinking Water by Liquid-Solid Extraction and HPLC
with Coupled Ultraviolet and Fluorescence Detection ... 143
551 Determination of Chlorination Disinfection Byproducts
and Chlorinated Solvents in Drinking Water by Liquid-
Liquid Extraction and Gas Chromatography with Electron-
Capture Detection 169
552 Determination of Haloacetic Acids in Drinking Water
by Liquid-Liquid Extraction, Derivatization, and Gas
Chromatography with Electron Capture Detection 201
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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 Arleen M.
Ciampone for providing outstanding secretarial and word processing support, and
for format improvements in presentation of the manual.
In addition, William L. Budde, Director of the Chemistry Research Division,
is recognized for his significant contributions. Robert L. Graves directed some
early research which led to the development of some of the methods contained in
this manual. Jimmie W. Hodgeson and James W. Eichelberger reviewed and edited
each of the individual methods and directed the publication of the manual. Fred
K. Kawahara, Arnold L. Cohen, Jeffery D. Collins, Winslow J. Bashe, and Terryl
V. Baker performed the major portion of the laboratory support necessary to
develop the methods. John P. Donnelly provided electronic engineering support
during several of the development projects. Appreciation is also extended to the
scientists in the Technical Support Division of the Office of Drinking Water for
their constructive and beneficial review of the analytical methods contained in
this manual.
The Quality Assurance Research Division of the Environmental Monitoring
Systems Laboratory - Cincinnati, also provided invaluable assistance by reviewing
all the methods, and producing second laboratory accuracy and precision data for
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 appreciation is due to
Thomas A. Clark, Director of the Environmental Monitoring Systems Laboratory -
Cincinnati, and Joseph Cotruvo, former Director of the Criteria and Standards
Division, Office of Drinking Water, for their cooperation and support during this
project.
VI
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ANALYTE - METHOD CROSS REFERENCE
Analvte Method No.
Acenaphthene 550, 550.1
Acenaphthylene 550, 550.1
Anthracene 550, 550.1
Benz(a)anthracene 550, 550.1
Benzo(b)fluoranthene 550, 550.1
Benzo(k)fluoranthene 550, 550.1
Benzo(g,h,i)perylene 550, 550.1
Benzo(a)pyrene 550, 550.1
Bis(2-ethylhexyl)adipate 506
Bis(2-ethylhexyl)phthalate 506
Bromochloroacetic Acid 552
Bromochloroacetonitrile 551
Bromodichloromethane 551
Bromoform 551
Butyl benzyl phthalate 506
Carbon Tetrachloride 551
Chloral Hydrate 551
Chloroform 551
Chloropicrin 551
Chrysene 550, 550.1
Dibenz(a,h)anthracene 550, 550.1
Dibromoacetic Acid 552
Dibromoacetonitrile 551
Dibromochloromethane 551
r,2-Dibromo-3-chloropropane(DBCP) 551
l,2-Dibromoethane(EDB) , 551
Dichloroacetic Acid 552
Dichloroacetonitrile 551
2,4-Dichlorophenol 552
l,l-Dichloropropanone-2 551
Diethyl phthalate 506
Dimethyl phthalate 506
Di-n-butyl phthalate 506
Di-n-octyl phthalate 506
Diquat 549
Endothall 548
Fluoranthene 550, 550.1
Fluorene 550, 550.1
Glyphosate 547
Indeno(l,2,3-cd)pyrene 550, 550.1
Monobromoacetic Acid 552
Monochloroacetic Acid 552
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Analvte
Naphthalene
Paraquat
Phenanthrene
Pyrene
2,3,7,8-Tetrachlorodi benzo-p-di oxi n
Tetrachloroethylene
Trichloroacetic Acid
Trichloroacetonitrile
1,1,1-Tri chloroethane
Tri chloroethylene
2,4,6-Trichlorophenol
1,1,l-Trichloropropanone-2
Method No.
550,
550,
550.1
549
550, 550.1
550.1
513
551
552
551
551
551
552
551
viii
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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 which may
have potential adverse effects upon human health. This manual provides nine,
analytical methods for 54 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. The current manual is a supplement to the earlier version,
providing, for the most part, methods for analytes which appear at a later time
in the regulatory framework. Efforts have been made herein to provide a manual
and methods format, which is consistent with the earlier version.
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 bases 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 contaminants in
drinking water which 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 (MCL's) with compliance
determined by regulatory monitoring or by the application 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
of 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 (VOC) were promulgated in June 1987 (see 52 FR 25690 and 51 FR 11396).
Analytical methods for these eight as well as other unregulated VOC's were
published in the December 1988 manual (EPA Methods 502.1, 502.2, 503.1, 524.1 and
524.2). Regulations for thirty synthetic organic chemicals (SOC's) were proposed
May 22, 1989 (54 FR 22062) and scheduled for promulgation by December 1990. Note
that this group included six SOC's 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 thirty compounds are by the VOC methods above or SOC methods also
included in the 1988 manual.
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The current manual provides analytical methods for many of the remaining
contaminants on the original list of 83 - namely adipates, diquat, endothajl,
glyphosate, polycyclic aromatic hydrocarbons (PAH's), phthalates and dioxin.
Phase V of EPA regulations for these and eleven other remaining SOC's from the
list of 83 is scheduled for proposal in June 1990 and promulgation in March 1992.
Analytical methods for the latter eleven were included in the December 1988
manual. Methods 551 and 552 of this manual cover the most important classes of
organic chlorination disinfection byproducts. These contaminants were included
in the first EPA priority list of additional substances, which may require
regulation under the Act (see 53 FR 1892). At least some of these will be
regulated by EPA's phase IV disinfectants and disinfectant byproducts, rule
scheduled for proposal early in 1992.
GENERAL COMMENTS
The current manual provides methods, which are in the same format and cast
in the same terminology as the December 1988 manual. The introduction to the
earlier manual discusses general method features on format, sample matrices,
method detection limits, 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 are designed for drinking water and drinking water
sources and not for more complex matrices such as waste water, hazardous waste
effluents or biological fluids. The method detection limits provided were
determined by replicate analyses of. fortified reagent water over a relatively
short period of time. As such, these are somewhat idealized limits, but
nevertheless provide a useful index of method performance. Reporting limits for
reliable quantitative data may be considerably higher.
The quality assurance sections are uniform and contain minimum requirements
for operating a reliable monitoring program - initial demonstration of
performance, routine analyses of reagent blanks, analyses of fortified reagent
blanks and fortified matrix samples, and analyses of quality control (QC)
samples. Other quality control 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 includes seven methods for synthetic organic chemicals and two
methods for chlorination disinfection byproducts. In general, the analytes may
by classified as nonvolatile and three of the methods employ separation by high
performance liquid chromatography (HPLC). The remainder utilize capillary column
gas chromatography (GC). Two of the methods use convenient liquid-solid
extraction (LSE) methods for analyte isolation, and two others offer LSE as an
option. By contrast to the original manual, four of the methods are for single
analytes - 2,3,7,8-tetrachlorobenzo-p-dioxin, glyphosate, endothall and diquat.
These analytes are not readily amenable to generic methods. Each method provides
an adequate summary statement. Some additional comments are germane here.
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Method 506 for phthalates and adipates offers both liquid-liquid extraction
(LLE) and LSE options. After capillary column GC separation, a photoionization
detector is required for detection and MDL's are limited to approximately
10 jug/L. Phthalates and adipates are among the most common contaminants
encountered in the laboratory and extreme care must be taken to ensure clean
reagent blanks.
Dioxin may be an extremely toxic chemical and water concentrations of a few
parts per trillion (pg/L) are of concern. In addition to extreme sensitivity,
unambiguous identification is an analytical requirement. Thus, Method 513 is an
inherently complex method, which employs LLE or LSE, extract cleanup, a sample
concentration factor of 10 , capillary column GC separation and analysis by high
resolution mass spectrometry. In addition, labeled isotopes of dioxin are
employed as surrogate analyte and internal standard to aid in identification and
quantitation and to compensate for analyte losses during the complex sample
handling procedure.
Methods 547, 548 and 549 are single analyte procedures for glyphosate,
endothall and diquat. Paraquat is a non-regulated ionic herbicide quite similar
to diquat and may be analyzed simultaneously. These may be characterized as
difficult analytes because of their high water solubility and low volatility. In
addition, glyphosate and endothall require derivatization prior to detection.
Glyphosate is analyzed by direct aqueous HPLC injection and undergoes post-
column derivatization prior to fluorescence detection. Endothall must be
transferred from the aqueous phase to an acetic acid matrix for derivatization,
followed by analysis by GC with electron capture detection (ECD). As with
glyphosate, no preconcentration factor is involved. Method 549 provides for the
extraction and concentration of diquat and paraquat in the base forms by LSE with
a C-8 cartridge, preconditioned to operate in the reverse phase mode. The
analytes are eluted with an acidic solvent and, after addition of ion-pairing
reagent, are separated by HPLC. The analytes are detected by ultraviolet
absorption (UV) with confirmation provided by a photodiode array spectrometer.
Methods 550 and 550.1 provide HPLC alternatives for polycyclic aromatic
hydrocarbons. Method 550 employs a conventional serial LLE approach while 550.1
uses a LSE procedure similar to Method 525. Dual UV and fluorescence detectors
are employed, with considerably lower MDL's reported for the latter.
Halogenated organic byproducts, other than the regulated trihalomethanes,
account for most of the total organic halogen formed by the chlorination of water
supplies. The most important classes in terms of occurrence are the neutral
analytes of Method 551 and the halo.acetic acids of Method 552. Method 551 is
quite similar to Method 504, employing a simple one step LLE and direct injection
of the extract into a capillary GC with ECD detection. The haloacetic method is
a serial LLE with analysis by GC-ECD and is quite similar to, but simpler than,
Method 515.1 for acid herbicides. Both employ diazomethane for methylation with
a micromolar generation procedure, which avoids the hazards associated with
handling concentrated diazomethane. These two methods are unique in that
significant background concentrations will always be present in chlorinated
supplies. When determining fortified matrix recoveries as required in the
quality assurance (QA) section, these background levels must be taken into
account when deciding upon fortification concentrations. In addition, the
uncertainty in measuring the background level should be considered when
establishing control limits, as called for in the quality assurance section.
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METHOD 506. Determination of Phthalate and Adi pate Esters in Drinking Water
by Liquid-Liquid Extraction or Liquid-Solid Extraction and Gas
Chromatography with Photoionization Detection
July 1990
F. K. Kawahara
J. W. Hodgeson
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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METHOD 506
Determination of Phthalate and Adipate Esters in Drinking Water
by Liquid-Liquid Extraction or Liquid-Solid Extraction and
Gas Chromatography with Photoionization Detection
1. SCOPE AND APPLICATION
1.1 This method describes a procedure for the determination of certain
phthalate and adipate esters in drinking water by liquid/liquid or
liquid/solid extraction. The following analytes can be determined by
this method:
PARAMETER CAS NO.
Bis (2-ethylhexyl) phthalate 117-81-7
Butyl benzyl phthalate 85-68-7
Di-n-butyl phthalate 84-74-2
Diethyl phthalate 84-66-2
Dimethyl phthalate 131-11-3
Bis(2-ethylhexyl) adipate 103-23-1
Di-n-octyl phthalate 117-81-7
1.2 This is a capillary column gas chromatographic (GC) method applicable
to the determination of the compounds listed above in ground water and
finished drinking water. When this method is used to analyze
unfamiliar samples for any or all of the compounds listed above,
compound identifications should be supported by at least one
additional qualitative technique. Method 525 provides gas
chromatograph/mass spectrometer (GC/MS) conditions appropriate for the
qualitative and quantitative confirmation of results for all the
analytes listed above, using the extract produced by this method.
1.3 This method has been validated in a single laboratory, and method
detection limits (MDLs) (1) have been determined for the analytes
above (Table 2). Observed detection limits may vary among waters,
depending upon the nature of interferences in the sample matrix and
the specific instrumentation used.
1.4 This method is restricted to use by or under the supervision of
analysts experienced in the use of GC, and in the interpretation of
gas chromatograms obtained by a computerized system. Each analyst
must demonstrate the ability to generate acceptable results with this
method using the procedure described in Sect. 10.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1-L, is extracted with a
ternary solvent consisting of methylene chloride, hexane and ethyl
acetate using a glass separatory funnel. The solvent extract is
isolated, dried and concentrated to a volume of 5 ml or less. The
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extract is further concentrated by gentle use of nitrogen gas blowing
to a volume of 1 mL or less. The analytes in the extract are
separated by means of capillary column gas chromatography using
temperature programming and the phthalate and adipate esters are then
measured with a photoionization detector (2-4). Alternatively a
measured volume of sample is extracted with a liquid-solid extraction
(LSE) cartridge or disk. The LSE cartridge or disk is eluted with
methylene chloride. The eluant is then concentrated using a gentle
stream of nitrogen or clean air to a volume of 1 ml or less.
3. DEFINITIONS
3.1 Laboratory reagent blank (LRB) — An aliquot of reagent water that is
treated exactly as a sample including exposure to all glassware
equipment, solvents, reagents, internal standards, and surrogates that
are used with other samples. The LRB is used to determine if method
analytes or other interferences are present in the laboratory
environment* the reagents, or the apparatus.
3.2 Field reagent blank (FRB) — Reagent water placed in a sample
container in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage, preservation
and all analytical procedures. The purpose of the FRB is to determine
if method analytes or other interferences are present in the field
, environment.
3.3 Laboratory fortified blank (LFB) - An aliquot of reagent water to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the methodology is in control, and
whether the 1aboratory is capable of making accurate and precise
measurements at the required method detection limit.
3.4 Laboratory fortified sample matrix (LFM) — An aliquot of an
environmental sample to which known quantities of the method analytes
are added in the laboratory. The LFM is analyzed exactly like a
sample, and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be determined
in a separate aliquot and the measured values in the LFM corrected for
background concentrations.
3.5 Stock standard solution — A concentrated solution containing a single
certified standard that is a method analyte, or a concentrated
solution of a single analyte prepared in the laboratory with an
assayed reference compound. Stock standard solutions are used to
prepare primary dilution standards.
3.6 Primary dilution standard solution — A solution of several analytes
prepared in the laboratory from stock standard solutions and diluted
as needed to prepare calibration solutions and other needed analyte
solutions.
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3.7 Calibration standard (CAL) — a solution prepared from the primary
dilution standard solution and stock standard solutions of the
internal standards and surrogate analytes. The CAL solutions are used
to calibrate the instrument response with respect to analyte
concentration.
3.8 Quality control sample (QCS) -- a sample matrix containing method
analytes or a solution of method analytes in a water miscible solvent
which is used to fortify reagent water or environmental samples. The
QCS is obtained from a source external to the laboratory, and is used
to check laboratory performance with externally prepared test
materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in water, solvents,
reagents, glassware, and sample processing hardware. These lead to
discrete artifacts and/or elevated baselines in gas chromatograms.
All of these materials must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running
laboratory reagent blanks (10.2).
4.1.1 Phthalate esters are contaminants in many products found in the
laboratory. It is particularly important to avoid the use of
plastics because phthalates are commonly used as plasticizers
and are easily extracted from plastic materials. Great care
must be exercised to prevent contamination. Exhaustive clean
up of reagents and glassware must be required to eliminate
background phthalate that is not derived from the sample.
4.1.2 Glassware must be scrupulously cleaned. Clean all glassware
as soon as possible after use by thoroughly rinsing with the
last solvent used. Follow by washing with hot water and
detergent and thorough rinsing with tap and reagent water.
Drain dry and heat in an oven or muffle furnace at 400°C for
1 hour. Do not heat volumetric glassware. Thorough rinsing
with acetone may be substituted for the heating. After
cooling, the glassware should be sealed with aluminum foil and
stored in a clean environment to prevent accumulation of dust
and other contaminants.
4.1.3 The use of high purity reagents and solvents helps to minimize
interference problems. Purification of solvents by
distillation in an all glass system may be required. WARNING:
When a solvent is purified, stabilizers added by the
manufacturer are removed thus potentially making the solvent
hazardous. Also, when a solvent is purified, preservatives
added by the manufacturer are removed thus potentially reducing
the shelf-life.
8
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extrlrtPd f^nn, 7hnCeS T beTUcaused ^ contaminants that are co-
varv S£ tnn thf Sampl6' Ihe 6xtent of matr1x interferences will
n? tht TL?T"it0 S°irce> dePendent "Pon the nature and diversity
of the industrial complex or municipality being sampled. Clean UD
procedures can be used to overcome many of these interferences
SAFETY
5*'1
E! no^h1^ °r carcn1n°9enicity of each reagent used in this method
b ^ treatedenasP7CooStepnytdeifi!;edi;t!;TVe^ each Chem1cal comP°und ™°t
De treated as a potential health hazard. Accordingly, exposure to
these chemicals must be reduced to the lowest possible level The
0™n^'l™S*mS™5'for maintaini"9 * Current awareness file of
OSHA regulations regarding the safe handling of the chemicals
ICp}r±IinHthi1S I?™0!' A r?frence file of material safety Sata
sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety Irl
1" f°r the InfoStSn S the
6. APPARATUS AND MATERIAI S
6.1 Sampling Equipment
6.1.1 Grab Sample Bottle-1-L or 1-qt amber glass, fitted with a
screw cap lined with Teflon. Foil may be substituted for
liah?nif amhfv.Wti6 1S n0t C,orros1ve- Protect samples from
light if amber bottles are not available. The bottle and cap
liner must be washed, rinsed with acetone or methylene chloride
and dried before use in order to minimize contamination. (See
'*r • 1 * 1 • y
6.2 Glassware
6.2.1 Separatory Funnel— 2-L with Teflon stopcock.
6.2.2 Drying Column--Chromatographic column-300 mm long x 10 mm ID
with Teflon stopcock and coarse frit filter disc at bottom
6.2.3 Concentrator Tube-Kuderna-Danish, 10 ml, graduated
calibration must be checked at the volumes employed in the
test. Tight ground glass stopper is used to prevent
evaporation of extracts. prevent
6.2.4 Evaporative Flask-Kuderna-Danish,
concentrator tube with springs.
500 ml, attach to
6.2.5 Snyder Column— Kuderna-Danish, three-ball macro size
6.2.6 Snyder Column— Kuderna-Danish, 2 or 3 ball micro size
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7.
6.2.7 Vials--10 to 15 ml, amber glass with Teflon-lined screw cap.
628 Boiling Chips-Approximately 10/40 mesh. Heat to 400°C for 30
Sin! or extract with methylene chloride in a Soxhlet apparatus.
6.2.9 Flask, Erlenmeyer—250 ml.
6.2.10 Chromatography column similar to 6.2.2.
6.2.11 Pasteur Pipets (and Bulb)
6.2.12 Autosampler Vials-Equipped with Teflon-lined septum and
threaded or crimp top caps.
6.3 Water Bath-Heated (with concentric ring covers) capable of
temperature control (± 2'C). The water bath should be used in a
ventilating hood.
6.4 Balance-Analytical, capable of weighing accurately to nearest
0.0001 gm.
6.5 Gas Chromatograph-An analytical system complete with temperature
programmable GC fitted with split-splitless injection mode system,
suitable for use with capillary columns and all required accessory
syringes, analytical columns, gases, detector and st"jchart recorder.
A data system for processing chromatographic data is recommended.
6.5.1 Column, Fused Silica Capillary-DB-5 or equivalent, 30 m long
x 0.32 mm ID with a film thickness of 0.25 micron.
6.5.2 The alternate column, Fused Silica Capi 11 ary-30 m long x
0.32 mm ID with a film thickness of 0.25 micron, DB-1 or
equivalent.
6 5.3 Detector - A high temperature photoionization detector
equipped for 10.0 electron volts and capable of operating from
250°C to 350°C is required.
6.5.4 An automatic injector system is suggested, but was not used for
the development of this method.
6.6 Vacuum pump, 110 VAC, capable of maintaining a vacuum of 8-10 mm Hg.
REAGENTS AND CONSUMABLE MATERIALS
7 1 Reagent Water—Reagent water is defined as water in which an
interfering substance is not observed at the MDL of the parameters of
interest Reagent water used to generate data in this method was
distilled water obtained from the Millipore L/A-7044 system comprised
of prefiltration, organic adsorption, deiomzation and Millipore
filtration columnar units. Any system may be used if it generates
10
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acceptable reagent water.
7.2 Acetone, hexane, methylene chloride, ethyl acetate, ethyl ether and
iso-octane — Pesticide quality or equivalent to distillation in glass
quality.
7.3 Sodium Sulfate—(ACS) Granular, anhydrous. Several levels of
purification may be required in order to reduce background phthalate
levels towards acceptance: 1) Heat 4 h at 400°C in a shallow tray,
2) Soxhlet extract with methylene chloride for 48 h.
7.4 Florisil—PR grade (60/100 mesh). To prepare for use, place 100 g of
Florisil into a 500-mL beaker and heat for approximately 16 h at 40°C.
After heating transfer to a 500-mL reagent bottle. Tightly seal and
cool to room temperature. When cool, add 3 ml of reagent water. Mix
thoroughly by shaking or rolling for 10 min. and let it stand for at
least 2 h. Store in the dark in glass containers with ground glass
stoppers or foil-lined screw caps.
7.5 Sodium Chloride—(ACS) Granular. Heat 4 h at 400°C in a shallow tray.
When cool, keep in tightly sealed bottle.
7.6 Ethyl Ether—(ACS) reagent grade.
7.7 Sodium Thiosulfate (Na2S203)—(ACS) reagent grade.
7.8 Alumina—Neutral activity Super I, W200 series (ICN Life Sciences
Group, No. 404583). To prepare for use, place 100 g of alumina into
a 500-mL beaker and heat for approximately 16 h at 400°C. After
heating transfer to a 500-mL reagent bottle. Tightly seal and cool
to room temperature. When cool, add 3 mL of reagent water. Mix
thoroughly by shaking or rolling for 10 min. and let it stand for at
least 2 h. Keep the bottle sealed tightly.
7.9 Liquid-solid extraction (LSE) cartridges. Cartridges are inert non-
leaching plastic, for example polypropylene, or glass, and must not
contain plasticizers, such as phthalate esters or adipates, that leach
into methylene chloride. The cartridges are packed with about 1 gram
of silica, or other inert inorganic support, whose surface is modified
by chemically bonded octadecyl (C18) groups. The packing must have a
narrow size distribution and must not leach organic compounds into
methylene chloride. One liter of water should pass through the
cartridge in about 2 hrs with the assistance of a slight vacuum of
about 13 cm (5 in.) of mercury. The extraction time should not vary
unreasonably among LSE cartridges.
7.10 Liquid-solid extraction disks, C-18, 47 mm. Disks are manufactured
with Teflon and should contain very little contamination.
7.11 Helium carrier gas, as contaminant free as possible.
11
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7.12 Stock standard solutions (1.00 |ug//iL) - Stock standard solutions can
be prepared from pure standard materials or purchased as certified
solutions.
7.12.1 Prepare stock standard solutions by accurately weighing about
0.0100 g of pure material. Dissolve the material in isooctane
and dilute to volume in a 10-mL volumetric flask. Larger
volumes can be used at the convenience of the analyst. When
compound purity is assayed to be 96% or greater, the weight can
be used without correction to calculate the concentration of
the stock standard. Commercially prepared stock standards can
be used at any concentration if they are certified by the
manufacturer or by an independent source.
7.12.2 Transfer the stock standard solutions into Teflon-sealed screw-
cap bottles. Store at 4°C and protect from light. Stock
standard solutions should be checked frequently for signs of
degradation or evaporation, especially just prior to preparing
calibration standards from them.
7.12.3 Stock standard solutions must be replaced after six months, or
• sooner if comparison with check standards indicates a problem.
Butyl benzyl phthalate is especially vulnerable to autoxidation.
7.13 Laboratory control sample concentrate - See Sect. 10.3.1.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 Grab samples must be collected in amber glass containers (Sect.6.1).
Conventional sampling practices should be followed (8,9); however, the
bottle must not be prerinsed with sample before collection.
8.2 SAMPLE PRESERVATION AND STORAGE
8.2.1 For sample dechlorination, add 60 mg sodium thiosulfate to the
sample bottle at the sampling site or in the laboratory before
shipping to the sampling site.
8.2.2 After the sample is collected in a bottle containing preserv-
ative^), seal the bottle and shake vigorously for 1 min.
8.2.3 The samples must be iced or refrigerated at 4°C free from light
from the time of collection until extraction. Limited holding
studies have indicated that the analytes thus stored are stable
up to 14 days or longer. Analyte stability may be affected by
the matrix; therefore, the analyst should verify that the
preservation technique is applicable to the particular samples
under study.
8.3 Extract Storage — Extracts should be stored at 4°C in absence of
light. A 14-day maximum extract storage time is recommended. The
12
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analyst should verify appropriate extract holding times applicable to
the samples under study. • . •
9. CALIBRATION
9.1 Establish gas chromatograph operating conditions equivalent to those
given in Table 1. The gas chromatographic system can be calibrated
using the external standard technique (Section 9.2).
9.1.1 Performance of the detector should be checked daily by a
specified procedure given in the gas chromatograph operation
manual. If the response is weak, the ultraviolet lamp is
removed carefully following disconnection of the power supply.
It is cleaned and then placed into its original position with
the aid of a leak detector.
9.2 External standard calibration procedure:
9.2.1 Prepare calibration standards at a minimum of three
concentration levels for each analyte of interest by adding
volumes of one or more stock standards to a volumetric flask
and diluting to volume with n-hexane. One of the external
standards should be at a concentration near, but above, the MDL
(Table 2) and the other concentrations should correspond to the
expected range of concentrations found in real samples or
should define the working range of the detector.
9.2.2 Using injection of 1 to 2 nl, analyze each calibration standard
according to Sect. 11.5 and tabulate peak height or area
responses against the mass injected. The results can be used
to prepares calibration curve for each compound. Alterna-
tively, if the ratio of response to amount injected
(calibration factor) is a constant over the working range (<10%
relative standard deviation, RSD), linearity through the origin
can be assumed and the average ratio or calibration factor can
be used in place of a calibration curve.
10. QUALITY CONTROL
10.1 Minimum quality control (QC) requirements are initial demonstration
of laboratory capability, analysis of laboratory reagent blanks,
laboratory fortified samples, laboratory fortified blanks, and QC
samples. Additional quality control practices are recommended.
10.2 Laboratory Reagent Blanks (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 of interest the LRB produces a peak that
would prevent the determination of that analyte using a known
13
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standard, determine the source of contamination and eliminate the
interference before processing samples.
10.3 Initial Demonstration of Capability.
10.3.1 Select a representative spike concentration, about 10 times
MDL or at the regulatory Maximum Contaminant Level (MCL),
(whichever is lower) for each analyte. Prepare a laboratory
control sample concentrate (in methanol) containing each
analyte at 1000 times selected concentration. With a syringe,
add 1 ml of the concentrate to each of seven 1-L aliquots of
reagent water, and analyze each aliquot according to procedures
in Sect. 11.1 or 11.2, and 11.3 and 11.4.
10.3.2 For each analyte, the mean recovery value should fall in the
range of R ± 30% (or within R ± 3Sr if broader) using the
values for R and S for reagent water in Table 3 or Table 4.
For those compounds that meet the acceptance criteria,
performance is considered acceptable and sample analysis may
begin. For those compounds that fail these criteria, initial
demonstration procedures should be repeated.
10.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples via a new,
unfamiliar method prior to obtaining some experience with it.
It is expected that, as laboratory personnel gain experience
with this method, the quality of data will improve beyond those
required here.
10.4 The analyst is permitted to modify GC columns, GC detectors, GC
conditions, concentration techniques, internal standards or surrogate
compounds. Each time such method modifications are made, the analyst
must repeat the procedures in Sect. 10.3.
10.5 Assessing Laboratory Performance - Laboratory Fortified Blank
10.5.1 The laboratory must analyze at least one laboratory fortified
blank (LFB) sample per sample set (all samples extracted within
a 24-h period). The spiking concentration of each analyte in
the LFB should be 10 times MDL or the MCL, whichever is less.
Calculate accuracy as percent recovery, R. If the recovery
of any analyte falls outside the control limits (see Sect.
10.5.2), that analyte is judged out of control, and the source
of the problem should be identified and resolved before
continuing analyses.
10.5.2 Until sufficient internal data become available, usually a
minimum of results from 20 to 30 analyses, the laboratory
should assess laboratory performance against the control limits
in Sect. 10.3.2 that are derived from the control limits
developed during the initial demonstration of capability
(10.3). When sufficient internal performance data becomes
14
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available, develop control limits from the mean percent
recovery, R, and standard deviation, Sp, of the percent
UPPER CONTROL LIMIT = R + 3S
r
LOWER CONTROL LIMIT = R - 3Sp
After each five to ten new recovery measurements, new control
limits should be calculated using only the most recent 20-30
data points.
10. 6 Assessing Analyte Recovery -- Laboratory Fortified Sample Matrix
10,6.1 The laboratory must fortify each analyte to a minimum of 10%
of the routine samples or one fortified sample per set
whichever is greater. The fortified concentration should not
be less^ than the background concentration of the sample
selected for fortifying. Ideally, this concentration should
be the same as that used for the laboratory fortified blank
(Sect. 10.5). Over time, samples from all routine sample
sources should be fortified.
10,6.2 Calculate the accuracy as percent recovery, R, for each
analyte, corrected for background concentrations measured in
the unfortified sample, and compare these values to the control
limits established in Sect. 10.5.2 from the analyses of LFBs.
10.6.3 If the recovery of any such analyte falls outside the
designated range, and the laboratory performance for that
analyte is shown to be in control (Sect. 10.5), the recovery
problem encountered with the dosed sample is judged to be
matrix related, not system related. The result for that
analyte in the unspiked sample is labeled suspect/matrix to
inform the data user that the results are suspect due to matrix
effects.
10,7 QUALITY CONTROL SAMPLES (QCS) - Each quarter, the laboratory should
analyze one or more QCS (if available). If criteria provided with the
are not met, corrective action should be taken and documented.
lO.SThe laboratory may adopt additional quality control practices for use
with this method. The specific practices that are most productive
depend upon the needs of the laboratory and the nature of the samples
For example, field or laboratory duplicates may be analyzed to assess
the precision of the environmental measurements.
11. PROCEDURE
11.1 LIQUID-LIQUID EXTRACTION
15
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11 1 1 Mark the water meniscus on the side of the sample bottle for
later determination of sample volume. Pour the entire sample
into a 2-L separatory funnel containing 50 g of NaCl.
11 1 2 Add 60 ml CH,C1, to the sample bottle. Seal, and shake gently
to rinse the2 inner walls of the bottle. Transfer the solvent
to the separatory funnel. . Extract the sample by shaking the
funnel for 2 min with initial and periodic venting to release
excess pressure. Allow the organic layer to separate for a
minimum of 10 min from the water phase. If the emulsion
interface between layers is more than one-third the volume ot
the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum
technique depends upon the sample, but may include stirring,
filtration of the emulsion through glass wool, centrifugation,
or other physical methods. Collect the solvent extract in a
250-mL Erlenmeyer flask.
11 1 3 Add a second 60-mL volume of methylene chloride to the sample
bottle and repeat the extraction procedure a second time,
combining the extracts in the Erlenmeyer flask. Perform a
third extraction in the same manner. Then extract with 40-mL
• of hexane, which extract (top phase) is added to the total.
11.1.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a
10-mL concentrator tube to a 500-mL evaporative flask. Other
concentration devices or techniques may be used in place of the
K-D concentrator, provided the concentration factor attained
in 11.1.6 - 11.1.8 is achieved without loss of analytes.
11.1.5 Pour the combined extract through a drying column (6.2.2)
containing about 10 cm of prerinsed anhydrous sodium sulfate,
and collect the extract in the K-D concentrator. Rinse the
Erlenmeyer flask and column with 20 to 30 ml of methylene
chloride to complete the quantitative transfer.
11 1 6 Add one or two clean boiling chips to the evaporative flask and
attach a three-ball Snyder column. Prewet the Snyder column
by adding about 1 ml of methylene chloride to the top. Place
the K-D apparatus on a hot water bath (60 to 65°C) so that the
concentrator tube is partially immersed in the hot water, and
the entire lower rounded surface of the flask is bathed with
hot vapor. Adjust the vertical position of the apparatus and
the water temperature as required to complete the concentration
in 40 min. At the proper rate of distillation the balls of
the column will actively chatter but the chambers will not
flood with condensed solvent. When the apparent volume of
liquid reaches approximately 7 ml, remove the K-D apparatus and
allow it to drain and cool for at least 10 min.
11.1.7 Increase the temperature of the hot water bath to about 85°C.
Remove the Snyder column, rinse the column and the 500-mL
16
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evaporative flask with 1 - 2 ml of hexane. Replace with a
micro column and evaporative flask. Concentrate the extract
as in Sect. 11.1.6 to 0.5 - 1 ml. The elapsed time of
concentration should be approximately 15 min.
11.1.8 Remove the micro Snyder column and rinse the column by flushing
with hexane using a 5-mL syringe. Concentrate to a volume of
1 ml by purging the liquid surface with a gentle flow of
nitrogen or clean air. If an autosampler is to be used,
transfer the extract to an autosampler vial with a Pasteur
pi pet. Seal the vial with a threaded or crimp top cap. Store
in refrigerator if further processing will not be performed.
If the sample extract requires no further cleanup, proceed with
gas chromatographic analysis (Sect. 11.5). If the sample
requires further cleanup, proceed to Sect. 11.4.
11.1.9 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the liquid to a 1000-mL
graduated cylinder. Record the sample volume to the nearest
5 mL.
11.2 LIQUID-SOLID EXTRACTION
11.2.1 This method is applicable to a wide range of organic compounds
that are efficiently partitioned from the water sample onto a
C18 organic phase chemically bonded to a solid inorganic matrix,
and are sufficiently volatile and thermally stable for gas
chromatography (10). Particulate bound organic matter will not
be partitioned, and more than trace levels of particulates in
the water may disrupt the partitioning process. Single
laboratory accuracy and precision data have been determined at
a single concentration for the analytes listed in 1.1 fortified
into reagent water and raw source water.
11.2.2 Set up the extraction apparatus shown in Figure 1A. An
automated extraction system may also be used. The reservoir is
not required, but recommended for convenient operation. Water
drains from the reservoir through the LSE cartridge and into
a syringe needle which is inserted through a rubber stopper
into the suction flask. A slight vacuum of 13 cm (5 in.) of
mercury is used during all operations with the apparatus. With
this extraction apparatus, sample elution requires
approximately 2 hours. Acceptable new cartridge and extraction
disk technology have recently become available, which allow
significantly faster elution rates.
11.2.3 Mark the water meniscus on the side of the sample bottle
(approximately 1 liter) for .later determination of sample
volume. Pour the water sample into the 2-L separatory funnel
with the stopcock closed.
17
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11.2.4 Flush each cartridge with two 10 ml aliquots of methylene
chloride, followed by two 10 ml aliquots of methanol, letting
the cartridge drain dry after each flush. These solvent
flushes may be accomplished by adding the solvents directly to
the solvent reservoir in Figure 1A. Add 10-mL of reagent water
to the solvent reservoir, but before the reagent water level
drops below the top edge of the packing in the LSE cartridge,
open the stopcock of the separatory funnel and begin adding
sample water to the solvent reservoir. Close the stopcock when
an adequate amount of sample is in the reservoir.
11.2.5 Periodically open the stopcock and drain a portion of the
sample water into the solvent reservoir. The water sample
will drain into the cartridge, and from the exit into the
suction flask. Maintain the packing material in the cartridge
immersed in water at all times. After all of the sample has
passed through the LSE cartridge, wash the separatory funnel
and cartridge with 10 ml of reagent water, and draw air through
the cartridge for about 10 min.
11.2.6 Transfer the 125-mL solvent reservoir and LSE cartridge (from
Figure 1A) to the elution apparatus (Figure IB). The same
125 mL solvent reservoir is used for both apparatus. Wash the
2-liter separatory funnel with 5 mL of methylene chloride and
collect the washings. Close the stopcock on the 100-mL
separatory funnel of the elution apparatus, add the washings
to the reservoir and enough additional methylene chloride to
bring the volume back up to 5 mL and elute the LSE cartridge.
Elute the LSE cartridge with an additional 5 mL of methylene
chloride (10-mL total). A small amount of nitrogen positive
pressure may be used to elute the cartridge. Small amounts of
residual water from the LSE cartridge will form an immiscible
layer with the methylene chloride in the 100-mL separatory
funnel. Open the stopcock and allow the methylene chloride to
pass through the drying column packed with anhydrous sodium
sulfate (1-in)' and into the collection vial. Do not allow the
water layer to enter the drying column. Remove the 100 mL
separatory funnel and wash the drying column with 2 mL of
methylene chloride. Add this to the extract. Concentrate the
extract to 1 mL under a gentle stream of nitrogen. The extract
is now ready for gas chromatography (11.4) or additional
cleanup (11.3).
11.3 DISK SAMPLE EXTRACTION
11.3.1 Preparation of disks.
11.3.1.1 Insert the disk into the 47 mm filter apparatus.
Wash the disk with 5 mL methylene chloride (MeClJ by
adding the MeCl, to the disk, pulling about Tialf
through the disk and allowing it to soak the disk for
about a minute, then pulling the remaining MeCl2
18
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through the disk. With the vacuum on, pull air
through the disk for a minute.
11.3.1.2 Pre-wet the disk with 5 ml methanol (MeOH) by adding
the MeOH to the disk, pulling about half through the
disk and allowing it to soak for about a minute, then
pulling most of the remaining MeOH through. A layer
of MeOH must be left on the surface of the disk,
which shouldn't be allowed to go dry from this point
until the end of the sample extraction. THIS IS A
CRITICAL STEP FOR A UNIFORM FLOW AND GOOD RECOVERY.
11.3.1.3 Rinse the disk with 5 ml reagent water by adding the
water to the disk and pulling most through, again
leaving a layer on the surface of the disk.
11.3.2 Add 5 mL MeOH per liter of water sample. Mix well.
11.3.3 Add the water sample to the reservoir and turn on the vacuum
to begin the filtration. Full aspirator vacuum may be used.
Particulate-free water may filter in as little as 10 minutes
or less. Filter the entire sample, draining as much water
from the sample container as possible.
11.3.4 Remove the filtration top from the vacuum flask, but don't
disassemble the reservoir and fritted base. Empty the water
from the flask and insert a suitable sample tube to contain
the eluant. The only constraint on the sample tube is that
it fit around the drip tip of the fritted base. Reassemble
the apparatus.
Add 5 mL of acetonitrile (CH3CN) to rinse the sample bottle.
Allow the CH3CN to settle to the bottom of the bottle and
transfer to the disk with a dispo-pipet, rinsing the sides of
the glass filtration reservoir in the process. Pull about half
of the CH3CN through the disk, release the vacuum, and allow the
disk to soak for a minute. Pull the remaining O-LCN through the
disk.
Repeat the above step twice, using MeCl2 instead of CH3CN. Pour
the combined eluates thru a small funnel with filter paper
containing 3 grams of anhydrous sodium sulfate. Rinse the test
tube and sodium sulfate with two 5 mL portions of MeCl,.
Collect the filtrate in a concentrator tube.
11.3.5 With the concentrator tube in a 28°C heating block, evaporate
the eluate with a stream of N2 to 0.5 mL.
11.4 EXTRACT CLEANUP - Cleanup procedures may not be necessary for a
relatively clean sample matrix, such as most drinking waters. If
particular circumstances demand the use of a cleanup procedure, the
19
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analyst may use either procedure below or any other appropriate
procedure. However, the analyst first must demonstrate that the
requirements of Sect. 10.3 and 10.5 can be met using the method as
revised to incorporate the cleanup procedure.
11.4.1 Florisil column cleanup for phthalate esters:
11.4.1.1 Place 10 g of Florisil (see 7.4) into a
chromatographic column. Tap the column to settle
the Florisil and add 1 cm of anhydrous sodium sulfate
to the top.
11.4.1.2 Preelute the column with 40 ml of hexane. Discard
the eluate and just prior to exposure of the sodium
sulfate layer to the air, quantitatively transfer
the sample extract (11.1.8 or 11.2.6) onto the
column, using an additional 2 ml of hexane to
complete the transfer. Just prior to exposure of the
sodium sulfate layer to the air, add 40 mL of hexane
and continue the elution of the column. Discard this
hexane eluate.
11.4.1.3 Next, elute the column with 100 ml of 20% ethyl ether
in hexane (V/V) into a 500-mL K-D flask equipped
with a 10-mL concentrator tube. Elute the column at
a rate of about 2 mL/min for all fractions.
Concentrate the collected fraction as in Section
11.1. No solvent exchange is necessary. Adjust the
volume of the cleaned extract to 1 ml in the
concentrator tube and analyze by gas chromatography.
11.4.2 Alumina column cleanup for phthalate esters:
11.4.2.1 Place 10 g of alumina into a chromatographic column.
Tap the column to settle the alumina and add 1 cm of
anhydrous sodium sulfate to the top.
11.4.2.2 Preelute the column with 40 ml of hexane. The rate
for all elutions should be about 2 mL/min. Discard
the eluate and just prior to exposure of the sodium
sulfate layer to the air, quantitatively transfer the
sample extract (11.1.8 or 11.2.6) onto the column,
using an additional 2 mL of hexane to complete the
transfer. Just prior to exposure of the sodium
sulfate layer to the air, add 35 mL of hexane and
continue the elution of the column. Discard this
hexane eluate.
11.4.2.3 Next, elute the column with 140 mL of 20% ethyl ether
in hexane (V/V) into a 500-mL K-D flask equipped with
a 10-mL concentrator tube. Concentrate the collected
fraction as in Section 11.1. No solvent exchange is
20
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necessary. Adjust the volume of the cleaned extract
to 1 ml in the concentrator tube and analyze by gas
chromatography.
11.5 GAS CHROMATOGRAPHY
11.5.1 Table 1 summarizes the recommended operating conditions for the
gas chromatograph. Included are retention data for the primary
and confirmation columns. Other capillary (open-tubular)
columns, chromatographic conditions, or detectors may be used
if the requirements of Section 10 are met.
11.5.2 Calibrate the system daily as described in Sect. 9.
11.5.3 Inject 1 to 2 ^L of the sample extract or standard into the gas
chromatograph. Smaller (1.0 *iL) volumes may be injected if
automatic devices are employed. For optimum reproducibillty,
an autoinjector is recommended.
11.5.4 Identify the analytes in the sample by comparing the retention
times of the peaks in the sample chromatogram
with those of the peaks in standard chromatograms. The width
of the retention time window used to make identifications should
be based upon measurements of actual retention time variations
of standards over the course of a day. Three times the standard
deviation of a retention time for a compound can be used to
calculate a suggested window size; however, the experience of
the analyst should weigh heavily in the interpretation of
chromatograms.
11.5.5 If the response for a peak exceeds the working range of the
system, dilute the extract and reanalyze.
11.5.6 If the measurement of the peak response is prevented by the
presence of interferences, further cleanup is required.
11.5.7 The calibration curves should be linear over the range of
concentrations in Tables 2-5.
12, CALCULATIONS
1.2.1 Calculate the amount of material injected from the peak response using
the calibration curve or calibration factor determined in Section
9.2.2. The concentration in the sample can be calculated from
Equation 2.
Equation 2.
Concentration (/ig/L) =
21
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where:
A - Amount of material injected (ng).
V, s Volume of extract injected (/^L).
V\ - Volume of total extract (p.L).
Ms - Volume of water extracted (ml).
12.2Report results in p.g/1 without correction for recovery data. All QC
data obtained should be reported with the sample results.
13. METHOD PERFORMANCE
Single laboratory accuracy and precision data were obtained by replicate
liquid-liquid extraction analyses of reagent water fortified at two sets
of concentrations of method analytes. The data are given in Tables 2 and
3. Accuracy and precision data by liquid-solid extraction of reagent
water fortified at a single concentration are given in Table 4. Finally,
method validation data obtained by the analyses of fortified tap water and
raw source water are given in Tables 5-7.
14. REFERENCES
1. Glaser, O.V., D.L. Foerst, 6.D. McKee, S.A. Quave, and W.L. Budde,
"Trace Analysis for Waste Waters," Environ. Sci. Technol. 15, 1426,
1981.
2. "Determination of Phthalates in Industrial and Municipal Wastewaters,"
EPA-600/4-81-063, U.S. Environmental Protection Agency, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio 45268, October
1981.
3. Giam, C.S., Chan, H.S. and Nef, G.S. "Sensitive Method for
Determination of Phthalate Ester Plasticizers in Open-Ocean Biota
Samples," Anal. Chem.. 47, 2225 (1975).
4. Giam, C.S., and Chan, H.S. "Control of Blanks in the Analysis of
Phthalates in Air and Ocean Biota Samples," U.S. National Bureau of
Standards, Special Publication 442, pp. 701-708, 1976.
5. "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, August 1977.
6. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206 (Revised,
January 1976).
7. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
22
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8. ASTM Annual Book of Standards, Part 31, D3694-78. "Standard Practices
tor "reparation of Sample Containers and for Preservation of Oraanir
PMladelpMa!" '"""* S°Cl6ty f°r Test1"9 '"d Materials,
9. ASTM Annual Book of Standards, Part 31, D3370. "Standard Practices
Phnad'eTphfa9. '" ^^ S°c1ety for Test1"9 and StartK.
10. J.W. Eichelberger, T.D. Behymer and W.L. Budde "Determinatinn nf
Organic Compounds in Drinking Water By Liquid-Soli!I ExSaction and
• u i.?^ Lumn "as Cnromatography/Mass Spectrometry", EPA Method —
" ™WL&r,^!,£P™%^ !* ?."»"!lc ^P""nds ii
EP
D
„,-, ^00/4-88/039. Envrroniiental MoniVing Systems Lab r or^
1988, pVplr03n25-356. Pr°tect1on ^^ Cincinnati,90hio 45268, December
23
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TABLE 1. RETENTION DATA AND CHROMATOGRAPHIC CONDITIONS
Parameter
Dimethyl phthalate
Diethyl phthalate
Di-n-butyl phthalate
Butyl benzyl phthalate
Bis(2-ethylhexyl) adipate
Bis(2-ethylhexyl) phthalate
Di-n-octyl phthalate
Column 1: DB-5, fused silica capillary, 30 m x 0.32 mm I.D.,
0.25 micron film thickness, Helium linear velocity = 30 cm/s.
Column 2: DB-1, fused silica capillary, 30 m x 0.32 mm I.D.,
0.25 micron film thickness, Helium linear velocity = 30 cm/s.
Column 1
^=r:^=^=^==^=^=
17.23
20.29
27.57
34.19
34.85
37.51
41.77
Column 2
___ ir^^^^ - • '-'— "
17.89
21.13
28.67
35.34
36.76
39.58
44.44
Chromatographic Conditions:
Injector temperature = 295°C
Detector temperature = 295°C
Program - 1 min hold at 60°C,
6°C/min to 260°C, 10 min hold.
Splitless injection with 45 s
delay
24
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TABLE 2. ACCURACY, PRECISION, AND METHOD DETECTION LIMIT DATA FROM
SIX LIQUID-LIQUID EXTRACTION ANALYSES OF FORTIFIED REAGENT WATER
Analyte
Dimethyl phthalate
Diethyl phthalate
Di-n-butyl phthalate
Butyl benzyl phthalate
Bis(2-ethylhexyl) adipate
Bis(2-ethylhexyl) phthalate
Di-n-octyl phthalate
True
Cone.
2.02
1.51
2.62
6.00
6.03
5.62
17.18
Mean
Meas.
Cone.
1.42
1.16
1.78
3.27
3.94
2.92
7.96
Std.
Dev.
0.38
0.28
0.41
0.89
1.44
0.75
2.14
Mean
Accuracy
% of True
Cone.
70.3
76.8
67.9
54.5
65.3
52.0
46.3.
MDL
1.14
0.84
1.23
2.67
11.82
2.25
6.42
25
-------
TABLE 3. ACCURACY AND PRECISION DATA FROM SEVEN LIQUID-LIQUID
EXTRACTION ANALYSES OF FORTIFIED REAGENT WATER
Analyte
Dimethyl phthalate
Di ethyl phthalate
Di-n-butyl phthalate
Butyl benzyl phthalate
Bis(Z-ethylhexyl) adipate
Bis(2-ethylhexyl) phthalate
Di-n-octyl phthalate
True
Concentration
M9/L
15
15
15
15
30
30
30
Mean Accuracy
% of True
Concentration
73
71
68
71
69
67
62
Standard
Deviation
%
16
16
15
15
18
21
23
26
-------
TABLE 4. ACCURACY AND PRECISION DATA FROM SIX LIQUID-SOLID
EXTRACTION ANALYSES OF FORTIFIED REAGENT WATER
Analyte
True
Concentration
Relative
Mean Accuracy Standard
% of True Deviation
Concentration %
Dimethyl phthalate
Di ethyl phthalate
Di-n-butyl phthalate
Butyl benzyl phthalate
Bis(2-ethylhexyl) adipate
Bis(2-ethylhexyl) phthalate
Di-n-octyl phthalate
15
15
15
15
30
30
30
74
85
74
72
84
101
85
11
10
11
14
11
13
13
27
-------
TABLE 5. ACCURACY AND PRECISION DATA FROM SIX LIQUID-LIQUID
EXTRACTION ANALYSES OF FORTIFIED TAP WATER
Analyte
Dimethyl phthalate
Di ethyl phthalate
Di-n-butyl phthalate
Butyl benzyl phthalate
Bis(2-ethylhexyl) adipate
Bis(2-ethylhexyl) phthalate
Di-n-octyl phthalate
True
Concentration
M9/L
5
5
5
5
5
5
5
Mean Accuracy
% of True
Concentration
103
106
94
93
87
93
72
Relative
Standard
Deviation
%
10.0
10.0
6.8
9.1
12.0
4.9
26.0
28
-------
TABLE 6. ACCURACY AND PRECISION DATA FROM SIX LIQUID-LIQUID
EXTRACTION ANALYSES OF FORTIFIED RAW SOURCE WATER
True
Concentration
Analyte /ug/L
Dimethyl phthalate
Di ethyl phthalate
Di-n-butyl phthalate
Butyl benzyl phthalate
Bis(2-ethylhexyl) adipate
Bis(2-ethylhexyl) phthalate
Di-n-octyl phthalate
5
5
5
5
5
5
5
Relative
Mean Accuracy Standard
% of True Deviation
Concentration %
59
78
99
72
115
91
54
51
45
29
23
32
35
24
29
-------
TABLE 7. ACCURACY AND PRECISION DATA FROM SIX LIQUID-SOLID
EXTRACTION ANALYSES OF FORTIFIED RAW SOURCE WATER
Analyte
True
Concentration
M9/L
Relative
Mean Accuracy Standard
% of True Deviation
Concentration %
Dimethyl phthalate
Di ethyl phthalate
Di-n-butyl phthalate
Butyl benzyl phthalate
Bis(2-ethylhexyl) adipate
Bis(2-ethylhexyl) phthalate
Di-n-octyl phthalate
5
5
5
5
5
5
5
110
111
95
82
65
60
53
20
32
30
20
24
21
15
30
-------
2 liter
separator/
funnel
HO
125ml
solvent
reservoir
ground glass
114/35
r
n LSS cartridge
C)
12Sml
solvent
reservoir
ground glass T 1 4/3 S
rubber stopper
No. 1@-2O90er-lot<
syringe needle
10OmI
separatory
ifunnei
1 iter
vacuum flask
drying
cokimn
1. 2 cm x 40 cm
f
1Oml
graduated
via*
A. Extraction apparatus
31
8. Ekitfora apparatus
-------
TIKE (HIM.)
Peaks obttlntd by Injecting 5 ng for the 1st, 2nd, 4th and 5th
compounds, 10 ng for the 6th, 7th and 8th compounds, and 2.5 ng
for the 3rd compound. (Table 1)
FIGURE 2
32
-------
METHOD 513.
DETERMINATION OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN
IN DRINKING WATER BY GAS CHROMATOGRAPHY WITH HIGH
RESOLUTION MASS SPECTROMETRY
July 1990
This method is taken from the SW-846 Methods Manual,
Method 8280, and adapted to drinking water.
A. Alford-Stevens
James W. Eichelberger
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
33
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METHOD 513
DETERMINATION OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN IN DRINKING WATER BY
GAS CHROMATOGRAPHY WITH HIGH-RESOLUTION MASS SPECTROMETRY
SCOPE AND APPLICATION
1.1 This method provides procedures for identification and measurement
of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, CASRN 1746-01-6) at
concentrations of 20 pg/L to 2 ng/L in water sample extracts. The
minimum measurable concentration will vary among samples, depending
on the presence or absence of interfering compounds in a particular
sample.
1.2 A water sample may contain floating, suspended, and settled
particulate matter, which should not be removed by filtering before
extraction. The estimated solubility of 2,3,7,8-TCDD in water is
<50 ng/L (1), but larger measured concentrations can be caused by
TCDD associated with particulates.
1.3 Because 2,3,7,8-TCDD may be extremely toxic, safety procedures
described in Section 5 should be followed to prevent exposure of
laboratory personnel to materials containing this compound.
SUMMARY OF METHOD
2.1
An entire 1-L water sample is transferred to a separatory funnel,
and two isotopically-labeled analyte analogs, CT4-2,3,7,8-TCDD
(surrogate compound, SC) and 1 CJ2-2,3,7,8-TCDD (internal standard,
IS), are added to the water. The sample container is rinsed with
methylene chloride, which is then added to the water sample. The
water sample is extracted sequentially with three 60-mL portions of
methylene chloride. AN optional liquid-solid extraction procedure
using Empore disk technology is also described in this method. When
using this option, all surrogate compounds and internal standards
and other solutions are added just as in the liquid-liquid extraction
procedure.The combined extract is subjected to column chromatographic
procedures to remove sample components that may interfere with
detection and measurement of TCDD. A 10-jLtL aliquot of a solution
containing 13C.,2-1,2,3,4-TCDD, which is used as a recovery standard
(RS), is addedto the extract before concentration and analysis.
The sample extract is concentrated to 10 /zL, and a l-/zL or Z-fj.1
aliquot is injected into a gas chromatograph (GC) equipped with a
fused silica capillary column and interfaced with a high resolution
mass spectrometer (MS). Selected characteristic ions are monitored
with high resolution MS (10,000 resolving power). Identification
of a sample component as TCDD is based on detection of two
characteristic ions (m/z 320 and 322) in the molecular ion cluster,
measurement of acceptable relative abundance of those two ions, and
34
-------
relative to the IS, 13Cir2,3,7,8-TCDD. Because the IS is a labeled
analog of the analyte, the procedure presumes that IS losses during
method procedures are equal to unlabeled TCDD losses. Therefore,
each calculated sample TCDD concentration has been compensated for
losses during sample preparation.
3. DEFINITIONS
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
Calibration limits -- the minimum (20 pg/L) and maximum (2 ng/L)
concentration of 2,3,7,8-TCDD in solutions used to calibrate detector
response. In some samples, <20 pg/L of 2,3,7,8-TCDD may be detected
but measured concentrations will only be estimated concentrations.
In other samples, interferences may prevent identification and
measurement of 20 pg/L.
Concentration calibration solution — a solution containing known
amounts of the analyte (unlabeled 2,3,7,8-TCDD), the IS (13C '
2,3,7,8-TCDD), the SC (37CT4-2,3,7,8-TCDD), and the RS (%,-1,2,3,¥-
TCDD); it is used to determine instrument response to the analyte
SC, and RS relative to response to the IS.
Field blank — a portion of reagent water that has been shipped to
the sampling site and exposed to conditions that samples have
experienced.
Internal standard (IS) -- 13C12-2,3,7,8-TCDD, which is added to every
sample and is present at the same concentration in every blank,
quality control sample, and concentration calibration solution. It
is added to the water sample before extraction and is used to
measure the concentration of unlabeled TCDD.
Laboratory reagent blank -- a blank prepared in the laboratory by
performing all analytical procedures except a 1-L aliquot of reagent
water is extracted rather than a sample.
Performance check solution ~ a solution containing a mixture of
known amounts of selected standard compound; it is used to
demonstrate continued acceptable 6C/MS system performance.
Recovery standard (RS) — A compound (13C12-1,2,3,4-TCDD) that is
present in every calibration solution and is added to each extract
just before analysis. It is used to measure the recovery of the
internal standard.
Response factor (RF) — response of the mass spectrometer to a known
amount of analyte relative to a known amount of internal standard.
Signal-to-noise ratio (S/N) — the ratio of the area of the analyte
signal to the area of the random background signal; it is determined
by integrating the signal for a characteristic ion in a region of
the selected ion current profile where only random noise is observed
and relating that area to the area measured for a positive response
35
-------
for the same ion. The same number of scans must be integrated for
both areas. (The ratio of peak heights may be used instead of peak
areas.)
3.10 Surrogate compound (SC) -- a compound (37C14-2,3,7,8~TCDD) that is
present in each calibration solution and is added at a low
concentration (20 pg/L) to each sample and blank before extraction.
Successful detection and measurement of the SC in each sample
provides some assurance that unlabeled 2,3,7,8-TCDD would be
detectable if present in the sample at >20 pg/L.
4. INTERFERENCES
4.1 An organic compound that has approximately the same GC retention
time 2,3,7,8-TCDD (within a few scans of the IS) and produces the
ions that are monitored to detect 2,3,7,8-TCDD is a potential
interference. Most frequently encountered interferences are other
sample components that are extracted along with TCDD; some potential
interferences are listed in Table 1. To minimize interference, high
purity reagents and solvents must be used and all equipment must be
meticulously cleaned. Laboratory reagent blanks must be analyzed
to demonstrate lack of contamination that would interfere with
2,3,7,8-TCDD measurement. Column chromatographic procedures are
used to remove some sample components; these procedures must be
performed carefully to minimize loss of 2,3,7,8-TCDD during attempts
to enrich its concentration relative to other sample components.
4.2 False positive identifications are produced only when an interfering
compound elutes from the GC column within 3 sec of the IS and
produces ions with exact masses and relative abundances very similar
to those for 2,3,7,8-TCDD. The specified GC column separates
2,3,7,8-TCDD from all 21 other TCDD isomers.
SAFETY
5.1
5.2
Because 2,3,7,8-TCDD has been identified as an animal carcinogen and
a possible human carcinogen, exposure to this compound and its
isotopically labeled analogs must be minimized (2,3). The
laboratory is responsible for maintaining a file of current OSHA
regulations regarding the safe handling of chemicals specified in
this method. A reference file of material data handling sheets
should also be made available to all personnel involved in analyses.
Each laboratory must develop a strict safety program for handling
2,3,7,8-TCDD. The following laboratory practices are recommended:
5.2.1 Minimize laboratory contamination by conducting all
manipulations in a hood.
5.2.2 Effluents of GC sample splitters and GC/MS vacuum pumps
should pass through either a column of activated carbon or
36
-------
be bubbled through a trap containing oil or high-boilinq
alcohols.
5.2.3 Liquid waste should be dissolved in methanol or ethanol and
irradiated with ultraviolet light at a wavelength <290 nm
for several days. Analyze the liquid wastes and dispose of
the solutions when 2,3,7,8-TCDD can no longer be detected.
5.3 The following precautions for safe handling of 2,3,7,8-TCDD in the
laboratory are presented as guidelines only. These precautions are
necessarily general in nature, because detailed specific
recommendations can be made only for the particular exposure and
circumstances of each individual use. Assistance in evaluating the
health hazards of particular conditions may be obtained from certain
consulting laboratories or from state Departments of Health or of
Labor, many of which have an industrial health service. Although
2,3,7,8-TCDD is extremely toxic to certain kinds of laboratory
animals, it has been handled for years without injury in analytical
and biological laboratories. Techniques used to handle radioactive
and infectious materials are applicable to 2,3,7,8-TCDD.
5.3.1 Protective equipment: Laboratory hood adequate for
radioactive work, safety glasses, and disposable plastic
gloves, apron or lab coat.
5.3.2 Training: Workers must be trained in the proper method of
removing contaminated gloves and clothing without contacting
the exterior surfaces.
5.3.3 Person hygiene: Thorough washing of hands and forearms
after each manipulation and before breaks (coffee, lunch
and shift). '
5.3.4 Confinement: Isolated work area, posted with signs,
segregated glassware and tools, plastic-backed absorbent
paper on benchtops.
5.3.5 Waste: Good technique includes minimizing contaminated
waste. Plastic liners should be used in waste cans.
5.3.6 Disposal of Wastes: Refer to the November 7, 1986, issue
of the Federal Register on Land Ban Rulings for details
concerning handling wastes containing dioxin.
5.3.7 Decontamination: Personnel - any mild soap with plenty of
scrubbing action. Glassware, tools, and surfaces - rinse
with 1,1,1-trichloroethane, then wash with any detergent and
water. Dish water may be disposed to the sewer after
percolation through a carbon filter. Solvent wastes should
be minimized, because they require special disposal through
commercial sources that are expensive.
37
-------
5.3.8
5.3.9
Laundry: Clothing known to be contaminated should be
disposed with the precautions described under "Disposal of
Hazardous Wastes". Laboratory coats or other clothing worn
in 2,3,7,8-TCDD work area may be laundered. Clothing should
be collected in plastic bags. Persons who convey the bags
and launder the clothing should be advised of the hazard and
trained in proper handling. The clothing may be put into
a washer without contact if the launderer knows the problem.
The washer should be run through one full cycle before being
used again for other clothing.
Wipe tests: A useful method to determine cleanliness of
work surfaces and tools is to wipe a surface area of
2 in. X 1 ft. with an acetone-saturated laboratory wiper
held in a pair of clean stainless steel forceps. Combine
wipers to make one composite sample in an extraction jar
containing 200 mL acetone. Place an equal number of unused
wipers in 200 mL acetone and use as a control. Extract each
jar with a wrist-action shaker for 20 min. Transfer extract
to a Kuderna-Danish (K-D) apparatus fitted with a
concentrator tube and a three-ball Snyder column. Add two
boiling chips and concentrate the extract to an apparent
volume of 1.0 mL with the same techniques used for sample
extracts. Add 100 fj.1 of the sample fortification solution
that has not been diluted with acetone or 1.5 mL of the
acetone-diluted solution (Section 7.14), and continue all
extract preparation steps and analytical procedures
described for samples. If any 2,3,7,8-TCDD is detected,
report the result as a quantity (picograms) per wipe test.
A lower limit of calibration of 20 pg/composite wipe test
is expected. A positive response for the control sample is
8 pg/wipe test. When the sample contains >25 pg, steps must
be taken to correct the contamination. First vacuum the
working places (hoods, benches, sink) using a vacuum cleaner
equipped with a high-efficiency particulate absorbent filter
and then wash with a detergent. Analyze a new set of wipers
before personnel are allowed in work in the previously
contaminated area.
Inhalation: Any procedure that may produce airborne
contamination must be carried out with good ventilation.
Gross losses to a ventilation system must not be allowed.
Handling the dilute solutions normally used in analytical
and animal work presents no significant inhalation hazards
except in case of an accident.
5 3 11 Accidents: Remove contaminated clothing immediately, taking
precautions not to contaminate skin or other articles. Wash
exposed skin vigorously and repeatedly until medical
attention is obtained.
5.3.10
38
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6. APPARATUS AND EQUIPMENT
6.1 Computerized GC/MS System
6.1.1 The GC must be capable of temperature programming and be
equipped with all required accessories, such as syringes,
gases, and capillary columns. The GC injection port must
be designed for capillary columns. Splitless or on-column
injection technique is recommended. With this method, a
2-/iL injection is used consistently. A 1-fj.l injection
volume can be used, but the injection volume should be
constant throughout analyses of calibration solutions and
related blanks, sample extracts, and quality control
samples.
6.1.2 GC/MS interface components should withstand temperatures up
to 280°C. The interface should be designed so that
separation of 2,3,7,8-TCDD from all other TCDD isomers
achieved in the GC column is not appreciably degraded. Cold
spots or active surfaces (adsorption sites) in the interface
can cause peak tailing and broadening. The GC column should
be inserted directly into the MS ion source without being
exposed to the ionizing electron beam. Graphite ferrules
should be avoided in the injection port because they may
adsorb TCDD. Vespel or equivalent ferrules are recommended.
6.1.3 The static resolving power of the MS must be maintained at
£10,000 (10% valley). The MS must be operated in a selected
ion monitoring (SIM) mode, and data must be acquired for the
ions listed in Table 2 during a total cycle time (including
instrument overhead time) of <1 s. Selection of the lock-
mass ion is left to the performing laboratory. Recommended
MS tuning conditions are provided in Section 9.1. The ADC
zero setting must allow peak-to-peak measurement of baseline
noise for every monitored channel and allow good estimation
of instrument resolving power.
6.1.4 A dedicated data system is used to control rapid SIM data
collection. Quantitation data (peak areas or peak heights)
must be acquired continuously and stored. The data system
must be capable of producing selected ion current profiles
(SICPs, which are displays of ion intensities as a function
of time) for each monitored ion, including the lock-mass
ion. Quantitation may be based on computer-generated peak
areas or on measured peak heights. The data system must be
capable of acquiring data for > five ions and generating
hard copies of SICPs for selected GC retention time
intervals and permit measurement of baseline noise.
6.2 GC Column. Two narrow bore, fused silica capillary columns coated
with phenyl cyanopropyl silicone are recommended; one is a 60-m SP-
39
-------
2330 and the other is a 50-m CP-SIL 88. Any capillary column that
separates 2,3,7,8-TCDD from all other TCDD isomers may be used, but
this separation must be demonstrated. At the beginning of each 12-h
period during which analyses are to be performed, column operating
conditions must be demonstrated to achieve the required separation
on the column to be used for samples. Operating conditions known
to produce acceptable results with the recommended columns are shown
in Table 3.
6.3 Miscellaneous Equipment.
6.3.1 Nitrogen evaporation apparatus with variable flow rate.
6.3.2 Balances capable of accurately weighing to 0.01 g and
0.0001 g.
6.3.3 Centrifuge.
6.3.4 Water bath equipped with concentric ring covers and capable
of being temperature controlled within +2°C.
6.3.5 Glove box.
6.3.6 Drying oven.
6.3.7 Minivials — 1-mL amber borosilicate glass with
conical-shaped reservoir and screw caps lined with
Teflon-faced silicone disks.
6.3.8 Pipets, disposable, Pasteur, 150 mm X 5 mm i.d.
6.3.9 Separatory funnels, 2 L with Teflon stopcock.
6.3.10 Kuderna-Danish concentrator, 500 ml, fitted with 10-mL
concentrator tube and three-ball Snyder column.
6.3.11 Teflon boiling chips washed with hexane before use.
6.3.12 Chromatography column, glass, 300 m X 10.5 mm i.d., fitted
with Teflon stopcock.
6.3.13 Adapters for concentrator tubes.
6.3.14 Continuous liquid-liquid extractor (optional).
6.3.15 Glass funnels, appropriate size to accommodate filter paper
used to filter extract (volume of approximately 170 ml).
6.3.16 Desiccator.
6.4 CAUTION: To avoid the risk of using contaminated glassware, all
glassware that is reused must be meticulously cleaned as soon as
40
-------
possible after use. Rinse glassware with the last solvent used in
it. Wash with hot water containing detergent. Rinse with copious
amounts of tap water and several portions of distilled. Drain dry
and heat in a muffle furnace at 400°C for 15-30 min. Volumetric
glassware must not be heated in a muffle furnace. Some thermally
stable materials (such as PCBs) may not be removed by heating in a
muffle furnace. In these cases, rinsing with high-purity acetone
and hexane may be substituted for muffle-furnace heating. After the
glassware is dry and cool, rinse it with hexane and store it
inverted or capped with solvent-rinsed aluminum foil in a clean
environment.
6.5 TCDD concentrations of concern in water are much lower than those
of concern in many other sample types. Extreme care must be taken
to prevent cross-contamination between water and other samples. The
use of separate glassware for water samples is recommended.
6.6 Empore extraction disks, C-18, 47mm.
6.7 Millipore Standard Filter Apparatus (or equivalent) to hold disks,
all glass
REAGENTS AND CONSUMABLE MATERIALS
7.1 Alumina, acidic (BioRad Lab. #132-1240 or equivalent). Extract in
a Soxhlet apparatus with methylene chloride for 6 h (> 3 cycles/h)
and activate it by heating in a foil-covered glass container for
24 h at 190°C.
7.2 Carbon, (Amoco PX-21 or equivalent).
7.3 Glass wool. Extract with methylene chloride and hexane and air-dry
before use. Store in a clean glass jar.
7.4 Potassium hydroxide, ACS grade.
7.5 Potassium silicate. Slowly dissolve 56 g of reagent grade potassium
hydroxide in 300 mL of anhydrous methanol in a 1-L round bottom
flask. Add slowly with swirling 100 g silica gel (prewashed and
activated). With a rotary evaporation apparatus with no vacuum
applied, rotate the flask and heat to 55°C for 90 min. After the
mixture cools to room temperature, pour it into a large glass column
containing a plug of glass wool at the end. Wash the mixture into
the column with methanol, and then add 200 mL of methanol. When the
methanol level reaches the bed of sorbent, add 200 mL of methylene
chloride to the column. Push the methylene chloride through the
column of sorbent by applying nitrogen pressure to dry or partially
dry the sorbent, which is then activated at 130°C overnight.
7.6 Silica gel, high purity grade, type 60, 70-230 mesh. Extract in a
Soxhlet apparatus with methylene chloride for 6 h (>3 cycles/h) and
41
-------
activate by heating in a foil-covered glass container for 24 h at
130°C.
7.7 Silica gel impregnated with 40% (w/w) sulfuric acid. Add two parts
(by weight) concentrated sulfuric acid to three parts (by weight)
silica gel (extracted and activated), mix with a glass rod until
free of lumps, and store in a screw-capped glass bottle.
7.8 Silica gel/Carbon. To a 20-g portion of silica gel add 500 mg
carbon, and blend until the mixture is a uniform color.
7.9 Sodium sulfate, granular, anhydrous.
7.10 Solvents, high purity, distilled-in-glass, or highest available
purity: methylene chloride, hexane, benzene, methanol, tridecane,
isooctane, toluene, cyclohexane, and acetone.
7.11 Sulfuric acid, concentrated, ACS grade, specific gravity 1.84.
7.12 Concentration Calibration Solutions (Table 4) — Five (or more)
tridecane solutions (CAL 1-5) containing unlabeled 2,3,7,8-TCDD and
isotopically labeled TCDDs. All five solutions contain unlabeled
2,3,7,8-TCDD at varying concentrations and the IS ( C12-2,3,7,8-
and the RS (13C12-1,2,3,4-TCDD) each at a
Three of these solutions also contain the
37Cl4-2,3,7,8-TCDD, CASRN 85508-50-5) at
All standards required for preparing CALs
are"commercially available but must be verified by comparison with
the National Bureau of Standards certified solution SRM-1614, which
contains 67.8 ng/mL of unlabeled 2,3,7,8-TCDD and 65.9 ng/mL of 13C-
labeled 2,3,7,8-TCDD at 23°C. Note: CALs can be prepared by
diluting calibration solutions used in Contract Laboratory Program
procedures for 2,3,7,8-TCDD determinations with low resolution MS;
to obtain appropriate IS concentrations for CAL 4, however, solvent
containing the IS must be used for dilution. Calibration solutions
used for USEPA Method.8290 can also be used to determine RFs for
2,3,7,8-TCDD; with those solutions the lower calibration
concentration may be higher (25 pg/L rather than 20 pg/L) or lower,
depending on injected volume of calibration solution. Because
Method 8290 solutions do not contain the SC, however, one or three
additional solutions containing that compound will be necessary to
measure its RF relative to the IS. Assuming adequate
reproducibility of RF measurements, triplicate analyses of one
solution (recommended SC concentration of 1.2 pg/AiL) or single
analysis of three solutions (0..6 to 1.8 pg//xL, Table 4) are
acceptable.
7.12.1 Each of CALs 1-5 contains the IS at a concentration of
50 pg/AtL; if 100% of the IS is extracted, 10 //L of this
solution is equivalent to a 10-fj.l extract of a 1-L sample
to which 500 pg of IS was added before extraction. If 100%
TCDD, CASRN 80494-19-5)
constant concentration.
surrogate compound (SC,
varying concentrations.
42
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of the analyte is extracted, CALs 1-5 contain unlabeled
2,3,7,8-TCDD at concentrations that are equivalent to 10-/iL
extracts of 1-L samples containing 20 pg to 2 ng.
7.12.2 CALs 1-3 contain the SC (37C1-2,3,7 ;8-TCDD) at a
concentration of 0.6 pg/juL, 1.2 pg/juL, and 1.8 pg//zL,
respectively; 10 juL of those solutions are equivalent to
10 ML extracts containing 30%, 60%, and 90%, respectively,
of the amount of SC added to each 1-L sample before
extraction.
7.12.3 Store CALS in 1-mL amber minivials at room temperature in
the dark.
7.13 Column Performance,Check Solution — contains a mixture of TCDDs
including the IS, the SC, unlabeled 2,3,7,8-TCDD, 1,2,3,4-TCDD
(CASRN 30746-58-8), 1,4,7,8-TCDD(CASRN 40581-94-0), 1,2,3,7,-TCDD
(CASRN 67028-18-6), 1,2,3,8-TCDD (CASRN 53555-02-5), 1,2,7,8-TCDD
(CASRN 34816-53-0), and 1,2,6,7-TCDD (CASRN 40581-90-6). Other
TCDDs can be present. Except for the IS and SC, solution component
concentrations are not critical. The IS concentration should be
10 ± 1 pg/ML and the SC concentration should be 0.6 ± 0.1 pg/L,
because ions produced by these compounds will be used to check
signal-to-noise ratios.
7.14 Sample Fortification Solution. A solution containing the IS at a
concentration of 5 to 25 pg/juL and the SC at a concentration of 0.2
to 1 pg//L6L, but with the ratio of IS to SC always 25:1. The
solution solvent is not critical; mix 20 to 100 pi, as appropriate
to produce needed IS and SC concentrations (50 pg/L and 2 pg/L,
respectively) of this solution with 1.5 mL of acetone. Add the
resulting solution to each sample and blank before extraction.
7.15 Recovery Standard Solution. A tridecane solution containing the
recovery standard, 13C12-1,2,3,4-TCDD at a concentration of 50 pg/juL.
A ip-/*L aliquot of this solution is added to each sample and blank
extract before concentrating the extract to its final volume for
analysis (Section 11.4.1).
8, SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 Samples must be collected in glass containers. The container should
not be rinsed with sample before collection.
8.2 Samples may be stored under ambient conditions as long as
temperature extremes (below freezing or >90°F) are avoided. To
.prevent photo-decomposition, samples must be protected from light
from the time of collection until extraction.
8.3 Ail samples must be extracted within 90 days after collection and
completely analyzed within 40 days after extraction.
43
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9. 6C/HS SYSTEM CALIBRATION.
9.1 MS Performance.
9.1.1 The MS must be operated in the electron ionization mode, and
a static resolving power of >10,000 (10% valley definition)
at >m/z 334 must be demonstrated before any analysis is
performed. The resolving power must be documented by
recording the mass profile of the reference peak. The
format of the peak profile representation must allow manual
determination of resolution (i.e., the horizontal axis must
be a calibrated mass scale (amu or ppm per division). The
peak width at 5% peak height must appear on the hard copy
and cannot exceed 100 ppm. Static resolving power must be
checked at the beginning and end of each 12-h period of
operation. A visual check of static resolution is
recommended before and after each analysis.
9.1.2 Chromatography time may exceed the long-term mass stability
of the high resolution MS, and mass drift of a few ppm can
affect the accuracy of measured masses. Therefore, a mass
drift correction is required. A lock mass ion from the
reference compound (high boiling perfluorokerosene, PFK, is
recommended) is used to calibrate the MS. An acceptable
lock mass is an ion with mass larger than the lightest mass
monitored but less than the heaviest ion monitored. The
amount of PFK introduced into the ion chamber during
analysis should be adjusted so that the amplitude of the
lock mass ion is <10% full scale. Excessive PFK may cause
noise problems and ion source contamination.
9.1.3 Using a PFK molecular leak, tune the MS to obtain resolving
power of >10,000 (10% valley) at m/z 334. Using a
reference peak near m/z 320, verify that the exact mass of
the reference peak is within 5 ppm of the known mass. The
low- and high-mass reference ions must be selected to
provide the voltage jump required to detect ions from m/z
320 through m/z 334. (Note: With a qualitative
confirmation option in Section 11.5.5, detected ion range
will be m/z 257 to m/z 334.)
9.1.4 MS resolving power must be demonstrated by recording the
mass peak profile of the high-mass reference signal obtained
using the low-mass ion as a reference. The minimum
resolving power of 10,000 must be demonstrated on the high-
mass ion while it is transmitted at a lower accelerating
voltage than the low-mass reference ion, which is
transmitted at full sensitivity. The peak profile
representation must allow manual determination of the
resolution (i.e., the horizontal axis must be a calibrated
mass scale in amu or ppm per division. The measured peak
44
-------
it 5% of the peak maximum must appear on the hard conv
and cannot exceed 100 ppm at the high mass. Py
9.2 Initial Calibration
9.2.1 GC .column performance. The laboratory must verify GC
conditions necessary for required separation of 2,3,7,8-TCDD
rhp!V°chftTCDD \somers- InJect 2 ML of the performance
check so ution and acquire SIM data for the five ions in
Table 2 (nominal m/z 320, 322, 328, 332, and 334) within a
total cycle time of <1 s. Acquire at least five scans for
*rn,,i^n&Cr?-SS e?ch GC Peak and use the same data
acquisition time for each ion monitored. The oeak
representing 2,3,7,8-TCDD and peaks representing any other
ILUU isomers must be resolved with a valley of <25% (Figure
rrAn Jey • = 10° x/y' where y is Peak height of 2,3,7 8-
Trnn aa«HX-JS measured ans snown in Figure 1 between 2378-
TCDD and its closest eluting isomer. CAUTION: The same
data acquisition parameters must be used to analyze al?
calibration and performance check solutions.
1 9'2'2 ?oVr]±^^ Ratio of integrated
rt3r iVhoioTo"^'"?'o'Tr'nrT; ^\ l"u °°'t Produced by the IS
( L-labeled 2,3,7,8-TCDD) must be >0.67 and <0.87 The S/N
ffm? f°r+miZ 328 Pr°duced by the~SC (13C-la~beled 1 3 4-
Trnn^ m,,ct hQ .o c ,^ the S/N ratio V Ieu i,',*,*
9.2.3
9.2.4
Using the same GC and MS conditions, analyze a 2-/xL aliquot
of the medium concentration CAL (CAL 3). Check ion ratios
specified, in Section 9.2.2. If 'criteria are met ° analyze
a 2-fj.L aliquot of each of the four (or more) remaining CALS?
For each CAL, ensure that ion ratios (Section 9.2 2) are
acceptable. For CAL 1 (the lowest concentration CAL) dat^
ensure that each ion produces a signal-to-noise (S/N ?at?o
nLrih iD\SPlay.-a SIC/ for a re9ion of the chromatogram
near the elution time of 2,3,7,8-TCDD but where no analyte
or interference peak is present. The preferred width of the
"b°-t .10-X JLul lwidth at Salf he19ht of the IS
thn°!is.e ils th? Jheight (meas^ec> ^om the lowest
uJhe dlsPlay window) of the largest signal not
attributable to any eluting substance.
9.2.5 RF Measurements. Using
45
-------
9.3
where A = the sum of integrated ion abundances of m/z
x 320 and 322 for unlabeled 2,3,7,8-TCDD, the
abundance of m/z 328 for the SC^ or the
abundances of m/z 332 and 334 for the RS.
A = the sum of integrated ion abundances of m/z
18 332 and 334 for the IS,
Q.s = injected quantity of IS, and
Q = injected quantity of unlabeled 2,3,7,8-TCDD,
x the SC, or the RS.
RF is a unitless number; units used to express
quantities must be equivalent.
926 For each compound (unlabeled 2,3,7,8-TCDD the SC and the
RS), calculate a mean RF and the relative standard deviation
(RSD) of the five measured RFs. When IRSD exceeds 20%,
analyze additional aliquots of appropriate CALs to obtain
an acceptable RSD of RFs over the entire concentration
range, or take action.to improve GC/MS performance.
Routine Calibration. If a laboratory operates during only one <12-h
oeriod (shift) each day, routine calibration procedures must be
Kormed a the beginning (after mass calibration and successful
analysis of the performance check solution to ensure adequate
sensitivity and acceptable ion ratios) of that shift, and the
performance check solution must be analyzed again atthe> endhjf that
shift to validate data acquired during the shift. If the laboratory
o eStls during consecutive shifts, routine calibrat on Procedures
must be performed at the beginning of each shift, but analysis OT
the performance check solution at the beginning of each shift and
at the end of the final 12-h period is sufficient.
9.3.1 Inject a 2-jaL aliquot of CAL 3, and analyze with the same
conditions used during Initial Calibration.
932 Demonstrate acceptable performance for ions abundance
ra?io1, and demonstrate that each measured RF for unlabeled
2 3 7 8-TCDD, the SC, and the RS is within 20% of the
appropriate mean RF measured during initial calibration.
If one or more of these criteria are not met, up to two
additional attempts can be made before remedial action is
necessary and the entire initial calibration process is
repeated. Corrective action may include increasing the
detector sensitivity, baking the GC column, clipping a short
lenqth (about 0.3-0.5 m) of the injector side of the GC
column, washing or replacing the GC column, and cleaning the
46
-------
ion source. If degradation of the standards in CALs is
suspected, a fresh set of CALs should be used for repeating
initial calibration procedures.
10. QUALITY CONTROL
10.1 Laboratory Reagent Blank. Perform all steps in the analytical
procedure using all reagents, standards, equipment, apparatus,
glassware, and solvents that would be used for a sample analysis
but omit a water sample, and substitute 1 L of reagent water.
10.1.1 Analyze two laboratory reagent blanks (LRBs) before sample
analyses begin and when a new batch of solvents or reagents
is used for sample extraction. Do not add any IS, SC or RS
to one blank; this will allow demonstration that reagents
contain no impurities producing any ion current above the
level of background noise for monitored ions.
10.1.2 Criteria for acceptable LRBs.
10.2
10.3
10.1.2.1
10.1.2.2
When no IS, SC, or RS is present, no ion current
above the level of background S/N is detected for
any monitored ion within 20 s of the retention
times previously measured for labeled 1,2,3,4-
TCDD or for unlabeled and labeled 2,3,7,8-TCDD.
When the IS is present, no ion current for m/z
259, 320, or 322 is observed that is >2% of the
abundance of m/z 332 within 5 scans of the IS
peak maximum.
10.1.3
Corrective action for unacceptable LRB. Check solvents,
reagents, apparatus, and glassware to locate and eliminate
the source of contamination before any samples are extracted
and analyzed. Purify or discard contaminated reagents and
solvents.
Field Blanks. An acceptable field blank must meet criteria in
Section 10.1.2.2. When results for a field blank are acceptable,
analysis of an LRB is not needed with that sample batch. When field
blank results are not acceptable, analysis of an LRB is needed; if
•t- il6^1*,8 are aucceptable' data for samples associated with the
field blank must be accompanied by pertinent information about the
nature and amount of contamination observed in the field blank.
Corrective action for unacceptable performance check solution data.
When thetlS sensitivity requirement (Section 9.2.2) is not met at
the end of a 12-h period in which sample extracts were analyzed all
related sample extracts must be reanalyzed after criteria have been
met. When other criteria (ion ratios or GC resolution) are not met,
all sample extracts that produced positive results or potential
47
-------
positive results must be reanalyzed after calibration criteria have
been met.
11. PROCEDURE
11.1 Sample Extraction — Liquid-Liquid Extraction
11 1 1 Mark the water meniscus on the side of the 1-L sample bottle
for later determination of the exact sample volume. Pour
the entire sample (approximately 1 L) into a 2-L separatory
funnel. A continuous liquid-liquid extractor may be used
instead of a separatory funnel.
11.1.2 Add 1.5 ml of the sample fortification solution (Section
7.14) to the sample in the separatory funnel.
11 1.3 Add 60 mL of methylene chloride to the sample bottle, seal
and shake 30 s to rinse the inner surface. Transfer the
solvent to the separatory funnel and extract the sample by
shaking the funnel for 2 min with periodic venting. Allow
the organic layer to separate from the water phase for a
minimum of 10 min. If an emulsion interface between layers
exists, the analyst may use mechanical techniques to
complete the phase separation. Collect the methylene
chloride layer directly into a 500-mL Kuderna Danish (K-D)
concentrator (mounted with a 10-raL concentrator tube) by
passing the sample extract through a filter funnel packed
with a glass wool plug and 5-g of anhydrous sodium sulfate.
Repeat the extraction with two additional 60-mL portions of
methylene chloride, filtering each extract before adding it
to the K-D concentrator. After the third extraction, rinse
the sodium sulfate with an additional 30 ml. of methylene
chloride to ensure quantitative transfer, and add rinse to
composite extract.
11 1 4 Add one or two clean boiling chips to the evaporative flask
and attach a Snyder column. Prewet the Snyder column by
adding about 1 mL of methylene chloride to the top. Place
the K-D apparatus on a hot water bath (60-65°C) so that the
concentrator tube is partially immersed in the hot water,
and the entire lower rounded surface of the flask is bathed
with hot vapor. Concentrate the extract until the apparent
volume of the liquid reaches 1 mL. Remove the K-D apparatus
and allow it to drain and cool for at least 10 min. Remove
the Snyder column, add 50 mL of hexane and a new boiling
chip and reattach the Snyder column. Increase the water
bath temperature to 85-90°C and concentrate the extract to
approximately 1 mL. Rinse the flask and the lower joint
with 1-2 mL hexane. Concentrate the extract to 1 mL under
a gentle stream of nitrogen. If further extract processing
is to be delayed, the extract should be quantitatively
48
-------
transferred to a Teflon-sealed, amber, screw-cap vial and
stored refrigerated and protected from light.
11.1.5 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the liquid to a 1000 ml
graduated cylinder. Record the sample volume to the nearest
5 ml.
11.2 Sample Extraction --Liquid-Solid Extraction
11.2.1 Preparation of disks
11.2.1.1 Insert the disk into the 47mm filter apparatus.
Wash the disk with about 10 mL of benzene by
adding the solvent to the disk, pulling about
half through the disk and allowing it to soak the
disk for about a minute, then pulling the
remaining rinse solvent through the disk. With
the vacuum on, pull air through the disk for
about one minute.
11.2.1.2 Pre-wet the disk with 10 ml methanol (MeOH) by
adding the MeOH to the disk, pulling about half
through the disk and allowing it to soak for
about a minute, then pulling MOST of the MeOH
through. A layer of MeOH should 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 an important step to
ensure uniform flow and good analyte recoveries.
11.2.1.3 Rinse the disk with 10 ml reagent water by adding
the water to the disk and pulling most through,
again leaving a layer on the surface of the disk.
11.2.2 Mark the water miniscus on the side of the 1-L sample bottle
for later determination of the exact sample volume.
11.2.3 Add the water sample, to which all necessary surrogate
compounds and internal standards have been added according
to Section 11.1.2, to the reservoir and turn on the vacuum
to begin the extraction. Aspirator vacuum should be adjusted
to allow the sample to pass through the disk in
approximately 20 minutes. Extract the entire sample,
draining as much water as possible from the sample
container. After all the sample has passed through, draw
air through the disk for about 10 minutes to remove some of
the residual water.
11.2.4 Remove the filtration top from the apparatus, but do not
disassemble the reservoir and fritted base. Empty the
49
-------
11.2.6
11.2.7
water from the flask and insert a suitable sample tube to
contain the eluate. The only constraint on the sample tube
is that it fit around the drip tip of the fritted base.
Reassemble the apparatus.
11.2.5 Add 5 ml benzene to the sample bottle and rinse the inside
of the container. Transfer this benzene to the disk with a
dispo- pipet or other suitable vessel, rinsing the sides of
the filtration reservoir in the process. Pull about half of
the benzene through the disk, release the vacuum, and allow
the disk to soak for about a minute. Pull the remaining
benzene through the disk.
Repeat the above step twice. Pour the combined eluates
through a small funnel containing about 3 grams of anhydrous
sodium sulfate. The sodium sulfate may be contained in a
prerinsed filter paper, or by a plug of prerinsed glass wool
in the stem of the funnel. Rinse the sodium sulfate with a
5 ml aliquot of benzene.
Quantitatively transfer the combined eluate to a suitable
graduated concentrator tube, and rinse the test tube with
benzene. Using micro-Kuderna-Danish or nitrogen blowdown,
concentrate the eluate almost to dryness, then add hexane
to bring the volume to 1 ml for sample extract cleanup.
11.2.8 Determine the original sample volume by refilling the sample
bottle to the mark, and transferring the liquid to a 1000
ml graduated cylinder. Record the sample volume to the
nearest 5 ml.
11.3 Sample Extract Cleanup
11.3.1 Chromatography columns 1 and 2, described below, are
recommended for every sample extract. A third column
containing silica gel and carbon may be useful for removal
of interferences from some sample extracts and may be used
at the analyst's discretion. Because each cleanup procedure
increases the chances of analyte loss, such procedures
should be minimized. Criteria for predicting when the
carbon column will be needed are not available, but that
column is probably not needed for finished drinking water
samples that have been filtered through granular activated
carbon.
11.3.2 Column Preparation
11.3.2.1 Column 1. Place 1.0 g of silica gel (See NOTE)
into a 1.0 cm X 20 cm column and tap the column
gently to settle the silica gel. Add 2 g
potassium hydroxide impregnated silica gel, 1 g
silica gel, 4.0 g of sulfuric acid impregnated
50
-------
11.3.3
silica gel, and 2 g silica gel. Tap column
gently after each addition. NOTE: The silica
gel for this application is partially deactivated
with 1% water immediately before packing the
column.
11.3.2.2 Column 2. Place 6.0 g of alumina into a 1.0 cm
X 30 cm column and tap the column gently to
settle the alumina. Add a 1-cm layer of purified
sodium sulfate to the top of the alumina.
11.3.2.3 Add hexane to each column until the packing is
free of channels and air bubbles. A small
positive pressure (5 psi) of clean nitrogen can
be used if needed.
Quantitatively transfer the sample extract to the top of the
silica gel in column 1. Rinse the concentrator tube with
two 0.5 ml portions of hexane; transfer rinses to Column 1
With 90 ml of hexane, elute the extract from Column 1
directly into Column 2.
11.3.4 Add an additional 20 mL of hexane to Column 2 and elute
until the hexane level is just below the top of the sodium
sulfate; discard the eluted hexane.
11.3.5 Add 20 ml of 20% methylene chloride/80% hexane (v/v) to
Column 2 and collect the eluate.
11.3.6 If carbon column cleanup is selected, proceed with Section
11.3.7. If not, proceed with Section 11.3.8.
11.3.7 Optional cleanup with Column 3. Reduce the volume of eluate
from Column 2 to about 1 ml in a K-D apparatus. Transfer
the concentrated eluate from Column 2 to a 4 mm X 200 mm
column (2 mL disposable pipette) containing 200 mg silica
gel/carbon. Elute with 15 ml methylene chloride and 15 ml
80/o methylene chloride/20% benzene (v/v) in forward
direction of flow. Discard these fractions^ Elute TCDD
with 15 ml toluene in a reverse direction flow. Collect
this eluate.
11.3.8 Concentrate the eluate (either the toluene fraction from
Section 11.3.7 or the methylene chloride/hexane fraction
from Section 11.3.5) to a small volume (<0.5 ml) and
tran|fer to a 1-mL minivial. Store the extract in the dark
at 4 C until just before analysis. Note: The final volume
is adjusted to 10 /zL immediately before GC/MS analysis.
11.4 GC/MS Analysis of Extracts
51
-------
11.4.1 Remove the sample or blank extract from storage and allow
it to warm to ambient laboratory temperature. Add a 10-^1
aliquot of the RS solution (Section 7.15) to the extract and
reduce the extract volume to 10 p.1 with a stream of dry,
purified nitrogen.
11.4.2 Inject a 2-p.l aliquot of the extract into the GC, operated
under conditions previously used to produce acceptable
results with the performance check solution.
11.4.3 Acquire SIM data using the same analytical conditions
previously used to determine RFs.
11.5 Identification Criteria
11.5.1 Obtain SICPs for each ion monitored.
11 5 2 The abundance of m/z 332 relative to m/z 334 produced by the
IS must be >0.67 and <0.87, and these ions must maximize
within 1 scan of each other. Retention time should be
within ±5 scans of that observed during the most recent
acceptable calibration. For good performance, the retention
time of the IS must be reproducible to ±5 scans from one
injection to the next. Over the course of a 12-h work
period, the IS retention time should be reproducible within
±10 scans. Less reproducible IS retention times indicate
serious chromatography problems that should be corrected
before further sample analyses.
11.5.3 The sample component must produce a signal for both ions
monitored to detect and measure unlabeled 2,3,7,8-TCDD, and
the abundance of m/z 320 relative to m/z 322 must be >0.67
and <0.87. All ions must maximize within 1 scan of each
other and within 3 sec of the IS.
11 5 4 The S/N ratio for each unlabeled TCDD and SC ion must be
>2.5 and must not have saturated the detector; the S/N ratio
for each IS and RS ion must be >10 and must not have
saturated the detector.
11.5.5
Additional qualitative confirmation can be obtained by
monitoring m/z 257 and 259 (fragment ions produced by loss
of COC1 from the analyte) along with ions previously
specified or by reanalysis of an aliquot of the extract to
monitor m/z 257 and 259 along with m/z 268 and 270, fragment
ions produced by loss of COC1 from the IS. The relative
abundance of m/z 257 to 259 and m/z 268 to 270 should be 0.9
to 1.1, and the abundance of 259 to 270 should be the same
as the ratio of 322 to 334 measured in the previous
injection. Although variable with instrumental conditions,
the abundance of fragment ions relative to molecular ions
52
-------
30-45X for each compound; therefore, the
for these 1ons w111 be
12. CALCULATIONS
12.1 From appropriate SICPs of nominal m/z 259, 320 and 322 obtain and
.'s^^.^asr^jirr'.^^tr^"1^
12.2 Calculate the concentration using the formula:
Cx = (Ax ' Qis)/(Ais . RF • V)
where Cx = concentration (picograms per liter),
A
'is
= sum of areas for m/z 320 and m/z 322 produced by
the sample component,
= sum of areas for m/z 332 and m/z 334 produced by
tne is,
Qjs = quantity (picograms) of IS added to the sample,
RF = mean RF measured for unlabeled 2,3,7,8-TCDDdurinq
initial calibration, and
V = Volume (liters) of water extracted.
12.3 When fortified samples of known composition are analyzed calculate
the percent method bias using the equation: calculate
B » 100 (Cs - Ct)/ Ct
where Cs = measured concentration (in picograms per liter),
Ct = theoretical concentration (i.e., the concentration
resulting from fortification plus any concen-
tration measured in the sample when an unfortified
sample extract was analyzed).
NOTE: The bias value retains a positive or negative sign.
12.4 Calculate the IS concentration using the formula:
c,-s - (A,. ' RF . Qrs)/(Ars V)
where Cis = concentration (picograms per liter),
sum of areas for nominal m/z 332 and 334 produced by
the IS, '
A
'is
53
-------
A = sum of areas for m/z 332 and m/z 334 produced by the
RS,
Qrs = quantity (picograms) of RS added to the sample,
RF - mean RF measured for the RS relative to the IS during
initial calibration, and
V = Volume (liters) of water extracted.
12 5 Report calculated concentrations with three significant figures when
12'5 measured concentration is >100 pg/L and with two significant^fjgure
when value is <100 pg/L. The recovery of the IS should be >40/o and
«••
*
+5 scans of the IS peak is determined as previously described and
*s multiplied by 2.5. With the following formula, the product is
related to the estimated unlabeled TCDD concentration required to
produce a signal equivalent of 2.5 S/N.
EMPC
2.5 * Bx ' Q18 / A
is
RF ' V
A,-
is
12.7
12.8
= background (height or area) for either nominal
m/z 320 or 322 within ±5 scans of the IS peak,
= peak height or area (depending on selection for
B ) for nominal m/z 332 when m/z 320 is used to
determine Bx or nominal m/z 334 when m/z 322 is
used to determine Bx, and
Q.s, RF, and V retain previous definitions.
An interference results when sample a component elutes in the
retention time window for 2,3,7,8-TCDD and produces both monitored
TCDD ions but measured relative .abundances do not meet
identification criteria. Any ion with S/N of <2.5 should be
ianored Ions producing S/N of >2.5 but with unacceptable relat ve
abundance should be treated as an interference and a quantitative
estimate of that interference should be calculated using the
equation in Section 12.2. Interferences observed in blanks and also
present in samples should not be reported as a sample interference
but should be reported as a blank interference.
Table 5 lists results of analyses of fortified reagent water samples
carried out using the Empore disk extraction method according to the
procedure detailed in Section 11.2. Even though this method was
developed for only 2,3,7,8-TCDD, since the other dioxins and furans
54
-------
had been studied, the results were included. The fortifying levels
were 0.16 ng/L for the tetra isomers, 0.8 ng/L for the penta, hexa,
and hepta isomers, and 1.6 ng/L for the octa isomers. The average
recovery for all isomers in all replicate analyses is 80% with an
ll/o relative standard deviation. No clean up was done on these
samples.
13. REFERENCES
1. "Mater Solubility of 2,3,7,8-Tetrachlorodibenzo-p-dioxin," L. Marple,
R. Brunck, and L. Throop, Environ. Sci. and Techno! . 1986, 20(2), 180-
2. "Carcinogens --Working with Carcinogens," Department of Health,
centers
3> DSM?tyJ-n Ac£Ldemi.c Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Ed., 1979.
4' ?«* SS* M-j7M0ck>*D1S>1ni^LaJys1s» So11/Sediment and Water Matrices,
IFB WA86-K357, September 1986.
55
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TABLE 1. POTENTIAL INTERFERENCES
Compound
Heptachlorobi phenyl
Nonachlorobi phenyl
Tetrachl oromethoxy-
bi phenyl
Tetrachl orobenzyl -
phenyl ether
DDT
DDE
Pentachl orobenzyl -
phenyl ether
Tetrachl oroxanthene
Hydroxytetrachl oro-
dibenzofuran
Tetrachl orophenyl-
benzoquinone
Interferina Ion
Formul a m/z
______ ===-==============================
M+ - 2 35C1 321.867
M+ - 4 35C1 319.8521
M* - 3 35C137C1 321.8491
M* 319.9329
M+ 321.9299
M+ 319.9329
M+ 321.9300
M+ - H35C1 319.9321
M+ - H35C1 321.9292
M* 319.9321
• M+ 321.9292
M+ - H35C1 319.9143
M+ - H35C1 321.9114
M+ 319.9143
M+ 321.9114
M+ 319.8966
M+ 321.8936
M+ 319.8966
M+ 321.8936
Required
Resolution
================
12,476
7,189
7,233
8,805
8,848
8,813
, 8,843
9,006
9,050
9,011
9,050
18,043
18,104
18,043
18,104
^
—
56
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TABLE 2. IONS TO BE MONITORED
Accurate
Mass
Elemental
Composition
Compound
258.9298
319.8965,
321.8936
327.8847
331.9368
and
333.9339
and
35,
C12H435C1337C102
13C12H435C1337C102
Unlabeled2,3,7,8-TCDD
Unlabeled 2,3,7,8-TCDD
Unlabeled2,3,7,8-TCDD
37,
Cl4-2,3,7,8-TCDD(SC)
13C12-2,3,7,8-TCDD (IS)
13C12-1,2,3,4-TCDD (RS)
13C12-2,3,7,8-TCDD (IS)
13C12-1,2S3,4-TCDD (RS)
57
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TABLE 3. GC OPERATING CONDITION GUIDELINES
Column coating
Film thickness
Column dimensions
Helium* linear
velocity
Initial temperature
Initial time
Temperature program
Retention time of
2,3,7,8-TCDD
SP-2330
0.2 urn
60 m X 0.24 mm
28-29 cm/sec
at 240°C
70°C
4 min
Rapid increase to 200°C;
200°C to 240°C at
4°C/min
24 min
CP-SIL 88
0.22 urn
50 m X 0.22 mm
28-29 cm/sec
at 240°C
45°C
3 miri
Rapid increase to 190°C;
190°C to 240°C at
5°C/min
26 min
*Hydrogen is an acceptable carrier gas,
58
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TABLE 4. COMPOSITION OF CONCENTRATION CALIBRATION SOLUTIONS
CAL #
1
2
3
4
5
Analyte
Unlabeled
2,3,7,8-TCDD
2 pg/fj.1
Surrogate Cmpd. Internal Std. Recovery Std.
10
50
100
200
37ci4-
2,3,7,8-TCDD
0.6
1.2
1.8
0
0
2,3,7^8-1000 1,2,3,1|-TCDD
50
50
50
50
50
30 pg//iL
30
30
30
30
59
-------
TCDF
TCDD
PCDF
PCDD
HxCDF
HxCDD
HpCDF
HpCDD
OCDF
OCDD
2
2
4
2
8
6
4
2
2
2
72
75
78
86
83
80
77
80
91
82
6
0
11
5
16
11
23
10
15
11
* Fortifying levels were 0.16 ng/L for the tetra isomers, 0.8 ng/L
for the pent! and hexa isomers, and 1.6 ng/L for the octal sonars.
Analyses were carried out using the procedure described in Section 11.2
of this method.
60
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61
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METHOD 547. DETERMINATION OF GLYPHOSATE IN DRINKING WATER
BY DIRECT-AQUEOUS-INJECTION HPLC, POST-COLUMN
DERIVATIZATION, AND FLUORESCENCE DETECTION
July 1990
T. W. Winfield
W. J. Bashe (Technology Applications Inc.)
T. V. Baker (Technology Applications Inc.)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
63
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METHOD 547
DETERMINATION OF GLYPHOSATE IN DRINKING WATER BY DIRECT-AQUEOUS-INJECTION
HPLC, POST-COLUMN DERIVATIZATION, AND FLUORESCENCE DETECTION
1. SCOPE AND APPLICATION
1.1 This method describes a procedure for the identification and
measurement of Glyphosate (N-phosphonomethyl glycine) in drinking
water matrices. Single laboratory accuracy and precision data have
been determined for this method.
1.2 Glyphosate was found to rapidly decompose in chlorinated waters (1).
It is therefore unlikely that the analyte will be evidenced in tap
water except as separate glycine and N-phosphonomethyl moieties,
neither of which is applicable to this method.
Chemistry Abstract Services
Analvte Registry Number
Glyphosate 1071-83-6
1.3 The method detection limits (MDL, defined in Section 13) for
glyphosate are listed in Table 1 (2). The MDLs for a specific
sample may differ from those listed.
2. SUMMARY OF METHOD
2.1 A water sample is filtered and a 200 p,L aliquot injected into a
cation exchange HPLC column. Separation is achieved by using an
isocratic elution. After elution from the analytical column at
65°C, the analyte is oxidized with calcium hypochlorite. The product
(glycine) is then coupled with o-phthalaldehyde-2-mercaptoethanol
complex at 38°C to give a fluorophor, which is detected by a
fluorometer with excitation at 340 nm and detection of emission
measured at > 455 nm (1,3).
3. DEFINITIONS
3.1 LABORATORY DUPLICATES (LD1 and LD2) - Two sample aliquots taken in
the analytical laboratory and analyzed separately with identical
procedures. Analyses of LD1 and LD2 give a measure of the precision
associated with laboratory procedures, but not with sample
collection, preservation, or storage procedures.
3.2 FIELD DUPLICATES (FD1 and FD2) - Two separate samples collected at
the same time and place under identical circumstances and treated
exactly the same throughout field and laboratory procedures.
Analyses of FD1 and FD2 give a measure of the precision associated
64
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with sample collection, preservation and storage, as well as with
laboratory procedures.
3.3 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water that is
treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates
that are used with other samples. The LRB is used to determine if
method analytes or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.4 FIELD REAGENT BLANK (FRB) - Reagent water placed in a sample
container in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB
is to determine if method analytes or other interferences are
present in the field environment.
3.5 LABORATORY PERFORMANCE CHECK SOLUTION (LPC) - A solution of method
analytes, surrogate compounds, and internal standards used to
evaluate the performance of the instrument system with respect to a
defined set of method criteria.
3.6 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the method is in control, and
whether the laboratory is capable of making accurate and precise
measurements at the required method detection limit.
3.7 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) - An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3.8 STOCK STANDARD SOLUTION - A concentrated solution containing a
single certified standard that is a method analyte, or a
concentrated solution of a single analyte prepared in the laboratory
with an assayed reference compound. Stock standard solutions are
used to prepare primary dilution standards.
3.9 CALIBRATION STANDARD (CAL) - A solution prepared from the primary
dilution standard solution and stock standard solutions of the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.1.0 QUALITY CONTROL SAMPLE (QCS) - A sample matrix containing method
analytes or a solution of method analytes in a water miscible
65
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4.
solvent which is used to fortify reagent water or environmental
samples. The QCS is obtained from a source external to the
laboratory, and is used to check laboratory performance with
externally prepared test materials.
INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware, and other sample processing hardware that lead
to discrete artifacts and/or elevated baselines in the
chromatograms. All of these materials must be routinely
demonstrated to be free from interferences under the conditions of
the analysis by analyzing laboratory reagent blanks as required by
Section 10.2.
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. Glassware should then be drained dry,
and heated in a laboratory oven at 4008C for several hours
before use. After drying and cooling, glassware should be
stored in a clean environment to prevent any accumulation of
dust or other contaminants.
4.1.2 The use of high purity reagents and solvents helps to
minimize interference problems. Purification of solvents by
distillation in all-glass systems may be required to achieve
necessary purity.
4.2 Samples may become contaminated during shipment or storage. Field
blanks must be analyzed to determine that sampling and storage
procedures have prevented contamination.
4.3 The extent of matrix interferences may vary considerably from source
to source, depending upon the nature and diversity of the matrix
being sampled. No interferences have been observed in the matrices
studied.
4.4 The extent of interferences that may be encountered using liquid
chromatographic techniques has not been fully assessed. Although
the HPLC conditions described allow for a unique resolution of the
compound covered in this method, other matrix components may
interfere.
5. SAFETY
5.1 The toxicity or carcinogenicity of chemicals used in this method has
not been precisely 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
66
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this method (5). A reference file of material data handling sheets
should be made available to all personnel involved in the chemical
analysis.
6. APPARATUS AND EQUIPMENT
6.1 SAMPLING EQUIPMENT (for discrete or composite sampling).
6.1.1 Grab sample bottle - 60 mL screw cap bottles (Pierce No.
13075 or equivalent) and caps equipped with a teflon-faced
silicone septa (Pierce No. 12722 or equivalent). Prior to
use, wash vials and septa as described in Section 4.1.1.
6.2 GLASSWARE
6.2.1 Autosampler vials - glass, 3.7 mL, with teflon-lined septa
and screw caps. (Supelco, #2-3219, or equivalent)
6,2.2 Volumetric flask - 1000 mL and 100 mL
6.3 BALANCE - analytical, capable of accurately weighing 0.0001 g.
6.4 pH METER - capable of measuring pH to 0.01 units.
6.5 FILTRATION APPARATUS
6.5.1 Macrofiltration - to filter mobile phase and derivatization
solutions used in HPLC system. Membrane filter, 0.2 n mesh,
47 mm diameter, Nylon 66 (Alltech, #2034 or equivalent)
6.5.2 Microfiltration - to filter samples prior to HPLC analysis.
Use 0.45 p, filters (Gelman Acrodisc - CR or equivalent)
6.5.3 Helium, for degassing solutions and solvents.
6.6 SYRINGES
6.6.1 One 250 juL glass syringe, with blunt tip needle for manual
injection.
6.6.2 3 - 5 mL disposable hypodermic syringes with Luer-Lok tip.
6.6.3 Micro syringes, various sizes.
6.7 INSTRUMENTATION - A schematic diagram of the analytical system
is shown in Figure 1.
6.7.1 A high performance liquid chromatograph (HPLC) capable of
injecting 200 juL aliquots and utilizing an isocratic pumping
system with constant flow rate of 0.5 mL/min.
67
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6.7.2 Column - 250 x 4 mm, Bio-Rad, Aminex A-9. Column
specifications: K+ form, packed at 65°C, pH = 1.9. This
column was used to generate the method performance statements
in Section 13. Different HPLC columns may be used if
requirements described in Section 10.3 are met. Use of guard
columns is recommended.
6.7.3 Guard Column - C18 packing - (Dupont, Zorbax Guard Column or
equivalent). An alternative guard column similar in
composition to the analytical column may also be used
provided the requirements of Section 10.3 are met.
6.7.4 Column Oven, (Fiatron, Model CH-30 and controller, Model TC-
50, or equivalent).
6.7.5 Post Column Reactor (PCR) - Capable of mixing reagents into
the mobile phase. Reactor to be equipped with pumps to
deliver 0.5 mL/min of each reagent; mixing tees; two 1.0 ml
delay coils, both thermostatted at 38°C; and constructed
using teflon tubing. (Kratos Model URS 051 and URA 200 or
equivalent).
6.7.6 Fluorescence Detector - Capable of excitation at 340 nm and
detecting of emission > 455 nm. A Schoeffel Model 970
fluorescence detector was used to generate the validation
data presented in this method.
6.7.7 Data System - A strip chart recording of the detector
response must be provided as a minimum requirement. The use
of a data system to calculate retention times and peak areas
is recommended but not required.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 HPLC MOBILE PHASE
7.1.1 REAGENT WATER - reagent water is defined as water of very
high purity, equivalent to distilled-in-glass solvents.
7.1.2 MOBILE PHASE - 0.005 M KH2P04 (0.68 gm) in 960 mL reagent
water, add 40 mL HPLC grade methanol, adjust pH of solution
to 1.9 with concentrated phosphoric acid then filter with
0.22 /LI filter and degas with helium before use.
7.2 POST COLUMN DERIVATIZATION SOLUTIONS
7.2.1 Calcium hypochlorite solution - Dissolve 1.36 g
KHoPO,
--. ~ . -, j .-..-_ _ '24'
11!6 g NaCfand 6.4 g NaOH in 500 mL deionized water. Add 15
mg Ca(C10)2 dissolved in 50 mL deionized water and dilute
68
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solution to 1000 mL with deionized water. Filter solution
through 0.22 IJL membrane filter and degas with helium before
use. It is recommended that this solution be made fresh
daily.
7.2.2 0-phthalaldehyde (OPA) reaction solution
7.2.2.1 2-Mercaptoethanol (1+1) - Mix 10.0 ml of
2-mercaptoethanol and 10.0 ml of acetonitrile. Cap
and store in hood. (Caution - stench)
7.2.2.2 Sodium borate (0.05N) - Dissolve 19.1 g of sodium
borate (Na2B40/10 H?0) in 1.0 L of reagent water.
The sodium borate will completely dissolve at room
temperature if prepared a day before use.
7.2.2.3 OPA Reaction Solution - Dissolve 100 + 10 mg of o-
phthalaldehyde (mp 55-58°C) in 10 ml of methanol.
Add to 1.0 L of 0.05 N sodium borate. Mix, filter
through 0.45 /i membrane filter, and degas. Add
100 p,l of 2-mercaptoethanol (1+1) and mix. Make up
fresh solution daily unless the reagent solution is
protected from atmospheric oxygen. The solution
can be stored in glass bottles under atmospheric
conditions at 4°C for up to two weeks without
appreciable increases in background fluorescence or
stored under nitrogen for indefinite periods.
Note: Fluoraldehyde (Pierce Chemical), a
commercially formulated OPA reaction solution, may
be substituted for Steps 7.2.2.1 through 7.2.2.3.
7.3 SAMPLE PRESERVATION REAGENTS
7.3.1 Sodium thiosulfate - granular - ACS grade or better (Fisher,
S-446).
7.4 STOCK STANDARD SOLUTION (1.00 jug/mL)
7.4.1 Accurately weigh and dissolve 0.1000 g of pure glyphosate in
1000 mL of deionized water. Larger or smaller volumes may be
used at the convenience of the analyst. If compound purity
is certified at 96% of greater, the weight may be used
without correction to calculate the concentration of the
stock standard.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 Collect samples in glass containers (6.1.1). Conventional sampling
practices (6) are to be followed.
69
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8.2 SAMPLE PRESERVATION - Treatment of samples to remove residual
chlorine will eliminate the possibility of glyphosate losses due to
chlorine during storage. Chlorine is destroyed by adding 100 mg/L
of sodium thiosulfate to the sample.
8.3 SAMPLE STORAGE - Samples should be stored at 4°C away from light and
analyzed within 2 weeks. A preservation study (7) has demonstrated
the stability of glyphosate in frozen samples for up to 18 months.
The analyst should verify appropriate sample holding times
applicable to the sample under study.
9. CALIBRATION
9.1 Establish liquid chromatographic operating conditions indicated in
Table 1.
9.2 Prepare a minimum of three calibration standards of glyphosate by
serial dilution of the stock standard solution in deionized water.
One of the calibration standards should correspond to a glyphosate
concentration near to, but above the MDL. The other concentrations
should comprise the range of concentrations expected for the
samples, or, otherwise, define the working range of the detector.
9.3 Analyze each calibration standard and tabulate peak area against
concentration (in /jg/L) injected. The results may be used to
prepare a calibration curve for glyphosate.
Alternatively, if the ratio of response to concentration (response
factor) is constant over the working range (< 10% relative standard
deviation), linearity through the origin can be assumed and the
average ratio or response factor can be used in place of a
calibration curve.
9.4 The working calibration curve must be verified on each working day
by the measurement of a minimum of two calibration check standards,
one at the beginning and one at the end of the analysis day. These
check standards should be at two different concentration levels to
verify the calibration curve. For extended periods of analysis
(greater than 8 hr), it is strongly recommended that check standards
be interspersed with samples at regular intervals during the course
of the analyses. If the response for the analyte varies from the
predicted response by more than ± 20%, the test must be repeated
using a fresh calibration standard. If the results still do not
agree, generate a new calibration curve.
10. QUALITY CONTROL
10.1 Minimum quality control (QC) requirements are initial demonstration
of laboratory capability, analysis of laboratory reagent blanks,
laboratory fortified matrix samples, laboratory fortified blanks and
QC samples.
70
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10.2 LABORATORY REAGENT BLANKS. Before processing any samples, the
analyst must demonstrate that all glassware and reagent
interferences are under control. Each time a set of samples is
extracted or reagents are changed, a laboratory reagent blank (LRB)
must be analyzed. If within the retention time window of the
analyte of interest the LRB produces a peak that would prevent the
determination of that analyte, determine the source of contamination
and eliminate the interference before processing samples.
10.3 INITIAL DEMONSTRATION OF CAPABILITY
10.3.1 Prepare laboratory fortified blanks (LFBs) at an analyte
concentration of 250 >g/L. With a syringe, add .250 mL of
the stock standard (Section 7.4) to at least four - 100 mL
aliquots of reagent water and analyze each aliquot according
to procedures beginning in Section 11.
10.3.2 The glyphosate recovery (R) values determined in 10.3.1
should be within ±30% of the R values listed in Table 2 for
at least three of four consecutive samples. The relative
standard deviation (Sr) of the mean recovery (R) should be
less than 30%. If the analyte of interest meets the
acceptance criterion, performance is judged acceptable and
sample analysis may begin. For analytes that fail this
criterion, initial demonstration procedures should be
repeated.
10.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples via a
new, unfamiliar method prior to obtaining some experience
with it. It is expected that as laboratory personnel gain
experience with this method the quality of the data will
improve beyond the requirements stated in Section 10.3.2.
10.4 The analyst is permitted to modify HPLC column, HPLC conditions, or
detectors to improve separations or lower analytical costs. Each
time such method modifications are made, the analyst must repeat the
procedures in Section 10.3.
10.5 LABORATORY FORTIFIED BLANKS
10.5.1 The laboratory must analyze at least one laboratory fortified
blank (LFB) sample per sample set (all samples analyzed
within a 24-h period). The fortified concentration of
glyphosate in the LFB should be 10 times the MDL. Calculate
accuracy as percent recovery (R). If R falls outside the
control limits (See Section 10.5.2'.), the analysis is judged
out of control, and the source of the problem must be
identified and resolved before continuing analyses.
71
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10.5.2 Until sufficient data become available from within their own
laboratory, usually a minimum of results from 20 to 30
analyses, the laboratory should assess laboratory performance
against the control limits in Section 10.3.2. When
sufficient internal performance data become available,
develop control limits from the mean percent recovery (R) and
SR of the percent recovery. These data are used to establish
upper and lower control limits as follows:
UPPER CONTROL LIMIT = R + 3SR
LOWER CONTROL LIMIT = X - 3SR
After each five to ten new recovery measurements, new control
limits should be calculated using only the most recent 20 -
30 data points.
10.6 LABORATORY FORTIFIED SAMPLE MATRIX
10.6.1 The laboratory must add a known fortified concentration to a
minimum of 10% of the routine samples or one fortified sample
per set, whichever is greater. The fortified concentration
should not be less then the background concentration of the
original sample. Ideally, the fortified concentration should
be the same as that used for the laboratory fortified blank
(Section 10.5). Over time, samples from all routine samples
sources should be fortified.
10.6.2 Calculate the accuracy as R for the analyte, corrected for
background concentrations measured in the original sample,
and compare these values to the control limits established in
Section 10.5.2 from the analyses of LFBs.
10.6.3 If the recovery of any sample falls outside the designated
range, and the laboratory performance for the analyte is
shown to be in control (Section 10.5), the recovery problem
encountered with the dosed sample is judged to be matrix
related, not system related. The result for the analyte in
the original sample is labeled suspect/matrix to inform the
data user that the results are suspect due to matrix effects.
10.7 QUALITY CONTROL SAMPLES (QCS) - Each quarter the laboratory should
analyze at least one QCS (if available). If criteria provided with
the QCS are not met, corrective action should be taken and
documented.
10.8 The laboratory may adopt additional quality control practices for
use with this method. The specific practices that are most
productive depend upon the needs of the laboratory and the nature of
the samples. For example, field or laboratory duplicates may be
analyzed to assess the precision of the environmental measurements
or field reagent blanks may be used to assess contamination of
samples under site conditions, transportation and storage.
72
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11. PROCEDURE
11.1
SAMPLE CLEANUP - The cleanup procedure for this direct aqueous
injection HPLC method is limited to the filtration procedure
described in Section 11.2.3. Applying only filtration, no
interferences were evidenced in the analysis of tap water, ground
water and municipal effluent. If particular circumstances demand
the use of an alternative cleanup procedure, the analyst must
demonstrate that the recovery of the analyte is within limits
specified by the method.
11.2 ANALYSIS
11.2.1 Table 1 details the recommended HPLC-PCR operating
conditions. An example of the chromatography achieved under
these conditions is shown in Figure 2.
11.2.2 Calibrate the system daily as described in Section 9.
11,2.3 Filter samples using 0.45 \i Acrodisc filters (6.5.2) and
inject 200 [iL of sample into the HPLC-PCR system for
analysis.
11.2.4 Record resulting peak sizes in area units.
11.2.5 If the response for a glyphosate peak in a sample
chromatogram exceeds the working calibration range, dilute
the sample with reagent water and reanalyze.
11.2.6 Some changes in analyte retention time may be observed
following the analysis of matrices with moderate to high
ionic strength. The equilibration of the analytical column
with the mobile phase will minimize this problem.
NOTE: The use of alternative analytical columns is
mentioned in Section 6.7.2.
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. If
the retention time of an unknown compound corresponds, within
limits (11.3.2), to the retention time of the standard, then
identification is considered positive.
11.3.2 The width of the retention time window used to make
identification should be based upon measurements of actual
retention time variations of standards over the course of a
day. Three times the standard deviation in retention time
can be used to calculate a suggested window size for a
compound. However, the experience of the analyst should
weigh heavily in the interpretation of chromatograms.
73
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11.3.3 Identification requires expert judgement when sample
components are not resolved chromatographically. When peaks
obviously represent more then one sample component (i.e.,
broadened peak with shoulder(s) or valley between two or more
maxima), or any time doubt exists over the identification of
a peak in a chromatogram, appropriate confirmatory techniques
such as use of an alternative detector which operates on a
physical/chemical principle different from that originally
used, e.g., mass spectrometry, or the use of an alternative
separation technology, e.g., anion exchange chromatography,
must be employed.
12. CALCULATIONS
12.1
Determine the concentration (C) of glyphosate in the sample by
direct comparison with the calibration curve described in Section 9,
or alternatively, by means of the equation below derived from the
calibration data.
A_
RF
where:
12.2
A - Area of glyphosate peak in sample
RF = Response factor derived from calibration data
For samples processed as part of a set where laboratory fortified
blank and/or laboratory fortified matrix recoveries fall outside
control limits in Section 10.5 and 10.6, data for the affected
samples must be labeled as suspect.
13. METHOD PERFORMANCE
13.1 METHOD DETECTION LIMITS - The method detection limit (MDL) is
defined as the minimum concentration of a substance that can be
measured and reported with 99% confidence that the value is above
the background level (2). The concentrations listed in Table 1 were
obtained using reagent water, ground water and dechlorinated tap
water.
13.2 Single-laboratory precision and accuracy results at several
concentrations in drinking water matrices are presented in Table 2.
14. REFERENCES
1. Bashe, W. J., T. V. Baker, "Analysis of Glyphosate in Drinking Water
by Direct Aqueous Injection HPLC with Post Column Derivatization," in
preparation, Technology Applications, Inc., 1988.
74
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2. 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.
3. Cowell, J. E., "Analytical Residue Method for N-phosphonomethyl
Glycine and Aminomethyl phosphonic Acid in Environmental Water,"
Monsanto Company, Method Number 86-63-1, 1987.
4. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for
Preparation of Sample Containers and for Preservation, "American
Society for Testing and Materials, Philadelphia, PA, p. 679, 1980.
5. "OSHA Safety and Health Standards, General Industry," (29CRF1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised,
January 1976).
6. ASTM Annual Book of Standards, Part II, Volume 11.01, D3370-82,
"Standard Practice for Sampling Water", American Society for Testing
and Materials, Philadelphia, PA, 1986.
7. Cowell, J. E., "Storage Stability of Glyphosate in Environmental
Water," Monsanto Company, 1988.
75
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TABLE 1. ANALYTICAL CONDITIONS AND METHOD DETECTION LIMITS FOR 6LYPHOSATE
Matrix
1
Retention Time (min)
RW
GW
TW-T
Conditions:
Column:
13.5
13.7
11.8
250 x 4 mm,
6.00
8.99
5.99
Bio-Rad, Aminex A-9 (Specification
as per Subsection 6.7) thermostatted at 65°C.
0.005 M KH2PO, - water:methanol (24:1) buffered
at pH = 1.9 (Section 7).
Isocratic
0.5 mL/min.
200 /iL
Calcium Hypochlorite flow rate = 0.5 mL/min.,
OPA solution flow rate = 0.5 mL/min., reactor
temperature = 38°C.
Excitation wavelength at 340 nm and detection
emission at 455 nm.
1 RW - reagent water, GW = ground water, TW-T = tap water "spiked after
dechlorination treatment.
2 All MDL data were generated from spiked samples at 25 /zg/L.
Mobile Phase:
Elution Mode:
Flow Rate:
Injection Volume:
PCR:
Detector:
76
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TABLE 2. RECOVERY OF GLYPHOSATE IN REPRESENTATIVE DRINKING WATER MATRICES
Fortified
Concentration
(M9/L)
2500
700
250
25
Matrix1
RW
GW
TW-T
RW
GW
TW-T
RW
GW
TW-T
RW
GW
TW-T
Number
of
Replicates
8
8
8
8
8
8
8
8
8
8
8
8
Mean
Recovery
%
102
103
99.2
101
98.7
96.4
95.6
101
98.0
96.0
96.0
108
Relative
Standard
Deviation
%
1.96
1.25
1.74
2.65
2.01
1.80
3.91
1.77
1.75
9.07
12.3
6.57
1RW = Reagent water, GW = Ground water, TW-T = Tap water spiked after
. dechlorination treatment
77
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HPLC
Pump
'HvpochlorUt^
Reservoir
Autolnjector
Fluorescence
Detector
mm*m
Nelson
Analytical
0»U
S/ste«
Computer
Quint1tat1on
Figure 1. HPLC, Post-Column Reactor System
78
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iiiinimmrimmnmii
79
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METHOD 548. DETERMINATION OF ENDOTHALL IN DRINKING WATER BY
AQUEOUS DERIVATIZATION, LIQUID-SOLID EXTRACTION, AND
GAS CHROMATOGRAPHY WITH ELECTRON-CAPTURE DETECTION
July 1990
J. W. Hodgeson
Merlin Bicking (Twin City Testing, St. Paul, Minnesota)
W. J. Bashe (Technology Applications, Incorporated)
David Becker (Technology Applications, Incorporated)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
81
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METHOD 548
DETERMINATION OF ENDOTHALL IN DRINKING WATER BY AQUEOUS DERIVATIZATION,
LIQUID-SOLID EXTRACTION, AND GAS CHROMATOGRAPHY WITH ELECTRON-CAPTURE DETECTION
1.
SCOPE AND APPLICATION
1.1
This method covers the determination of endothall in drinking water
sources and finished drinking water. The following analyte can be
determined by this method:
Analvte
Endothall
Chemical Abstract Services
Registry Number
145-73-3
12 This is a gas chromatographic (GC) method applicable to the
determination of the compound listed above. When this method is
used to analyze unfamiliar samples, compound identification should
be supported by at least one additional qualitative technique. A
gas chromatograph/mass spectrometer (GC/MS) may be used for the
qualitative confirmation of results for endothall using the extract
produced by this method.
1 3 The method detection limit1 (MDL, defined in Section 13) for
endothall is listed in Table 1. The MDL for a specific sample may
differ from the listed value, depending upon the nature of
interferences in the sample matrix and the amount of sample used in
the procedure.
1.4 The endothall-pentafluorophenylhydrazine derivative employed for
chromatographic detection is not available commercially. Thus, this
method employs procedural standards, in which endothall calibration
solutions (9.2.1) are processed through the analysis procedure
(11.2).
1 5 This method is restricted to use by or under the supervision of
analysts experienced in the use of gas chromatography and in the
interpretation of gas chromatograms. Each analyst must demonstrate
the ability to generate acceptable results with this method using
the procedure described in Section 11.
SUMMARY OF METHOD
21 A 5.0 mL volume of liquid sample is placed in a Kuderna-Danish tube
and the volume is reduced to less than 0.5 mL using a heating block.
The tube is charged with glacial acetic acid and sodium acetate,
followed by a solution of the derivatization reagent, penta-
fluorophenylhydrazine (PFPH), in glacial acetic acid. After heating
at 150°C for 90 minutes the derivative is extracted by a solid
82
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sorbent from the reaction solution, followed by elution with 5 0 ml
of methyl-tert-butyl ether (MTBE). The MTBE extract is analyzed by
gas chromatography with electron capture detection (6C/ECD).
3. DEFINITIONS
3.1 INTERNAL STANDARD - A pure analyte(s) added to a solution in known
amount(s) and used to measure the relative responses of other method
analytes and surrogates that are components of the same solution.
The internal standard must be analyte that is not a sample
component.
3.2 SURROGATE ANALYTE - A pure analyte(s), which is extremely unlikely
to be found in any sample, and which is added to a sample aliquot in
known amount(s) before extraction and is measured with the same
procedures used to measure other sample components. The purpose of
a surrogate analyte is to monitor method performance with each
sample.
3.3 LABORATORY DUPLICATES (LD1 and LD2) - Two sample aliquots taken in
the analytical laboratory and analyzed separately with identical
procedures. Analyses of LD1 and LD2 give a measure of the precision
associated with laboratory procedures, but not with sample
collection, preservation, or storage procedures.
3.4 FIELD DUPLICATES (FD1 and FD2) - Two separate samples collected at
the same time and place under identical circumstances and treated
exactly the same throughout field and laboratory procedures.
Analyses of FD1 and FD2 give a measure of the precision, associated
with sample collection, preservation and storage, as well as with
laboratory procedures.
3.5 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water that is
treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates
that are used with other samples. The LRB is used to determine if
method analytes or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.6 FIELD REAGENT BLANK (FRB) - Reagent water placed in a sample
container in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB
is to determine if method analytes or other interferences are
present in the field environment.
3.7 LABORATORY PERFORMANCE CHECK SOLUTION (LPC) - A solution of method
.. analytes, surrogate compounds, and internal standards used to
evaluate the performance of the instrument system with respect to a
defined set of method criteria.
83
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3.8 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise
measurements at the required method detection limit.
3.9 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) - An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3.10 STOCK STANDARD SOLUTION - A concentrated solution containing a
single certified standard that is a method analyte, or a
concentrated solution of a single analyte prepared in the laboratory
with an assayed reference compound. Stock standard solutions are
used to prepare primary dilution standards.
3.11 PRIMARY DILUTION STANDARD SOLUTION - A solution of several analytes
prepared in the laboratory from stock standard solutions and diluted
as needed to prepare calibration solutions and other needed analyte
solutions.
3.12 CALIBRATION STANDARD (CAL) - A solution prepared from the primary
dilution standard solution and stock standard solutions of the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.13 QUALITY CONTROL SAMPLE (QCS) - A sample matrix containing method
analytes or a solution of method analytes in a water miscible
solvent which is used to fortify reagent water or environmental
samples. The QCS is obtained from a source external to the
laboratory, and is used to check laboratory performance with
externally prepared test materials.
4. INTERFERENCES
4.1 Method interference may be caused by contaminants in solvents,
reagents, glassware, and other sample processing hardware that lead
to discrete artifacts and/or elevated baselines in the
chromatograms. All of these materials must be routinely
demonstrated to be free from interferences under the conditions of
the analysis by running laboratory reagent blanks as described in
Section 10.2.
4.1.1 Glassware must be scrupulously clean2. Clean all glassware
as soon as possible after use by rinsing with the last
84
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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 409C for several hours before use
Solvent rinses with methanol may be substituted for the oven
heating. After drying and cooling, glassware should be
stored in a clean environment to prevent any accumulation of
dust or other contaminants.
4.1.2 The use of high purity reagents and solvents is absolutely
necessary to minimize interference problems. Purification of
solvents by distillation in all-glass systems immediately
prior to use is highly recommended.
4.2 Matrix interferences may be caused by contaminants that are
contracted from the sample. The extent of matrix interferences
will vary considerably from source to source, depending upon the
nature and diversity of the matrix being sampled. If significant
interferences occur in subsequent samples, some additional cleanup
may be necessary to achieve the MDL listed in Table I.
4.3 The extent of interferences that may be encountered using gas
chromatographic techniques has not been fully assessed. Although
the GC conditions described allow for a unique resolution of the
specific compound covered by this method, other matrix components
may interfere.
5. SAFETY
5.1 The toxicity or carcinogen!city of each reagent used in this method
«hn,,inH h ? P»lec;sely defined; however, each chemical compound
should be treated as a potential health hazard. From this
viewpoint exposure to these chemicals must be reduced to the lowest
possible level by whatever means available. The laboratory is
responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemical specified in
ctnnir i K A rferenc.e file of material data handling sheets
should also be made available to all personnel involved in the
chemical analysis. Additionally references to laboratory safety are
aVctl I 3D I 6 *
6. APPARATUS AND MATERIALS
6.1 SAMPLING EQUIPMENT (for discrete or composite sampling).
6.1.1 Grab sample bottle - Amber glass fitted with screw caps lined
with Teflon. If amber bottles are not available, protect
samples from light. The container must be washed, rinsed
with methanol, and dried before use to minimize contamination
85
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6.2 GLASSWARE
6.2.1 Volumetric flasks - 5 mL, 25 ml
6.2.2 Vials - glass, 1 mL, with Teflon-lined caps
6.2.3 Glass syringes, 250 /*L, 500 p.1
6.2.4 Pipets - 1 ml, 4 ml
6.3 BALANCE - analytical, capable of accurately weighing 0.0001 g.
SOLID SORBENT CARTRIDGES - C-18
6.4
6.5
Vacuum manifold for extraction using solid sorbent cartridges -
Supelco 5-7030 or equivalent
6.6 Kuderna-Danish (K-D) concentrator tubes - 10 or 25 mL graduated
6.6.1 Snyder column, Kuderna-Danish -2- ball micro
Tube heater for 25 mL K-D tubes
6.7
6.8
6.9
Boiling chips - carborundum, #12 granules Heat at 400°C for 30
minutes prior to use. Cool and stored in dessicator.
Gas chromatographic system capable of temperature programming
6.9.1 Autosampler
6.9.2 Electron capture detector
6.9.3 Column 1: Supelco SPB-5, 0.25 mm x 30 m or equivalent
Column 2: J&W DB-1, 0.32 mm x 30 mm or equivalent
694 Strip-chart recorder compatible with detector. Use of a data
system with printer for measuring and recording peak areas
and retention times is recommended.
7. REAGENTS AND SOLUTIONS
7 1 REAGENT WATER - reagent water is defined as a water of very high
purity, equivalent to distilled in glass solvents
7.2 PENTAFLUOROPHENYLHYDRAZINE (PFPH) - Aldrich
7.3 SODIUM ACETATE - anhydrous
7.4 SODIUM THIOSULFATE
86
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7.5 ACETIC ACID - glacial
7.6 METHYL-TERT-BUTYL ETHER (MTBE) - distilled in glass
7.7 ENDOTHALL-PFPH DERIVATIVE - See Appendix for synthesis procedure
7.8 ENDOSULFAN I
7.9 ENDOTHALL, monohydrate
7.10 STOCK STANDARD SOLUTIONS
7.10.1 Endothall - 10 //g/mL in reagent water
7.10.2 Endothall - 50 //g/mL in reagent water
7.10.3 Stock standard solutions must be replaced after six months
or sooner, if comparison with check standards indicates a
problem.
7.11 REACTION SOLUTIONS
7.11.1 PFPH solution - 4 mg/mL in glacial acetic acid
7'n'2 i™rrnal standard stock solution - 10 Mg/mL endosulfan I in
MTBE
8- SAMPLE COLLECTION. PRESERVATION. AND HANDLING
8.1 Grab samples must be collected in glass containers. Conventional
sampling practices should be followed, except that the bottle must
cSLfn Prewashed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of Tygon tubing and other'potential
sources of contamination.
8.2 The samples must be iced or refrigerated at 4°C from the time of
Sl^il^.""^!^1.^.1?**10"-. The ™alyte measured here is not
to light and
8.3 Some samples are likely to be biologically active and the stability
of samples upon storage will be different for each matrix. All
samples should be derivatized within 7 days of collection and
analysis completed within 1 day of derivatization. If these
criteria are not met, the analyst must demonstrate the stability of
the stored sample by performing suitable holding time studies
87
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9. CALIBRATION
9.1 Establish gas chromatographic operating parameters to Produce a
retention time equivalent to that indicated in Table 1. The
chromatographic system can be calibrated using the internal standard
technique (Section 9.2).
911 Due to the complex nature of the sample chromatogram, the
" ' analyst should periodically inject a solution containing only
pure endothall-PFPH (See Appendix) to verify the retention
time of the derivative.
9.2 INTERNAL STANDARD CALIBRATION PROCEDURE:
921 Use 250 and 500 /xL syringes to add sufficient quantities of
7 10 1 or 7.10.2 stock solutions to reagent water in 25 mL
volumetric flasks to produce endothall standard solutions at
the following concentrations in /*g/L: 500 (250 /*L of 7.1U.J
stock), 200 (100 ML of 7.10.2 stock), 100 (50 /*L of 7.10.2
stock) and 50 (125 /iL of 7.10.1 stock).
922 Process each standard as per Section 11.2. The internal
standard is added as described in Section 11.2.7. It is
recommended that triplicate samples of each standard be
processed.
9.2.3 Before analyzing matrix samples, the analyst must process a
series of calibration standards to validate elution patterns
and the absence of interferences from reagents.
924 Analyze each calibration standard and tabulate the ratio of
the area of the endothall-PFPH derivative peak versus that of
the internal standard against endothall concentration. The
results may be used to prepare a calibration curve for
endothall.
925 The working calibration curve must be verified on each
working day by processing and analyzing one or more
calibration standards. If the response varies from the
previous response by more than ± 20%, the test must be
repeated using a fresh calibration standard. Should the
retest fail, a new calibration curve must be generated.
10. QUALITY CONTROL
10 1 Each laboratory that uses this method is required to operate a
formal quality control (QC) program. The minimum QC requirements
are initial demonstration of laboratory capability, analysis of
laboratory reagent blanks, laboratory fortified blanks, laboratory
fortified matrix samples and QC check standards.
88
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10.2 LABORATORY REAGENT BLANKS. Before processing any samples, the
analyst must demonstrate that all glassware and reagent
interferences are under control. Each time a set of samples is
analyzed or reagents are changed, a method blank must be analyzed.
For this method, the method blank is filtered reagent water. If
within the retention time window of an anafyte of interest the
method blank produces a peak which prevents the measurement of that
analyte, determine the source of contamination and eliminate the
interference before processing samples.
10.3 INITIAL DEMONSTRATION OF CAPABILITY
10.3.1 Select a representative fortified concentration (about 10
times MDL) for endothall. Prepare a concentrate (in reagent
water) containing the analyte at 10 times the selected
concentration. Using a pipet, add 1.00 mL of the concentrate
to each of at least four 10 mL aliquots of reagent water and
analyze each aliquot according to procedures beginning in
Section 11.
10.3.2 The recovery value should for at least three out of four
consecutively analyzed samples fall in the range of R + 30%
(or within R + 3SR, if broader) using the values for R and S
for reagent water (Table 2). If the recovery value meets the
acceptance criteria, performance is acceptable and sample
analysis may begin. If the recovery value fails these
criteria, initial demonstration of capability should be
repeated.
10.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples by a
new, unfamiliar method prior to evidencing a basal level of
skill at performing the technique. It is expected that as
laboratory personnel gain experience with this method the
quality of the data will improve beyond the requirements
stated in Section 10.3.2.
10.4 The analyst is permitted to modify GC columns, GC conditions or
detectors to improve separations or lower analytical costs. Each
time such method modifications are made, the analyst must repeat the
procedures in Section 10.3.
10.5 Assessing the Internal Standard - In using the IS calibration
procedure, the analyst is expected to monitor the IS response (peak
area or peak height) of all samples during each analysis day. The
IS response for any sample chromatogram should not deviate from the
calibration standard IS response by more than 30%.
10.5.1 If a deviation of greater than 30% is encountered for a
sample, reinject the extract.
89
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10.5.1.1 If acceptable IS response is achieved for the re-
injected extract, then report the results for that
sample.
10.5.1.2 If a deviation of greater than 30% is obtained for
the reinjected extract, analysis of the sample
should be repeated beginning with Section 11,
provided the sample is still available. Otherwise,
report results obtained from the reinjected extract,
but annotate as suspect.
10.5.2 If consecutive samples fail the IS response acceptance
criterion, immediately analyze a calibration check standard.
10.5.2.1 If the check standard provides a response factor
(RF) within 20% of the predicated value, then
follow procedures itemized in Section 10.5.1 for
each sample failing the IS response criterion.
10.5.2.2 If the check standard provides a response factor
(RF) with deviates more than 20% of the predicted
value, then the analyst must recalibrate, as
specified in Section 9.2.
10.6 ASSESSING LABORATORY PERFORMANCE
10.6.1 The laboratory must analyze at least one LFB per sample set
(all samples analyzed within a 24 hour period). The
fortifying concentration in the LFB should be 10 times the
MDL. Calculate accuracy as percent recovery (X,-). If the
recovery falls outside the control limits (See Section
10.6.2), the system is judged out of control, and the source
of the problem must be identified and resolved before
continuing analyses.
10.6.2 Until sufficient LFB data become available, usually a minimum
of results from 20 to 30 analyses, the laboratory should
assess its performance against the control limits described in
Section 10.3.2. When sufficient laboratory performance data
becomes available, develop control limits from the mean
percent recovery (X) and standard deviation (S) of the percent
recovery. These data are used to establish upper and lower
control limits as follows:
Upper Control Limit = X + 3S
Lower Control Limit = X - 3S
After each group of five to ten new recovery measurements,
control limits should be recalculated using only the most
recent 20 to 30 data points.
90
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10.6.3 It is recommended that the laboratory periodically determine
.and document its detection limit capabilities for endothall.
10.6.4 Each quarter the laboratory should analyze QCS (if available)
If criteria provided with the QCS are not met, corrective
.action should be taken and documented.
10.7 ASSESSING ANALYTE RECOVERY
10.7.1 The laboratory must add a known fortified concentration to a
minimum of 10% of the routine samples or one fortified sample
per set, whichever is greater. The fortified concentration
should not be less than the background concentration of the
sample selected for spiking. The fortified concentration
should be the same as that used for the LFB (Section 10.6).
Over time, samples from all routine sample sources should be
fortified.
10.7.2 Calculate the percent recovery (Rf) for endothall, corrected
for background concentrations measured in the unfortified
sample, and compare these values to the control limits
established in Section 10.6.2 for the analyses of LFBs.
10.7.3 If the recovery falls outside the designated range, and the
laboratory performance for that sample set is shown to be in
control (Section 10.6), the recovery problem encountered with
the dosed 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.
11. PROCEDURE
11.1 CLEANUP AND SEPARATION - Cleanup procedures may not be necessary for
a relatively clean sample matrix. If particular circumstances
demand the use of an alternative cleanup procedure, the analyst must
demonstrate that the recovery of endothall is within the limits
specified by the method.
11.1.1 If the sample is not clean, or the complexity is unknown, the
entire sample should be centrifuged at 2500 rpm for 10
minutes. The supernatant is decanted from the centrifuge
bottle and passed through glass fiber filter paper into a
container which can be tightly sealed.
11.1.2 Store all samples at 4°C.
11.2 SAMPLE EXTRACTION AND ANALYSIS
11.2.1 Measure out a 5.0 mL aliquot of the sample and place it in a
10 or 25 mL K-D tube. Add boiling chips.
91
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11.2.2 Place on tube heater at maximum setting and concentrate
sample to less than 0.5 ml.
11.2.3 Add 4 ml glacial acetic acid, 200 mg sodium acetate and 1 ml
of glacial acetic acid containing 4 mg PFPH. Use glass
stirring rod to break-up the sodium acetate solid. Place a
Micro Snyder column on each K-D tube.
11.2.4 Heat at 150°C for 90 minutes.
11.2.5 Dilute the reaction mixture with reagent water and decant
into a 50 ml beaker or flask. Wash the K-D tube and residue
with aliquots of reagent water and add to the beaker until
the total aqueous volume is 40-45 ml.
11.2.6 Assemble the vacuum manifold. Rinse the solid sorbent
cartridge by passing 5 ml of reagent water though the
cartridge. Discard the water. Extract the aqueous sample
from 11.2.5 by passing the sample through the solid sorbent
cartridge at a rate of 5-6 ml per minute.
11.2.7 Wash the cartridge with 5 ml reagent water. Elute the
cartridge with two 2 ml aliquots of MTBE. Combine the
eluates with .05 ml of the internal standard stock solution
(7.11.2) and dilute to 5 ml in a volumetric flask with MTBE.
11.2.8 Analyze the eluates by 6C/ECD using conditions described in
Table 1. This table includes the retention time and MDL that
were obtained under these conditions. Sample chromatograms
of an endothall standard and a LRB both with internal
standard are represented in Figures 1 and 2. Other columns,
chromatographic conditions, or detectors may be used if the
requirements of Section 10.3 are met.
11.3 IDENTIFICATION OF THE ANALYTE
11.3.1 Identify endothall by comparison of its retention time to the
retention time of a reference chromatogram. If the retention
time of the unknown compound corresponds, within limits, to
the retention time of a standard endothall, then
identification is considered positive. However, positive
identifications should be confirmed by retention time
comparisons on the second GC column, or by using 6C/MS.
11.3.2 The width of the retention time window used to make
identifications should be based upon measurements of actual
retention time variations of standards over the course of a
day. Three times the standard deviation of a retention time
can be used to calculate a suggested window size for a
compound. However, the experience of the analyst should
weigh heavily in the interpretation of chromatograms.
92
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11.3.3 Identification requires expert judgement when sample
components are not resolved chromatographically, that is,
when GC peaks from interferences are present. Any time doubt
1 exists over the identification of the endothall peak,
appropriate techniques such as use of an alternative detector
which operates on a chemical/physical principle different
from that originally used, e.g., mass spectrometry, or the
use of a second chromatography column must be used.
11.4 If the peak area exceeds the linear range of the calibration curve,
a smaller sample volume should be used. Alternatively, the final
solution may be diluted with MTBE and reanalyzed.
11.5 If the peak area measurement is prevented by the presence of
interferences, further cleanup is required.
12. CALCULATIONS
12.1 Determine the peak area ratio for endothall in the injected sample.
12.1.1 Calculate the concentration of endothall injected using the
calibration curve in Section 9.2. The concentration in a
liquid sample can be calculated from Equation 1:
Equation 1 Concentration, /zg/L = (AUVF)
(VS)
where:
A = Concentration of endothall in extract, in jug/L
VF = Final volume of MTBE, in ml
VS = Sample volume, in mL
12.2 Report results as micrograms per liter. When duplicate and
fortified samples are analyzed, report all data obtained with the
sample results.
12.3 For samples processed as part of a set where the laboratory
fortified sample recovery falls outside of the control limits
established in Section 10.6, data must be labeled as suspect.
13. METHOD PERFORMANCE
13.1 METHOD DETECTION LIMITS - The MDL is defined as the minimum
concentration of a substance that can be measured and reported with
99% confidence that the value is above the background level. The
estimated MDL concentration listed in Table 1 was obtained using
reagent water. Similar results were achieved using representative
matrices.
93
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13.2 This method has not been tested for linearity of recovery from
fortified reagent water.
13.3 In a single laboratory using dechlorinated tap and reagent water
fortified matrices, the average recoveries presented in Table 2 were
obtained. The standard deviation of the percent recovery is also
included in Table 2.
14. REFERENCES
1. 40 CFR Part 136, Appendix B.
2. ASTM Annual Book of Standards, Part 31, D3694-78. "Standard
Practices for Preparation of Sample Containers and for Preservation
of Organic Constituents", American Society for Testing and
Materials, Philadelphia, PA.
94
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TABLE 1. GAS CHROMATGGRAPHY CONDITIONS AND METHOD DETECTION LIMITS
Analvte Ret. Time (min.) MDL (ttg/L)
Endothall 42.3 11.5
GC conditions: 0.25 mm x 30 m SPB-5 column; 2 fj.1
injection; hold one minute at 60°C, program to
300°C at 4°C/minute, hold at 300°C for 15 minutes.
95
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TABLE 2. SINGLE OPERATOR ACCURACY AND PRECISION
Analyte
Matrix
Type
Average
Percent
Recovery
Standard
Deviation
(percent)
Fortified
Cone.
(M9/L)
Number
of
Analyses
Endothall
Reagent
Water
120
108
25.3
15.3
15
150
8
8
Dechlorinated
Tap 84.0
Water 94.0
13.8
13.3
15
150
8
8
100 mg/L sodium thiosulfate (Na2S203) added to prior to fortifying
with endothall
96
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APPENDIX
Preparation of Endothall-Pentafluorophenylhydrazine
1. Prepare solution A of endothall by dissolving 0.204 g of endothall
monohydrate (1.0 mmol) in 14 ml of methylene chloride and 3.6 ml of dry
tetrahydrofuran (THF).
2. Prepare solution B of dicyclohexylcarbodiimide (DCC) by dissolving 0.206 g
(1.0 mmol) in 3.4 ml of dry THF.
3. Mix solutions A and B and cover with a watchglass. (Note: a white
precipitate will form in 3 to 5 minutes).
4. Gently stir the mixture from Step 3 with a magnetic stirrer for 4.5 hours
at ambient temperature.
5. Prepare solution C by dissolving 0.206 g of DCC and 0.198 g of
pentafluorophenylhydrazine (PFPH) in 18 ml of dry THF.
6. Mix solution C with the mixture from step 4, cover with a watchglass and
stir the mixture overnight (16 hours) at ambient temperature.
7. Filter the mixture and dry the filtrate under reduced pressure to yield a
beige powder.
8. Recrystallize the beige powder with 20 ml of warm (40°C) methanol: H20 (8:2
v/v).
9. Filter the solution from Step 8 to remove the insoluble material.
10. Allow the filtrate from Step 9 to cool to room temperature. A precipitate
will form immediately upon cooling.
11. Filter and wash the precipitate formed in Step 10 with two 1 ml portions of
cold methanol: H20 (8:2). Save the filtrate.
12. Allow the filtrate from Step 11 to stand overnight covered with a
watchglass at ambient temperature. A precipitate will form on standing.
13. Filter and wash the precipitate from Step 12 with two 1 mL portions of cold
methanol: H20 (8:2).
14. Recrystallize the off white precipitate from Step 13 with 20 ml of warm
methanol: H20 (8:2). Filter the warm solution and allow the filtrate to
cool, producing a white, crystalline precipitate.
15. Filter the white precipitate from Step 14, wash with two 1 ml portions of
cold methanol: H20 (8:2) and dry under vacuum.
97
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16. Determine the melting point of the precipitate of Step 15. The melting
point of the endothall-pentafluorophenylhydrazine derivative is 201.0°C.
If the melting point of the precipitate is not within 1.0 C of this melting
point, recrystallize again as per Steps 14 - 15.
98
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METHOD 549. DETERMINATION OF DIQUAT AND PARAQUAT IN, DRINKING WATER
BY LIQUID-SOLID EXTRACTION AND HPLC WITH ULTRAVIOLET DETECTION
July 1990
J. W. Hodgeson
W. J. Bashe (Technology Applications Inc.)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
101
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METHOD 549
DETERMINATION OF DIQUAT AND PARAQUAT IN DRINKING WATER
BY LIQUID-SOLID EXTRACTION AND HPLC WITH ULTRAVIOLET DETECTION
1.
SCOPE AND APPLICATION
1.1
'his is a high performance liquid chromatography (HPLC) method for
;he determination of diquat (l,r-ethylene-2,2'-bipyridilium
libromide salt) and paraquat (l.l'-dimethyl-M1- bipyridilium
i • _ i. -i _ : _i ^ _ _ T 4. \ ~-. J «-•!** I* •!***•« *.i*\4- sM/t t*f\it^r+f\f> ^mf\ -Pi n 1 O noH Hv*T nl/1 Flfl
Thi:
the
dibromide , _._ . . . . . . . .
dichloride salt) in drinking water sources and finished drinking
water (1,2).
Analvtes
Diquat
Paraquat
Chemistry Abstract Services
Registry Number
85-00-7
1910-42-5
1.2
1.3
1.4
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.
The method detection limits (MDL, defined in Section 13) (3) for
diquat and paraquat are listed in Table 1. The MDLs for a specific
sample may differ from those listed.
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 Section 10.3.
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 Cg solid
sorbent cartridge which has been specially prepared for the
reversed-phase, ion-pair mode. The 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).
2.2 Analysis of diquat and paraquat is complicated by their ionic
nature. All sources of adsorption, i.e. glassware, should be
102
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avoided when possible or 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 that is
treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates
that are used with other samples. The LRB is used to determine if
method analytes or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.2 FIELD REAGENT BLANK (FRB) - Reagent water placed in a sample
container in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB
is to determine if method analytes or other interferences are
present in the field environment.
3.3 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water to
which known quantities of the method analytes are added in "the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise
measurements at the required method detection limit.
3.4 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) - An aliquot of an
environmental sample to which known quantities of the method
analytes are added in the laboratory. The LFM is analyzed exactly
like a sample, and its purpose is to determine whether the sample
, matrix contributes bias to the analytical results. The background
concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM
corrected for background concentrations.
3.5 STOCK STANDARD SOLUTION - A concentrated solution containing a
single certified standard that is a method analyte, or a
concentrated solution of a single analyte prepared in the laboratory
with an assayed reference compound. Stock standard solutions are
used to prepare primary dilution standards.
3.6 PRIMARY DILUTION STANDARD SOLUTION — A solution of several analytes
prepared in the laboratory from stock standard solutions and diluted
as needed to prepare calibration solutions and other needed analyte
solutions.
3.7 CALIBRATION STANDARD (CAL) - A solution prepared from the primary
dilution standard solution and stock standard solutions of the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
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4.
3.8 QUALITY CONTROL SAMPLE (QCS) - A sample matrix containing method
analytes or a solution of method analytes in a water miscible
solvent which is used to fortify reagent water or environmental
samples. The QCS is obtained from a source external to the
laboratory, and is used to check laboratory performance with
externally prepared test materials.
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 Section 10.2.
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 Section 4.1.1 should be followed. Upon drying,
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.
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.
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.
SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined. Each chemical compound should be
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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.
6. APPARATUS AND EQUIPMENT
6.1 SAMPLING EQUIPMENT, discrete or composite sampling.
6.1.1 Grab sample bottle - Amber polyvinylchloride (PVC) high
;.:.. density, one-liter, 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.
6.5.2 Manual injector or automatic injector, capable of delivering
200 ML
6.5.3 Analytical column
; 6.5.3.1 Hamilton PRP-1, (5 #m, 150 mm x 4.1 mm), or
equivalent
6.5.3.2 Guard column, C8 packing
6.5.3.3 Column Oven (Fiatron, Model CH-30 and
controller, Model TC-50, or equivalent)
6.5.4 Photodiode array detector (LKB 2140 Rapid Spectral Detector
or equivalent)
6.5.5 Data system - Use of a data system to report retention times
and peak areas is recommended but not required.
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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 Vacuum pump, 100 VAC, capable of maintaining a vacuum of
8-10 mm of Hg.
6.6.4 Membrane Filters, 0.45 urn pore-size, 47 mm diameter, Nylon
7. REAGENTS AND CONSUMABLES
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 Section 10 are met.
7.2 METHANOL - HPLC grade or higher purity
7.3 ORTHOPHOSPHORIC ACID, 85% (w/v) - reagent grade
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.
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7.14.2 Conditioning solution B. Dissolve 10.0 g of 1-
hexanesulfonic 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
500 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 Cartridge eluting solution. Add 13.,5 mL of orthophosphoric
acid and 10.3 ml of diethyl amine 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-hexanesulfonic
acid in 15 ml of the cartridge eluting solution and dilute
to 25 ml in a volumetric flask with cartridge eluting
solution.
7.15 STOCK STANDARD SOLUTIONS
7.15.1 Diquat dibromide
Paraquat dichloride
7.15.2 Stock diquat and paraquat solutions (1000 mg/L). Dry diquat
and paraquat salts in an oven at 110°C for 3 hours. 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 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 (Section
7.15.2) were taken to be diquat dibromide, monohydrate and
paraquat dichloride, tetrahydrate (5). The drying procedure
described in Section 7.15.2 will provide these hydration
levels, regardless of formulae referenced by manufacturers.
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-hexahesulfonic acid, sodium salt. Mix and
dilute with deionized water to a final volume of 1 L.
8. SAMPLE 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
107
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9.
must be free as possible of adsorption sites which might extract the
sample.
8.2 The samples must be iced or refrigerated at 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 su'lfuric acid to
a pH = 2 in order to prevent adsorption of method analytes by the
humectant material.
8.4 Sample storage stability may depend on the matrix tested. Storage
stability of representative drinking water matrices have been listed
in Table 3. All samples must be extracted within 7 days of
collection. Extracts must be analyzed within 21 days of extraction
(1). If these criteria are not met, the analyst must demonstrate
the stability of the stored sample by performing suitable holding
times studies.
CALIBRATION
9.1
Establish HPLC operating conditions indicated in Table 1. The
chromatographic system can be calibrated using the external standard
technique.
9.2 In order to closely match calibration standards to samples, process
standards by the following method: Using C8 cartridges conditioned
according to Section 11.2.1, pass 250 ml of reagent water through
the cartridge and discard the water. Dry the cartridge by passing
5 ml of methanol through the cartridge. Discard the methanol. Pass
4.0 ml of the cartridge eluting solution through the cartridge and
catch in a 5 ml silanized volumetric flask. Fortify the eluted
solution with 100 /iL of the ion-pair concentrate and with 500 /iL of
the stock standard and dilute to the mark with cartridge 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.
9.3 Analyze a minimum of three calibration standards prepared by the
procedure described in Section 9.2 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 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 concentration injected. The results may be used to prepare
calibration curves for diquat and paraquat.
108
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9,4 The working calibration curve must be verified on each working day
by measurement of a minimum of two calibration check standards, one
at the beginning and one at the end of the analysis day. These
check standards should be at two different concentration levels to
verify the calibration curve. For extended periods of analysis
(greater than 8 hr), it is strongly recommended that check standards
be interspersed with samples at regular intervals during the course
of the analyses. If the response for 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.
10. QUALITY CONTROL
10.1 Minimum quality control (QC) requirements are initial demonstration
of laboratory capability, analysis of laboratory reagent blanks,
laboratory fortified matrix samples, and laboratory fortified
blanks. The laboratory must maintain records to document the
quality of the data generated. Additional quality control practices
are recommended.
10.2 LABORATORY REAGENT BLANKS (LRB) - Before processing any samples, the
analyst must analyze a 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 (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.
10.3 INITIAL DEMONSTRATION OF CAPABILITY
10.3.1 Prepare laboratory fortified blanks (LFBs) at analyte
concentrations of 100 jug/L. With a syringe, add 25 juL of
the stock standard (Sec. 7.14.2) to at least four - 250 mL
aliquots of reagent water and analyze each aliquot according
to procedures beginning in Section 11.2.
10.3.2 The recovery (R) values determined in 10.3.1 should be
within ± 30% of the R values listed in Table 2 for at least
three of four consecutive samples. The relative standard
deviation (Sr) of the mean recovery should be less than 30%.
If the analyte of interest meets the acceptance criterion,
performance is judged acceptable and sample analysis may
begin. For analytes that fail this criterion, initial
demonstration procedures should be repeated.
10.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples via a
new, unfamiliar method prior to obtaining some experience
with it. It is expected that as laboratory personnel gain
109
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experience with this method the quality of the data will
improve beyond the requirements stated in Section 10.3.2.
10.4 The analyst is permitted to 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 Section 10.3.
10.5 LABORATORY FORTIFIED BLANKS
10.5.1 The laboratory must analyze at least one laboratory
fortified blank (LFB) sample per sample set (all samples
analyzed within a 24-h period). The fortified concentration
of each analyte in the LFB should be 10 times the MDL.
Calculate accuracy as percent recovery (R). If the recovery
of either analyte falls outside the control limits (Section
10.5.2), that analyte is judged out of control, and the
source of the problem must be identified and resolved before
continuing analyses.
10.5.2 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 Section
10.3.2. When sufficient internal performance data become
available, develop control limits from the mean percent
recovery (R) and standard deviation (Sr) of the percent
recovery. These data are used to establish upper and lower
control limits as follows:
UPPER CONTROL LIMIT = R + 3S_
LOWER CONTROL LIMIT = R - 3Sr
After each five to ten new recovery measurements, new
control limits should be calculated using only the most
recent 20-30 data points.
10.6 LABORATORY FORTIFIED SAMPLE MATRIX
10.6.1 The laboratory must add a known fortified concentration to
a minimum of 10% of the routine samples or one fortified
sample per set, whichever is greater. The fortified
concentration should not be less then the background
concentration of the original sample. Ideally, the
fortified concentration should be the same as that used for
the laboratory fortified blank (Section 10.5). Over time,
samples from all routine samples sources should be
fortified.
10.6.2 Calculate the accuracy as percent recovery (R) for each
analyte, corrected for background concentrations measured in
the original sample, and compare these values to the control
110
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limits established in Section 10.5.2 from the analyses of
LFBs.
10.6.3 If the recovery of any such analyte falls outside the
designated range, and the laboratory performance for that
analyte is shown to be in control (Section 10.5), the
recovery problem encountered with the dosed sample is judged
to be matrix related, not system related. The result for
that analyte in the original sample is labeled
suspect/matrix to inform the data user that the results are
suspect due to matrix effects.
10.7 QUALITY CONTROL SAMPLES (QCS) - Each quarter the laboratory should
analyze one or more QCS (if available). If criteria provided with
the QCS are not met, corrective action should be taken and
documented.
10.8 The laboratory may adopt additional quality control practices for
use with this method. The specific practices that are most
productive depend upon the needs of the laboratory and the nature of
the samples. For example, field or laboratory duplicates may be
analyzed to assess the precision of the environmental measurements
or field reagent blanks may be used to assess contamination of
samples under site conditions, transportation and storage.
11. PROCEDURE
a
11.1 SAMPLE CLEANUP - Cleanup procedures may not be necessary for a
relatively clean sample matrix. The cleanup procedures recommended
in this method have been used for the analysis of various sample
types. If particular circumstances demand the use of an alternative
cleanup procedure, the analyst must demonstrate that the recovery of
the analytes is within the limits specified by the method.
11.1.1 If the sample contains particulates, or the complexity is
unknown, the entire sample should be passed through a
0.45 urn 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 SAMPLE EXTRACTION AND ANALYSIS
11.2.1 Before sample extraction, the C8 extraction cartridges must
be conditioned by the following procedure.
11.2.1.1 Place a C8 cartridge on the solid phase
extraction system manifold.
11.2.1.2 Elute the following solutions through the
cartridge in the stated order. Take special
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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.
11 2 2 The CR cartridges should not be prepared more than 48 hours
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
Section 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 Cs cartridge on the solid phase
extraction vacuum manifold. Attach a 60 ml reservoir to the
CR 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
11.2.7
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.
Remove the 5 mL volumetric flask with the extract. Fortify
the extract with 100 p.1 of the ion-pair concentrate. Adjust
the volume to the mark with cartridge eluting solution, mix
thoroughly, and seal tightly until analyzed.
112
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11.2.8 Analyze sample by HPLC using conditions described in
Table 1. Integration and data reduction must be consistent
with that performed in Section 9.3. Figure 1 presents a
representative, sample chromatogram.
11.3 IDENTIFICATION OF ANALYTES
11.3.1 Identify a sample component by comparison of its retention
time to the retention time of a reference chromatogram. If
the retention time of an unknown compound corresponds
within limits (11.3.2), to the retention time of a standard
compound, then identification is considered positive.
11.3.2 The width of the retention time window used to make
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.
11.3.3 Identification requires expert judgement when sample
components are not resolved chromatographically. When peaks
obviously represent more then one sample component M 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
tails 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.
11.3.4 If the peak area exceeds the linear range of the calibration
curve, a smaller sample volume should be used. Alter-
natively, the final solution may be diluted with mobile
phase and reanalyzed.
12. CALCULATIONS
12.1 Determine the concentration of the analytes in the sample.
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12 1.1 Calculate the concentration of each analyte injected from
the peak area using the calibration curves in Section 9.3
and the following equation.
Concentration, p.g/1 = (A) x (VF)
(VS)
where:
A = Concentration of analyte in sample extract, in ng/L
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 reagent water.
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 are presented in
Table 2.
14. REFERENCES
1
Bashe, W. J., "Determination of Diquat and Paraquat in Drinking Waters
by High Performance Liquid Chromatography with Ultraviolet Detection,
in preparation, Technology Applications, Inc., 1988.
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.
Glaser, J. A., D. L. Foerst, 6. M. McKee, S. A. Quave, and W L.
Budde, "Trace Analyses for Wastewaters," Environ. Sci . Technol.. ib,
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.
3.
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5.
Worbey, B. L., "Analytical Method for the Simultaneous Determination
of Diquat and Paraquat Residues in Potatoes by High Pressure Uau?d
Chromatography, "Pesti^_Sci 18(41, 245, 1987. pressure Liquid
6< £ln£T™l^ "Standard Practice for
7.
^
Honitonng and Support Laboratory, Cincinnati, Ohio 45268, March ^979.
115
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TABLE 1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
CONDITIONS AND METHOD DETECTION LIMITS
Analyte
Retention Time (min)
Diquat
Paraquat
2.1
2.3
0.44
0.80
HPLC Conditions:
Column:
Column Temperature:
Flow Rate:
Hamilton PRP-1, 5/i, 4.1 mm x 150 mm
35.0 C
2.0 mL/min., Ion-Pair Mobile Phase
(Section 7.16)
Injection Volume: 200 /iL
LKB Photodiode Array Detector Settings:
Wavelength Range: 210 - 370 nm
1 scan/sec.
1 nm
1 sec.
5.0 min.
Sample Rate:
Wavelength Step:
Integration Time:
Run Time:
Quantitation:
Wavelengths:
Diquat - 308 nm
Paraquat - 257 nm
a MDL data were obtained from samples fortified at 2 fj.g/1 (diquat)
and 2.3 jug/L (paraquat), n = 6
116
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TABLE 2. SINGLE OPERATOR ACCURACY AND PRECISION
Analyte
Diquat
Paraquat
Matrix
Type
Reagent
Water
Ground
Water
Tapa
Water
Reagent
Water
Ground
Water
Tapa
Water
Number
of
Analyses
6
6
7
7
6
6
6
7
. 7
6
6
Fortified
Concentration
2.0
10
100
1000
100
100
2,3
11
113
113
113
Rel at i ve
Accuracy
(Recovery)
85.6
92.1
96.2
90.0
102.2
91.3
87.6
99.7
94.4
92.1
74.2
Relative
Standard
Deviation
5 1
w * A
7.3
5.6
9.8
3.7
4.7
9.1
6.9
12.0
3 4
*^ • ~
1 8
J. • \j
Dechlorinated with Na2S203 (100 mg/L)
117
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TABLE 3. 14-DAY SAMPLE HOLDING/PRESERVATION DATA3
Analvte Matrix
Percent Recovery
Dav 14
R
R
Dlquat
Paraquat
RWb
TWC
6Wd
RW
TW
GW
98.8
84.1
84.9
90
72
98
.8
.1
.1
+ 8.
+ 1.
± 6,
+ 4
+ 0
+ 1
.6
.0
.6
.4
.8
.4
93.2 +
94.1 +
87.5 ±
86
86
72
.8 +
.7 +
.5 +
1.4
5.8
3.1
4.4
4.7
4.8
101.9 ± 2.9
94.4 + 12.0
72.
89
84
66
.4 ±
.2 +
.7 +
.4 +
4.5
3.9
2.9
7.9
Data is average of 4 samples for each matrix. All matrices were
preserved with H2S04 (pH = 2). Concentration of each analyte was
100 /zg/L.
RW s Reagent Water
TW - Tap Water - Dechlorinated with Na2S203 (100 mg/L)
GW - Groundwater
118
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titnl
M«l* HPLC san)P1e chromatograms of dlquat
iil S !1?) a2d Pflra?uat (>." 257 nra). Retention
t me of dlquat (C- 10 ug/L) is 2.03 min.; retention
time of paraquat (C * 11 ug/L) 1s 2.25 min.
119
-------
Figure 2. UV spectra of dlquat at 10 ug/L
and paraquat at 11 ug/L.
120
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METHOD 550, DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS IN
DRINKING WATER BY LIQUID-LIQUID EXTRACTION AND HPLC
WITH COUPLED ULTRAVIOLET AND FLUORESCENCE DETECTION
July 1990
J. W. Hodgeson
W. J. Bashe (Technology Applications Inc.)
T. V. Baker (Technology Applications Inc.)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
121
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METHOD 550
DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS IN DRINKING WATER BY LIQUID-
LIQUID EXTRACTION AND HPLC WITH COUPLED ULTRAVIOLET AND FLUORESCENCE DETECTION
1. SCOPE AND APPLICATION
1.1 This method describes a procedure for determination of certain
polycyclic aromatic hydrocarbons (PAH) in drinking water sources and
finished drinking water. The following analytes can be determined
by this method:
Chemical Abstract Services
Analvte Registry Number
Acenaphthene 83-32-9
Acenaphthylene 208-96-8
Anthracene ll^~lcl
Benzo(a)anthracene 56-55-3
Benzo(a)pyrene 59"H"«
Benzo(b)fluoranthene 205-99-2
Benzo(g,h,i)perylene Jni~no~Q
Benzo(k)fluoranthene 207-08-9
Chrysene 218-01-9
Dibenzo(a,h)anthracene 53-70-3
Fluoranthene 206-44-0
Fluorene ,«S6lJ3;J
Indeno(l,2,3-cd)pyrene 193-39-05
Naphthalene -91-20-3
Phenanthrene 8 «i~?
Pyrene 129-00-0
1.2 This is a high performance liquid chromatography (HPLC) method
applicable to the determination of the compounds listed above. When
this method is used to analyze unfamiliar samples, compound
identifications should be supported by at least one qualitative
technique. Method 525 provides gas chromatographic/mass
spectrometer (GC/MS) conditions appropriate for the qualitative and
quantitative confirmation of results for the above analytes, using
the extract produced by this method.
1 3 The method detection limit(l) (MDL, defined in Section 13) for each
analyte is listed in Table 1. The MDL for a specific matrix may
differ from those listed, depending on the nature of interferences
in the sample matrix.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is serially
extracted with methylene chloride using a separatory funnel. The
methylene chloride extract is dried and concentrated to a volume of
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1 ml. A 3.0 ml portion of acetonitrile is added to the extract and
concentrated to a final volume of 0.5 ml. The extract analytes are
then separated by HPLC. Ultraviolet adsorption (UV) and
fluorescence detectors are used with HPLC to quantitatively measure
the PAHs.
3. DEFINITIONS
3.1 INTERNAL STANDARD - A pure analyte(s) added to a solution in known
amounts(s) and used to measure the relative responses of other
method analytes and surrogates that are components of the same
solution. The internal standard must be an analyte that is not a
sample component.
3.2 SURROGATE ANALYTE - A pure analyte(s), which is extremely unlikely
to be found in any sample, and which is added to a sample aliquot in
known amount(s) before extraction and is measured with the same
procedures used to measure other sample components. The purpose of
a surrogate analyte is to monitor method performance with each
sample.
3.3 LABORATORY DUPLICATES (LD1 and LD2) - Two sample aliquots taken in
the analytical laboratory and analyzed separately with identical
procedures. Analyses of LD1 and LD2 give a measure of the precision
associated with laboratory procedures, but not with sample
collection, preservation, or storage procedures.
3.4 FIELD DUPLICATES (FD1 and FD2) - Two separate samples collected at
the same time and place under identical circumstances and treated
exactly the same throughout field and laboratory procedures.
Analyses of FD1 and FD2 give a measure of the precision associated
with sample collection, preservation and storage, as well as with
laboratory procedures.
3.5 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water that is
treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates
that are used with other samples. The LRB is used to determine if
method analytes or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.6 FIELD REAGENT BLANK (FRB) - Reagent water placed in a sample
container in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB
is to determine if method analytes or other interferences are
present in the field environment.
3.7 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the method is in control, and
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whether the laboratory is capable of making accurate and precise
measurements at the required method detection limit.
3.8 LABORATORY FORTIFIED MATRIX SAMPLE (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.
3.9 STOCK STANDARD SOLUTION - A concentrated solution containing a
single certified standard that is a method analyte, or a
concentrated solution of a single analyte prepared in the laboratory
with an assayed reference compound. Stock standard solutions are
used to prepare primary dilution standards.
3.10 PRIMARY DILUTION STANDARD SOLUTION - A solution of several analytes
prepared in the laboratory from stock standard solutions and diluted
as needed to prepare calibration solutions and other needed analyte
solutions.
3.11 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. One of these standards, usually of middle
concentration, can be used as the calibration check standard.
3.12 QUALITY CONTROL SAMPLE (QCS) - A sample matrix containing method
analytes or a solution of method analytes in a water miscible
solvent which is used to fortify reagent water or environmental
samples. The QCS is obtained from a source external to the
laboratory, and is used to check laboratory performance with
externally prepared test materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware, and other sample processing hardware that lead
to discrete artifacts and/or elevated baselines in the
chromatograms. All of these materials must be routinely
demonstrated to be free from interferences under the conditions of
the analysis by running laboratory reagent blanks as described in
Section 10.2.
4.1.1 Glassware must be scrupulously cleaned(2). Clean all
glassware as soon as possible after use by rinsing with the
last solvent used in it. Solvent rinsing should be followed
by detergent washing with hot water, and rinses with tap
water and distilled water. The glassware should then be
124
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drained dry, and heated in a muffle furnace at 400°C for 15
to 30 minutes. Some thermally stable materials, such as
PCBs, may not be eliminated by this treatment. Solvent
rinses with acetone and pesticide quality hexane may be
substituted for the muffle furnace heating. Thorough
rinsing with such solvents usually eliminates PCB
interference. Volumetric glassware should not be heated in
a muffle furnace. After drying and cooling, glassware
should be sealed and stored in a clean environment to
prevent any accumulation of dust or other contaminants.
Store inverted or capped with aluminum foil.
4.1.2 The use of high purity reagents and solvents helps to
minimize interference problems. Purification of solvents by
distillation in all-glass systems may be required.
4.2 Matrix interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix interferences
will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality
being sampled. The cleanup procedure suggested in Section 11.1 can
be used to overcome many of these interferences, but unique samples
may require additional cleanup approaches to achieve the MDLs listed
in Table 1.
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 a unique resolution of the
specific PAH covered by this method, other PAHs may interfere.
4.4 Matrix interferences have been found for benzo(a)anthracene,
benzo(a)pyrene and benzo(g,h,i)perylene. The nature of the
interferences has not been fully assessed.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest
possible level by whatever means available. The laboratory is
responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified
in this method. A reference file of material data handling sheets
should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are
available and have been identified for the information of the
analyst.(3-5)
5.2 The following analytes covered by this method have been tentatively
classified as known or suspected, human or mammalian carcinogens:
benzo(a)anthracene, benzo(a)pyrene, and dibenzo(a,h)anthracene.
125
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Pr-imary standards of these toxic compounds should be prepared in a
hood. A NOISH/MESA approved toxic gas respirator should be worn
when the analyst handles high concentrations of these toxic
compounds.
6. APPARATUS AND EQUIPMENT (All specifications are suggested. Catalog
numbers are included for illustration only).
6.1 SAMPLING EQUIPMENT, for discrete or composite sampling.
6.1.1 Grab sample bottle - 1 L or 1 qt, amber glass, fitted with
a screw cap lined with Teflon. Foil may be substituted for
Teflon if the sample is not corrosive. If amber bottles are
not available, protect samples from light. The bottle and
cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
6.2 GLASSWARE
6.2.1 Separatory funnels - 2 L, with Teflon stopcock, 125 mL, with
Teflon stopcock.
6.2.2 Drying column - Chromatographic column, approximately 250 mm
long x 19 mm ID, with coarse frit filter disc.
6.2.3 Concentrator tube, Kuderna-Danish - 10 mL, graduated
Calibration must be checked at the volumes employed in the
test. Ground glass stopper is used to prevent evaporation
of extracts.
6.2.4 Evaporative flask, Kuderna-Danish - 500 ml. Attach to
concentrator tube with springs.
6.2.5 Synder column, Kuderna-Danish - Three-ball macro
6.2.6 Vials - 10 to 15 mL, amberglass, with Teflon-lined screw
cap.
6.2.7 Boiling chips - carborundum, #12 granules Heat at 400°C for
30 minutes prior to use. Cool and store in dessicator.
6.3 EVAPORATION EQUIPMENT
6.3.1 Water bath - Heated, with concentric ring cover, capable of
temperature control (± 2°C). The bath should be used in a
hood.
6.3.2 Nitrogen evaporation manifold - 12 port (Organomation,
N-EVAP, Model III or EQUIVALENT.)
6.4 BALANCE - Analytical, capable of accurately weighing O.OOOlg.
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6.5 HIGH PERFORMANCE LIQUID CHROMATOGRAPH - An analytical system
complete with liquid pumping system, column supplies, temperature
controlled column oven, injector, detectors, and a compatible strip-
chart recorder. A data system is highly recommended for measuring
peak areas and retention times.
6.5.1 Gradient pumping system - constant flow
6.5.2 Analytical reverse-phase column - Supelco LC-PAH, 5 micron
particle diameter, in a 25 cm x 4.6 mm ID stainless steel
column or EQUIVALENT. This column was used to develop the
method performance statements in Section 13.
6.5.3 Detectors - Fluorescence and UV detectors. The fluorescence
detector is used for excitation at 280 nm and emission
greater than 389 nm cut-off (Schoeffel FS970 or EQUIVALENT.)
Fluorometer should have dispersive optics for excitation and
can utilize either filter or dispersive optics at the
emission detector. The UV detector is used at 254 nm
(Waters Assbc. Model 450) and should be coupled to the
fluorescence detector. These detectors were used to develop
the method performance statements in Section 13.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 REAGENT WATER - Reagent water is defined as a water in which an
interferant is not observed at the MDL of the analytes 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 bottles with Teflon lined screw
caps.
7.2 SODIUM THIOSULFATE - (ACS) Granular
7.3 METHYLENE CHLORIDE - Pesticide quality or equivalent
7.4 ACETONITRILE - HPLC quality, distilled in glass
7,5 SODIUM SULFATE - (ACS) Granular, anhydrous. Purify by heating at
400°C for 4 hours in a shallow tray.
7.6 STOCK STANDARD SOLUTIONS (1.00 ng/nl) - Stock standard solutions can
be prepared from pure standard materials or purchased as certified
solutions.
7.6.1 Prepare stock standard solutions by accurately weighing
about 0.0100 g of pure material. Dissolve the material in
acetonitrile and dilute to volume in a 10 mL volumetric
flask. Larger volumes can be used at the convenience of the
analyst. When compound purity is assayed at 96% or greater,
the weight can be used without correction to calculate the
concentration of the stock standard. Certified,
127
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commercially prepared stock standards can be used at any
concentration.
7.6.2 Transfer the stock standard solutions into Teflon-sealed
screw cap bottles. Store at 4°C and protect from light.
Stock standard solutions should be checked frequently for
signs of degradation or evaporation, especially just prior
to preparing calibration standards from them.
7.6.3 Stock standard solutions must be replaced after six months,
or sooner if comparison with check standards indicates a
problem.
7.7 LABORATORY CONTROL SAMPLE CONCENTRATE - See Section 10.3.1.
8. SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Grab samples must be collected in glass containers. Conventional
sampling practices should be followed, except that the bottle must
not be pre-rinsed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program.
8.2 All samples must be iced or refrigerated at 4°C from the time of
collection until extraction. PAHs are known to be light sensitive;
therefore, samples, extracts, and standards should be stored in
amber or foil-wrapped bottles in order to minimize photolytic
decomposition. Fill the sample bottles and, if residual chlorine is
present, add 100 mg of sodium thiosulfate per liter of sample and
mix well. EPA Methods 330.4 and 330.5 may be used for measurement
of residual chlorine. Field test kits are available for this
purpose. Adjust the pH of the sample to < 2 with 6N HC1 to inhibit
biological activity.
8.3 All samples must be extracted within 7 days of collection and
completely analyzed within 30 days of extraction.(6) Polycyclic
aromatic hydrocarbons are known to be light sensitive. Therefore
sample extracts and standards should be stored in amber vials
in the dark in a refrigerator or freezer in order to minimize
photolytic decomposition.
9. CALIBRATION
9.1 Use liquid chromatographic operating conditions given in Table 1.
The chromatographic system can be calibrated using the external
standard technique (Section 9.2) or the internal standard technique
(Section 9.3.) Note: Calibration standard solutions must be
prepared such that no unresolved analytes are mixed together.
Special care must be taken so that analyte concentrations in
standard solutions are not so high as to cause peak fusing or
overlap.
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9.2 EXTERNAL STANDARD CALIBRATION PROCEDURE:
9.2.1 Prepare calibration standards at a minimum of three
(recommend five) concentration levels for each analyte by
adding volumes of one or more primary dilution standard
solutions (3.10) to a volumetric flask and diluting to
volume with acetonitrile. One of the external standards
should be at a concentration near, but above the MDL (Table
1) and the other concentrations should bracket the expected
range of concentrations found in real samples or should
define the working range of the detector.
9.2.2 Using injections of 5 to 100 ML, analyze each calibration
standard according to Section 11.3. Tabulate peak area or
height responses against the mass injected. The results can
be used to prepare a calibration curve for each compound.
Alternatively, if the ratio of response to amount injected,
(calibration factor) is a constant over the working range
[< 10% relative standard deviation (RSD)], linearity through
the origin can be assumed and the average ratio or
calibration factor can be used in place of a calibration
curve.
9.3 INTERNAL STANDARD (IS) CALIBRATION PROCEDURE - To use this approach,
.the analyst must select one or more internal standards that are
similar in analytical behavior to the compounds of interest. The
analyst must further demonstrate that the measurement of the
internal standard is not affected by method or matrix interferences.
Because of these limitations, no internal standard can be suggested
that is applicable to all samples.
9.3.1 Prepare calibration standards at a minimum of three
(recommend five) concentration levels for each analyte of
interest by adding volumes, of one or more primary dilution
standard solutions (3.10) to a volumetric flask. To each
calibration standard, add a known amount of one or more
internal standards, and dilute to volume with acetonitrile.
One of the standards should be at a concentration near but
above, the MDL and the other concentrations should bracket
the analyte concentrations found in the sample concentrates
or should define the working range of the detector.
9.3.2 Using injections of 5 to 100 /zL analyze each calibration
standard according to Section 11.3. Tabulate peak height or
area responses against concentration for each compound and
internal standard. Calculate response factor (RF) for each
compound using Equation 1.
Equation 1
RF = FAsI FCisI
[Ais] [CsJ
129
-------
where:
As - Response for the analyte to be measured
Ais = Response for the internal standard
Cis - Concentration of the internal standard (/xg/L)
Cs = Concentration of the analyte to be measured (M9/L)
If RF value over the working range is constant (< 10% RSD),
the RF can be assured to be invariant and the average RF can
be used for calculations. Alternatively, the results can be
used to plot a calibration curve of response ratios, As/Ais
vs. Cs/Cis.
9.4 The working calibration curve, calibration factor, or RF must be
verified on each working day by the measurement of one or more
calibration standards. If the response for any analyte varies from
the predicted response by more than ± 20%, the test must be repeated
using fresh calibration standard. If the fresh calibration standard
also deviates by more ± 20%, a new calibration curve must be
prepared for that compound.
9.4.1 Daily calibration requirements using the external standard
calibration procedure are a minimum of two calibration check
standards, one at the beginning and one at the end of the
analysis day. These check standards should be at two
different concentration levels to verify the calibration
curve. For extended periods of analysis (> 8 hrs), it is
strongly recommended that check standards be interspersed
with samples at regular intervals during the course of the
analysis.
9.4.2 Minimum daily calibration requirements using the internal
standard calibration procedure consist of initial analyses
of a calibration check standard followed by verification of
the internal standard response of each sample applying
criterion described in Section 10.4.
9.5 Before using .any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interferences from reagents.
10. QUALITY CONTROL
10.1 Each laboratory that uses this method is required to operate a
formal quality control (QC) program. The minimum QC requirements
are initial demonstration of laboratory capability, analysis of
laboratory reagent blanks, laboratory fortified blanks, laboratory
fortified matrix samples and QC samples. The laboratory must
maintain records to document the quality of the data generated.
Additional quality control practices are recommended.
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10.2 LABORATORY REAGENT BLANKS (LRB). Before processing any samples, the
analyst must demonstrate that all glassware and reagent
interferences are under control. Each time a set of samples is
analyzed or reagents are changed, a LRB must be analyzed. For this
method, the LRB is filtered reagent water. If within the retention
time window (11.4.2) of an analyte of interest, the LRB produces a
peak that interferes with analyte determination, determine the
source of contamination and eliminate the interference before
processing samples.
10.3 INITIAL DEMONSTRATION OF CAPABILITY
10.3.1 Select a representative spike concentration (about 10 times
MDL) for each analyte. Prepare a laboratory control sample
concentrate (in acetonitrile) from the stock standard
solution containing each analyte at 1000 times the selected
concentration. Using a pipet, add 1.00 mL of the
concentrate to each of at least four 1 L aliquots of reagent
water and analyze each aliquot according to procedures
beginning in Section 11.2.
10.3.2 For each analyte, the recovery value must for at least three
out of four consecutively analyzed samples fall in the range
of R ± 30% (or within R ± 3 Sr, if broader) using the values
for R and Sp for reagent water in Table 2 (fortification
level 1). The relative standard deviation of the mean
recovery measured in 10.3.1 should be < 30% or 3S
(whichever is greater), using the values for S (level 1) in
Table 2. For those compounds that meet the acceptance
criteria, performance is acceptable and sample analysis may
begin. For those compounds that fail these criteria,
initial demonstration of capability must be repeated.
10.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples by a
new, unfamiliar method prior to evidencing a basic level of
skill at performing the technique. It is expected that as
laboratory personnel gain experience with this method the
quality of the data will improve beyond the requirements
stated in Section 10.3.2.
10.3.4 The analyst is permitted to modify 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 Section
J. U • O •
10.4 ASSESSING THE INTERNAL STANDARD - When using the IS calibration
procedure, the analyst is expected to monitor the IS response (peak
area or peak height) of all samples during each analysis day. The
131
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IS response for any sample chromatogram should not deviate from the
daily mean IS response by more than 30%.
10.4.1 If a deviation of > 30% is encountered for a sample, re-
inject the extract.
10.4.1.1 If acceptable IS response is achieved for the
re-injected extract, then report the results for
that sample.
10 4.1.2 If a deviation of > 30% is obtained for the re-
injected extract, analysis of the sample should
be repeated beginning with Section 11.2,
provided the sample is still available.
Otherwise, report results obtained from the re-
injected extract, but annotate as suspect.
10.4.2 If consecutive samples fail the IS response acceptance
criterion, immediately analyze a calibration check standard.
10 4 2.1 If the check standard provides a response factor
(RF) within 20% of the predicted value, then
follow procedures itemized in Section 10.4.1 for
each sample failing the IS response criterion.
10.4.2.2. If the check standard provides a response factor
(RF) which deviates more than 20% of the
predicted value, then the analyst must
recalibrate, as specified in Section 9.3.
10.5 LABORATORY FORTIFIED BLANK
10 5 1 The laboratory must analyze at least one laboratory
fortified blank (LFB) per sample set (all samples prepared
for analysis within a 24 hour period). The fortified
concentration of each analyte in the LFB should be at least
10 times the MDL. Calculate accuracy as percent recovery
(R) If the recovery of any analyte falls outside the
control limits (See Section 10.5.2), that analyte is judged
out of control, and the source of the problem must be
identified and resolved before continuing analyses.
10 5.2 Until sufficient LFB data become available, usually a
minimum of results from 20 to 30 analyses, the laboratory
must assess its performance against the control limits
described in Section 10.3.2. When sufficient laboratory
performance data becomes available, develop control limits
from the mean percent recovery (R) and standard deviation
(S) of the percent recovery. These data are used to
establish upper and lower control limits as follows:
132
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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.
10,6 LABORATORY FORTIFIED MATRIX SAMPLE
10.6.1 The laboratory must add a known fortified concentration to
a minimum of 10% of the routine samples or one fortified
sample per set, whichever is greater. The fortified
concentration should not be less than the background
concentration of the original sample. Ideally, the
fortified concentration should be the same as that used for
the LFB (Section 10.5). Over time, samples from all routine
sample sources should be fortified.
10.6.2 Calculate the percent recovery (R) for each analyte,
corrected for background concentrations measured in the
original sample, and compare these values to the control
limits established in Section 10.5.2 from the analyses of
LFBs.
10.6.3 If the recovery of any analyte falls outside the designated
range, and the laboratory performance for that analyte is
shown to be in control (Section 10.5), 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 must be labelled suspect/matrix to
inform the data user that the results are suspect due to
matrix effects.
10.7 Quality Control Samples (QCS) - Each quarter the laboratory should
analyze one or more QCS (if available). If criteria provided with
the QCS are not met, corrective action should be taken and
documented.
10.8 The laboratory may adopt additional quality control practices for
use with this method. The specific practices that are most
productive depend upon the needs of the laboratory and the nature of
the samples. For example, field or laboratory duplicates may be
analyzed to assess the precision of the environmental measurements
or field reagent blanks may be used to assess contamination of
samples under site conditions, transportation and storage.
11. PROCEDURE
11.1 SAMPLE CLEANUP - Cleanup procedures may not be necessary for a
relatively clean sample matrix. If particular circumstances demand
the use of a cleanup procedure, the analyst first must demonstrate
that the requirements of Section 10.5 can be met using the method as
133
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revised to incorporate the cleanup procedure. EPA Method 610
describes one possible cleanup procedure for this analyte list.
11.2 SAMPLE EXTRACTION - LIQUID-LIQUID EXTRACTION
11.2.1 Mark the water meniscus on the side of the sample bottle for
later determination of sample volume. -Pour the entire
sample into a 2 L separatory funnel.
11.2.2 Add 60 mL of methylene chloride to the sample bottle, seal,
and shake for 30 seconds to rinse the inner surface.
Transfer the solvent to the separatory funnel and extract
the sample by shaking the funnel for 2 minutes with periodic
venting to release excess pressure. Allow the organic layer
to separate from the water phase for a minimum of 10
minutes. If the emulsion interface between layers is more
than one-third the volume of the solvent layers, the analyst
must employ mechanical techniques to complete the phase
separation. The optimum technique depends upon the sample,
but may include stirring, filtration of emulsion through
glass wool, centrifugation, or other physical methods.
Collect the methylene chloride extract in a 250 mL
Erlenmeyer flask.
11.2.3 Add a second 60 mL volume of methylene chloride to the
sample bottle and repeat the extraction procedure a second
time, combining the extracts in the Erlenmeyer flask.
Perform a third extraction in the same manner.
11.2.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a
10 mL concentrator tube to a 500 mL evaporative flask.
Other concentration devices or techniques may be used in
place of the K-D concentrator if the requirements of Section
10.6 are met.
11.2.5 Pour the combined extract through a solvent-rinsed drying
column containing about 10 cm of anhydrous sodium sulfate,
and collect the extract in the K-D concentrator. Rinse the
Erlenmeyer flask and column with 20 - 30 mL of methylene
chloride to complete the quantitative transfer.
11.2.6 Add one or two clean boiling chips to the evaporative flask
and attach a three-ball Snyder column. Prewet the Snyder
column by adding about 1 mL of methylene chloride to the
top. Place the K-D apparatus on a hot water bath (60 to
65°C) so that the concentrator tube is partially immersed in
the hot water, and the entire lower rounded surface of the
flask is bathed with hot vapor. Adjust the vertical
position of the apparatus and the water temperature as
required to complete the concentration in 15 to 20 minutes.
At the proper rate of distillation the balls of the column
will actively chatter but the chambers will not flood with
134
-------
condensed solvent. When the apparatus volume of liquid
reaches 0.5 ml, remove the K-D apparatus and allow it to
drain and cool for at least 10 minutes.
11.2.7 Remove the Synder column and rinse the flask and its lower
joint into the concentrator tube with 1 to 2 ml of methylene
chloride. A 5 ml syringe is recommended for this operation.
Stopper the concentrator tube and store refrigerated (4°C)
if further processing will not be performed immediately. If
the extract will be stored longer than two days, it should
be transferred to a Teflon-sealed screw-cap vial and
protected from light.
11.2.8 Evaporate the extract with a gentle stream of N2 flow to a
volume of 1.0 ml. Add 3.0 ml of acetonitrile (MeCN) and
concentrate with the N2 flow to a final volume of 0.5 ml_.
Stopper the concentrator tube and store refrigerated if
further processing will not be performed immediately. If
the extract will be stored longer than two days, it should
be transferred to a Teflon-sealed screw cap vial and
protected from light. If the sample extract requires no
further cleanup, proceed with liquid chromatographic
analysis (Section 11.3).
11.2.9 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the liquid to a 1000 ml
graduated cylinder. Record the sample volume to the nearest
5 ml.
11.3 SAMPLE ANALYSIS
11.3.1 Table 1 summarizes the recommended operating conditions for
the HPLC. Included in this table are retention times and
MDLs that can be achieved under these conditions. The UV
detector is recommended for the determination of
naphthalene, acenaphthylene, acenaphthene and fluorene. The
fluorescence detector is recommended for the remaining PAHs.
An example for the separation achieved by this HPLC column
is shown in Figure 1. Other HPLC columns, chromatographic
conditions, or detectors may be used if the requirements of
Section 10.5 are met.
11.3.2 Calibrate the system daily as described in Section 9.
11.3.3 If the internal standard calibration procedure is being
used, the internal standard must be added to the sample
extract and mixed thoroughly immediately before injection
into the instrument.
11.3.4 Inject 5 to 100 juL of the sample extract or standard into
the HPLC using a high pressure syringe or a constant volume
sample injection loop. Record the volume injected to the
135
-------
nearest 0.1 juL, and the resulting peak size in area or peak
height units. Re-equilibrate the HPLC column at the initial
gradient conditions for at least 10 minutes between
injections.
11.3.5 If the response for a peak exceeds the working range of the
system, dilute the extract with acetonitrile and reanalyze.
11.4 IDENTIFICATION OF ANALYTES
11.4.1 Identify a sample component by comparison of its retention
time to the retention time in reference chromatogram. If
the retention time of an unknown compound corresponds,
within limits, to the retention time of a standard compound,
then identification is considered positive.
11.4.2 The width of the retention time window used to make
identifications should be based upon measurements of actual
retention time variations of standards over the course of a
day. Three times the standard deviation of a retention time
can be used to calculate a suggested window size for a
compound. However, the experience of the analyst should
weigh heavily in the interpretation of chromatograms.
11.4.3 Identification requires expert judgement when sample
components are not resolved chromatographically, that is,
when GC peaks obviously represent more than one sample
component (i.e., broadened peak with shoulder(s) or valley
between two or more maxima). Any time doubt exists over the
identification of a peak in a chromatogram, appropriate
confirmatory techniques need to be employed such as use of
an alternative detector which operates on a
chemical/physical principle different from that originally
used, e.g., mass spectrometry, or the use of a second
chromatography column.
12. CALCULATIONS
12.1 Determine the concentration of individual compounds in the sample as
fol1ows.
12.1.1 If the external standard calibration procedure is used,
calculate the amount of material injected from the peak
response using the calibration curve or calibration factor
determined in Section 9.2.2. The concentration in the
sample can be calculated from Equation 2.
Equation 2
Concentration (M9/L) = (A) (Vt)
(V,-) (Vs)
136
-------
where:
A = Amount of material injected (ng).
Vi = Volume of extract injected (/xL).
Vt - Volume of total extract (;uL).
Vs = Volume of water extraction (ml).
12.1.2 If the internal standard calibration procedure is used
calculate the concentration in the sample using the response
factor (RF) determined in Section 9.3.2 and Equation 3
Equation 3
Concentration (/zg/L) = (A ) (I )
(A,.)
(VJ
where:
As = Response for the parameter to be measured
Ais = Response for the internal standard.
= Amount of internal standard added to each extract
(M9).
= Volume of water extracted (L).
Is
Vo
12.2
Report results in jug/L without correction for recovery data
data obtained should be reported with the sample results.
All QC
13. METHOD PERFORMANCE
13.1 The method detection limit (MDL) is defined as the minimum
S£ ?V°n °f ua substance that can be measured and reported with
99/c confidence that the value is above zero. The MDL is equal to
the level calculated by multiplying the standard deviation of N
replicate measurements times the students' t test critical value for
a 99 percent confidence level at N - 1 degrees of freedom.
13.2 In a single laboratory, analyte recoveries from reagent water were
determined at two concentration levels. Results were used to
determine analyte MDLs and demonstrate method range. Analytes were
7nwlded into three spiking sets: compounds measured by UV detection
(UV) and two groups of compounds measured by fluorescence detection
(FD-A and FD-B), and analyzed separately. MDL values are given in
Table 1 Precision and accuracy data obtained for the two
concentration levels in reagent water are presented in Table 2.
13.3 In a single laboratory, analyte recoveries from dechlorinated tap
water were determined at one concentration level. Results were used
to demonstrate method performance capabilities for a finished
drinking water matrix. As with Section 13.2, analytes were grouped
into three spiking sets (UV, FD-A and FD-B). Precision and accuracy
results for the dechlorinated tap water are shown in Table 3
137
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14. REFERENCES
Glaser, J. A., D. L. Foerst, G. D. McKee, S. A. Qwave and W. L. Budde,
"Trace Analysis of Wastewaters", Environ. Sen. Techno!^ 15, 1425,
1981.
ASTM Annual Book of Standards, Part 31, D3694. "Standard Practices
for Preparation of Sample Containers and for Preservation of Organic
Constituents", American Society for Testing and Materials,
Philadelphia, PA, p. 679, 1980.
"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.
"OSHA Safety and Health Standards, General Industry", (29 CFR 1910),
Occupational Safety and Health Administration, OSHA 2206 (Revised
1976).
"Safety in Academic Chemistry Laboratories", American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
"Determination of Polynuclear Aromatic Hydrocarbons in Industrial and
Municipal Wastewaters", EPA-600/4-82-025, U.S. : Environmental
Protection Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio 45268, September 1982.
138
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TABLE 1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY CONDITIONS
AND METHOD DETECTION LIMITS
Method
Detection
Retention
Analyte
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fl uoranthene
Pyrene
Benzo (a) anthracene
Chrysene
Benzo ( b) f 1 uoranthene
Benzo ( k) f 1 uoranthene
Benzo(a)pyrene
Dibenzo( a, h) anthracene
Benzo(g,h,i)perylene
Indeno(l,2,3-cd)pyrene
Sample
Set
UV
UV
UV
UV
FD-B
FD-A
FD-B
FD-A
FD-B
FD-A
FD-B
FD-A
FD-B
FD-B
FD-B
FD-A
Time
min
12.5
13.8
15.4
15.6
16.8
17.6
18.7
19.4
21.9
22.3
24.2
25.0
26.0
27.1
27.8
28.3
Method
Limit
Detection Fortification
Limit
jug/L (a)
3.3
2.3
3.0
0.25
0.162
0.079
0.026
0.126
0.002
0.063
0.003
0.002
0.029
0.019
0.014
0.011
Level
10.0
10.0
10.0
1.00
0.500
0.625
0.025
0.625
0.010
0.625
0.010
0.0125
0.050
0.125
0.050
0.125
HPLC column conditions: Reverse-phase LC-PAH, 5 micron particle size, in a 25 cm
x 4.6 mm ID stainless steel column. Isocratic elution for 2 min. using
acetonitrile/water (3.5 : 6.5), then linear gradient elution to 100% acetonitrile
over 22 min. at 2.0 mL/min. flow rate.
(a) The MDL for naphthalene, acenaphthylene, acenaphthene, and fluorene were
determined using a UV detector. All others were determined using a
fluorescence detector.
139
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TABLE 2. SINGLE-LABORATORY ACCURACY AND PRECISION FROM SEVEN
REPLICATE ANALYSES OF FORTIFIED REAGENT WATER
Concentration
Level
1
Analyte
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fl uoranthene
Pyrene
Benzo (a) anthracene
Chrysene
Benzo (b) f 1 uoranthene
Benzo ( k) f 1 uoranthene
Benzo(a)pyrene
Dibenzo (a, h) anthracene
Benzo (g,h,i)perylene
Indeno(l»2,3-cd)pyrene
M9/L
10.0
10.0
10.0
1.0
0.5
0.625
0.025
0.625
0.01
0.625
0.01
0.0125
0.05
0.125
0.05
0.125
R(a)
96.0
95.5
94.5
91.0
72.5
89.6
113
93.6
99.0
94.4
99.0
77.6
85.7
81.6
108
72.4
Concentration
Level
2(c)
Sr(b)
10.5
7.0
9.5
8.0
10.3
4.0
33.2
6.4
10.5
3.2
10.5
6.0
18.3
4.8
9.0
2.8
^g/L
2.0
2.0
2.0
0.2
0.1
0.125
0.005
0.125
0.002
0.125
0.002
0.0025
0.01
0.025
0.01
0.025
R
83.3
98.5
47.3
92.0
69.5
74.4
140
82.4
50.0
94.0
50.0
100
41.7
78.0
50.0
66.0
Sr
17.4
15.2
6rt
.2
7.8
17.0
13.2
50.0
9.6
10.0
11.6
25.0
40.0
16.7
12.0
10.0
8.0
(a) R - Mean Recovery, %
S_ = Relative Standard Deviation of R, %
(b) S = Relative Standard Deviation or K, 7,
(c) Spike Level 2 = Concentration for analytes which yield a signal-
noise ratio of approximately 10 in the extract (25 /zL injection)
signal-to-
140
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TABLE 3. SINGLE-LABORATORY ACCURACY AND PRECISION FROM NINE
REPLICATE ANALYSES OF FORTIFIED TAP WATER (a)
Analyte
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzp(a)anthracene
Chrysene
Benzo(b)fluoranthene
Benzo ( k) f 1 uoranthene
Benzo(a)pyrene
Dibenzo( a, h) anthracene
Benzo (g,h,i)perylene
Indeno (1,2 , 3-cd) pyrene
Fortified
Concentration
Level
M9/L
10.0
10.0
10.0
1.0
0.5
0.625
0.025
0.625
0.01
0.625
0.006
0.0125
0.05
0.125
0.05
0.125
Relative
Accuracy
(Recovery)
%
76.0
71.4
76.6
89.4
77.4
97.0
103.0
86.0
91.3
91.1
74.7
101.0
87.0
94.2
86.0
100.0
Relative
Standard
Deviation
V
/o
5 4
w • T
11- 1
A -L*» J.
9.9
5 5
*J • \J
5 6
w • W
6.3
8.3
10 1
J. W • X
10.5
10 9
J. V •
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i»-<) ozuag
auamuejonu (Q) ozuag
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•-aji auaqiuejontj
1
cn
CM
CO
CM
(M
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CM
s
ru
CM
5
CM .-.
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vt
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ai
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O C
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142
-------
550.1.
Mft£ WM-YCYCLIC AROMATIC HYDROCARBONS
uTr WATER BY LIWID-SOLID EXTRACTION AND HPLC
WITH COUPLED ULTRAVIOLET AND FLUORESCENCE DETECTION
July 1990
J. W. Hodgeson
W. J. Bashe (Technology Applications Inc.)
T. V. Baker (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 550.1
DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS IN DRINKING WATER BY LIQUID-
SOLID EXTRACTION AND HPLC WITH COUPLED ULTRAVIOLET AND FLUORESCENCE DETECTION
1. SCOPE AND APPLICATION
1.1
This method describes a procedure for determination of certain
polycyclic aromatic hydrocarbons (PAH) in drinking water sources
and finished drinking water. The following analytes can be
determined by this method:
Chemical Abstract Services
Registry Number
Analvte
Acenaphthene
Acenaphthylene
Anthracene
Benzo(a)anthracene
Benzo(a)pyrene
Benzo(b) fluoranthene
Benzo(g,h,i)perylene
Benzo(k)f1uoranthene
Chrysene
Dibenzo(a,h)anthracene
Fluoranthene
Fluorene
Indeno(l,2,3-cd)pyrene
Naphthalene
Phenanthrene
Pyrene
83-32-9
208-96-8
120-12-7
56-55-3
50-32-8
205-99-2
191-24-2
207-08-9
218-01-9
53-70-3
206-44-0
86-73-7
193-39-05
91-20-3
85-01-8
129-00-0
12 This is a high performance liquid chromatography (HPLC) method
applicable to the determination of the compounds listed above. When
this method is used to analyze unfamiliar samples, compound
identifications should be supported by at least one qualitative
technique. Method 525 provides gas chromatographic/mass spectrometer
(GC/MS) conditions appropriate for the qualitative and quantitative
confirmation of results for the above analytes, using the extract
produced by this method. Note: To utilize Method 525, the standards
must be in acetonitrile also.
1 3 The method detection limit(l) (MDL, defined in Section 13) for each
analyte is listed in Table 1. The MDL for a specific matrix may
differ from those listed, depending on the nature of interferences
in the sample matrix.
SUMMARY OF METHOD
2 1 Polycyclic aromatic hydrocarbons and internal standards, if used,
are extracted from a water sample by passing 1 liter of sample
144
-------
through a cartridge containing about 1 gram of a solid
SiTdxJt;Si£^?taAct11'
a
3. DEFINITIONS
3.1
3.2
3.6
™St be 3" a"alyte that 1s "
sample
analyte(s), which is extremely unlikely
, and which is added to a sample aliquot
extraction and is measured with the same
other sample components. The purpose of
o monitor method performance with each
collection, preservation, °o7 storage "procedures. "Ot Wlth Samp1e
3'4 theLsaSftimeTaSnd(FDl1acaend F°2) •" Tw°- Separate samPles collected at
of FD and
give a measure of the precson
. as
3'5 IsTfrtid eSy\BsLAaNlsaiLle)in *" ?11qUOt °f r6agent W3ter that
envTronment, the reagents, or the apparatus. 'amatory
FIELD REAGENT BLANK (FRB) - Reagent water placed in a sample
container in the laboratory and treated as a sample in all resects
including exposure to sampling site conditions, storage, preservation
and all analytical procedures. The purpose of the FRB Is tS
™
145
-------
3 7 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent .water to
which known quantities of the method analytes are added in the
laboratory The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the method is in control .and whether
the laboratory is capable of making accurate and precise measurements
at the required method detection limit.
An aliquot of an
sasrs
38 LABORATORY FORTIFIED MATRIX SAMPLE (LFM)
Q
ar. i£. :»
corrected for background concentrations.
STOCK STANDARD SOLUTION - A concentrated solution containing a single
certified standard that is a method analyte, or a concentrated
solution of a single analyte prepared in the laboratory with an
Assayed reference compound. Stock standard solutions are used to
prepare primary dilution standards.
PRIMARY DILUTION STANDARD SOLUTION - A solution of several analytes
Dreoared in the laboratory from stock standard solutions and diluted
as XSed to prepare calibration solutions and other needed analyte
solutions.
CALIBRATION STANDARD (CAL) - A solution prepared from the primary
dllltlSn standard solution and stock standard solutions of the
internal standards and surrogate analytes. Th e CALjwl ut i"™ J™
used to calibrate the instrument response with respect to analyte
concentration.
3 12 QUALITY CONTROL SAMPLE (QCS) - A sample matrix containing method
analytes or a solution of method analytes in a water miscible solvent
which is used to fortify reagent water or environmental samples.
?he QCS is obtained from a source external to the laboratory, and
iJ used to check laboratory performance with external ly prepared test
materi al s .
4. INTERFERENCES
3 10
3
3'
4.1
Method interferences may be caused by contaminants in solvents,
reaaents qlassware, and other sample processing hardware that lead
todiscretl artifacts and/or elevated baselines in the chromatograms
All of these materials must be routinely demonstrated to be free from
interferencer under the conditions of the analysis by running
laboratory reagent blanks as described in Section 10.2.
4 1 1 Glassware must be scrupulously cleaned(2). Clean all
glassware as soon as possible after use by rinsing with the
146
-------
last solvent used in it. Solvent rinsing should be followed
by detergent washing with hot water, and rinses with tap
water and distilled water. The glassware should then be
drained dry, and heated in a muffle furnace at 400°C for 15
to 30 minutes. Some thermally stable materials, such as
PCBs, may not be eliminated by this treatment. Solvent
rinses with acetone and pesticide quality hexane may be
substituted for the muffle furnace heating. Thorough
rinsing with such solvents usually eliminates PCB
interference. Volumetric glassware should not be heated in
\"1U1!,flu furnace- After drying and cooling, glassware
should be sealed and stored in a clean environment to
prevent any accumulation of dust or other contaminants.
Store inverted or capped with aluminum foil.
4.1.2 The use of high purity reagents and solvents helps to
minimize interference problems. Purification of solvents by
distillation in all-glass systems may be required.
4'2 rnlv^ l^erferences may be caused by contaminants that are
contracted from the sample. The extent of matrix interferences
will vary considerably from source to source, depending upon the
hpinnM^iJ1^51^ °f the industrial complex or municipality
being sampled. The cleanup procedure suggested in Section 11 1 can
be used to overcome many of these interferences, but unique samples
may require additional cleanup approaches to achieve the MDLs listed
in iaoie i.
4.3 The extent of interferences that may be encountered using liquid
JJro!"j|tographic techniques has not been fully assessed. Although
cnL-?- conditions described allow for a unique resolution of the
specific PAH covered by this method, other PAHs may interfere.
4.4 Matrix interferences have been found for benzo(a)anthracene,
benzo(a)pyrene and benzo(g,h,i)perylene. The nature of the
interferences has not been fully assessed.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method
chlil?\ I1 P^j^y defined; however, each chemical compound
°" *J6 *£*J « « P0^13! health ha»rd, From'this
azar. rom tis
viewpoint, exposure to these chemicals must be reduced to the lowest
KS™l®hi* y *fh*t?ver means available. The laboratory is
responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified
chnn?iS "! K' A/efere"ce file of material data handling sheets
should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are
Jn!J 3f oa"cd> haVe been ident1f1ed for the information of the
anaiysu. (o-oj
147
-------
5 2 The following analytes covered by this method have been tentatively
classified as known or suspected, human or mammalian carcinogens:
benzo(a)anthracene, benzo(a)pyrene, and dibenj°ia'h)^^cf"e:
Primary standards of these toxic compounds should be prepared in a
hood. A NIOSH/MESA approved toxic gas respirator should be worn
when the analyst handles high concentrations of these toxic compounds.
6. APPARATUS AND EQUIPMENT (All specifications are suggested.
numbers are included for illustration only.)
Catalog
6.1
6.2
6.3
SAMPLING EQUIPMENT (for discrete or composite sampling).
6.1.1
Grab sample bottle - 1 L or 1 qt, amber glass, fitted with
a screw cap lined with Teflon. Foil may be substituted for
Teflon if the sample is not corrosive. If amber bottles are
not available, protect samples from light. The bottle and
cap liner must be washed, rinsed with acetone or methylene
chloride, and dried before use to minimize contamination.
GLASSWARE
6.2.1
6.2.2
6.2.3
6.2.4
Separatory funnels - 2 L, with Teflon stopcock, 125 mL, with
Teflon stopcock.
Drying column - Chromatographic column, approximately 250 mm
long x 19 mm ID, with coarse frit filter disc.
Concentrator tube, Kuderna-Danish - 10 mL, graduated
Calibration must be checked at the volumes employed in the
test. Ground glass stopper is used to prevent evaporation
of extracts.
Vials - 10 to 15 mL, amber glass, with Teflon-lined screw
cap.
EVAPORATION EQUIPMENT
6.3.1
Water bath - Heated, with concentric ring cover, capable of
temperature control (± 2°C). The bath should be used in a
hood.
6.3.2 Nitrogen evaporation manifold - 12 port (Organomation, N-
EVAP, Model III or equivalent.)
6.4 BALANCE - Analytical, capable of accurately weighing O.OOOlg.
6.5 HIGH PERFORMANCE LIQUID CHROMATOGRAPH - An analytical system
complete with liquid pumping system, column supplies, injector,
detectors, and a compatible strip-chart recorder. A data system is
highly recommended for measuring peak areas and retention times.
6.5.1 Gradient pumping system - constant flow
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6.5.2 Analytical reverse-phase column - Supel.co LC-PAH, 5 micron
particle diameter, in a 25 cm x 4.6 mm ID stainless steel
column (Supelco #5-8318 or equivalent). This column was
used to develop the method performance statements in Section
J.O *
6.5.3 Detectors - Fluorescence and UV detectors. The fluorescence
detector is used for excitation at 280 nm and emission
greater than 389 nm cut-off (Schoeffel FS970 or equivalent )
Fluorometer should have dispersive optics for excitation and
can utilize either filter or dispersive optics at the
emission detector. The UV detector is used at 254 nm (Waters
Assoc. Model 450) and should be coupled to the fluorescence
detector. These detectors were used to develop the method
performance statements in Section 13.
6.6 EXTRACTION APPARATUS
6.6.1 Liquid-Solid Extraction (LSE) cartridges, C-18, approximately
luuu mg/b.u mL.
6.6.2 Liquid-Solid Extraction System, Baker - 10 SPE or
equivalent. '
6.6.3 Vacuum pump, 100 VAC, capable of maintaining capable of
maintaining a vacuum of 8-10 mm Hg.
6.6.4 Empore Extraction Disks, C-18, 47 mm.
6.6.5 Millipore Standard Filter Apparatus to hold disk, all glass.
7- REAGENTS AND CONSUMABLE MATERIALS
7.1 REAGENT WATER - Reagent water is defined as a water in which an
interferant is not observed at the MDL of the analytes of interest
Prepare reagent water by filtering tap water through a bed containing
ca. 0.5 kg of activated carbon, or by using commercially available
water purification systems. Any source of reagent water which passes
the requirements of Section 10 may be used. Store in clean bottles
with teflon-lined screw caps.
7.2 SODIUM THIOSULFATE - (ACS) Granular
7.3 METHYLENE CHLORIDE - Pesticide quality or equivalent
7.4 ACETONITRILE - HPLC quality, distilled in glass
7'5 ^rV"1^ " (^CS) Granular> anhydrous. Purify by heating at
400 C for 4 hours in a shallow tray.
149
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7 6 STOCK STANDARD SOLUTIONS (1.00 ug/uL) - Stock standard. solutions
can be prepared from pure standard materials or purchased as
certified solutions.
7 6 1 Prepare stock standard solutions by accurately weighing
about 0.0100 g of pure material, dissolve the material in
acetonitrile and dilute to volume in a 10 ml volumetric
flask. Larger volumes can be used at the convenience of
the analyst. When compound purity is assayed at 96% or
greater, the weight can be used without correction to
calculate the concentration of the stock standard.
Certified, commercially prepared stock standards can be used
at any concentration.
762 Transfer the stock standard solutions into Teflon-sealed
screw cap bottles. Store at 4 C and protect from light.
Stock standard solutions should be checked frequently for
signs of degradation or evaporation, especially just prior
to preparing calibration standards from them.
763 Stock standard solutions must be replaced after six months,
or sooner if comparison with check standards indicates a
problem.
7.7 LABORATORY CONTROL SAMPLE CONCENTRATE - See Section 10.3.1.
7 8 Fortification Solution of Internal Standards - Prepare a solution
of internal standards in methanol or acetone at concentrations of
0 5 - 2.0 mg/mL. This solution may be used for the preparation of
the calibration solutions specified in 9.3.1. Dilute an aliquot of
the solution to 50 - 100 Mg/mL and use this solution to fortify the
actual water samples as directed in 11.2.2.
8. SAMPLE COLLFCTIQN. PRESFRVATION AND STORAGE
8.1
8 2
Grab samples must be collected in glass containers. Conventional
sampling practices should be followed, except that the bottle must
not be pre-rinsed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of Tygon tubing and other potential
sources of contamination.
All samples must be iced or refrigerated at 4°C from the time of
collection until extraction. PAHs are known to be light sensitive;
therefore, samples, extracts, and standards should be stored in amber
or foil-wrapped bottles in order to minimize photolytic
decomposition. Fill the sample bottles and, if residual chlorine
is present, add 100 mg of sodium thiosulfate per liter of sample and
mix well. EPA Methods 330.4 and 330.5 may be used for measurement
of residual chlorine. Field test kits are available for this
150
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With 6N-HC1 to
8.3 All samples must be extracted within 7 days of collection
completely analyzed within 40 days of extraction (6).
9. CALIBRATION
9.2 EXTERNAL STANDARD CALIBRATION PROCEDURE:
9.2.1
9.3
Prepare calibration standards at a minimum of three
concentration levels for each analyte by adding volumes of
one or more primary dilution standard solutions (3 oTto a
volumetric flask and diluting to volume with acetonitHle
One,°f *ne external standards should be at a concentrat on
.near but above the MDL (Table 1) and the othe?
concentrat ons should bracket the expected rSnge of
concentrations found in real samples or should define the
working range of the detector.
9'2'2 SiS8iiHect1o5?'of*5 to 10° ^L> ana]yze each calibration
standard according to Section 11. Tabulate peak area o?
hr?,cLre*SP°nSeS aga1nst the mass Ejected. The results can
be used to prepare a calibration curve for each compound
Alternatively, if the ratio of response to amount inSed
? J?rroVT ^i ^,a C°nstant over the ™* "9 range
the oriain r!nStahndard deviatio» (RSD)], linearity through
tne origin can be assumed and the average ratio or
calibration factor can be used in place of a9 calibration
INTERNAL STANDARD (IS) CALIBRATION PROCEDURE - To use this approach
the analyst must select one or more internal standard! that a^
similar in analytical behavior to the compounds of Interest The
fSir i T^ fU/^her Demonstrate 'thit the measurement of thl
internal standard is not affected by method or matrix
93>1
Jali^atTrStandards at a minimum of three
v *10" l6Vels for each anal^te of interest by adding
volumes of one or more primary dilution standard solutions
151
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9.3.2
(3.10) to a volumetric flask. To each calibration standard,
add a known amount of one or more internal standards and
dilute to volume with acetonitrile. One of the standards
inould be at a concentration near but above, the MDL and the
other concentrations should bracket the analyte
concentrations found in the sample concentrates or should
define the working range of the detector.
Using injections of 5 to 100 p,L analyze each calibration
standard according to Section 11. Tabulate peak height or
area responses against concentration for each compound and
internal standard. Calculate response factor (RF) for each
compound using Equation 1.
Equation 1
RF
TAsI FCisI
[Ais] [Cs]
where:
As = Response for the analyte to be measured
Ais = Response for the internal standard
Cis = Concentration of the internal standard
Cs = Concentration of the analyte to be measured 0*g/L)
If RF value over the working range is constant (< 10% RSD),
the RF can be assured to be invariant and the average RF can
be used for calculations. Alternatively, the results can be
used to plot a calibration curve of response ratios, AS/Ais
vs. Cs/Cis.
••• ass's
also9 deviates bj more ± 20%, a new calibration curve must be
prepared for that compound.
9 4 1 Daily calibration requirements using the external standard
9 ca bration procedure are a minimum of two calibration check
standards, one at the beginning and one at the end of the
analysis day. These check standards should be at two
different concentration levels to verify the ca i bration
rurve For extended periods of analysis (> 8 hrs), it is
strongly recommended that check standards be interspersed
with samples at regular intervals during the course of the
analysis.
9.4.2 Minimum daily calibration requirements using the internal
standard calibration procedure consist of initial analyses
of a calibration check standard followed by verification of
152
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the internal standard response of each sample applying
criterion described in Section 10.4.
9.5 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interferences from reagents.
10. QUALITY CONTROL
10.1 Each laboratory that uses this method is required to operate a
formal quality control (QC) program. The minimum QC requirements
are initial demonstration of laboratory capability, analysis of
laboratory reagent blanks, laboratory fortified blanks, laboratory
fortified matrix samples and QC samples. Additional quality control
practices are recommended.
10.2 LABORATORY REAGENT BLANKS (LRB) - Before processing any samples, the
analyst must demonstrate that all glassware and reagent
interferences are under control. Each time a set of samples is
analyzed or reagents are changed, a LRB must be analyzed. For this
method, the LRB is filtered reagent water. If within the retention
time window of an analyte of interest, the LRB produces a peak that
interferes with analyte determination, determine the source of
contamination and eliminate the interference before processing
samples.
10.3 INITIAL DEMONSTRATION OF CAPABILITY
10.3.1 Select a representative spike concentration (about 10 times
MDL) for each analyte. Prepare a laboratory control sample
concentrate (in acetonitrile) from the stock standard
solutions containing each analyte at 1000 times the selected
concentration. Using a pipet, add 1.00 mL of the
concentrate to each of at least four 1 L aliquots of reagent
water and analyze each aliquot according to procedures
beginning in Section 11.2.
10.3.2 For each analyte, the recovery value must for at least three
out of four consecutively analyzed samples fall in the range
of R + 30% (or within R ± 3 Sr, if broader) using the values
for R and Sr for reagent water in Table 2 (fortification
level 1). The relative standard deviation of the mean
recovery measured in 10.3.1 should be ± 30% or 3ST
(whichever is greater), using the values of S (level 1)) in
Table 2. For those compounds that meet the acceptance
criteria, performance is acceptable and sample analysis may
begin. For those compounds that fail these criteria,
initial demonstration of capability must be repeated.
10.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples by a
153
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new, unfamiliar method prior to evidencing a basal level of
skill at performing the technique. It is expected that as
laboratory personnel gain experience with this method the
quality of the data will improve beyond the requirements
stated in Section 10.3.2.
10.3.4 The analyst is permitted to modify 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 Section
10.3.
10.4 ASSESSING THE INTERNAL STANDARD - When using the IS calibration
procedure, the analyst is expected to monitor the IS response (peak
area or peak height) of all samples during each analysis day. The
IS response for any sample chromatogram should not deviate from the
daily mean IS response by more than 30%.
10.4.1 If a deviation of > 30% is encountered for a sample, re-
inject the extract.
10.4.1.1 If acceptable IS response is achieved for the
re-injected extract, then report the results for
that sample.
10.4.1.2 If a deviation of > 30% is obtained for the re-
injected extract, analysis of the sample should
be repeated beginning with Section 11.2,
provided the sample is still available.
Otherwise, report results obtained from the re-
injected extract, but annotate as suspect.
10.4.2 If consecutive samples fail the IS response acceptance
criterion, immediately analyze a calibration check standard.
10.4.2.1 If the check standard provides a response factor
(RF) within 20% of the predicted value, then
follow procedures itemized in Section 10.4.1 for
each sample failing the IS response criterion.
10.4.2.2. If the check standard provides a response factor
(RF) which deviates more than 20% of the
predicted value, then the analyst must
recalibrate, as specified in Section 9.3.
10.5 LABORATORY FORTIFIED BLANK
10.5.1 The laboratory must analyze at least one laboratory
fortified blank (LFB) per sample set (all samples analyzed
within a 24 hour period). The concentration of each analyte
in the LFB should be 10 times the MDL. Calculate accuracy
as percent recovery (R). If the recovery of any analyte
154
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falls outside the control limits (See Section 10.5.2), that
analyte is judged out of control, and the source of the
problem must be identified and resolved before continuing
analyses. 3
10.5.2 Until sufficient LFB data become available, usually a
minimum of results from 20 to 30 analyses, the laboratory
XcrVhSS,f" lts Perf°™ance against the contro1 Umlts
described in Section 10.3.2. When sufficient laboratory
performance data becomes available, develop control limits
from the mean percent recovery (R) and standard deviation
( V u-?f ,_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!
9™up of five to ten new recovery measurements,
r«rn 9nTlt!nS]10.Uld *? recal cl^ ated using only the most
recent to to 30 data points.
10.6 LABORATORY FORTIFIED MATRIX SAMPLE (LFM)
concentration to
or one fortified
The fortified
the background
Ideally, the
as that used for
from all routine
10.6.2
10.6.1 The laboratory must add a known fortified
a minimum of 10% of the routine samples
sample per set, whichever is greater.
concentration should not be less than
concentration of the original sample.
fortified concentration should be the same
the LFB (Section 10.5). Over time, samples
sample sources should be fortified.
Calculate the 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 Section 10.5.2 for the analyses of
LFBs.
If the recovery of any analyte falls outside the designated
range, and the laboratory performance for that analyte is
shown to be in control (Section 10.5), 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 must be labelled suspect/matrix to
inform the data user that the results are suspect due to
matrix effects.
10.7 QUALITY CONTROL SAMPLES (QCS) - Each quarter, the laboratory should
?nf yoZrVTrp°rnTe QC*S °f available>- If Criteria provided with
documented corrective action should be taken and
10.6.3
155
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10.8 The laboratory may adopt additional quality Control practices for
use with this method. The specific practices that are most
productive depend upon the needs of the laboratory and the nature of
the samples. For example, field or laboratory duplicates may be
analyzed to assess the precision of the environmental measurements
or field reagent blanks may be used to assess contamination of
samples under site conditions, transportation and storage.
11. PROCEDURE
11 1 SAMPLE CLEANUP - Cleanup procedures may not be necessary for a
relatively clean sample matrix. If particular circumstances demand
the use of a cleanup procedure, the analyst first must demonstrate
that the requirements of Section 10.5 can be met using the method as
revised to incorporate the cleanup procedure EPA Method 610
describes one possible cleanup procedure for this analyte list.
11.2 SAMPLE EXTRACTION - LIQUID-SOLID EXTRACTION (LSE)
11.2.1 Preparation of liquid-solid extraction cartridges.
11.2.1.1 Wash each C-18 (l.Og) cartridgewith four 10 mL
aliquots of methylene chloride (MeCl2). Let the
cartridge drain after each wash.
11 2 1 2 Wash each cartridge with four 10 mL aliquots of
' ' methanol (MeOH), letting the cartridge drain
after each wash.
11 2 1 3 Wash the cartridges with two, 10 mL aliquots of
reagent water. Allow the first 10 mL portion to
wash through letting the cartridge drain dry.
Next wash the last 10 mL portion through keeping
the cartridge wet. (Water level just above the
packing).
11 2 2 Mark the water meniscus on the side of the sample bottle
(approximately 1 L) for later determination of sample
volume. Pour the entire sample into a 2 L separatory
funnel. Add an aliquot of the fortification solution of
internal standards (50-100 w/ml) described in 7.8. The
addition of a 100 pi aliquot will yield internal standard
concentrations of 5-10 jug/L in aqueous solution. The
optimum internal standard concentrations employed will
depend upon the UV absorbance and/or fluorescence Properties
of the compounds. Concentrations should be selected which
yield peak area counts equivalent to the upper range of
analyte concentrations.
11.2.3 Attach a prepared C-18 cartridge (Section II-2-1) on a.1 ^
vacuum flask. Attach a 75 mL reservoir to the C-18
156
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cartridge with an appropriate adaptor. Position the 2 L
separatory funnel with the sample so that the sample can be
run into the 75 ml reservoir. Connect the vacuum source
(hose of vacuum pump) to the 1 L vacuum flask and filter the
entire sample through the cartridge extraction train.
Adjust vacuum to 8-10 mm Hg.
11.2.4 Wash the cartridge with 10 ml of reagent water. Continue to
draw vacuum through the cartridge for an additional 10
minutes to dry the cartridge. Release the vacuum and
discard the sample waste.
11.2.5 Elute the sample from the cartridge with two 5 ml portions
of MeCl2. Wash the 2 L separatory funnel with 2 ml of MeCl2
and add to the cartridge extract. Note: All glass surfaces
coming in contact with the aqueous sample must be washed
with methylene chloride (1 ml per container) and added to
the column eluate.
11.2.6 Prepare a chromatographic column by packing it with 1 inch
of anhydrous sodium sulfate. Wet the sodium sulfate by
passing 10 ml of methylene chloride through the column.
Pour the cartridge extract and washings from Section 11.2.5
through the chromatographic column and collect into a
calibrated 10 ml Kuderna-Danish concentrator tube.
11.2.7 Rinse the drying column with an additional 2 ml of MeCl2 and
collect in the concentrator tube. Stopper the concentrator
tube and store refrigerated (4°C) if further processing will
not be performed immediately. If the extract will be stored
longer than two days, it should be transferred to a Teflon-
sealed screw-cap vial and protected from light.
11.2.8 Evaporate the eluate with a stream of N2 to a volume of 1.0
mL. Add 3.0 ml of acetonitrile (MeCN) and concentrate to a
final volume of 0.5 ml. Stopper the concentrator tube and
store refrigerated if further processing will not be
performed immediately. If the extract will be stored longer
than two days, it should be transferred to a Teflon-sealed
screw cap vial and protected from light. If the sample
extract requires no further cleanup, proceed with liquid
chromatographic analysis (Section 11.4).
11.2.9 Determine the original sample volume by refilling the sample
bottle to the mark and transferring the liquid to a 1000 mL
graduated cylinder. Record the sample volume to the nearest
5 ml.
11.3 SAMPLE EXTRACTION - DISK EXTRACTION
11.3.1 Preparation of disks. ,
157
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11.3.1.1 Insert the disk into the 47 mm filter apparatus.
Wash the disk with 5 ml methylene chloride
(MeCl2) by adding the MeCl2 to the disk, pulling
about half through the disk and allowing it to
soak the disk for about a minute, then pulling
the remaining MeCl2 through the disk. With the
vacuum on, pull air through the disk for a
minute.
11.3.1.2 Pre-wet the disk with 5 ml methanol (MeOH) by
adding the MeOH to the disk, pulling about half
through the disk and allowing it to soak for
about a minute, then pulling most of the
remaining MeOH through. A layer of MeOH must be
left on the surface of the disk, which shouldn't
be allowed to go dry from this point until the
end of the sample extraction. THIS IS A
CRITICAL STEP FOR A UNIFORM FLOW AND GOOD
RECOVERY.
11.3.1.3 Rinse the disk with 5 mL reagent water by adding
the water to the disk and pulling most through,
again leaving a layer on the surface of the
disk.
11.3.2 Add 5 mL MeOH per liter of water sample. Mix well.
11.3.3 Add the water sample to the reservoir and turn on the vacuum
to begin the filtration. Full aspirator vacuum may be used.
Particulate-free water may filter in as little as 10 minutes
or less. Filter the entire sample, draining as much water
from the sample container as possible.
11.3.4 Remove the filtration top from the vacuum flask, but don't
disassemble the reservoir and fritted base. Empty the water
from the flask and insert a suitable sample tube to contain
the eluant. The only constraint on the sample tube is that
it fit around the drip tip of the fritted base. Reassemble
the apparatus.
Add 5 mL of acetonitrile (CH3CN) to rinse the sample bottle.
Allow the CH,CN to settle to the bottom of the bottle and
transfer to the disk with a dispo-pipet, rinsing the sides
of the glass filtration reservoir in the process. Pull
about half of the CH3CN through the disk, release the
vacuum, and allow the disk to soak for a minute. Pull the
remaining CH3CN through the disk.
Repeat the above step twice, using MeCl2 instead of CH3CN.
Pour the combined eluates thru a small funnel with filter
paper containing 3 grams of anhydrous sodium sulfate. Rinse
158
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the test tube and sodium sulfate with two 5 ml portions of
MeCl2. Collect the filtrate in a concentrator tube.
11.3.5 With the concentrator tube in a 28
evaporate the eluate with a stream of N
C heating block,
i2 to 0.5 ml.
11.4 SAMPLE ANALYSIS
11.4.1
Table 1 summarizes the recommended operating conditions for
the HPLC. Included in this table are retention times and
MDLs that can be achieved under these conditions. The UV
detector is recommended for the determination of
naphthalene, acenaphthylene, acenaphthene and fluorene. The
fluorescence detector is recommended for the remaining PAHs
An example for the separation achieved by this HPLC column
is shown in Figure 1. Other HPLC columns, chromatographic
conditions, or detectors may be used if the requirements of
Section 10.5 are met.
11.4.2 Calibrate the system daily as described in Section 9.
11.4.3 Inject 5 to 100 juL of the sample extract or standard into
the HPLC using a high pressure syringe or a constant volume
sample injection loop. Record the volume injected to the
nearest 0.1 /zL, and the resulting peak size in area or peak
height units. Re-equilibrate the HPLC column at the initial
gradient conditions for ,at least 10 minutes between
injections. *
11.4.4 Identify the analytes in the sample by comparing the
retention time of the peaks in the sample chromatogram with
those of the peaks in standard chromatograms. The width of
the retention time window used to make identifications
should be based upon measurements of actual retention time
variations of standards over the course of a day. Three
times the standard deviation of a retention time 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 Identification requires expert judgement when sample
components are not resolved chromatographically, that is,
when GC peaks obviously represent more than one sample
component (i.e., broadened peak with shoulder(s) or valley
between two or more maxima), or any time doubt exists over
the identification of a peak in a chromatogram, appropriate
confirmatory techniques need to be employed such as use of
an alternative detector which operates on a
chemical/physical principle different from that originally
used, e.g., mass spectrometry, or the use of a second
chromatography column.
159
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11.4.6 If the response for a peak exceeds the working range of the
system, dilute the extract with acetonitrile and reanalyze.
11.4.7 If the measurement of the peak response is prevented by the
presence of interferences, further cleanup is required.
12. CALCULATIONS
12.1 Determine the concentration of individual compounds in the sample.
12.1.1 If the external standard calibration procedure is used,
calculate the amount of material injected from the peak
response using the calibration curve or calibration factor
determined in Section 9.2.2. The concentration in the
sample can be calculated from Equation 2.
Equation 2
Concentration
= (A) (Vt)
(V=) (VJ
where:
A = Amount of material injected (ng).
Vi - Volume of extract injected (/zL).
Vt = VolumeN)f total extract (AIL).
Vs = Volume of water extraction (mL).
12.1.2 If the internal standard calibration procedure is used,
calculate the concentration in the sample using the response
factor (RF) determined in Section 9.3.2 and Equation 3.
Equation 3
\
Concentration (jug/L) = (As) (Is)
(Ais) (RF) (V0)
where:
As = Response for the parameter to be measured.
Ais = Response for the internal standard.
Is = Amount of internal standard added to each extract
(jug).
Vo = Volume of water extracted (L).
12 2 Report results in jug/L without correction for recovery data. All QC
data obtained should be reported with the sample results.
160
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13. METHOD PERFQRMANCF
13.1 The method detection limit (MDL) is defined as the minimum
99- P^cision and accuracy
results for the dechlonnated tap water are shown in Table 3
lable 4 contains precision and accuracy results from replicate
analyses of five well water samples using Empore disk liquid-solid
GXLVclCLlOn •
14. REFERENCES
ASTM Annual Book of Standards, Part 31, D3694. "Standard Practices
tor Preparation of Sample Containers and for Preservation of Organic
1. Glaser, J. A., D. L. Foerst, G. D. McKee, S. A. Quave and W. L. Budde,
Trace Analysis of Wastewaters", Environ. Sci. Techno!. 15, 1426
2.
3.
4.
5.
"Carcinogens - Working with Carcinogens", Department of Health,
Education and We fare. Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, August 1977.
"OSHA Safety and Health Standards, General Industry", (29 CFR 1910)
Occupational Safety and Health Administration, OSHA 2206 (Revised
"Safety in Academic Chemistry Laboratories", American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
161
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6 "Determination of Polynuclear Aromatic Hydrocarbons in Industrial and
Municipal Wastewaters", EPA-600/4-82-025, U. S. Envnronmental
Protection Agency, Environmental Monitoring Systems Laboratory,
Cincinnati, Ohio 45268, September 1982.
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TABLE 1. HIGH PERFORMANCE LIQUID CHRONATOGRAPHY CONDITIONS
AND METHOD DETECTION LIMITS
Method
Detection
Analyte
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo (a) anthracene
Chrysene
Benzo (b)fluoranthene
Benzo ( k) f 1 uoranthene
Benzo(a)pyrene
Di benzo (a , h) anthracene
Benzo (g , h , i ) peryl ene
Indeno(l,2,3-cd)pyrene
Sample
Set
UV
UV
UV
UV
FD-B
FD-A
FD-B
FD-A
FD-B
FD-A
FD-B
FD-A
FD-B
FD-B
FD-B
FD-A
Retention
Time
min
12.5
13.8
15.4
15.8
16.8
17.6
18.7
19.4
21.9
22.3
24.2
25.0
26.0
27.1
27.8
28.3
Method
Detection
Limit
M9/L (a)
2.20
1.41
2.04
0.126
0.150
0.140
0.009
0.126
0.004
0.160
0.006
0.003
0.016
0.035
0.020
0.036
Limit
Fortification
Level
10.0
10.0
10.0
1.00
0.500
0.625
0.025
0.625
0.010
0.625
0.010
0.0125
0.050
0.125
0.050
0.125
HPLC column conditions: Reverse-phase LC-PAH, 5 micron particle size, in a
25 cm x 4.6 mm ID stainless steel column. Isocratic elution for 2 min.
using acetonitrile/water (3.5 : 6.5), then linear gradient elution to 100%
acetonitrile over 22 min. at 2.0 mL/min. flow rate.
(a) The MDL for naphthalene, acenaphthylene, acenaphthene, and fluorene were
determined using a UV detector. All others were determined using a
fluorescence detector.
163
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TABLE 2. SINGLE-LABORATORY ACCURACY AND PRECISION FROM
SEVEN REPLICATE ANALYSES OF FORTIFIED REAGENT WATER
Concentration
Level
R(a)
SP(b)
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo (a) anthracene
Chrysene
Benzo (b)fluoranthene
Benzo(k)fluoranthene
Benzo(a)pyrene
Dibenzo (a, h) anthracene
Benzo (g,h,i)perylene
Indeno(l,2,3-cd)pyrene
(a) R - Mean Recovery %
(b) Sr « Standard Deviation
10.0
10.0
10.0
1.0
0.500
0.625
0.025
0.625
0.010
0.625
0.010
0.0125
0.050
0.125
0.050
0.125
of the %
70.5
78.0
79.0
74.5
66.9
72.8
90.2
88.8
76.0
93.6
87.5
81.2
76.5
78.4
81.5
75.2
7.0
4.5
6.5
4.0
9.3
7.2
12.0
6.4
14.0
8.0
18.5
7.2
10.3
8.8
13.0
9.2
164
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TABLE 3. SINGLE-LABORATORY ACCURACY AND PRECISION FROM
NINE REPLICATE ANALYSES OF FORTIFIED TAP WATER (a)
Fortified
Concentration
Level
Analyte M9/L R Sr
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo (a) anthracene
Chrysene
Benzo (b) f 1 uoranthene
Benzo (k)fl uoranthene
Benzo (a)pyrene
Dibenzo( a, h) anthracene
Benzo (g , h , i ) peryl ene
Indeno(l,2,3-cd)pyrene
10.0
10.0
10.0
1.0
0.5
0.625
0.025
0.625
0.01
0.625
0.006
0.0125
0.05
0.125
0.05
0.125
72.8
64.1
67.1
72.5
59.5
63.3
80.7
80.7
78.1
73.1
65.9
74.9
70.0
64.7
67.3
74.0
10.7
8.0
7.6
7.1
4.3
9.1
6.7
13.3
6.5
10.2
5.6
10.8
7.5
7.5
8.0
10.2
(a) Tap water was dechlorinated with sodium thiosulfate, according to the
method (100 mg/L), upon collection prior to spiking with analytes.
165
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TABLE 4. SINGLE-LABORATORY ACCURACY AND PRECISION FROM FIVE REPLICATE
ANALYSES OF FORTIFIED WELL WATER USING DISK LIQUID-SOLID EXTRACTION
Analyte
Concentration
Level 1
M9/L R(a)
Concentration
Level 2
SP(b)
R
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo (a) anthracene
Chrysene
Benzo(b)fl uoranthene
Benzo(k)f 1 uoranthene
Benzo(a)pyrene
Dibenzo(a,h)anthracene
Benzo (g,h,i)perylene
Indeno(l,2,3-cd)pyrene
11.0
22.0
11.0
2.2
1.1
1.1
2.2
1.1
1.1
1.1
2.2
1.1
1.1
2.2
2.2
1.1
49.6
57.8
53.0
71.4
87.0
62.8
89.4
96.0
89.6
98.4
78.5
90.2
87.0
88.8
100.4
105.2
45.1
30.3
33.5
26.1
14.3
18.1
9.8
13.3
12.4
11.2
14.3
11.5
4.9
10.3
15.7
14.8
110.0
220.0
110.0
22.0
11.0
11.0
22.0
11.0
11.0
11.0
22.0
11.0
11.0
22.0
22.0
11.0
75.2
77.0
74.2
80.0
72.2
66.8
69.2
52.8
62.2
60.8
82.0
73.2
54.7
65.0
59.5
85.2
17.9
11.8
16.3
13.9
12.7
8.6
9.6
9.3
13.9
14.2
10.7
11.9
9.8
13.0
8.1
17.9
(a) R « Mean Recovery %
(b) Sr » Standard Deviation of the %
(c) Concentration Level 2 = Concentration for analytes which yield a signal-to-
noise ratio of approximately 10 in the extract (25 /iL injection)
166
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0u9 j£d (p 'o-e 'g *t) ouapui
ft-o* 9U9t/U9d (?qB) ozuag
fr-OJ 9U999JlftUe (M *•) OZU9qiO
»uajAd {«) ozuog
ozutg
19-04 9U9lUUCJOni I (q) OZU98
rs-oj 9U999 jy^ue (e) ozuag
m
d
I
m
o
I
cu
o
esuodsaa
CD
'CU
h»
in
'OJ
^r
fU
CM
CXI
^ c
CD —
_«g Jj
•v o
* o>
CU J-»
r *
O)
CO
r*.
to
in
cu
+ Analysis set (See Section 14)
Figure 1. PAH HPLC Chromatogram using UV Detection.
Chromatographic Conditions are as Stated in Table 1.
167
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-------
METHOD 551. DETERMINATION OF CHLORINATION DISINFECTION BYPRODUCTS AND
CHLORINATED SOLVENTS IN DRINKING WATER BY LIQUID-LIQUID EXTRACTION
AND GAS CHROMATOGRAPHY WITH ELECTRON-CAPTURE DETECTION
July 1990
J. W. Hodgeson
A. L. Cohen
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
169
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METHOD 551
DETERMINATION OF CHLORINATION DISINFECTION BYPRODUCTS AND CHLORINATED
SOLVENTS IN DRINKING WATER BY LIQUID-LIQUID EXTRACTION AND GAS
CHROMATOGRAPHY WITH ELECTRON-CAPTURE DETECTION
1. SCOPE AND APPLICATION
1.1 This method (1-4) is applicable to the determination of the
following analytes in finished drinking water, drinking water during
intermediate stages of treatment, and raw source water:
ANALYTE CAS No.
Bromochloroacetonitrile 83463-62-1
Bromodichloromethane 75-27-4
Bromoform 75-25-2
Carbon Tetrachloride 56-23-5
Chloral Hydrate 75-87-6
Chloroform 67-66-3
Chloropicrin 76-06-2
Dibromoacetonitrile 3252-43-5
Dibromochloromethane 124-48-1
l,2-Dibromo-3-chloropropane [DBCP] 96-12-8
1,2-Dibromoethane [EDB] 106-93-4
Dichloroacetonitrile 3018-12-0
Trichloroacetonitrile 545-06-2
Tetrachloroethylene 127-18-4
1,1,1-Trichloroethane 71-55-6
Trichloroethylene 79-01-6
1,1,1-Tri chloro-2-propanone 918-00-3
l,l-Dichloro-2-propanone 513-88-2
1.2 This analyte list includes twelve commonly observed chlorination
disinfection byproducts (3,4) and six commonly used chlorinated
organic solvents - carbon tetrachloride, l,2-dibromo-3-chloropropane
(DBCP), 1,2-dibromoethane (EDB), tetrachloroethylene, 1,1,1-tri-
chloroethane and trichloroethylene.
1.3 This method is intended as a stand-alone procedure for the analysis
of only the trihalomethanes (THMs) or as a procedure for the total
analyte list. The dechlorination/preservation technique presented
in section 8 differs for the two modes of operation, with a simpler
technique available for the THM analysis. The six solvents may be
analyzed in the THM mode, since the same dechlorination reagents may
be employed.
1.4 The experimentally determined method detection limits (MDLs) (5) for
the above listed analytes are provided in Table 2. Actual MDL
values will vary according to the particular matrix analyzed and the
specific instrumentation employed.
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2. SUMMARY OF METHOD
2.1 A 35 ml sample aliquot is extracted with 2 ml of methyl-tert-butyl
ether (MTBE). Two //L of the extract is then injected into a GC
equipped with a fused silica capillary column and linearized
electron capture detector for separation and analysis. Aqueous
calibration standards are also extracted and analyzed in order to
compensate for any extraction losses. A typical sample can be
extracted and analyzed in 40 to 50 min using the primary column
chosen for this method (6,8.2.1). Confirmation of the eluted
compounds may be obtained using a dissimilar column (6.8.2.2) or by
the use of GC-MS.
3. DEFINITIONS
3.1 Internal standard — A pure analyte(s) added to a solution in known
amount(s) and used to measure the relative responses of other method
analytes and surrogates that are components of the same solution.
The internal standard must be an analyte that is not a sample
component.
3.2 Surrogate analyte — A pure analyte(s), which is extremely unlikely
to be found in any sample, and which is added to a sample aliquot in
known amount (s) before extraction and is measured with the same
procedures used to measure other sample components. The purpose of
a surrogate analyte is to monitor method performance with each
sample.
3.3 Laboratory duplicates (LD1 and LD2) — Two sample aliquots taken in
the analytical laboratory and analyzed separately with identical
procedures. Analyses of LD1 and LD2 give a measure of the precision
associated with laboratory procedures, but not with sample 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. Ana-
lyses of FD1 and FD2 give a measure of the precision associated with
sample collection, preservation and storage, as well as with lab-
oratory procedures.
3.5 Laboratory reagent blank (LRB) — An aliquot of reagent water that
is treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates
that are used with other samples. The LRB is used to determine if
method analytes or other interferences are present in the labora-
tory environment, the reagents, or the apparatus.
3.6 Field reagent blank (FRB) — Reagent water placed in a sample con-
tainer in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage,
171
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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 to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise
measurements at the required method detection limit.
3.8 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 — A concentrated solution containing a
single certified standard that is a method analyte, or a
concentrated solution of a single analyte prepared in the laboratory
with an assayed reference compound. Stock standard solutions are
used to prepare primary dilution standards.
3.10 Primary dilution standard solution — A solution of several analytes
prepared in the laboratory from stock standard solutions and diluted
as needed to prepare calibration solutions and other needed analyte
solutions.
3.11
3.12
Calibration standard (CAL) — a solution prepared from the primary
dilution standard solution and stock standard solutions of the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
Quality control sample (QCS) — a sample matrix containing method
analytes or a solution of method analytes in a water miscible
solvent which is used to fortify reagent water
samples. The QCS is obtained from a source
laboratory, and is used to check laboratory
externally prepared test materials.
or environmental
external to the
performance with
4.
INTERFERENCES
4.1 Impurities contained in the extracting solvent usually account for
the majority of the analytical problems. Solvent blanks should be
analyzed for each new bottle of solvent before use. An interference-
free solvent is a solvent containing no peaks yielding data at > MDL
(Table 2) and at the retention times of the analytes of interest.
Indirect daily checks on the extracting solvent are obtained by
172
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monitoring the laboratory reagent blanks (10.2). Whenever an
interference is noted in the sample blank, the analyst should
analyze another solvent blank. Low level interferences generally
can be removed by distillation or column chromatography (2).
4.2 Commercial lots of the MTBE extraction solvent often contain
observable amounts of chlorinated solvent impurities, e.g.,
chloroform, trichloroethylene, carbon tetrachloride. When present,
these impurities can normally be removed by a double distillation of
the MTBE.
4.3 This liquid/liquid extraction technique efficiently extracts a wide
boiling range of non-polar and polar organic components of the
sample. Thus, confirmation is quite important, particularly at
lower analyte concentrations. A confirmatory column (6.8.2.2) is
provided for this purpose. Alternatively, a more powerful technique
is confirmation by GC-MS.
5. SAFETY
5.1 The toxicity and carcinogenicity of chemicals used in this method
have not been precisely defined; each chemical should be treated as
a potential health hazard, and exposure to these chemicals should be
minimized. Each laboratory is responsible for maintaining awareness
of OSHA regulations regarding safe handling of chemicals used in
this method. Additional references to laboratory safety are
available (6-8) for the information of the analyst.
5.2 The following have been tentatively classified as known or suspected
human or mammalian carcinogens:
Chloroform, l,2-Dibromo-3-chloropropane, 1,2-Dibromoethane.
5.3 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. Therefore, protective
clothing and gloves should be used and MTBE should be used only in
a chemical fume hood or glove box. The same precaution applies to
pure standard materials.
6. APPARATUS AND EQUIPMENT (All specifications in Sections 6 and 7 are
suggested. Catalogue numbers are included for illustration only.)
6.1 SAMPLE CONTAINERS - 40 mL screw cap vials [Pierce #13075] or
equivalent each equipped with a PTFE-faced silicone septum (Pierce
#12722, Fisher TFE-lined #02-883-3F or equivalent). NOTE: Some
commercial 40 mL vials do not have adequate volume when salt is
added. (See Sect. 11.1.4). Prior to use, wash vials and septa with
detergent and rinse with tap water, followed by distilled water.
Allow the vials and septa to dry at room temperature, place the
vials in an oven and heat to 400°C for 30 min. After removal from
173
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the oven allow the vials to cool in an area known to be free of
organics.
6.2 VIALS - Autosampler, screw cap with septa, 1.8 ml, Van'an #96-000099
-00 or EQUIVALENT.
6.3 MICRO SYRINGES - 10 pi, 25 /iL, 50 /iL, 100 #L, 250 juL,
6.4 PIPETTES - 2.0 mL transfer, glass.
6.5 VOLUMETRIC FLASK - 10 mL, 100 mL, 250 mL glass stoppered.
6.6 DISPOSABLE PASTEUR PIPETS - Kimble No. 72050575 or equivalent.
6.7 STANDARD SOLUTION STORAGE CONTAINERS - 15 mL Boston round, amber
glass bottles with TFE-lined caps. Wheaton Cat. No. 220092 or
equivalent. TFE-lined caps must be purchased separately. Size 18-
400, Fisher TFE-lined screw cap No. 02-883-3D or EQUIVALENT.
6.8 GAS CHROMATOGRAPHY SYSTEM
6.8.1 The GC must be capable of temperature programming and should
be equipped with a linearized electron capture detector,
fused silica capillary column, and splitless injector
(splitless mode, 30 sec. delay). An auto-sampler/injector is
desirable.
6.8.2 Two GC columns are recommended. Column A should be used as
the primary analytical column unless routinely occurring
analytes are not adequately resolved. Column B is
recommended for use as a confirmatory column when GC/MS
confirmation is unavailable.
6.8.2.1
Column A - 0.32 mm ID x 30 m fused silica capillary
with chemically bonded methyl polysiloxane phase
(DB-1, 1.0 urn film thickness or equivalent). The
linear velocity of the helium carrier is
established at 23 cm/sec at 35°C. The oven is
programmed to hold at 35°C for 9 min, to increase to
40°C at l°C/min, and held for 3 min, to increase to
120°C at 6°C/min and held at 120°C until all
expected compounds have eluted. A temperature of
150°C is then maintained for 5 min. Injector
temperature: 200°C. Detector temperature:
(See Table 1 for retention time data).
290°C
6.8.2.2 Column B - 0.32 mm ID x 30 m with chemically bonded
50% trifluoropropyl phase (DB-210, SP-2401, 0.5 urn
film thickness or equivalent). The linear velocity
of the helium carrier gas is established at 27
cm/sec. The column temperature is programmed to
hold at 30°C for 11 min, to increase to 120°C at
174
-------
10 C/min and held at 120°C until all expected
compounds have eluted. A temperature of 150°C is
then maintained for 5 min. (See Table 1 for
retention data).
6.9 pH Meter - capable of accurate measurement of pH (± 0.2 units) in
the range, pH = 4-8. For laboratory or field measurement of sample
pH.
6.10 pH Paper - narrow ranges, pH = 3-5.5 and pH = 6.0-8 0 For
measurement of initial and adjusted sample pH in the field.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 REAGENTS
7.1.1 MTBE - High purity grade, It may be necessary to double
distill the solvent if impurities are observed which coelute
with some of the more volatile compounds.
7.1.2 Acetone - High purity, demonstrated to be free of analytes.
, . 7,1.3 Sodium Chloride, NaCl - ACS Reagent Grade. Before use
pulverize a batch of NaCl and place in muffle furnace,
increase temperature to 400°C and hold for 30 min. Store in
a capped bottle.
,7-2 REAGENT WATER - Reagent water is defined as purified water which
does not contain any measurable quantities of the analyte or anv
other interfering species.
7.2.1 A Millipore Super-Q water system or its EQUIVALENT may be
used to generate deionized reagent water. Distilled water
that has been charcoal filtered may also be suitable.
7.2.2 Test reagent water each day it is used by analyzing according
to Sect. 11.2.
7.3 STOCK STANDARD SOLUTIONS - These solutions may be obtained as
certified solutions or prepared from neat materials using the
following procedures:
7.3.1 Prepare stock standard solutions(5 mg/mL) for the THM's and
the six solvents by accurately weighing approximately 0.05 g
of pure material. Dilute to volume with methanol in a 10 mL
volumetric flask. Accurate standards for the more volatile
analytes may be prepared in the following manner.
7.3.1.1 Place about 9.8 mL of methanol into a 10-mL ground-
glass stoppered volumetric flask. Allow the flask
175
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to stand, unstoppered, for about 10 min and weigh
to the nearest 0.1 mg.
7.3.1.2 Use a 100-juL syringe and immediately add two or
more drops of standard material to the flask. Be
sure that the standard material falls directly into
the alcohol without contacting the neck of the
flask.
7.3.1.3 Reweigh, dilute to volume, stopper, then mix by
inverting the flask several times. Calculate the
concentration in micrograms per microliter from the
net gain in weight.
7.3.2 Prepare stock standard solutions (5.0 mg/mL) for the eight
remaining chlorination byproducts (1.1) by accurately
weighing about O.OSOOg of pure material. Dissolve the
material in acetone and dilute to volume in a 10-mL
volumetric flask. Acetone is employed because decomposition
has been observed during storage in methanol for the
dihaloacetonitriles, chloropicrin and 1,1,1-
tri chloropropanone-2.
7.3.3 Larger volumes of standard solution may be prepared at the
discretion of the analyst. When compound purity is assayed
to be 96% or greater, the weight can be used without
correction to calculate the concentration of the stock
standard. Commercially prepared stock standards can be used
at any concentration if they are certified by the
manufacturer or by an independent source.
7 3.4 Transfer the stock standard solutions into Teflon-lined screw
cap amber bottles. Store at 4°C and protect from light.
Stock standard solutions should be checked frequently for
signs of degradation or evaporation, especially just prior to
preparing calibration standards from them.
7.3.5 The stored THM stock standards in methanol are stable for up
to six months. The solvent standards in methanol are stable
at least four months. The other analytes stored in acetone
are stable for at least four months except for chloral
hydrate. Initially, fresh chloral hydrate standards should
be prepared weekly, until the stability of this analyte is
determined.
7 4 PRIMARY DILUTION STANDARDS—Prepare primary dilution standards by
combining and diluting stock standards in methanol (THMs and
solvents) or acetone (remaining disinfection byproducts). The
primary dilution standards should be prepared at concentrations that
can be easily diluted to prepare aqueous calibration standards
(Sect. 9.1) that will bracket the working concentration range.
176
-------
Store the primary dilution standard solutions at 4°C with minimal
headspace and check frequently for signs of deterioration or
evaporation, especially just before preparing calibration standards
t0
7'5 be™0 ANALYTES"~Known commercial sources of the analytes are given
ANALYIE SOURCES
Bromodichloromethane Columbia Chemicals
Camden, S.C.
Pfalz and Bauer
Bromochl oroacetoni tri 1 e Waterbury Co™ .
»"»"«"• Aldrlch
Carbon Tetrachloride
CM oral Hydrate S1gma
Chlorofo™
Dibromoacetonitrile "
Adrlch
Dibromochloromethane
1,2-Dibromoethane
1 , 2-Di bromo-3-chl oropropane
Dichloroacetonitrile Pfalz
l.l-Dichloropropanone-2 PfalZ Aidrich
Tetrachloroethylene A dr rh
Trichloroacetonitrile Aldrich
Columbia
1,1,1-Trichloroethane
Trichloroethylene
1 , 1 , 1-Tr i chl oro-2-propanone
7.6 Hydrochloric Acid Solutions, 0.2 and 1.0 N - Prepare solutions for
adjustment of sample pH by serial dilution of ACS reagent ar-de
hydrochloric acid (HC1). y •
7.7 Stock Solution of Internal Standard(s) - Prepare a solution of in-
ternal standard(s) in methanol at concentration(s) of 0.5-1.0 mq/mL
fU?nJ fo al iquinni0f ^ s°1jjtion w1th methanol by an appropriate
factor (e.g. 1:100) required for the internal standard fortification
solution used in preparing calibration standards (9.1.2) or for-
tifying aqueous samples (11.1.3).
177
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8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 SAMPLE COLLECTION, DECHLORINATION, AND PRESERVATION
8.1.1 The analyte list of section 1.1 may be conveniently divided
into three classes - the four THM's, the six halogenated
solvents(1.2) and the eight remaining organic disinfection
by-products. The halogenated solvents are quite stable
compounds by design and stability upon storage after collec-
tion is not an issue. Likewise, the THM's are preserved by
the addition of any of the following common dechlorination
reagents, sodium sulfite or thiosulfate, ascorbic acid and
ammonium chloride. If the sample assay is only for the THM's
and/or solvents, the acidification step in 8.1.3 should be
omitted and only dechlorination reagent added as specified in
8.1.2. Thiosulfate, sulfite and ascorbic acid promote the
decomposition of some members of the third class of analytes,
e.g. the dihaloacetonitriles and chloropicrin, and may not be
used as dechlorination reagents in their analysis. In
addition, many of these analytes require the acidification
step in 8.1.3 for storage stability. Thus analysis for the
total analyte list requires the use of ammonium chloride for
dechlorination and sample acidification. NOTE, however, the
possible exception of a separate sampling requirement for
chloral hydrate in 8.1.8.
8.1.2 Add the dechlorination reagent as the neat material to the 40
mL sample vials(6.1) immediately before shipment to the
field. The reagent amounts are 4 mg for sodium thiosulfate
or sulfite and ammonium chloride and 25 mg for ascorbic acid.
Alternatively, for the first three reagents, 100 /*L of a
freshly prepared solution at a concentration of 40 mg/mL may
be added to the sample vial just before sample collection
(8.1.4). Any of these reagents may be used for the THM's,
whereas ammonium chloride must be employed for the simultan-
eous measurement in a single sample of all the analytes
listed (1.1). As described in 8.1.8, the measurement of
chloral hydrate may require the collection of a separate
sample dechlorinated with sodium sulfite or ascorbic acid.
8.1.3 Adjustment of Sample pH - Prior to sample collection, the
amount of HC1 required to reduce the sample pH to the range,
4.5-5.0 must be measured. Collect 40 mL samples and add to
100 mL beakers containing 10 mg ammonium chloride. Measure
the initial pH with the narrow range pH paper, 6.0-8.0
(6.10), or a pH meter. Initially, adjust the sample pH to
the range 4.5-5.0 with the 0.2 N HC1 solution by dropwise
addition with a Pasteur pipet (6.6). Measure the pH during
addition with the narrow range pH paper, 3.0-5.5, or a pH
meter. If greater than 10 drops are required (ca. 0.1 mL),
178
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measure the amount of 1.0 N HC1 solution required and use
this amount for sample acidification: Care should be
exercised not to adjust the sample pH below the carbonic acid
endpoint, pH « 4.2. Below the endpoint, the pH will decrease
rapidly with dropwise acid addition. Some of the analytes
may not be stable below pH = 4.0. Add the required volume of
HC1 solution to the 40 ml sample vials(6.1) immediately
before collection (8.1.4).
8.1.4 Collect all samples in duplicate. Fill sample bottles to
just overflowing but take care not to flush out the
dechlorination and preservation reagents. No air bubbles
should pass through the sample as the bottle is filled, or be
trapped in the sample when the bottle is sealed.
8.1.5 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.
8.1.6 When sampling from an open body of water, fill a 1-quart
, wide-mouth bottle or 1-liter beaker with sample from a
representative area, and carefully fill duplicate sample
vials from the 1-quart container.
8.1.7 The samples must be chilled to 4°C on the day of collection
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 suffi-
cient ice to ensure that they will be at 4°C on arrival at
the laboratory.
8.1.8 In some matrices dechlorinated with ammonium chloride, forti-
fied matrix recoveries of chloral hydrate have been lower
than expected by 50 percent or greater, when compared to the
same sample dechlorinated with ascorbic acid or sodium
sulfite. In other matrices, recoveries have been normal.
The reason for these differences has not been determined.
Any analyst employing this method must demonstrate that
ammonium chloride is a suitable dechlorination agent for
chloral hydrate in the matrix of concern by determining
matrix recoveries as outlined in 10.6. If problems are
encountered, a separate sample, dechlorinated with 100 mg/L
sodium sulfite or 625 mg/L ascorbic acid, must be collected
for the analysis of chloral hydrate. Limited field data
obtained to date have indicated better precision for chloral
hydrate analyses in samples dechlorinated with sodium sulfite
than with ascorbic acid.
179
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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.
8.2.2 Analyze all samples within 14 days of collection. Samples
not analyzed within this period must be discarded and
replaced.
9. CALIBRATION
9.1 PREPARATION OF CALIBRATION STANDARDS
9.1.1 At least three calibration standards are needed. One should
contain the analytes at a concentration near to but greater
than the method detection limit (Table 2) for each compound;
the other two should bracket the concentration range expected
in samples. For example, if the MDL is 0.1 ^g/L, and a
sample expected to contain approximately 1.0 p,g/l is to be
analyzed, aqueous standards should be prepared at
concentrations of 0.2 jug/L, 1.0 /ug/L, and 2.0 /zg/L.
9.1.2 To prepare a calibration standard, add an appropriate volume
of a primary dilution standard to a 35-mL aliquot of reagent
water in a 40-mL vial. Use a 25-/LtL micro syringe and rapidly
inject the standard into the middle point of the water
volume. Remove the needle as quickly as possible after
injection. If required (9.3), add an appropriate volume of
the internal standard fortification solution (7.7) in the
same manner. The aqueous concentration of internal
standard(s) should yield area counts or peak heights
equivalent to the medium to upper ranges of analyte
concentrations. Mix by inverting the sample vial three times
without shaking. Aqueous standards must be prepared fresh
daily and extracted immediately after preparation (Section
11.2).
9.1.3 Alternatively, add an appropriate volume of primary dilution
standard and internal standard solution to reagent water in
a 100 mL volumetric flask and fill to the mark. Mix by
inverting three times as in 9.1.2. Weigh a 35 mL aliquot of
this standard into a pre-calibrated 40-mL vial.
9.2 EXTERNAL STANDARD CALIBRATION PROCEDURE
9.2.1 Extract and analyze each calibration standard according to
Section 11 and tabulate peak height or area response versus
the concentration of the standard. The results are used to
prepare a calibration curve for each compound by plotting the
peak height or area response versus the concentration.
Alternatively, if the ratio of response to concentration
180
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(response factor) is constant over the working range (< 10%
relative standard deviation,[RSD]), linearity to the origin
can be assumed, and the average ratio or response factor can
be used in place of a calibration curve.
9.2.2 Single point calibration is sometimes an acceptable alterna-
tive to a calibration curve. Prepare single point standards
from the primary dilution standard solutions. The single
point calibration standard should be prepared at a concentra-
tion that produces a response close (± 20%) to that of the
unknowns.
9.3 INTERNAL STANDARD (IS) CALIBRATION PROCEDURE - To use this approach
the analyst must select one or more internal standards that are
similar in analytical behavior to the compounds of interest. The
?nJiJ i T*,! /*her dem?nstrate that the measurement of the
internal standard is not affected by method or matrix interferences.
Specific internal standard are not recommended in this method. The
method validation data reported in Section 13 were obtained by the
external calibration procedure. y
9.3.1 Extract and analyze each calibration standard according to
Section 11. Tabulate peak height or area responses against
concentration for each compound and internal standard.
Calculate response factor (RF) for each compound using
tquation l.
9.4
Equation 1
where
RF = TAsl FCis]
[Ais] [Cs]
As = Response for the analyte to be measured
Ais - Response for the internal standard
Cis = Concentration of the internal standard
Cs = Concentration of the analyte to be measured
It RJ;rvalueuover the working range is constant (< 10% RSD),
the RF can be assumed to be invariant and the average RF can
be used for calculations. Alternatively, the results can be
used to plot a calibration curve of response versus analyte
ratios, As/Ais vs. Cs/Cis.
The working calibration curve, calibration factor, or RF must be
verified on each working day by the measurement of one or more
calibration standards. If the response for any analyte varies from
the predicted response by more than ± 20%, the test must be repeated
using fresh calibration standard. If the fresh calibration standard
181
-------
also deviates by more ± 20%, a new calibration curve must be
prepared for that compound.
9 4 1 Daily calibration requirements using the external standard
calibration procedure are a minimum of two calibration check
standards, one at the beginning and one at the end of the
analysis day. These check standards should be at two diff-
erent concentration levels to verify the calibration curve.
For extended periods of analysis (> 8 hrs), it is strongly
recommended that check standards be interspersed with samples
at regular intervals during the course of the analysis.
9.4.2 Minimum daily calibration requirements using the internal
standard calibration procedure consist of initial analyses ot
a calibration check standard followed by verification of the
internal standard response of each sample applying criteria
described in Section 10.4.
10. QUALITY CONTROL
10 1 Each laboratory that uses this method is required to operate a
formal quality control (QC) program. Minimum QC requirements are
Initial Demonstration of laboratory capability ,™rntoring 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 quality
control practices are recommended.
10.2
LABORATORY REAGENT BLANKS (LRB). Before processing any samples the
analyst must analyze at least one LRB to d6"10"5^6^.?1.1^9^":
ware 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 of any
analyte (11.3.5), the LRB produces a peak that would prevent the
determination of that analyte, determine the source of contamination
and eliminate the interference before processing samples.
10.3 INITIAL DEMONSTRATION OF CAPABILITY
10 3 1 Select a representative fortified concentration for each of
' ' the target anal ytes. Concentrations near analyte levels in
Table 4 are recommended. Prepare a laboratory control (LC)
sample concentrate in acetone or methanol 1000 times more
concentrated than the selected concentration. The LC sample
concentrate must be prepared independently from the standards
used to prepare the calibration curve (9.1). With a syringe,
add 100 ML of the LC sample concentrate to each of four to
seven 100 mL aliquots of reagent water. Analyze the aliquots
according to the method beginning in Section 11, but use
calibration curves based upon non-extracted standards as
called for in Section 10.3.2.
182
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10.3.2 Calculate the mean percent recovery (R) and the standard
deviation of the recovery (Sr). The recovery is determined
as the ratio of the measured concentration to the actual
fortified concentration. The measured concentration must be
based upon absolute or non-extracted standards, rather than
the extracted aqueous standards called for in 9.2.1 or
9.3.1. Prepare absolute calibration curves by injecting
known standards in MTBE, which span the range of fortified
concentrations measured. For each analyte, the mean recovery
value must fall in the range of R ±30% or within R ± 3Sr if
broader, using the values for R and S for reagent water in
Table 4. The standard deviation should be less than ± 30%
or 3Sr, 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, this procedure must be repeated using a
minimum of five fresh samples until satisfactory performance
has been demonstrated.
10.3.3 The initial demonstration of capability is used primarily to
preclude a laboratory from analyzing unknown samples via a
new, unfamiliar method prior to obtaining some experience
:, with it. It is expected that as laboratory personnel gain
experience with this method, the quality of data will
improve beyond those required here.
10.3.4 The analyst is permitted to modify GC columns, GC
conditions, internal standard or surrogate compounds. Each
time such method modifications are made, the analyst must
repeat the procedures in Sect. 10.3.1.
10.4 ASSESSING THE INTERNAL STANDARD
10.4.1 When using the internal standard calibration procedure, the
analyst is expected to monitor the IS response (peak area or
peak height) of all samples during each analysis day. The
IS response for any sample chromatogram should not deviate
from daily calibration standard's IS response by more than
30/0.
10.4.2 If >30% deviation occurs with an individual extract
optimize instrument performance and inject a second aliquot
of that extract. M
10.4.2.1 If the reinjected aliquot produces an acceptable
internal standard response, report results for
that aliquot.
10.4.2.2 If a deviation of greater than 30% is obtained
for the reinjected extract, analysis of the
samples should be repeated beginning with Sect.
183
-------
11, provided the sample is still available.
Otherwise, report results obtained from the
reinjected extract, but annotate as suspect.
10 4.3 If consecutive samples fail the IS response acceptance
criterion, immediately analyze a calibration check
standard.
10 4 3.1 If the check standard provides a response factor
(RF) within 20% of the predicted value, then
follow procedures itemized in Sect. 10.4.2 for
each sample failing the IS response criterion.
10.4.3.2 If the check standard provides a response factor
which deviates more than 20% of the predicted
value, then the analyst must recalibrate, as
specified in Sect. 9.
10.5 LABORATORY FORTIFIED BLANK
10 5.1 The laboratory must analyze at least one laboratory
fortified blank (LFB) sample with every 20 samples or one
per sample set (all samples extracted within a 24-hr
period), whichever is greater. Fortified concentrations
near those in Table 4 are recommended. The LFB sample must
be prepared from a standard mix, which is prepared
separately and independently from the standards used to
prepare the calibration curve. Calculate the mean accuracy
(R), based upon extracted standards as called for in
Sections 9.2.1 and 9.3.1. If the accuracy for any analyte
falls outside the control limits (see Sect. 10.5.2), that
analyte is judged out of control, and the source of the
problem should be identified and resolved before continuing
analyses.
10 5.2 Prepare control charts based on mean upper and lower control
limits, R ± 3 S , from accuracy and precision data collected
over a period of time. The initial demonstration of
capability (10.3) may be used to estimate the initial
limits, after correction of recovery data to an accuracy
basis. After each four to six 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.
184
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10.6 LABORATORY FORTIFIED SAMPLE MATRIX
10.6.1 The laboratory must add known fortified concentrations of
analytes to a minimum of 10% of the routine samples or one
fortified 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
u h u°vfr t?'me' san)Ples fr°m an routine sample sources
should be fortified.
10.6.2 Calculate the mean percent accuracy, 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.: ft
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 (10.5).
10.6.3 If the analysis of the unfortified sample reveals the
absence of measurable background concentrations, and the
added concentrations are those specified in Sect. 10.5, then
the appropriate control limits would be the acceptance
limits in Sect. 10.5.
10.6.4 If the sample contains measurable background concentrations
of analytes, calculate mean accuracy of the fortified con-
centration, R, for each such analyte after correcting for
the background concentration.
R = 100 (A - B)/C
Compare these values to reagent water accuracy data, R* at
comparable fortified concentrations from Tables 3-5. Re-
sults are considered comparable if the measured accuracies
fall within the range,
R* ± 3SC,
where Sc is the estimated percent relative standard devi-
ation in the measurement of the fortified concentration
By contrast to the measurement of accuracies in reagent
V?«er (10-5-2) or matrix samples without background
(10.6.3), the relative standard deviation, S , must be
expressed as the statistical sum of variation from two
185
-------
sources, the measurement of the total concentration as well
as the measurement of background concentration. In this
case, variances defined as Sz, are additive and Sc can be
expressed as
2 + S 2
+ ^
or
2x1/2
b I '
where S and Sb are the percent relative standard deviations
of the3total measured concentration and the background
concentration respectively. The value of Sa may be esti-
mated from the mean measurement of A above or from data at
comparable concentrations from Tables 3-5. Likewise, Sb can
be measured from repetitive measurements of the background
concentration or estimated from comparable concentration
data from Tables 3-5.
10.6.5 If the accuracy of any such analyte falls outside the
designated range, and the laboratory performance for that
analyte is shown to be in control (Sect. 10.5), the 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.
10 7 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.
10.8 The laboratory may adapt additional quality control practices for
use with this method. The specific practices that are most
productive depend upon the needs of the laboratory and the nature of
the samples. For example, field or laboratory duplicates may be
analyzed to assess the precision of the environmental measurements
or field reagent blanks may be used to assess contamination of
samples under site conditions, transportation and storage.
11. PROCEDURE
11.1 SAMPLE PREPARATION
11.1.1 Remove samples from storage and allow them to equilibrate to
room temperature.
11 1.2 Remove the vial caps. Discard a 5-mL volume of the sample.
Replace the vial caps and weigh the containers with contents
to the nearest 0.1 g and record these weights for subsequent
sample volume determination. (See Sect. 11.2.4 for
continuation of weighing and calculation of true volume).
186
-------
Alternatively, the sample vials may be precalibrated by
weighing in 35 ml of water and scoring the meniscus on the
bottle. This will eliminate the gravimetric step above and
in 11.2.4.
11.1.3 Inject an aliquot of the internal standard fortification
solution (7.7) into the sample. The aqueous concentration
of internal standard(s) must be the same as that used in
preparing calibration standards (9.1.2).
11.1.4 Remove the vial cap of each sample and add 8 g NaCl (Sect.
7.1.3) to the sample vial. Recap and dissolve the NaCl by
inverting and shaking the vial vigorously (approx. 20 sec.).
11.2 SAMPLE EXTRACTION
11.2.1 Remove the vial cap and add 2 ml of MTBE with a transfer or
automatic dispensing pi pet. Recap and shake by hand for 1
mm. Invert the vial and allow the water and MTBE phases to
separate (approx. 2 min).
11.2.2 By using a disposable Pasteur pipet (6.6), transfer a
portion of the solvent phase from the 40 ml vial to an
autosampler vial. Be certain no water has carried over onto
the bottom of the autosampler vial. If a dual phase appears
in the autosampler vial, the bottom layer can be easily
removed and discarded by using a Pasteur pipet. The
remaining MTBE phase may be transferred to a second
autosampler vial for a subsequent analysis. Approximately
1.5 ml of the solvent phase can be conveniently transferred
from the original 2 ml volume.
11.2.3 Discard the remaining contents of the sample vial. Shake
off the last few drops with short, brisk wrist movements.
11.2.4 Reweigh the empty vial with the original cap and calculate
the net weight of sample by difference to the nearest 0.1 g
(Sect. 11.1.2 minus Sect. 11.2.4). This net weight (in
grams) is equivalent to the volume of water (in ml)
extracted, V_.
o
11.2.5 The sample extract may be stored at 4 deg C for a maximum of
seven days before chromatographic analysis if required.
11.3 SAMPLE ANALYSIS AND IDENTIFICATION
11.3.1 The recommended GC operating conditions are described in
6.8.2.1 and 6.8.2.2 along with recommended primary and
confirmation columns. Retention data for the primary and
confirmation columns are given in Table 1 and examples of
separations attained with the primary column are shown in
Figures 1 and 2. Other GC columns, chromatographic
187
-------
conditions, or detectors may be used if the requirements of
Section 10 are met.
11.3.2 Calibrate the system daily as described in Section 9. The
standards and extracts must be in MTBE.
11.3.3 Inject 1-2 /uL of the sample extract and record the resulting
peak size in area units. For optimum performance and
precision, an autosampler for sample injection and a data
system for signal processing are strongly recommended.
11.3.4 Identify sample components by comparison of retention times
to retention data from a reference chromatogram. If the
retention time of an unknown compound corresponds, within
limits (11.3.5), to the retention time of a standard
compound, then identification is considered positive.
11.3.5 The width of the retention time window used to make
identifications should be based upon measurements of actual
retention time variations of standards over the course of a
day. Three times the standard deviation of a retention time
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.
11.3.6 Identification requires expert judgment when sample
components are not resolved chromatographically, that is,
when GC peaks obviously represent more than one sample
component (i.e., broadened peak with shoulder(s) or valley
between two or more maxima). Whenever doubt exists over the
identification of a peak in a chromatogram, confirmation is
required by the use of a dissimilar column or by GC-MS.
11.3.7 If the peak area exceeds the linear range of the calibration
curve, the final extract should be diluted with MTBE and
reanalyzed.
12. CALCULATIONS
12 1 Calculate the uncorrected concentrations (Ci) of each analyte in the
sample from the response factors or calibration curves generated in
9.2.1 or 9.3.1.
12.2 Calculate the corrected sample concentration as:
Concentration, M9/L = Ci x 35 ,
Vs
where the sample volume, Vs in mL, is equivalent to the net sample
weight in grams determined in 11.1.2 and 11,2.4.
188
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13. METHOD PERFORMANCE
13.1 Single laboratory (EMSL-Cincinnati) recovery and precision data at
three concentrations in a reagent water matrix are presented in
Tables 3-5. Accuracy and precision data based on extracted
standards for fortified tap water, raw source water and groundwater
are presented in Tables 6-8.
14. REFERENCES
1. Glaze, W.W., Lin, C.C., "Optimization of Liquid-Liquid Extraction
Methods for Analysis of Organics in Water", EPA-600/S4-83-052, U.S.
Environmental Protection Agency, January 1984.
2. Richard, J.J., Junk, G.A., "Liquid Extraction for Rapid Determination
of Halomethanes in Water," Journal AWWA. 69, 62, 1977.
3. Reding, R., P.S. Fair, C.J. Shipp, and H.J. Brass, "Measurement of
Dihaloacetonitriles and Chloropicrin in Drinking Water",
" Disinfection Byproducts: Current Perspectives ", AWWA, Denver,CO
1989.
4. Hodgeson, J.W., Cohen, A.L. and Collins, J. P., "Analytical Methods
for Measuring Organic Chlorination Byproducts" Proceedings Water
Quality Technology Conference (WQTC-16), St. Louis, MO, Nov. 13-17,
1988, American Water Works Association, Denver, CO, pp. 981-1001.
5. Glaser, J.A., Foerst, D.L., McKee, G.D., Quave, S.A. and Budde, W.L.
"Trace Analysis for Wastewaters", Environ. Sci. Techno!.. 15, 1426,
1981. ~
6. "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, Georgia, August 1977.
7. "OSHA Safety and Health Standards, General Industry", (29CFR1910),
OSHA 2206, Occupational Safety and Health Administration, Washington,
D.C. Revised January 1976.
8. "Safety in Academic Chemistry Laboratories", 3rd Edition, American
Chemical Society Publication, Committee on Chemical Safety,
Washington, D.C., 1979.
189
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TABLE 1. RETENTION DATA
Analyte
Chloroform
1,1, 1-Tri chl oroethane
Carbon Tetrachloride
Tri chl oroaceton i tri 1 e
Di chl oroacetoni tri 1 e
Bromodi chl oromethane
Trichloroethylene
Chloral Hydrate
1 , 1 , -di chl oropropanone-2
Chloropicrin
Di bromochl oromethane
Bromochl oroaceton i tr i 1 e
1,2-Dibromoethane (EDB)
Tetrachl oroethyl ene
1,1, 1-Tri chl oropropanone
Bromoform
Dibromoacetonitrile
1 , 2-Di bromo-3-Chl oropropane (DBCP)
Column A
Retention
Time (min)
5.25
6.37
7.29
7.59
8.72
9.02
9.13
9.70
10.73
15.80
16.40
16.77
17.40
19.57
21.36
23.54
24.03
32.32
Column B
Time (min)
3.09
2.04
3.41
5.03
9.09
4.21
4.38
6.56
11.19
39.94
6.40
14.43
9.71
6.94
15.66
10.73
17.45
20.35
Column A: DB-1, 0.32 mm x 30 m, 1 micron film thickness
Column B: DB-210, 0.32 mm x 30 m, 0.5 micron film thickness
190
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FIGURE 1
Chiorination Byproducts - DB-1 Primary Column
1. Chloroform
2. TCAN
3. DCAN
4. BDCM
5. MTBE Contaminant
6. CH
7. DCP
8. CP
9. DBCM
10. BCAN
11. EDB
12. TCP
13. Bromoform
14. DBAN
15. DBCP
Concentration
fug/LI
13.8
10.8
2.4
2.4
17.5
10.0
3.2
9.9
4.7
18.7
11.9
5.3
2.2
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METHOD 552. DETERMINATION OF HALOACETIC ACIDS IN DRINKING WATER
BY LIQUID-LIpUID EXTRACTION, DERIVATIZATION, AND GAS
CHROMATOGRAPHY WITH ELECTRON CAPTURE DETECTION
July 1990
Jimmie W. Hodgeson
J. Collins (Technology Applications Inc.)
R. E. Barth (Technology Applications Inc.)
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
201
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METHOD 552
DETERMINATION OF HALOACETIC ACIDS IN DRINKING WATER BY LIQUID-LIQUID EXTRACTION,
DERIVATIZATION, AND GAS CHROMATOGRAPHY WITH ELECTRON CAPTURE DETECTION
1. SCOPE AND APPLICATION
1.1 This is a gas chromatographic (GC) method (1-4,11) applicable to the
determination of thfe listed halogenated acetic acids in drinking
water, ground water, raw water and any intermediate treatment stage.
In addition, the chlorinated phenols listed may be analyzed by 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
2,4-Dichlorophenol 120-83-2
2,4,6-Trichlorophenol 88-06-2
1.2 This method is applicable to the determination of these analytes
over the concentration ranges typically found in drinking water
(1,2,4), subject to the method detection limits (MDL) listed in
Table 2. The detection limits 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 normally within the spiking
level ranges in Tables 2-5.
1.3 Tribromoacetic acid has not been included because of problems with
extraction and chromatography by this method. The mixed
bromochloroacetic acids have recently been synthesized. The
bromochloroacetic acid is present in chlorinated supplies and method
validation data are provided herein. However, neat material for
this compound is not readily available. The mixed trihalogenated
acids may also be present. These are not included because of
current problems with sample purity and the chromatography for these
two compounds.
1.4 The 2-chlorophenol has not been included as a method analyte in the
above list, primarily because its realistic detection limit in
environmental samples is likely to be above the odor threshold.
Poor precision is usually obtained for this compound at even higher
levels. In addition, this analyte displays instability under the
dechlorination/preservation conditions described herein.
Nevertheless, some method validation data are given in Tables 2-7.
202
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1.5 This method is designed for analysts skilled in liquid-liquid
extractions, extract concentration techniques, derivatization
procedures and the use of GC and interpretation of gas
chromatograms.
1.6 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 GC/mass spectroscopy (MS)
or by GC using dissimilar columns.
2. SUMMARY OF METHOD
2.1 A 100 ml volume of sample is adjusted to pH 11.5 and extracted with
methyl-tert-butyl ether (MTBE) to remove neutral and basic organic
compounds. The aqueous sample is then acidified to pH 0.5 and the
acids are extracted into MTBE. After the extract is dried and
concentrated, the acids are converted to their methyl esters with
diazomethane (DAM). Excess DAM is removed and the methyl esters are
determined by capillary GC using an electron capture detector (ECD).
An alternative microextraction procedure is also offered in which a
30-mL sample is extracted without cleanup with a single 3-mL aliquot
of MTBE for direct analysis by GC-ECD after methylation. Samples
containing high concentrations of haloacetic acids and other
disinfection byproducts, or other potentially interfering organic
compounds, may require the sample cleanup.
3. DEFINITIONS
3.1 Internal standard — A pure analyte(s) added to a solution in known
amount(s) and used to measure the relative responses of other method
analytes and surrogates that are components of the same solution.
The internal standard must be an analyte that is not a sample
component.
3.2 Surrogate analyte — A pure analyte(s), which is extremely unlikely
to be found in any sample, and which is added to a sample aliquot in
known amount(s) before extraction and is measured with the same
procedures used to measure other sample components. The purpose of
a surrogate analyte is to monitor method performance with each
sample.
3.3 Laboratory duplicates (LD1 and LD2) — Two sample aliquots taken in
the analytical laboratory and analyzed separately with identical
procedures. Analyses of LD1 and LD2 give a measure of the precision
associated with laboratory procedures, but not with sample
collection, 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
203
-------
3.6
with sample collection, preservation and storage, as well as with
laboratory procedures.
3.5 Laboratory reagent blank (LRB) — An aliquot of reagent water that
is treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates
that are used with other samples. The LRB is used to determine if
method analytes or other interferences are present in the labora-
tory environment, the reagents, or the apparatus.
Field reagent blank (FRB) — Reagent water placed in a sample
container in the laboratory and treated as a sample in all respects,
including exposure to sampling site conditions, storage,
preservation and all analytical procedures. The purpose of the FRB
is to determine if method analytes or other interferences are
present in the field environment.
3.7 Laboratory fortified blank (LFB) -- An aliquot of reagent water to
which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its
purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise
measurements at the required method detection limit.
3.8 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.
Stock standard solution — A concentrated solution containing a
single certified standard that is a method analyte, or a
concentrated solution of a single analyte prepared in the laboratory
with an assayed reference compound. Stock standard solutions are
used to prepare primary dilution standards.
3.10 Primary dilution standard solution — A solution of several analytes
prepared in the laboratory from stock standard solutions and diluted
as needed to prepare calibration solutions and other needed analyte
solutions.
3.11 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. '
3.12 Quality control sample (QCS) — A sample matrix containing method
analytes or a solution of method analytes in a water miscible
3.9
204
-------
solvent which is used to fortify reagent water or environmental
samples. The QCS is obtained from a source external to the
laboratory, and is used to check laboratory performance with
externally prepared test materials.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware and other sample processing apparatus that lead
to discrete artifacts or elevated baselines in gas chromatograms.
All reagents and apparatus must be routinely demonstrated to be free
from interferences under the conditions of the analysis by analyzing
laboratory reagent blanks as described in Section 10.2. Subtracting
blank values from sample results is not permitted.
4.1.1 Glassware must be scrupulously cleaned (5). Clean all
glassware as soon as possible after use by thoroughly rinsing
with the last solvent used in it. Follow by washing with hot
water and detergent and thorough rinsing with tap water,
dilute acid, and reagent water. Drain and heat in an oven or
muffle furnace at 400°C for 1 hour. Do not heat volumetric
ware. Thermally stable materials such as PCBs might not be
eliminated by this treatment. Thorough rinsing with reagent
grade acetone may be substituted for the heating. After
drying and cooling, seal and store glassware in a clean
environment to prevent any accumulation of dust or other
contaminants. Store inverted or capped with aluminum foil.
4.1.2 The use of high purity reagents and solvents helps to
minimize interference problems. Purification of solvents by
distillation in all-glass systems may be required. The
extraction solvent, MTBE, may need to be redistilled.
4.2 Whereas the 2,4,6-trichlorophenol is converted quantitatively to the
corresponding anisole by the methylation procedure, (11.3), the 2,4
-dichlorophenol is only partially converted (10-20%). The 2,4
dichloroanisole partially coelutes with the 2,4,6-trichloroanisole
on the DB-1701 primary column with the chromatographic conditions
employed (Table 1). The 2,4-dichlorophenol is quantitated on the
phenol peak. The extent of interference of the dichloroanisole with
the 2,4,6-trichlorophenol analysis is insignificant (0.8%) when
these compounds are present at equal concentrations. For samples in
which the 2,4-dichlorophenol concentration appears significantly
higher than that of the 2,4,6-trichlorophenol, e.g. greater than a
factor of 15, analyses should be performed on the DB-210
confirmation column, on which these compounds are completely
resolved.
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 and glass wool must be acid-rinsed with
(1+9) hydrochloric acid, and the sodium sulfate must be acidified
205
-------
4.4
4.5
4.6
(see 7.6) with sulfuric acid prior to use to avoid analyte losses
due to adsorption.
Organic acids and phenols, especially chlorinated compounds, cause
the most direct interference with the determination. The addition
of base and subsequent extraction of the basic sample removes many
neutral and basic chlorinated hydrocarbons and phthalate esters that
might otherwise interfere with the electron capture analysis.
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.
Matrix interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix interferences
will vary considerably from source to source, depending upon the
water sampled. Positive 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 reduced to the lowest possible
level by whatever means available. The laboratory is responsible
for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this
method. A reference file of material data handling sheets should
also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available
and have been identified (6-8) for the information of the analyst.
5.2 Diazomethane is a toxic carcinogen and can explode under certain
conditions, when produced in a purified or highly concentrated form.
In this form, the following safety precautions must be followed.
5.2.1 Use only in a well ventilated hood — do not breathe vapors.
5.2.2 Use a safety screen. Wear protective clothing and a shielded
safety hood.
5.2.3 Use mechanical pipetting aides.
5.2.4 Do not heat above 90°C.
206
-------
5.2.5 Avoid grinding surfaces,
bearings, glass stirrers.
ground glass joints, sleeve
6.
5.3
5.2.6 Store away from alkali metals.
5.2.7 Solutions of diazomethane decompose rapidly in the presence
of solid materials such as copper powder, calcium chloride,
and boiling chips.
For the above reasons, the diazomethane generation apparatus used in
the esterification procedure specified in this method (3) produces
only micromolar amounts of diazomethane in very dilute solution
(11.3) to minimize safety hazards. In this form, the solution is
not explosive. Nevertheless, the following precautions should be
followed.
5.3.1 Use only in a well ventilated hood.
5.3.2 When handling the diazomethane solution, avoid contact with
skin. If contact is made, immediately wash the exposed area
with warm water,
5.3.3 Collect and store the diazomethane solution produced at 0°C
to minimize losses due to decomposition.
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.
APPARATUS AND EQUIPMENT (All specifications in Sections 6 and 7 are
suggested. Catalogue numbers are provided for illustration only.)
5.4
6.1
6.2
6.3
6.4
Separatory funnels, 250 ml, with TFE fluorocarbon stopcocks, ground
glass or TFE fluorocarbon stoppers.
Screw cap 40 ml vials (Pierce #13219 or equivalent). Screw caps
should have TFE fluorocarbon liners.
Balance, analytical, capable of weighing to 0.0001 g.
Diazomethane generator - The generator assembly is shown in Figure
1 along with the diazomethane collection vessel. There are some
diazomethane generating kits commercially available. One is the
Aldrich Diazald Kit, Part No. 110,025-0; also see Aldrichim Acta,
1983, 16(1),3 for a review of the preparation and reactions of
diazomethane.
6.5
Six or 12 position analytical concentrator.
Model #111/6917 or equivalent).
(Organomation, N-EVAP
207
-------
6.6
6.7
6.8
6.9
6.10
Gas chromatograph - Analytical system complete with gas
chromatograph equipped for electron capture detection,
split/splitless capillary injection, temperature programming,
differential flow control, and with all required accessories
including syringes, analytical columns, gases and strip-chart
recorder. A data system is recommended for measuring peak areas.
An autoinjector is recommended for improved precision of analyses.
The gases flowing through the election capture detector should be
vented through the laboratory fume hood system.
Vials - Amber glass, 7 to 10 ml capacity with TFE-fluorocarbon lined
screw cap.
Primary GC column - DB-1701 or equivalent bonded, fused silica
column, 30m x 0.32mm ID, 0.25 urn film thickness.
Confirmatory GC column - DB-210 or equivalent bonded, fused silica
column, 30 m x 0.32 mm ID, 0.50 1m film thickness.
Pasteur pipets, glass disposable, 5 3/4" length wide bore diameter.
(Baxter Scientific Products Giant-Pette-Pipets, Cat. No. P5240-1 or
equivalent)
6.11 Volumetric ware, 5 ml.
6.12 pH Meter - Wide range with the capability of accurate pH
measurements in the 0-1 and 11-12 ranges. The use of separate glass
pH electrode and calomel reference electrode facilitates this
measurement.
7. REAGENTS AND CONSUMABLE MATERIALS
7.1 Glass wool - Acid washed, Heat to 400°C for 1 hr.
7.2 Reagent water - Reagent water is defined as a water in which an
interference is not observed at the method detection limit of each
parameter of interest.
7.2.1 A Milli-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.
7.2.2 Test reagent water each day it is used by analyzing according
to Sect. 11.
7.3 Methanol - Pesticide quality or equivalent.
7.4 Ethyl ether - Nanograde, redistilled in glass if necessary. Ethers
must be free of peroxides as indicated by EM Quant test strips,
available from EM Science, Gibbstown, NJ. Procedures recommended
for removal of peroxides are provided with the test strips. Ethers
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must be periodically tested (monthly) for peroxide formation during
use.
7.5 Methyl-tert-butyl ether - Nanograde, redistilled in glass if
necessary. The same peroxide precautions as in 7.4 apply to this
ether.
7.6 Sodium sulfate - (ACS) granular, acidified, 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
methylene chloride in a Soxhlet apparatus for 48 hr. Acidify by
slurrying 100 g sodium sulfate with just enough ethyl ether to cover
the solid. Add 0.1 ml concentrated sulfuric acid and mix
thoroughly. Remove the ether under vacuum or allow to evaporate in
a loosely covered beaker in a hood. Mix 1 g of the resulting solid
with 5 ml of reagent water and measure the pH of the mixture. It
must be below pH 4. Store at 130°C.
7.7 Sulfuric acid solution (1+1) - Slowly add 50 ml H,S04 (sp. gr. 1.84)
to 50 ml of reagent water.
7.8 Sodium hydroxide (NaOH), IN - Dissolve 4 g ACS grade in reagent
water and dilute up to 100 ml in a 100 ml volumetric flask.
7.9 Potassium Hydroxide (KOH), 37% - Dissolve 37 g of ACS grade in
reagent water and dilute up to 100 ml in a 100 ml volumetric flask.
7.10 Carbitol - (Diethylene glycol monoethyl ether), ACS. Available from
Aldrich Chemical Co.
7.11 Diazald- (N-methyl-N-nitroso-p-toluenesulfonamide), ACS. Available
from Aldrich Chemical Co.
7.12 Diazald Solution - Prepare a solution containing 10 g Diazald in 100
ml of a 50:50 by volume mixture of ethyl ether and carbitol. This
solution is stable for one month or longer when stored at 4°C in an
amber colored bottle with a Teflon-lined screw cap.
7.13 Silica Gel - Chromatographic grade, nominal 100 mesh. Heat to 400°C
for 4 hr. Store at 130°C.
7.14 Acetone - ACS reagent grade or equivalent.
7.15 Ammonium Chloride - ACS reagent grade or equivalent.
7.16 Sodium Sulfite - ACS reagent grade or equivalent.
7.17 1,2,3-Trichloropropane, Aldrich Chemical, 99+%.
7.18 3,5-Dichlorobenzoic Acid, Aldrich Chemical, 99%.
7.19 Copper (II) Sulfate Pentahydrate - ACS reagent grade or equivalent.
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SAMPLE COLLECTION, PRESERVATION AND STORAGE
8.1
Grab samples must be collected in accordance with conventional
sampling practices (9) using 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 chlorine,
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
minute.
8.1.3
8.1.4
CALIBRATION
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.
Dried extract concentrates (11.3.6) should be stored at 0-4°C
away from light in glass vials with TFE-faced caps. Extracts
should be analyzed within 48 hours following preparation.
9.1 Establish GC operating parameters equivalent to specifications in
Table 1. The GC system must be calibrated using the internal
standard (IS) technique.
9.2 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
2-7, one internal standard (1,2,3-trichloropropane) was employed.
The concentration of the internal standard used in obtaining these
data was 0.4 pg/mL in the final 5.0 ml concentrate (11.3.3).
9.2.1 Prepare separate stock standard solutions for each compound
of interest at a concentration of 1-5 mg/mL in MTBE solvent.
Method analytes may be obtained as neat materials or
ampulized solutions (>99% purity) from a number of commercial
suppliers.
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9.2.2 Prepare primary dilution standard solutions by combining and
diluting stock calibration standards with MTBE. The primary
dilution standards are used to prepare calibration standards,
which comprise at least three concentration levels (optimally
five) of each analyte with the lowest standard being at or
near the method detection limit 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.
9.2.2.1 Calibration standards for 100-mL sample
extraction (11.1) — These standards are
prepared in the final 5-mL MTBE extract form and
thus are not subject to the extraction
procedure. These standards must be esterified
according to the procedure beginning in 11.3.3.
Thus, the individual calibration standards are
initially prepared in approximately 4 ml MTBE to
allow for the addition of diazomethane solution
and the final dilution to 5.0 ml as called for
in 11.3.3.2. NOTE: The concentrations of the
5 mL calibration standards must be equivalent,
after correction for the concentration factor,
to aqueous standards which span the
concentration range called for in 9.2.2.
9.2.2.2 Calibration Standards for 30-mL
(Microextraction) Samples (11.2) — In this
procedure, aqueous standards are prepared by
dilution of primary dilution standards with
reagent water. These aqueous standards are
treated and extracted in the same manner as the
samples according to 11.2. The final 2-mL
extract is esterified according to the procedure
beginning in 11.3.4.
9.2.3 Include a surrogate analyte within the calibration
standards prepared in Section 9.2.2. Both 3,5-dichloro-
benzoic acid and 2,3-dichloropropanoic acid have been
used as surrogate analytes in this method.
9.2.4 Inject 2 /zL of each standard and calculate the relative
response for each analyte (RRa) using the equation:
RRa = Aa/Ais
Where A^ is the peak area of the analyte and A,s the peak area
of the internal standard.
9.2.5 Generate a calibration curve of RRa versus analyte
concentration of the standards expressed in equivalent /tg/L
in the original aqueous sample. The working calibration
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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. QUALITY CONTROL
10.1
Minimum quality control (QC) requirements are initial demonstration
of laboratory capability, determination of surrogate compound
recoveries in each sample and blank, monitoring internal standard
peak area or height in each sample and blank, analysis of laboratory
reagent blanks, laboratory fortified blanks, laboratory fortified
sample matrices, and QC samples. Additional quality control
practices are recommended.
10.2 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 (11.4.4)
of any analyte, the LRB produces a peak that would prevent the
determination of that analyte, determine the source of contamination
and eliminate the interference before processing samples.
10.3 INITIAL DEMONSTRATION OF CAPABILITY
10.3.1 Select a representative fortified concentration for each of
the target analytes. Concentrations near level 3 (Table 4)
are recommended. Prepare a laboratory control (LC) sample
concentrate in methanol 1000 times more concentrated than
the selected concentration. With a syringe, add 100 pi of
the LC sample concentrate to each of four to seven 100 mL
aliquots of reagent water. Analyze the aliquots according
to the method beginning in Section 11 and calculate mean
recoveries and standard deviation for each analyte.
10.3.2 Calculate the mean percent recovery (R) and the, standard
deviation of the recovery (SR). For each analyte, the mean
recovery values for all must fall in the range of R ± 30%
(or within R ± 3SR if broader) using the values for R and
Sp for reagent water in Table 4. The standard deviation
should be less than ± 30% or 3SR, 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, this procedure
must be repeated using a minimum of five fresh samples
until satisfactory performance has been demonstrated.
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10.3.3 The initial demonstration of capability is used primarily
to preclude a laboratory from analyzing unknown samples via
a new, unfamiliar method prior to obtaining some experience
with it. It is expected that as laboratory personnel gain
experience with this method, the quality of data will
improve beyond those required here.
10.3.4 The analyst is permitted to modify GC columns, GC
conditions, 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.
10.3.1.
10.4 ASSESSING SURROGATE RECOVERY
10.4.1 When surrogate recovery from a sample or method 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.
10.4.2 If the extract reanalysis fails the 70-130% recovery
criterion, the problem must be identified and corrected
before continuing.
10.4.3 If the extract reanalysis meets the surrogate recovery
criterion, report only data for the analyzed extract. If
sample extract continues to fail the recovery criterion,
report all data for that sample as suspect.
10.4.4 Develop and maintain control charts on surrogate recovery
as described in 10.6.2. 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.
10.5 ASSESSING THE INTERNAL STANDARD
10.5.1 When using the internal standard calibration procedure, the
analyst is expected to monitor the IS response (peak area
or peak height) of all samples during each analysis day.
The IS response for any sample chromatogram should not
deviate from daily calibration standard's IS response by
more than 30%.
10.5.2 If >30% deviation occurs with an individual extract,
optimize instrument performance and inject a second aliquot
of that extract.
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10.5.2.1 If the reinjected aliquot produces an acceptable
internal standard response, report results for
that aliquot.
10.5.2.2 If a deviation of greater than 30% is obtained
for the reinjected extract, analysis of the
samples should be repeated beginning with Sect.
11, provided the sample is still available.
Otherwise, report results obtained from the
reinjected extract, but annotate as suspect.
10.5.3 If consecutive samples fail the IS response acceptance
criterion, immediately analyze a calibration check
standard.
10.5.3.1 If the check standard provides a response factor
(RF) within 20% of the predicted value, then
follow procedures itemized in Sect. 10.5.2 for
each sample failing the IS response criterion.
10.5.3.2 If the check standard provides a response factor
which deviates more than 20% of the predicted
value, then the analyst must recalibrate, as
specified in Sect. 9.
10.6 LABORATORY FORTIFIED BLANK
10.6.1 The laboratory must analyze at least one laboratory
fortified blank (LFB) sample with every 20 samples or one
per sample set (all samples extracted within a 24-hr
period), whichever is greater. Fortified concentrations
near level 3 (Table 4) are recommended. Calculate accuracy
as percent recovery (R). If the recovery of any analyte
falls outside the control limits (see Sect. 10.6.2), that
analyte is judged out of control, and the source of the
problem should be identified and resolved before continuing
analyses.
10.6.2 Prepare control charts based on mean upper and lower control
limits, R ± 3 SR. The initial demonstration of capability
(10.3) establishes the initial limits. After each four to
six 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
measurements. Add these results to the ongoing control
charts to document data quality.
10.7 LABORATORY FORTIFIED SAMPLE MATRIX
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10.7.1 The laboratory must add known concentrations of analytes to
a minimum of 10% of the routine samples or one concentration
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. 10.6). Over time,
samples from all routine sample sources should be fortified.
10.7.2 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 (10.6).
10.7.3 If the analysis of the unfortified sample reveals the
absence of measurable background concentrations, and the
added concentrations are those specified in Sect. 10.6, then
the appropriate control limits would be the acceptance
limits in Sect. 10.6.
10.7.4 If the sample contains measurable background concentrations
of analytes, calculate mean recovery of the fortified
concentration, R, for each such analyte after correcting for
the background concentration.
R = 100 (A - B)/C
Compare these values to reagent water recovery data, R*, at
comparable fortified concentrations from Tables 3-5.
Results are considered comparable if the measured
recoveries fall within the range,
R* ± 3SC,
where Sc is the estimated percent relative standard
deviation in the measurement of the fortified
concentration. By contrast to the measurement of
recoveries in reagent water (10.6.2) or matrix samples
without background (10.7.3), the relative standard
deviation, Sc, must be expressed 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 S , are
additive and Sc can be expressed as,
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= s.
or
where S and Sb are the percent relative standard deviations
of thea total measured concentration and , the background
concentration respectively. The value of Sa may be
estimated from the mean measurement of A above or from data
at comparable concentrations from Tables 3-5. Likewise, Sb
can be measured from repetitive measurements of the
background concentration or estimated from comparable
concentration data from Tables 2-5.
10.7.5 If the recovery of any such analyte falls outside the
designated range, and the laboratory performance for that
analyte is shown to be in control (Sect. 10.6), the recovery
problem encountered with the fortified sample is judged to
be matrix related, not system related. The result for that
analyte in the unfortified sample is labeled suspect/matrix
to inform the data user that the results are suspect due to
matrix effects.
10.8 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.
10.9 The laboratory may adapt additional quality control practices for
use with this method. The specific practices that are most
productive depend upon the needs of the laboratory and the nature of
the samples. For example, field or laboratory duplicates may be
analyzed to assess the precision of the environmental measurements
or field reagent blanks may be used to assess contamination of
samples under site conditions, transportation and storage.
11. PROCEDURE
11.1 SAMPLE PREPARATION — 100-mL SAMPLE: This procedure employs a
sample cleanup step, serial extraction with MTBE, extract
concentration and drying prior to esterification (11.3.3). In this
procedure, sample standards are prepared in the final 5-mL extract
form prior to esterification.
11.1.1 Remove the samples from storage (Sect. 8.1.3) and allow to
equilibrate to room temperature.
11.1.2 Transfer 100 mL of sample with a pi pet to a 250 mL
separatory funnel. Add 1 mL of 1.0 N NaOH solution.
Remove an aliquot and measure pH, which should be
approximately 11.5.
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11.1.3 OPTIONAL - Add 100 #L of surrogate fortifying solution (5
Mg/mL of 3,5-dichlorobenzoic acid or 2,3-dibromo-propanoic
acid in methanol) to each sample including standards and
blanks.
11.1.4 Return the aliquot to the separatory funnel. (NOTE: If
sufficient sample is available, use a separate 100 mL
sample to measure the basic pH and to determine the amount
of H2S04 required. The measurement of pH should be done
with the wide range pH meter described in 6.12. Add 30 mL
MTBE. Extract the sample one time by vigorously shaking
the funnel for 2 min with periodic venting to release
excess pressure. Allow the organic layer to separate from
the water phase for a minimum of 10 min. If the emulsion
interface between layers is more than one third the volume
of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum
technique depends upon the sample, but may include
stirring, filtration of the emulsion through glass wool,
centrifugation, or other physical methods. Discard the
organic phase and return the aqueous phase to the 250 mL
separatory funnel.
11.1.5 Add sufficient 1:1 H2S04 in reagent water (ca. 15-20 mL) to
adjust the pH to pH < 0.5. Add 15 mL of MTBE and extract
for 2 minutes as in 11.1.3. Allow the phases to separate
for 10 min. If an emulsion persists employ the same
procedures for separation as in 11.1.3. Separate the
phases and collect the MTBE phase in a 40 mL screw cap vial
(6.2). Add 15 mL of MTBE to the sample and repeat the
extraction a second time. Combine the extracts in the
40-mL vial.
11.1.6 Extract Concentration - Evaporate the solvent at room
temperature to a volume of 1-2 mL under a gentle stream of
dry nitrogen. Under these conditions the sample cools
during evaporation and some condensation will be observed
on the outside of the vial. Alternately place the vials in
a water bath maintained at 35°C and concentrate to a minimum
volume of 2 mL. The method validation data in Tables 2-7
were obtained with the former technique. These gentle
concentration conditions are necessary because of the
volatility of the monohaloacetic acids.
11.1.7 Extract Drying Technique
11.1.7.1 Prepare sodium sulfate drying tubes by inserting
a small piece of acid washed glass wool into the
bottom, restricted, end of a wide bore, Pasteur
pipet (6.10).
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11.1.7.2 Add a column of approximately 5 cm acidified
sodium sulfate. Tap pi pet gently to pack sodium
sulfate.
11.1.7.3 Immediately, using another Pasteur pipet,
transfer the 1-2 ml MTBE extract from the 40 ml
vial into the top of the drying tube. A small
amount of separated water phase will likely be
present in the bottom of the 40 ml vial. Avoid
transferring any of the water phase. Examine the
lower portion of the Pasteur pipet to see whether
a separate water phase is present. Collect dried
extract in a 5.0 ml volumetric flask.
11.1.7.4 Rinse sides of 40 ml sample tube with
approximately 0.7 ml of clean MTBE. Transfer
this MTBE into the drying tube using the same
pipet as in Step 3.
11.1.7.5 Repeat Step 4 until the volumetric flask contains
3.8 to 4.0 ml.
11.2 SAMPLE PREPARATION -- 30-mL SAMPLE: Without employing any sample
cleanup, a 30-mL aliquot is salted and extracted with a single
aliquot of MTBE. The extract is esterified directly, without a
prior drying step. Aqueous standards are also processed through the
complete procedure (11).
11.2.1 Remove the samples from storage and allow them to
equilibrate to room temperature.
11.2.2 Transfer 30 mL sample or standard with a pipet to a 40 mL
vial equipped with a Teflon-faced screw cap. A slightly
larger vial might be more suitable.
11.2.3 OPTIONAL - Add 30 fil of surrogate spiking solution
(10 jug/mL 2,3-dibromopropanoic acid in methanol) to each
sample including standards and blanks.
11.2.4 Add 1.5 - 3.0 mL concentrated sulfuric acid to lower
the pH to less than 0.5. The analyst must verify that the
pH is less than 0.5.
11.2.5 Add accurately 3.0 mL methyl tertiary butyl ether (MTBE)
using a pipet.
11.2.6 Add 3 g copper (II) sulfate pentahydrate, followed by 12g
acidified sodium sulfate, carefully to prevent splashing
the MTBE. The blue color of the copper sulfate solution
facilitates observation of the phase interface when the
organic extract is transferred in Section 11.2.10.
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11.2.7 Cap all vials immediately, and shake by hand to break up
clumps. Vent, recap, and lay vials on their sides until all
vials have been shaken. Clumps of undissolved salt will
cause loss of analytes.
11.2.8 Place vials in a mechanical shaker and shake for
approximately 30 min. Required shaking time will vary from
shaker to shaker. Shaking by hand is perfectly acceptable.
The required time for this will have to be established
during the initial demonstration of capability.
11.2.9 Remove vials from shaker and allow to stand for 5 min for
phase separation.
11,2.10 Transfer exactly 2.0 ml of the ether extract (top layer)
using a pipet into a 2.0 ml volumetric flask.
Be careful to not include any water.
11.2.11 Using a stream of clean, dry nitrogen, evaporate
approximately 0.3 ml of MTBE from the flask to make room
for the addition of diazomethane and internal standard
(11.3.4.2).
11.3 ESTERIFICATION OF ACIDS
11.3.1 Assemble the diazomethane generator shown in Figure 1 in a
hood. The collection vessel is a 10-15 ml vial, equipped
with a Teflon-lined screw cap and maintained at 0-5°C. It is
perfectly acceptable to use a commercially available
diazomethane generator in place of the one shown in Figure
X *
11.3.2 Add a sufficient amount of ethyl ether to tube 1 to cover
the first impinger. Add 5 ml of MTBE to the collection
vial. Set the nitrogen flow at 5-10 cm3/min. Add 2 ml
Diazald solution and 1.5 ml of 37% KOH solution to the
second impinger. Connect the tubing as shown and allow the
N2 flow to purge the diazomethane from the reaction vessel
into the collection vial for 30 min. Cap the vial when
collection is complete and maintain at 0-55C. When stored
at 0-5 C this diazomethane solution may be used over a period
of 48 hours.
11.3.3 Esterification of 100-mL Extract (from 11.1.7.5)
11.3,3.1 Fortify the sample (11.1.7.5) and standard
(9.2.2.1) extracts with identical volumes of the
internal standard(s). The appropriate amount of
internal standard is dependent on the calibration
range. As a general rule, the internal standard
response should be approximately equal to the
response produced by the middle trichloroacetic
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acid calibration standard. For the validation
data in Table 3-7, 20 /iL of a 100 ng/ml internal
standard solution in MTBE were added to the 5.0
ml concentrate to yield a concentration of 0.4
/jg/mL.
11.3.3.2 Add 100 /iL methanol and 500 ill of cold
diazomethane solution (11.3.2). A persistent
pale yellow color after the addition of
diazomethane indicates that an excess was
available for esterification of the analytes. If
this is not obtained, continue adding successive
50 ill aliquots of diazomethane solution until the
persistent yellow color is obtained. Dilute to
a final volume of 5.0 ml with MTBE.
11.3.4 Esterification of 30-mL extract (from 11.2.11).
11.3.4.1 Add 20 pi of a 20 /jg/mL solution of 1,2,3-
trichloropropane in methanol as the internal
standard to the extract from aqueous standards or
samples (11.2.11).
11 3 4.2 Add 250 ML of cold diazomethane solution
(11.3.2). A persistent yellow color representing
excess diazomethane should be observed in the
solution. The final extract volume should be
2.0 ml.
11.3.5 Allow the sample from 11.3.3.2 or 11.3.4.2 to remain in
contact with dizaoamethane for 30 minutes. Remove any
unreacted diazomethane by addition of 0.2 g silica gel.
Effervescence due to nitrogen evolution is a further
indication that excess diazomethane is present. Mix gently
by inverting once.
11.3.6 After a contact time of 15-20 minutes, transfer a portion of
the extract solution to an appropriate vial for injection
into the GC. A duplicate GC vial may be filled from excess
sample extract, if desired. Analyze the samples as soon as
possible. Alternatively, the sample extract, after removal
from the silica gel, may be stored for 48 hours at 0-4°C
away from light in glass vials with TFE-lined caps.
11.4 GAS CHROMATOGRAPHY
11 4.1 Table 1 summarizes the recommended operating conditions for
the GC. Included in Table 1 are the retention times
observed using this method. An example of the separation
achieved using these conditions is shown in Figure 2. Other
GC columns, chromatographic conditions, or detectors may be
used if the requirements of Section 10.3 are met.
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11.4.2 Calibrate the system daily as described in Section 9 The
standards and extracts must be in MTBE.
11.4.3 Inject 2 pLof the sample extract. Record the resulting
peak size in area units.
11.4.4 The width of the retention time window used to make
identifications should be based upon measurements of actual
retention time variations of standards over the course of a
day. Three times the standard deviation of a retention time
can be used to calculate a suggested window size for a
compound. However, the experience of the analyst should
weigh heavily in the interpretation of chromatograms.
11.4.5 If the response for the peak exceeds the working range of
the system, dilute the extract and reanalyze.
12. CALCULATIONS
12.1 Calculate analyte concentrations in the sample from the response for
the analyte relative to the internal standard (RR ) usinq the
equation in Section 9.2.4. a y
12.2 For samples processed as part of a set where recoveries falls
outside of the control limits established in Section 10, results for
the affected analytes must be labeled as suspect.
13. PRECISION AND ACCURACY
13.1 In a single laboratory (EMSL-Cincinnati), recovery and precision
data were obtained at four concentrations in reagent water (Tables
Z-5). Tables 6 and 7 give representative recovery and precision
data for fortified tap water, which had been chlorinated. The
Method Detection Limit (MDL) (10) data are given in Table 2, and
Tables 3-5 illustrate instrument range. These method validation
data were obtained by the 100-mL sample extraction procedure In
the calculation of MDL's, the mean observed concentrations were not
corrected for recovery. Method detection limits using the
microextraction sample preparation were determined from eight
replicate analyses of fortified reagent water. The data showed they
were not significantly different from those listed in Table 2
obtained using the large sample preparation procedure. Also, data
obtained from replicate analyses of a variety of drinking water
samples using the microextraction sample preparation procedure were
found to be essentially equivalent to the 100 mL procedure. The
data indicate that both sample preparation procedures presented for
the haloacetic acids provide good analytical results under routine
use for finished drinking waters. However, the microextraction
procedure has not been tested on samples from formation potential
testsj in which the total concentration of haloacetic acids may
exceed 100 jtg/L. In these cases, the cleanup steps presented in this
method may be necessary to eliminate interferences.
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14. REFERENCES
1. Quimby, B.D., Delaney, M.F., Uden. P.C. and Barnes, R.M. Anal. Chem.
51, 1980, pp. 259-263.
2. Uden, P.C. and Miller, J.W., J. Am. Water Works Assoc. 75, 1983, pp.
524-527.
3. Hodgeson, J.W. and Cohen, A.L. and Collins, J.D., "Analytical Methods
for Measuring Organic Chlorination Byproducts", Proceedings Water
Quality Technology Conference (WQTC-16), St. Louis, MO, Nov. 13-17,
1988, American Water Works Association, Denver, CO, pp. 981-1001.
4. Fair. P.S., Barth, R.C., Flesch, J. J. and Brass, H. "Measurement of
Disinfection Byproducts 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.
5. 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.
6. "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, Georgia, August 1977.
7. "OSHA Safety and Health Standards, General Industry", (29CFR1910),
OSHA 2206, Occupational Safety and Health Administration, Washington,
D.C. Revised January 1976.
8. "Safety In Academic Chemistry Laboratories", 3rd Edition, American
Chemical Society Publication, Committee on Chemical Safety,
Washington, D.C., 1979.
9. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water", American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
10. Glaser, J. A., Foerst, D. L., McKee, G. D., Quave, S. A. and Budde, W.
L., Environ. Sci. Techno!. 15, 1981, pp. 1426-1435.
11. Chinn, R. and Krasner, S. " A Simplified Technique for the Measurement
of Halogenated Organic Acids in Drinking Water by Electron Capture
Gas Chromatography". Presented at the 28th Pacific Conference on
Chemistry and Spectroscopy, Pasadena, CA, October, 1989
222
-------
TABLE I. RETENTION DATA AND CHROMATOGRAPHIC CONDITIONS
Retention Time, min.
Analyte
Monochloroacetic Acid
Monobromoacetic Acid
Dichloroacetic Acid
Trichloroacetic Acid
1,2,3-Trichloropropane a)
Bromochloroacetic Acid
Dibromoacetic Acid
2-Chlorophenol
2,4-Dichlorophenol
2 , 4 , 6-Tri chl orophenol
3,5-Dichlorobenzoic Acid b)
Column A
5.77
8.70
9.40
12.20
13.28
13.52
16.00
16.65
20.70
21.94
23.06
Column B
10.97
13.03
12.72
14.37
13.87
15.11
16.83
18.32
19.27
22.08
23.95
Column A: DB-1701, 30 m x 0.32 mm i.d., 0.25 /on film thickness, Injector
Temp. = 200 C, Detector Temp. = 290°C, Helium Linear Velocity
= 27 cm/sec, Splitless injection with 30 s delay
Program: Hold at 50°C for 10 min, to 210°C at 10°C/min. and hold 10 min.
Column B: DB-210, 30 m x 0.32 mm i.d., 0.50 /zm film thickness, Injector
Temp. = 200 C, Detector Temp. = 290°C, Linear Helium Flow = 25
cm/sec, splitless injection with 30 s delay.
Program: Hold at 50°C for 10 min., to 210°< at 10°C/min and hold 10 min.
(a) Internal Standard
(b) Surrogate Acid
223
-------
TABLE 2. ANALYTE RECOVERY AND PRECISION DATA
AND METHOD DETECTION LIMITS8
LEVEL 1 IN REAGENT WATER
Analyte
Monochloroacetic Acid
Honobromoacetic Acid
Dichloroacetic Acid
Trichloroacetic Acid
Bromochloro-
acetic acid
Dibromoacetic Acid
2-Chlorophenol
2,4-Dichlorophenol
2,4, 6-Tr i chl orophenol
Fortified
Cone.
M/L
0.050
0.050
0.050
0.050
0.100
0.050
0.200
0.250
0.050
Mean
Meas.
Cone.
M9/L
0.037
0.029
0.042
0.039
0.150
0.029
0.123
0.147
0.033
Std.
Dev.
W/L
0.014
0.002
0.004
0.023
0.063
0.004
0.038
0.085
0.006
Rel.
Std.
Dev.,%
38
7
10
59
41
14
31
58
18
Mean
Recovery
%
74
58
84
78
150
58
61
59
66
Method
Detection
Limit
09/L
0.052
0.0074
0.015
0.085
0.14
0.015
0.14
0.32
0.022
Produced by analysis of seven aliquots of fortified reagent water (Reference 10).
224
-------
TABLE 3. ANALYTE RECOVERY AND PRECISION DATA8
LEVEL 2 IN REAGENT WATER
Analyte
Monochloroacetic Acid
Monobromoacetic Acid
Dichloroacetic Acid
Trichloroacetic Acid
Bromochloroacetic Acid
Dibromoacetic Acid
2-Chlorophenol
2,4-Dichlorophenol
2,4, 6-Tri chl orophenol
Fortified
Cone.
W/L
1.0
1.0
2.5
0.50
0.50
1.25
2.50
1.00
0.50
Mean
Meas.
Cone.
/*9/L
0.81
0.61
2.53
0.30
0.51
0.81
1.79
0.74
0.43
Std.
Dev.
M9/L
0.065
0.046
0.15
0.032
0.041
0.033
0.62
0.072
0.032
Rel.
Std.
Dev., °X
8
8
6
11
8
4
35
10
7
Mean
Recovery
; %
=====
81
61
101
60
103
65
72
74
86
"Produced by the analysis of seven aliquots of fortified reagent water.
225
-------
TABLE 4. ANALYTE RECOVERY AND PRECISION DATA8
LEVEL 3 IN REAGENT WATER
Fortified
Cone.
Analyte
Monochloroacetic Acid
Monobromoacetic Acid
Dichloroacetic Acid
Trichloroacetic Acid
Bromochloroacetic Acid
Dibromoacetic Acid
2-Chlorophenol
2,4-Dichlorophenol
2 , 4, 6-Tri chl orophenol
W/L
5.0
5.0
12.50
2.50
1.00
2.50
6.25
5.00
2.50
Mean
Meas.
Cone.
3.47
2.85
11.84
2.18
0.90
1.84
5.66
5.12
2.47
Std.
Dev.
m'1
0.25
0.13
0.25
0.083
0.059
0.11
0.34
0.47
0.054
Rel.
Std.
Dev., .%
.-,- r, — ~
7
5
2
4
7
6
6
9.
2
Mean
Recovery
%
69
57
95
87
90
74
91
102
99
""Produced by the analysis of seven aliquots of fortified reagent water.
226
-------
TABLE 5. ANALYTE RECOVERY AND PRECISION DATA8
LEVEL 4 IN REAGENT WATER
Analyte
Monochloroacetic Acid
Monobromoacetic Acid
Dichloroacetic Acid
Trichloroacetic Acid
Bromochloroacetic Acid
Dibromoacetic Acid
2-Chlorophenol
2,4-Dichlorophenol
2,4,6-Trichlorophenol
Fortified
Cone.
09/L
10.0
10.0
25.0
5.00
5.00
5.00
12.50
10.00
5.00
Mean
Meas.
Cone.
W/L.
7.08
7.62
24.1
5.70
4.66
5.35
12.7
11.0
5.18
Std.
Dev.
pg/'L
0.16
0.18
0.41
0.11
0.22
0.096
0.66
0.57
0.072
Rel.
Std.
Dev., %
2.3
2.4
1.7
1.9
4.7
1.8
5.2
5.2
1.4
Mean
Recovery
%
71
76
96
114
93
107
102
110
104
"Produced by the analysis of seven aliquots of fortified reagent water.
227
-------
TABLE 6. ANALYTE RECOVERY AND PRECISION DATA8
LEVEL 1 IN TAP WATER
Back-
ground Fortified
Cone., Cone.
W/L
1.83
0.32
32.3
5.4
10.6
11.5
0
0
W/L-
3.60
1.20
36.0
10.0
15.0
45.0
10.0
2.00
Mean"
Meas.
Cone.
Mg/L
2.23
1.36
26.0
10.7
19.2
41.6
12.0
28.8
Std.
Dev.
ra/L
0.19
0.11
2.4
0.83
1.4
8.5
1.2
0.23
Rel.
Std . Mean
Dev. Recovery
8
8
9
8
7
20
10
8
K>
62
113
72
107
128
92
120
144
Analyte
n
Honochloroaceti c Acid
Monobromacetic Acid
Dichloroacetic Acid
Trichloroacetic Acid
Dibromoacetic Acid
2-Chlorophenol
2,4-Dichlorophenol
2,4,6-Tri chlorophenol
a Produced by the analysis of seven aliquots of fortified tap water.
b Background level subtracted.
228
-------
TABLE 7. ANALYTE RECOVERY AND PRECISION DATA0
iSLEVEL 2 IN TAP WATER
Back-
• ground
Cone.
Analyte /ig/L
Mpnochloroacetic Acid
Monobromacetic Acid
Dichloroacetic Acid
Trichloroacetic Acid
Dibromoacetic Acid
2rChlorophenol
2,4-Dichlorophenol
2 , 4 , 6-TH chl orophenol
1.44
0.27
27.9
49.2
11.0
11.0
0
0
Forti-
fied
Cone.
Atg/L
10.0
4.00
72.0
20.0
30.0
60.0
30.0
10.0
Mean"
Meas.
Cone.
pg/L
6.45
3.85
61.0
20.7
34.1
69.8
26.9
10.7
Std.
Dev.
/*g/L
0.26
0.20
2.9
1.0
0.89
6.0
1.5
0.27
Rel.
Std.
Dev.
%
40
5
5
5
3
9
55
2
Mean
Recovery
%
64
96
85
104
114
116
90
107
a Produced by the analysis of seven aliquots of fortified tap water.
b Background level subtracted.
229
-------
N, FLOW'
«— FLAT JOINT WITH 0 RING AND CLAMP
OIETHYL ETHER LEVEL
*—FLAT JOINT WITH 0 RING AND CLAMP
OtAZALO LEVEL
KOH LEVEL
METHYL TERTIARY
BUTYL ETHER LEVEL
FIGURE 1. DIAZOMETHANE GENERATOR
230
-------
FIGURE ?^
Spiked Reagent Water
1!
Honochloracetic Acid • 6.25 pg/L
Monobromacetic Acid - 6.25 pg/l
Dichloroacetlc Acid - 6.25 /ig/l
Trichloroacetic Acid • 1.6 pg/L
Internal Standard • 20 /ig/L
Dibromoacetic Acid -1.0 fig/I
2-Chloro-Phenol • 6.25 0g/L
2, 4-D1ch1orophenol - 6.25
2,4,6-Trlchlorophenol -1.6
FIGURC ?B
Representative Tap Water Sample
1) Monochloroacetic Acid - Sackground + 5 vg/L Spike
2) Honobromo-acetic Acid - Background 4 5 09/L Spike
3) Dichloroacetlc Acid - 13.4 »g/L
4) Trichloroacetic Acid • 3.7 /w/L
5) Internal Standard - 20 jig/L
6) Dibromoacetic Add -2.0
231
-------
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