x>EPA
United States
Environmental Protection
Agency
Office of Water and
Office of Research and Development
Washington, DC 20460
EPA815-R-00-014
August 2000
www.epa.gov/safewater
Methods for the
Determination of Organic and
Inorganic Compounds in
Drinking Water
Volume 1
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EPA 815-R-00-014
August 2000
METHODS FOR THE DETERMINATION
OF ORGANIC AND INORGANIC
COMPOUNDS IN DRINKING WATER
Volume I
Technical Support Center
Office of Ground Water and Drinking Water
and
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio
Printed on Recycled Paper
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DISCLAIMER
The methods contained in this manual were developed at either the Office of Research
and Development's National Exposure Research Laboratory or the Office of Ground Water and
DrinkingWater's Technical Support Center. All of these methods have been reviewed in
accordance with the U.S. EPA's peer review requirements and cleared by EPA management for
publication. Publication of these methods by the U.S. EPA does not infer anything about their
status of approval for compliance monitoring. The user should refer to current drinking water
regulations to determine which of these methods have been approved for compliance monitoring.
In addition, mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
ABSTRACT
Seven methods for the analysis of organic compounds and four methods for the analysis
of inorganic compounds in drinking water are contained in this manual. Many of these methods
have either already been approved for drinking water compliance monitoring or for performing
analysis required in the Unregulated Contaminant Monitoring Regulation. Other methods
included in this manual may be approved for compliance monitoring at a future date or are useful
for developing occurrence data. Methods for the analysis of inorganic and organic compounds
have been combined in this manual to facilitate their timely publication. Most of these methods
are also available at the Office of Water website, www.epa.gov/ogwdw/methods/sourcalt.html or
at the Office of Research and Development website, www.epa.gov/nerlcwww/ordmeth.htm.
u
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FOREWORD
Accurate and precise analytical measurements are essential to the determination of the
quality and character of drinking waters. Both the Office of Ground Water and Drinking Water's
Technical Support Center and the Office of Research and Development's National Exposure
Research Laboratory are dedicated to developing new and innovative methods that provide the
data quality required, while at the same time using technologies that can simplify analytical
methods, and thereby reduce the costs of performing drinking water analyses. This manual was
prepared to assemble under a single cover 7 new analytical methods for the determination of
organic compounds and 4 new analytical methods for the determination of inorganic compounds
in drinking water. We are pleased to provide this manual and believe that it will be of
considerable value to both public and private analytical laboratories that wish to monitor for
these compounds in drinking water.
David J. Munch
Chemistry Laboratory Manager
Technical Support Center
Standards and Risk Management Division
Office of Ground Water and Drinking Water
'' J •
»wr<
Thomas D. Behymer, Chief
Chemical Exposure Research Branch
Microbiological and Chemical Exposure
Assessment Research Division :
National Exposure Research Laboratory
in
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TABLE OF CONTENTS
Method
Number Title Revision Page
— Disclaimer ii
- Abstract ii
— Foreword iii
— Analyte - Method Gross Reference iv
300.1 Determination of Inorganic Anions in Drinking 1.0
Water by Ion Chromatography
314.0 Determination of Perchlorate in Drinking Water 1.0
using Ion Chromatography
317.0 Determination of Inorganic Oxhyalide Disinfection 1.0
By-Products in Drinking Water Using Ion
Chromatography with the Addition of a Postcolumn
Reagent for Trace Bromate Analysis
321.8 Determination of Bromate in Drinking Waters by 1.0
Ion Chromatography Inductively Coupled
Plasma/Mass Spectrometry
515.3 Determination of Chlorinated Acids in Drinking 1.0
Water by Liquid-Liquid Extraction, Derivatization
and Gas Chromatography with Electron Capture
Detection
526 Determination of Selected Semivolatile Organic 1.0
Compounds in Drinking Water by Solid Phase
Extraction and Capillary Column Gas Chromatography/
Mass Spectrometry (GC/MS)
iv
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TABLE OF CONTENTS (Continued)
Method
Number Title Revision
528 Determination of Phenols in Drinking Water by Solid 1.0
Phase Extraction and Capillary Column Gas
Chromatography/Mass Spectrometry (GC/MS)
532 Determination of Phenylurea Compounds in Drinking 1.0
Water by Solid Phase Extraction and High Performance
Liquid Chromatography with UV Detection
549.2 Determination of Diquat and Paraquat in Drinking Water 1.0
By Liquid-Solid Extraction and High Performance
Liquid Chromatography with Ultraviolet Detection
556 Determination of Carbonyl Compounds in Drinking Water 1.0
by Pentafluorobenzylhydroxylamine Derivatization
and Capillary Gas Chromatography with Electron
Capture Detection
556.1 Determination of Carbonyl Compounds in Drinking 1.0
Water by Fast Gas Chromatography
v
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ANALYTE - METHOD CROSS REFERENCE
ANALYTE
Acetaldehyde
Acetochlor
Acifluorfen/Lactofen
Bentazon
Benzaldehyde
Bromate
Bromide
Butanal
Chloramben
Chlorate
Chloride
Chlorite
4-Chloro-3-methylphenol
2-Chlorophenol
Crotonaldehyde
Cyanazine
Cyclohexanone
2,4-D
2,4-DB
Dacthal Acid Metabolites
Dalapon
Decanal
Diazinon
METHOD NO.
556, 556.1
526
515.3
515.3
556.1,556.
300.1,317.0,321.8
300.1,317.0,321.8
556,556.1
515.3
300.1,317.0
300.1,317.0
300.1,317.0
, 528
528
556, 556.1
526
556,556.1
.515.3
515.3
515.3
515.3
556,556.1
526
VI
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ANALYTE METHOD NO.
Dicamba 515.3
3,5-Dichlorobenzoic Acid 515.3
2,4-Dichlorophenol 526,528
Dichlorprop 515.3
Diflubenzuron 532
2,4-Dimethylphenol 528
2,4-Dinitrophenol 528
Dinoseb 515.3
1,2-Diphenylhydrazine 526
Diquat 549.2
Disulfoton 526
Diuron 532
Fluometuron 532
Fluoride 300.1,317.0
Fonofos 526
Formaldehyde 556,556.1
Glyoxal 556,556.1
Heptanal 556,556.1
Hexanal 556,556.1
5-Hydroxydicamba 515.3
Linuron 532
2-Methyl-4,6-Dinitrophenol 528
Methyl Glyoxal 556, 556.1
2-Methyphenol 528
vn
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ANALYTE METHOD NO.
Nitrate 300.1,317.0
Nitrite 300.1,317.0
Nitrobenzene 526
2-Nitrophenol 528
4-Nitrophenol 515.3,528
Nonanal 556,556.1
Octanal 556,556.1
Paraquat 549.2
Pentachlorophenol 515.3,528
Pentanal 556,556.1
Perchlorate 314.0
Phenol 528
Phosphate 300.1,317.0
Picloram 515.3
Prometon 526
Propanal 556,556.1
Propanil 532
SiduronA&B 532
Sulfate 300.1,317.0
'2,4,5-T 515.3
2,4,5-TP (Silvex) 515.3
Tebuthiuron 532
Terbufos 526
Thidiazuron 532
2,4,6-Trichlorophenol 526,528
Vlll
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METHOD 300.1 DETERMINATION OF INORGANIC ANIONS IN DRINKING
WATER BY ION CHROMATOGRAPHY
Revision 1.0
September 1997
John D. Pfaff, USEPA, ORD, NERL (Method 300.0,1993)
Daniel P. Hautman and David J. Munch, USEPA, Office of Water
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
300.1-1
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METHOD 300.1
DETERMINATION OF INORGANIC ANIONS IN DRINKING WATER
BY ION CHROMATOGRAPHY
1. SCOPE AND APPLICATION
1.1 This method covers the determination of the following inorganic anions in reagent
water, surface water, ground water, and finished drinking water. As a result of
different specified injection volumes (See conditions in Tables 1A and IB), these
anions are divided between the common anions listed in Part A and the inorganic
disinfection by-products listed in Part B. These different injection volumes are
required hi order to compensate for the relative concentrations of these anions in
drinking water and maintain good chromatographic peak shape throughout the
expected dynamic range of the detector. Bromide is included in both Part A, due to
its importance as a common anion, as well as Part B due to its critical role as a
disinfection by-product precursor.
PART A.— Common Anions
Bromide Nitrite
Chloride ortho-Phosphate-P
Fluoride Sulfate
Nitrate
PART B.— Inorganic Disinfection By-products
Bromate Chlorite
Bromide Chlorate
1.2 The single laboratory Method Detection Limits (MDL, defined in Sect. 3.11) for the
above analytes are listed in Tables 1A, IB and 1C. The MDL for a specific matrix
may differ from those listed, depending upon the nature of the sample and the
specific instrumentation employed.
1.2.1 In order to achieve comparable detection limits, an ion chromatographic
system must utilize suppressed conductivity detection, be properly
maintained and must be capable of yielding a baseline with no more than 5
nS noise/drift per minute of monitored response over the background
conductivity.
1.3 This method is recommended for use only by or under the supervision of analysts
experienced in the use of ion chromatography and in the interpretation of the
resulting ion chromatograms.
300.1-2
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1.4 When this method is used to analyze unfamiliar samples for any of the above anions,
anion identification should be supported by the use of a fortified sample matrix
covering the anions of interest. The fortification procedure is described in £>ect.
9.4.1. " '••' ' : ' '-'
1.5 Users of the method data should state the data-quality objectives prior to analysis.
Users of the method must demonstrate the ability to generate acceptable results with
this method, using the procedures described in Sect. 9.0.
1.6 Bromide and nitrite react with most oxidants employed as disinfectants. The utility
of measuring these anions in treated water should be considered prior to conducting
the analysis.
2. SUMMARY OF METHOD
2.1 A small volume of sample, 10 uL for Part A and 50 uL for Part B, is introduced into
an ion chromatograph. The anions of interest are separated and measured, using a
system comprised of a guard column, analytical column, suppressor device, and
conductivity detector.
2.2 The ONLY difference between Parts A and B is the volume of sample analyzed by
the ion chromatographic system. The separator columns and guard columns as well
as eluent conditions are identical.
3. DEFINITIONS
3.1 ANALYSIS BATCH -- A group of no more than 20 field samples (Field sample
analyses include only those samples derived from a field sample matrix. These
include the initial and duplicate field samples as well as all Laboratory Fortified
Sample Matrices). The analysis batch must include an Initial Calibration Check
Standard, an End Calibration Check Standard, Laboratory Reagent Blank, and a
Laboratory Fortified Blank. Within an ANALYSIS BATCH, for every group of ten
field samples, at least one Laboratory Fortified Matrix (LFM) and either a Field
Duplicate, a Laboratory Duplicate or a duplicate of the LFM must be analyzed.
When more than 10 field samples are analyzed, a Continuing Calibration Check
-Standard must be analyzed after the tenth field sample analysis.
3.2 CALIBRATION STANDARD (CAL) - A solution prepared from the primary
dilution standard solution or stock standard solutions and the surrogate analyte. The
CAL solutions are used to calibrate the instrument response with respect to analyte
concentration.
300.1-3
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3.2.1 INITIAL CALIBRATION STANDARDS - A series of CAL solutions used
to initially establish instrument calibration and develop calibration curves
for individual target anions.
3.2.2 INITIAL CALIBRATION CHECK STANDARD - An individual CAL
solution, analyzed initially, prior to any sample analysis, which verifies
previously established calibration curves.
3.2.3 CONTINUING CALIBRATION CHECK STANDARD - An individual
CAL solution which is analyzed after every tenth field sample analyses
which verifies the previously established calibration curves and confirms
accurate analyte quantitation for the previous ten field samples analyzed.
3.2.4 END CALIBRATION CHECK STANDARD -- An individual CAL solution
which is analyzed after the last field sample analyses which verifies the
previously established calibration curves and confirms accurate analyte
quantitation for all field samples analyzed since the last continuing
calibration check.
3.3 FIELD DUPLICATES — 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 field duplicates indicate the precision associated
with sample collection, preservation and storage, as well as with laboratory
procedures.
3.4 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) - A solution of one
or more method analytes, surrogates, or other test substances used to evaluate the
performance of the instrument system with respect to a defined set of criteria.
3.5 LABORATORY DUPLICATE - Two sample aliquots, taken in the laboratory from
a single sample bottle, and analyzed separately with identical procedures. Analyses
of LD1 and LD2 indicate precision associated specifically with the laboratory
procedures, removing any associated variables attributed by sample collection,
preservation, or storage procedures.
3.6 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water or other
blank matrices to which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its purpose is to
determine whether the methodology is in control, and whether the laboratory is
capable of making accurate and precise measurements.
3.7 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) -- An aliquot of an
environmental sample to which known quantities of the method analytes are added
300.1-4
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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 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or other
blank matrices that are treated exactly as a sample including exposure to all
glassware, equipment, solvents, reagents, and surrogates that are used with other
samples. The LRB is used to determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or the apparatus.
3.9 LINEAR CALIBRATION RANGE (LCR) - The concentration range over which
the instrument response is linear.
3.10 MATERIAL SAFETY DATA SHEET (MSDS) -- Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical properties, fire,
and reactivity data including storage, spill, and handling precautions.
3.11 METHOD DETECTION LIMIT (MDL) - The minimum concentration of an
analyte that can be identified, measured and reported with 99% confidence that the
analyte concentration is greater than zero.
3.12 MINIMUM REPORTING LEVEL (MRL) -- The minimum concentration that can
be reported for an anion in a sample following analysis. This defined concentration
can be no lower than the concentration of the lowest calibration standard and can
only be used if acceptable quality control criteria for this standard are met.
3.13 PERFORMANCE EVALUATION SAMPLE (PE) - A certified solution of method
analytes whose concentration is unknown to the analyst. Often^ an aliquot of this
solution is added to a known volume of reagent water and analyzed with procedures
used for samples. Results of analyses are used to determine statistically the accuracy
and precision that can be expected when a method is performed by a competent
analyst. .-..., ; •.
3.14 QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of known
concentrations that is used to fortify an aliquot of LRB or sample matrix. The QCS
is obtained from a source external to the laboratory and different from the source of
calibration standards. It is used to check laboratory performance with externally
prepared test materials. '...-,;
3.15 SURROGATE ANALYTE -- An analyte added to a sample, which is' unlikely to be
found in any sample at significant concentration, and which is added directly to a
300.1-5
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sample aliquot in known amounts before any sample processing procedures are
conducted. It is measured with the same procedures used to measure other sample
components. The purpose of the surrogate analyte is to monitor method
performance with each sample.
3.16 STOCK STANDARD SOLUTION (SSS) -- A concentrated solution containing one
or more method analytes prepared in the laboratory using assayed reference materials
or purchased from a reputable commercial source.
4. INTERFERENCES
4.1 Interferences can be divided into three different categories: direct chromatographic
coelution, where an analyte response is observed at very nearly the same retention
time as the target anion; concentration dependant coelution, which is observed when
the response of higher than typical concentrations of the neighboring peak overlap
into the retention window of the target anion; and, ionic character displacement,
where retention times may significantly shift due to the influence of high ionic
strength matrices (high mineral content or hardness) overloading the exchange sites
in the column and significantly shortening target analyte's retention times.
4.1.1 A direct chromatographic coelution may be solved by changing columns,
eluent strength, modifying the eluent with organic solvents (if compatible
with 1C columns), changing the detection systems, or selective removal of
the interference with pretreatment. Sample dilution will have little to no
effect. The analyst must verify that these changes do not negatively affect
performance by repeating and passing all the QC criteria in Section 9.
4.1.2 Sample dilution may resolve some of the difficulties if the interference is the
result of either concentration dependant coelution or ionic character
displacement, but it must be clarified that sample dilution will alter your
Minimum Reporting Limit (MRL) by a proportion equivalent to that of
the dilution. Therefore, careful consideration of project objectives should be
given prior to performing such a dilution. An alternative to sample dilution,
may be dilution of the eluent as outlined in 11.9.
4.1.3 Pretreatment cartridges can be effective as a means to eliminate certain
matrix interferences. Prior to using any pretreatment, the analyst should be
aware that all instrument calibration standards must be pretreated in exactly
the same manner as the pretreated unknown field samples. The need for
these cartridges have been greatly reduced with recent advances in high
capacity anion exchange columns.
4.1.3.1 Extreme caution should be exercised in using these pretreatment
cartridges. Artifacts are known to leach from certain cartridges
300.1-6
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which can foul the guard and analytical columns causing loss of
column capacity indicated by shortened retention times and
irreproducible results. Frequently compare your calibration
standard chromatograms to those of the column test
chromatogram (received when the column was purchased) to
insure proper separation and similar response ratios between the
target analytes is observed.
4.2 Method interferences may be caused by contaminants hi the reagent water, reagents,
glassware, and other sample processing apparatus that lead to discrete artifacts or
elevated baselines in an ion chromatogram. These interferences can lead to false
positive results for target analytes as well as reduced detection limits as a
consequence of elevated baseline noise.
4.3 Samples that contain particles larger than 0.45 microns and reagent solutions that
contain particles larger than 0.20 microns require filtration to prevent damage to
instrument columns and flow systems.
4.4 Any anion that is only weakly retained by the column may elute in the retention time
window of fluoride and potentially interfere. At concentrations of fluoride above 1.5
mg/L, this interference may not be significant, however, it is the responsibility of the
user to generate precision and accuracy information in each sample matrix.
4.5 Close attention should be given to the potential for carry over peaks from one
analysis which will effect the proper detection of analytes of interest in a second,
subsequent analysis. Normally, the elution of sulfate (retention time of 13.8 min.)
indicates the end of a chromatographic run, but, in the ozonated and chlorine dioxide
matrices, which were included as part of the single operator accuracy and bias study
(See Table 2B), a small response (200 nS baseline rise) was observed for a very late
eluting unknown peak at approximately 23 minutes. Consequently, a run time of 25
minutes is recommended to allow for the proper elution of any potentially interferant
late peaks. It is the responsibility of the user to confirm that no late eluting peaks
have carried over into a subsequent analysis thereby compromising the integrity of
the analytical results.
4.6 Any residual chlorine dioxide present in the sample will result in the formation of
additional chlorite prior to analysis. If any concentration of chlorine dioxide is
suspected in the sample, the sample must be purged with an inert gas (helium, argon
or nitrogen) for approximately five minutes or until no chlorine dioxide remains.
This sparging must be conducted prior to ethylenediamine preservation and at time
of sample collection.
300.1-7
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5. SAFETY
6.
5.1 The toxicity or carcinogenicity of each reagent used in this method have not been
fully established. Each chemical should be regarded as a potential health hazard and
exposure should be as low as reasonably achievable. Cautions are included for
known extremely hazardous materials or procedures.
5.2 Each laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A
reference file of Material Safety Data Sheets (MSDS) should be made available to
all personnel involved in the chemical analysis. The preparation of a formal safety
plan is also advisable.
5.3 The following chemicals have the potential to be highly toxic or hazardous, consult
MSDS.
5.3.1 Sulfuric acid - When used to prepared a 25 mN sulfuric acid regenerant
solution for chemical suppression using a Dionex Anion Micro Membrane
Suppressor (AMMS).
6.1 Ion chromatograph - Analytical system complete with ion chromatograph and all
required accessories including syringes, analytical columns, compressed gasses and a
conductivity detector.
6.1.1 Anion guard column: Dionex AG9-HC, 2 mm (P/N 52248), or equivalent.
This column functions as a protector of the separator column. If omitted
from the system the retention times will be shorter.
6.1.2 Anion separator column: Dionex AS9-HC column, 2 mm (P/N 52244), or
equivalent. The microbore (2 mm) was selected in the development of this
method as a means to tighten the bromate elution band and thus reduce the
detection limit. An optional column (2 mm or 4 mm) may be used if
comparable resolution of peaks is obtained, and the requirements of Sect.
9.0 can be met. The AS9-HC, 2 mm column using the conditions outlined
in Table 1A and IB produced the separation shown in Figures 1 through 4.
6.1.2.1 If a 4 mm column is employed, the inj ection volume should be
raised by a factor of four to 40 uL for Part A anions and 200 uL
for Part B anions in order to attain comparable detection limits. A
four fold increase in injection volume compensates for the four
300.1-8
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fold increase in cross sectional surface area of the 4 mm standard
bore column over the 2 mm microbore column.
6.1.2.2 Comparable results can be attained using the Dionex, AS9-HC, 4
mm column. MDLs for the part B, inorganic disinfection by-
products using this 4 mm column are displayed along with
analysis conditions in Table 1C.
6.1.3 Anion suppressor device: The data presented in this method were generated
using a Dionex Anion Self Regenerating Suppressor (ASRS, P/N 43187).
An equivalent suppressor device may be utilized provided comparable
detection limits are achieved and adequate baseline stability is attained as
measured by a combined baseline drift/noise of no more than 5 nS per
minute over the background conductivity.
6.1.3.1 The ASRS was set to perform electrolytic suppression at a current
setting of 100 mA using an external source DI water mode.
Insufficient baseline stability was observed using the ASRS in
recycle mode.
6.1.4 Detector — Conductivity cell (Dionex CD20, or equivalent) capable of
providing data as required in Sect. 9.2.
6.2 The Dionex Peaknet Data Chromatography Software was used to generate all the
data in the attached tables. Systems using a strip chart recorder and integrator or
other computer based data system may achieve approximately the same MDL's but
the user should demonstrate this by the procedure outlined in Sect. 9.2.
6.3 Analytical balance, ±0.1 mg sensitivity. Used to accurately weigh target analyte
salts for stock standard preparation.
6.4 Top loading balance, ±10 mg sensitivity. Used to accurately weigh reagents to
prepare eluents.
6.5 Weigh boats, plastic, disposable - for weighing eluent reagents.
6.6 Syringes, plastic, disposable, 10 mL - used during sample preparation.
6.7 Pipets, Pasteur, plastic or glass, disposable, graduated, 5 mL and 10 mL.
6.8 Bottles, high density polyethylene (HDPE), opaque or glass, amber, 30 mL, 125 mL,
250 mL. For sampling and storage of calibration solutions. Opaque or amber due to
the photoreactivity of chlorite anion.
6.9 Micro beakers, plastic, disposable - used during sample preparation.
300.1-9
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7. REAGENTS AND STANDARDS
7.1 Reagent water: Distilled or deionized water, free of the anions of interest. Water
should contain particles no larger than 0.20 microns.
7.2 Eluent solution : Sodium carbonate (CASRN 497-19-8) 9.0 mM. Dissolve 1.91 g
sodium carbonate (Na^jCOs) in reagent water and dilute to 2 L.
7.2. 1 This eluent solution must be purged for 1 0 minutes with helium prior to use
to remove dissolved gases which may form micro bubbles in the 1C
compromising system performance and adversely effecting the integrity of
the data.
7.3 Stock standard solutions, 1000 mg/L (1 mg/mL): Stock standard solutions may be
purchased as certified solutions or prepared from ACS reagent grade, potassium or
sodium salts as listed below, for most analytes. Chlorite requires careful
consideration as outline below in 7.3.5.1.
7.3.1 Bromide (Br) 1000 mg/L: Dissolve 0.1288 g sodium bromide (NaBr,
CASRN 7647-15-6) in reagent water and dilute to 100 mL in a volumetric
flask.
7.3 .2 Bromate (BrO3') 1 000 mg/L: Dissolve 0. 1 1 80 g of sodium bromate
(NaBrO3, CASRN 7789-38-0) in reagent water and dilute to 100 mL in a
volumetric flask.
7.3.3 Chlorate (C103~) 1000 mg/L: Dissolve 0.1275 g of sodium chlorate
(NaC103, CASRN 7775-09-9) in reagent water and dilute to 100 mL in a
volumetric flask.
7.3.4 Chloride (Cl") 1000 mg/L: Dissolve 0.1649 g sodium chloride (NaCl,
CASRN 7647-14-5) in reagent water and dilute to 100 mL in a volumetric
flask.
7.3.5 Chlorite (C102') 1000 mg/L: Assuming an exact 80.0 % NaC102 is
amperometrically titrated from technical grade NaC102 (See Sect. 7.3.5.1).
Dissolve 0. 1 676 g of sodium chlorite (NaC 1 02, CASRN 775 8- 1 9-2) in
reagent water and dilute to 100 mL in a volumetric flask.
7.3.5.1 High purity sodium chlorite (NaCIO 2) is not currently
commercially available due to potential explosive instability.
Recrystallization of the technical grade (approx. 80%) can be
300.1-10
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performed but it is labor intensive and time consuming. The
simplest approach is to determine the exact % NaCIO 2 using the
iodometric titration procedure (Standard Methods, 19th Ed.,
4500-C1O2.C). Following titration, an individual component
standard of chlorite must be analyzed to determine if there is any
significant contamination (greater than 1% of the chlorite weight)
in the technical grade chlorite standard from any of the Part B
components. These contaminants will place a high bias on the
calibration of the other anions if all four Part B components are
mixed in an combined calibration solution. If these other anions
are present as contaminants, a separate chlorite calibration needs
to be performed.
7.3.6 Fluoride (F") 1000 mg/L: Dissolve 0.2210 g sodium fluoride (NaF, CASRN
7681 -49-4) in reagent water and dilute to 100 mL in a volumetric flask.
7.3.7 Nitrate (NOyN) 1000 mg/L: Dissolve 0.6068 g sodium nitrate (NaNO3,
CASRN 7631-99-4) in reagent water and dilute to 100 mL in a volumetric
flask.
7.3.8 Nitrite (NOyN) 1000 mg/L: Dissolve 0.4926 g sodium nitrite (NaNO2,
CASRN 7632-00-0) in reagent water and dilute to 100 mL in a volumetric
flask.
7.3.9 Phosphate (PO43'-P) 1000 mg/L: Dissolve 0.4394 g potassium
dihydrogenphosphate (KH2PO4, CASRN 7778-77-0) in reagent water and
dilute to 100 mL in a volumetric flask.
7.3.10 Sulfate (SO42') 1000 mg/L: Dissolve 0.1814 g potassium sulfate (K2SO4,
CASRN 7778-80-5) in reagent water and dilute to 100 mL in a volumetric
flask.
NOTE: Stability of standards: Stock standards (7.3) for most anions are stable
for at least 6 months when stored at 4°C. Except for the chlorite
standard which is only stable for two weeks when stored protected from
light at 4°C, and nitrite and phosphate which are only stable for 1 month
when stored at 4°C. Dilute working standards should be prepared
monthly, except those that contain chlorite, or nitrite and phosphate
which should be-prepared fresh daily.
7.4 Ethylenediamine (EDA) preservation solution, 100 mg/mL: Dilute 2.8 mL of
ethylenediamine (99%) (CASRN 107-15-3) to 25 mL with reagent water. Prepare
fresh monthly.
300.1-11
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7.5 Surrogate Solution: 0.50 mg/mL dichloroacetate (DCA) prepared by dissolving
0.065 g dichloroacetic acid, potassium salt (C12CHCO2K, CASRN 19559-59-2) in
reagent water and dilute to 100 mL in a volumetric flask.
7.5.1 Dichloroacetate is potentially present in treated drinking waters as the
acetate of the organic disinfection byproduct, dichloroacetic acid (DCAA).
Typical concentrations of DCAA rarely exceed 50 ug/L, which, for this
worst case example, would represent only a five percent increase in the
observed response over the fortified concentration of 1.00 mg/L.
Consequently, the criteria for acceptable recovery (90% to 115%) for the
surrogate is weighted to 115% to allow for this potential background.
7.5.2 Prepare this solution fresh every 3 months or sooner if signs of degradation
are present.
8. SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Samples should be collected in plastic or glass bottles. All bottles must be
thoroughly cleaned and rinsed with reagent water. Volume collected should be
sufficient to insure a representative sample, allow for replicate analysis, if required,
and minimize waste disposal.
8.2 Special sampling requirements and precautions for chlorite.
8.2.1 Sample bottles used for chlorite analysis must be opaque to protect the
sample from light.
8.2.2 When preparing the LFM, be aware that chlorite is an oxidant and may react
with the natural organic matter in an untreated drinking water matrix as a
result of oxidative demand. If untreated water is collected for chlorite
analysis, and subsequently used for the LFM, EDA preservation will not
control this demand and reduced chlorite recoveries may be observed.
8.3 Sample preservation and holding times for the anions that can be determined by this
method are as follows:
PART A: Common Anions
Analvte Preservation Holding Time
Bromide None required 28 days
Chloride None required 28 days
Fluoride None required 28 days
Nitrate-N Cool to 4°C 48 hours
Nitrite-N Cool to 4°C 48 hours
ortho-Phosphate-P Coolto4°C 48 hours
Sulfate Coolto4°C 28 days
300.1-12
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PART B : Inorganic Disinfection By-products
Analyte Preservation Holding Time
Bromate 50mg/LEDA 28 days
Bromide None required 28 days
Chlorate 50 mg/L ED A 28 days
Chlorite 50 mg/L EDA, Cool to 4°C 14 days
8.4 When collecting a sample from a treatment plant employing chlorine dioxide, the
sample must be sparged with an inert gas (helium, argon, nitrogen) prior to addition
of the EDA preservative at time of sample collection.
8.5 All four anions, in Part B, can be analyzed in a sample matrix which has been
preserved with EDA. Add a sufficient volume of the EDA preservation solution
(Sect. 7.4) such that the final concentration is 50 mg/L in the sample. This would be
equivalent to adding 0.5 mL of the EDA preservation solution to 1 L of sample.
8.6 EDA is primarily used as a preservative for chlorite. Chlorite is susceptible to
degradation both through catalytic reactions with dissolved iron salts and reactivity
towards free chlorine which exists as hypochlorous acid/hypochlorite ion in most
drinking water as a residual disinfectant. EDA serves a dual purpose as a
preservative for chlorite by chelating iron as well as any other catalytically
destructive metal cations and removing hypochlorous acid/hypochlorite ion by
forming an organochloroamine. EDA preservation of chlorite also preserves the
integrity of chlorate which can increase in unpreserved samples as a result of chlorite
degradation. EDA also preserves the integrity of bromate concentrations by binding
with hypobromous acid/hypobromite which is an intermediate formed as by-product
of the reaction of either ozone or hypochlorous acid/hypochlorite with bromide ion.
If hypobromous acid/hypobromite is not removed from the matrix further reactions
may form bromate ion.
8.7 Degradation of ortho-phosphate has been observed in samples held at room
temperature for over 16 hrs (see table 3 A). Therefore, samples to be analyzed for
ortho-phosphate must not be held at room temperature for more than 12 cumulative
hours.
9. QUALITY CONTROL
9.1 Each laboratory using this method is required to operate a formal quality control
(QC) program. The requirements of this program consist of an initial demonstration
of laboratory performance, and subsequent analysis in each analysis batch (Sect. 3.1)
of a Laboratory Reagent Blank, Laboratory Fortified Blank, Instrument Performance
Check Standard, calibration check standards, Laboratory Fortified Sample Matrices
(LFM) and either Field, Laboratory or LFM duplicate sample analyses. This section
300.1-13
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details the specific requirements for each of these QC parameters. The laboratory is
required to maintain performance records that define the quality of the data that are
generated.
9.2 INITIAL DEMONSTRATION OF PERFORMANCE
9.2.1 The initial demonstration of performance is used to characterize instrument
performance (determination of accuracy through the analysis of the QCS)
and laboratory performance (determination of MDLs) prior to performing
analyses by this method.
9.2.2 Quality Control Sample (QCS) - When beginning the use of this method, on
a quarterly basis or as required to meet data-quality needs, verify the
calibration standards and acceptable instrument performance with the
preparation and analyses of a QCS. If the determined concentrations are not
within ±15% of the stated values, performance of the determinative step of
the method is unacceptable. The source of the problem must be identified
and corrected before either proceeding with the initial determination of
MDLs or continuing with on-going analyses.
9.2.3 Method Detection Limit (MDL) — MDLs must be established for all
analytes, using reagent water (blank) fortified at a concentration of three to
five times the estimated instrument detection limit.(6) To determine MDL
values, take seven replicate aliquots of the fortified reagent water and
process through the entire analytical method over at least three separate
days. Perform all calculations defined in the method and report the
concentration values in the appropriate units. Calculate the MDL as
follows:
MDL = (t) x (S)
where, t= Student's t value for a 99% confidence level and a standard
deviation estimate with n-1 degrees of freedom [t = 3.14 for
seven replicates].
S = standard deviation of the replicate analyses.
9.2.3.1 MDLs should be determined every 6 months, when a new
operator begins work or whenever there is a significant change in
the background, or instrument response.
9.3 ASSESSING LABORATORY PERFORMANCE
9.3.1 Laboratory Reagent Blank (LRB) ~ The laboratory must analyze at least one
LRB with each analysis batch (defined Sect 3.1). Data produced are used to
300.1-14
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assess contamination from the laboratory environment. Values that exceed
the MDL indicate laboratory or reagent contamination should be suspected
and corrective actions must be taken before continuing the analysis.
9.3.1.1 If conducting analysis for the Part B anions, EDA must be added
to the LRB at 50 mg/L. By including EDA in the LRB, any bias
as a consequence of the EDA which may be observed in the field
samples, particularly in terms of background contamination, will
be identified.
9.3.2 Laboratory Fortified Blank (LFB) - The LFB should be prepared at
concentrations similar to those expected in the field samples and ideally at
the same concentration used to prepare the LFM. Calculate accuracy as
percent recovery (Sect. 9.4.1.3). If the recovery of any analyte falls outside
the required concentration dependant control limits (Sect. 9.3.2.2), that
analyte is judged out of control, and the source of the problem should be
identified and resolved before continuing analyses.
9.3.2.1 If conducting analysis for the Part B anions, EDA must be added
to the LFB at 50 mg/L. The addition of EDA to all reagent water
prepared calibration and quality control samples is required not as
a preservative but rather as a means to^ normalize any bias
attributed by the presence of EDA in the field samples.
9.3.2.2 Control Limits for the LFB
Concentration range Percent Recovery Limits
MRL to lOxMRL 75 -125 %
1 OxMRL to highest calibration level 85 - 115 %
9.3.2.2.1 These control limits only apply if the MRL is established within a
factor of 10 times the MDL. Otherwise, the limits are set at 85%
to 115%.
9.3.2.3 The laboratory must use the LFB to assess laboratory performance
against the required control limits listed in 9.3.2.2. When
sufficient internal performance data become available (usually a
minimum of 20-30 analyses), optional control limits can be
developed from the percent mean recovery (x) and the standard
deviation (S) of the mean recovery. These data can be used to
establish the upper and lower control limits as follows:
UPPER CONTROL LIMIT = x + 3S
LOWER CONTROL LIMIT = x - 3S
300.1-15
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The optional control limits must be equal to or better than those
listed in 9.3.2.2. After each five to ten new recovery measure-
ments, new control limits can be calculated using only the most
recent 20-30 data points. Also, the standard deviation (S) data
should be used to establish an on-going precision statement for
the level of concentrations monitored. These data must be kept
on file and be available for review.
9.3.3 Instrument Performance Check Solution (IPC) — The Initial Calibration
Check Standard is to be evaluated as the instrument performance check
solution in order to confirm proper instrument performance. Proper
chromatographic performance must be demonstrated by calculating the Peak
Gaussian Factor (PGF), which is a means to measure peak symmetry and
monitoring retention time drift in the surrogate peak over time. Critically
evaluate the surrogate peak in the initial calibration check standard, and
calculate the PGF as follows,
1.83 x W(l/2)
PGF = -----------------------
where: W(l/2) is the peak width at half height
W(l/10) is the peak width at tenth height
9.3.3.1 The PGF must fall between 0.80 and 1.15 in order to demonstrate
proper instrument performance.
9.3.3.2 The retention time for the surrogate in the IPC must be closely
monitored on each day of analysis and throughout the lifetime of
the analytical column. Small variations in retention time can be
anticipated when a new solution of eluent is prepared but if shifts
of more than 2% are observed in the surrogate retention time, some
type of instrument problem is present. Potential problems include
improperly prepared eluent, erroneous method parameters
programmed such as flow rate or some other system problem. The
chromatographic profile (elution order) of the target anions
following an ion chromatographic analysis should closely replicate
the profile displayed in the test chromatogram that was shipped
when the column was purchased. As a column ages, it is normal to
see a gradual shift and shortening of retention times, but if after
several years of use, extensive use over less than a year, or use with
harsh samples, this retention time has noticeably shifted to any less
than 80% of the original recorded value, the column may require
300.1-16
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cleaning or replacement. Particularly if resolution problems are
beginning to become common between previously resolved peaks.
A laboratory must retain a historic record of retention times for the
surrogate and all the target anions to provide evidence of an
analytical columns vitality.
9.4 ASSESSING ANALYTE RECOVERY AND DATA QUALITY
9.4. 1 Laboratory Fortified Sample Matrix (LFM) — The laboratory must add a
known amount of analyte to a minimum of 10% of the field samples within
an analysis batch. The LFM sample must be prepared from a sample matrix
which has been analyzed prior to fortification. The analyte concentration
must be high enough to be detected above the original sample and should
adhere to the requirement of 9.4. 1 .2. It is recommended that the solutions
used to fortify the LFM be prepared from the same stocks used to prepare
the calibration standards and not from external source stocks. This will
remove the bias contributed by an externally prepared stock and focus on
any potential bias introduced by the field sample matrix.
9.4. 1.1 If the fortified concentration is less than the observed background
concentration of the unfortified matrix, the recovery should not be
calculated. This is due to the difficulty in calculating accurate
recoveries of the fortified concentration when the native sample
concentration is so high.
9.4. 1 .2 The LFM should be prepared at concentrations no greater than five
times the highest concentration observed in any field sample. If no
analyte is observed in any field sample, the LFM must be fortified
no greater than five times the lowest calibration level which as
outlined in 12.2 is the minimum reported level (MRL). For
example, if bromate is not detected in any field samples above the
lowest calibrations standard concentration of 5.00 ug/L, the highest
LFM fortified concentration allowed is 25.0 ug/L.
9.4. 1 .3 Calculate the percent recovery for each analyte, corrected for
concentrations measured in the unfortified sample. Percent
recovery should be calculated using the following equation:
CS-C
R= --------
where, R = percent recovery.
Cs = fortified sample concentration
C = sample background concentration
s = concentration equivalent of analyte added to sample.
300.1-17
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9.4. 1 .4 Until sufficient data becomes available (usually a minimum of 20
to 30 analysis), assess laboratory performance against recovery
limits of 75 to 125%. When sufficient internal performance data
becomes available develop control limits from percent mean
recovery and the standard deviation of the mean recovery. The
optional control limits must be equal to or better than the required
control limits of 75-125%.
9.4. 1 .5 If the recovery of any analyte falls outside the designated LFM
recovery range and the laboratory performance for that analyte is
shown to be hi control (Sect. 9.3), the recovery problem
encountered with the LFM is judged to be matrix induced and the
results for that sample and the LFM are reported with a "matrix
induced bias" qualifier.
9.4.2 SURROGATE RECOVERY - Calculate the surrogate recovery from all
analyses using the following formula
SFC
where, R = percent recovery.
SRC = Surrogate Recovered Concentration
SFC = Surrogate Fortified Concentration
9.4.2.1 Surrogate recoveries must fall between 90-1 15% for proper
instrument performance and analyst technique to be verified. The
recovery of the surrogate is slightly bias to 115% to allow for the
potential contribution of trace levels of dichloroacetate as the
halogenated organic disinfection by-product (DBF) dichloroacetic
acid (DCAA) Background levels of this organic DBF are rarely
observed above 50 ug/L (0.05 mg/L) which constitutes only 5% of
the 1.00 mg/L recommended fortified concentration.
9.4.2.2 If the surrogate recovery falls outside the 90- 115% recovery
window, a analysis error is evident and sample reanalysis is
required. Poor recoveries could be the result of imprecise sample
injection or analyst fortification errors.
9.4.3 FIELD OR LABORATORY DUPLICATES - The laboratory must analyze
either a field or a laboratory duplicate for a minimum of 10% of the
collected field samples or at least one with every analysis batch, whichever
is greater. The sample matrix selected for this duplicate analysis must
300.1-18
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contain measurable concentrations of the target anions in order to establish
the precision of the analysis set and insure the quality of the data. If none of
the samples within an analysis batch have measurable concentrations, the
LFM should be employed as a laboratory duplicate.
9.4.3.1 Calculate the relative percent difference (RPD) of the initial
quantitated concentration (Ic) and duplicate quantitated
concentration (Dc) using the following formula,
RPD = -------------- XI 00
([Ic + DJ/2)
9.4.3.2 Duplicate analysis acceptance criteria
Concentration range ' ~ RPD Limits
MRLtolOxMRL +/- 20 %
lOxMRL to highest calibration level +/- 10 %
9.4.3.3 If the RPD fails to meet these criteria, the samples must be
reported with a qualifier identifying the sample analysis result as
yielding a poor duplicate analysis RPD. This should not be a
chronic problem and if it frequently recurs (>20% of duplicate
analyses) it indicates a problem with the instrument or individual
technique.
9.4.4 , Where reference materials are available, they should be analyzed to provide
additional performance data. The analysis of reference samples is a valuable
tool for demonstrating the ability to perform the method acceptably.
9.4.5 In recognition of the rapid advances occurring in chromatography, the
. analyst is permitted certain options, such as the use of different columns,
injection volumes, and/or eluents, to improve the separations or lower the
cost of measurements. Each time such modifications to the method are
made, the analyst is required to repeat the procedure in Sect. 9.2 and adhere
to the condition of baseline stability found in Sect. 1 .2. 1 .
9.4.6 It is recommended that the laboratory adopt additional quality assurance
practices for use with this method. The specific practices that are most
. productive depend upon the needs of the laboratory and the nature of the
samples. Whenever possible, the laboratory should perform analysis of
quality control check samples and participate in relevant performance
evaluation sample studies.
300.1-19
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10. CALIBRATION AND STANDARDIZATION
10.1 Establish ion chromatographic operating parameters equivalent to those indicated in
Tables 1A or IB if employing a 2 mm column, Table 1C if employing a 4 mm
column.
10.2 Estimate the Linear Calibration Range (LCR) ~ The LCR should cover the expected
concentration range of the field samples and should not extend over more than 2
orders of magnitude in concentration (For example, if quantitating nitrate in the
expected range of 1.0 mg/L to 10 mg/L, 2 orders of magnitude would permit the
minimum and maximum calibration standards of 0.20 mg/L and 20 mg/L,
respectively.) The restriction of 2 orders of magnitude is prescribed since beyond
this it is difficult to maintain linearity throughout the entire calibration range.
10.2.1 If quantification is desired over a larger range, then two separate calibration
curves should be prepared.
10.2.2 For an individual calibration curve, a minimum of three calibration
standards are required for a curve that extends over a single order of
magnitude and a minimum of five calibration standards are required if the
curve covers two orders of magnitude. (For example, using the nitrate
example cited above in section 10.2, but in this case limit the curve to
extend only from 1.0 mg/L to 10 mg/L or a single order of magnitude. A
third standard is required somewhere in the middle of the range. For the
calibration range of 0.20 mg/L to 20 mg/L, over two orders of magnitude,
five calibrations standards should be employed, one each at the lower and
upper concentration ranges and the other three proportionally divided
throughout the middle of the curve.)
10.3 Prepare the calibration standards by carefully adding measured volumes of one or
more stock standards (7.3) to a volumetric flask and diluting to volume with reagent
water.
10.3.1 For the Part B anions, EDA must be added to the calibration standards at 50
mg/L. The addition of EDA to all reagent water prepared calibration and
quality control samples is required not as a preservative but rather as a
means to normalize any bias attributed by the presence of EDA in the field
samples.
10.3.2 Prepare a 10.0 mL aliquot of surrogate fortified calibration solution which
can be held for direct manual injection or used to fill an autosampler vial.
Add 20 uL of the surrogate solution (7.5) to a 20 mL disposable plastic
micro beaker. Using a 10.0 mL disposable pipet, place exactly 10.0 mL of
calibration standard into the micro beaker and mix. The calibration standard
300.1-20
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is now ready for analysis. The same surrogate solution that has been
employed for the standards should also be used in the section 11.3.2 for the
field samples.
10.4 Using a 2 mm column, inject 10 uL (Part A) or 50 uL (Part B) of each calibration
standard. Using a 4 mm column, inject 50 uL (Part A) or 200 uL (Part B) of each
calibration standard. Tabulate peak area responses against the concentration. The
results are used to prepare calibration curves using a linear least squares fit for each
analyte. Acceptable calibration curves are confirmed after reviewing the curves for
linearity and passing the criteria for the initial calibration check standard in section
10.5.1. Alternately, if the ratio of response to concentration (response factor) is
constant over the LCR (indicated by < 15% 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,
10.4.1 Peak areas are strongly recommended since they have been found to be more
consistent, in terms of quantitation, than peak heights. Peak height can tend
to be suppressed as a result of high levels of common anions in a given
matrix which can compete for exchange sites. Using peak areas, it is the
analyst responsibility to review all chromatograms to insure accurate
baseline integration of target analyte peaks since poorly drawn baselines will
more significantly influence peak areas than peak heights.
10.5 Once the calibration curves have been established they must be verified prior to
conducting any sample analysis using an initial calibration check standard (3.2.2).
This verification must be performed on each analysis day or whenever fresh eluent has
been prepared. A continuing calibration check standard (3.2.3) must be analyzed after
every tenth sample and at the end of the analysis set as an end calibration check
standard (3.2.4). The response for the initial, continuing and end calibration check
must satisfy the criteria listed in 10.5.1. If during the analysis set, the response differs
by more than the calibration verification criteria shown in 10.5.1., or the retention
times shift more than ± 5% from the expected values for any analyte, the test must be
repeated, using fresh calibration standards. If the results are still outside these criteria,
sample analysis must be discontinued, the cause determined and/or in the case of drift,
the instrument recalibrated. All samples following the last acceptable calibration
check standard must be reanalyzed.
10.5.1 Control limits for calibration verification
Concentration range Percent Recover
MRL to lOxMRL 75 - 125 %
lOxMRL to highest calibration level 85 - 115 %
300.1-21
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10.5.1.1 These control limits only apply if the MRL is established within a
factor of 10 times the MDL. Otherwise, the limits are set at 85% to
115%.
10.5.2 CALIBRATION VERIFICATION REQUIREMENT FOR PART B
As a mandatory requirement of calibration verification, the laboratory
MUST verify calibration using the lowest calibration standard as the initial
calibration check standard.
10.5.3 After satisfying the requirement of 10.5.2, the levels selected for the other
calibration check standards should be varied between a middle calibration
level and the highest calibration level.
11. PROCEDURE
11.1 Tables 1A and IB summarize the recommended operating conditions for the ion
chromatograph. Included in these tables are estimated retention times that can be
achieved by this method. Other columns, chromatographic conditions, or detectors
may be used if the requirements of Sect. 9.2 are met.
11.2 Check system calibration daily and, if required, recalibrate as described in Sect. 10.
11.3 Sample Preparation
11.3.1 For refrigerated or samples arriving to the laboratory cold, ensure the
samples have come to room temperature prior to conducting sample analysis
by allowing the samples to warm on the bench for at least 1 hour.
11.3.2 Prepare a 10.0 mL aliquot of surrogate fortified sample which can be held for
direct manual injection or used to fill an autosampler vial. Add 20 uL of the
surrogate solution (7.5) to a 20 mL disposable plastic micro beaker. Using a
10.0 mL disposable pipet, place exactly 10.0 mL of sample into the micro
beaker and mix. Sample is now ready for analysis.
11.3.2.1 The less than 1% dilution error introduced by the addition of the
surrogate is considered insignificant.
11.4 Using a Luer lock, plastic 10 mL syringe, withdraw the sample from the micro
beaker and attach a 0.45 urn particulate filter (demonstrated to be free of ionic
contaminants) directly to the syringe. Filter the sample into an autosampler vial (If
vial is not designed to automatically filter) or manually load the injection loop
injecting a fixed amount of well mixed sample. If using a manually loaded injection
300.1-22
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loop, flush the loop thoroughly between sample analysis using sufficient volumes of
each new sample matrix.
11.5 Using a 2 mm column, inject 10 uL (Part A) or 50 uL (Part B) of each sample.
Using a 4 mm column, inject 40 uL (Part A) or 200 uL (Part B) of each sample.
Tabulate peak area responses against the concentration. During this procedure,
retention times must be recorded. Use the same size loop for standards and samples.
Record the resulting peak size in area units. An automated constant volume
injection system may also be used.
11.6 The width of the retention time window used to make identifications should be
based upon measurements of actual retention time variations of standards over the
course of a day. Three times the standard deviation of a retention time can be used
to calculate a suggested window size for each analyte. However, the experience of
the analyst should weigh heavily in the interpretation of chromatograms.
11.7 If the response of a sample analyte exceeds the calibration range, the sample may be
diluted with an appropriate amount of reagent water and reanalyzed. If this is not
possible then three new calibration concentrations must be employed to create a
separate high concentration curve, one standard near the estimated concentration
and the other two bracketing around an interval equivalent to ± 25% the estimated
concentration. The latter procedure involves significantly more time than a simple
sample dilution therefore, it is advisable to collect sufficient sample to allow for
sample dilution or sample reanalysis, if required.
11.8 Shifts in retention time are inversely proportional to concentration. Nitrate,
phosphate and sulfate will exhibit the greatest degree of change, although all anions
can be affected. In some cases this peak migration may produce poor resolution or
make peak identification difficult.
11.9 Should more complete resolution be needed between any two coeluting peaks, the
eluent (7.2) can be diluted. This will spread out the run, however, and will cause
late eluting anions to be retained even longer. The analysts must verify that this
dilution does not negatively affect performance by repeating and passing all the QC
criteria in Section 9. As a specific precaution, upon dilution of the carbonate eluent,
a peak for bicarbonate may be observed within the retention time window for
bromate which will negatively impact the analysis.
11.9.1 Eluent dilution will reduce the overall response of an anion due to
chromatographic band broadening which will be evident by shortened and
broadened peaks. This will adversely effect the MDLs for each analyte.
300.1-23
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12. DATA ANALYSIS AND CALCULATIONS
12.1 Prepare a calibration curve for each analyte by plotting instrument response, as peak
area, against standard concentration. Compute sample concentration by comparing
sample response with the standard curve. If a sample has been diluted, multiply the
response by the appropriate dilution factor.
12.2 Report ONLY those values that fall between the lowest and the highest calibration
standards. Samples with target analyte responses exceeding the highest standard
should be diluted and reanalyzed. Samples with target analytes identified but
quantitated below the concentration established by the lowest calibration standard
should be reported as below the minimum reporting limit (MRL).
12.3 Report results for Part A anions in mg/L and for Part B anions in ug/L.
12.4 Report NO2' asN
NO3- asN
HPO4=asP
Br" in mg/L when reported with Part A
Br" hi ug/L when reported with Part B
13. METHODS PERFORMANCE
13.1 Tables 1A, IB, and 1C give the single laboratory (OW OGWDW TSC-Cincinnati)
retention times, standard conditions and MDL determined for each anion included in
the method. MDLs for the Part A anions were determined in reagent water on the 2
mm column (Table 1 A). MDLs for the Part B anions were conducted not only in
reagent water but also a simulated high ionic strength water (HIW) on the 2 mm
column (Table IB) and in reagent water on the 4 mm column (Table 1C). HIW is
designed to simulate a high ionic strength field sample. It was prepared from
reagent water which was fortified with the common anions of chloride at 100 mg/L,
carbonate at 100 mg/L, nitrate at 10.0 mg/L as nitrogen, phosphate at 10.0 mg/L as
phosphorous, and sulfate at 100 mg/L.
13.2 Tables 2A and 2B give the single laboratory (OW OGWDW TSC-Cincinnati)
standard deviation for each anion included in the method in a variety of waters for
the standard conditions identified in Table 1A and IB, respectively.
13.3 Tables 3A and 3B shown stability data for the Part A and B anions, respectively.
Each data point hi these tables represent the mean percent recovery following
triplicate analysis. These data were used to formulate the holding times shown in
Sect. 8.3.
300.1-24
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14. POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or eliminates the
quantity or toxicity of waste at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The EPA has established a
preferred hierarchy of environmental management techniques that places pollution
prevention as the management option of first choice. Whenever feasible, laboratory
personnel should use pollution prevention techniques to address their waste
generation. When wastes cannot be feasibly reduced at the source, the Agency
recommends recycling as the next best option.
14.2 Quantity of the chemicals purchased should be based on expected usage during its
shelf life and disposal cost of unused material. Actual reagent preparation volumes
should reflect anticipated usage and reagent stability.
14.3 For information about pollution prevention that may be applicable to laboratories
and research institutions, consult "Less is Better: Laboratory Chemical Management
for Waste Reduction," available from the American Chemical Society's Department
of Government Regulations and Science Policy, 1155 16th Street N.W., Washington
D.C. 20036,
(202) 872-4477.
15. WASTE MANAGEMENT
15.1 The Environmental Protection Agency requires that laboratory waste management
practices be conducted consistent with all applicable rules and regulations. Excess
reagents, samples and method process wastes should be characterized and disposed
of in an acceptable manner. The Agency urges laboratories to protect the air, water,
and land by minimizing and controlling all releases from hoods and bench
operations, complying with the letter and spirit of any waste discharge permit and
regulations, and by complying with all solid and hazardous waste regulations,
particularly the hazardous waste identification rules and land disposal restrictions.
For further information on waste management consult the "Waste Management
Manual for Laboratory Personnel," available from the American Chemical Society at
the address listed in Sect. 14.3.
16. REFERENCES
1. "Determination of Inorganic Disinfection By-Products by Ion Chromatography", J.
Pfaff, C. Brockhoff. J. Am. Water Works Assoc., Vol 82, No. 4, pg 192.
2. Standard Methods for the Examination of Water and Wastewater, Method 4110B,
"Anions by Ion Chromatography", 18th Edition of Standard Methods (1992).
300.1-25
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3. Dionex, System DX500 Operation and Maintenance Manual, Dionex Corp.,
Sunnyvale, California 94086,1996.
4. Method Detection Limit (MDL) as described in "Trace Analyses for Wastewater," J.
Glaser, D. Foer?t, G. McKee, S. Quave, W. Budde, Environmental Science and
Technology, Vol. 15, Number 12, page 1426, December, 1981.
5. American Society for Testing and Materials. Test Method for Anions in Water by
Chemically-Suppressed Ion Chromatography D4327-91. Annual Book of Standards,
Vol 11.01 (1993).
6. Code of Federal Regulations 40, Ch. l,Pt. 136, Appendix B.
7. Hautman, D.P. & Bolyard, M. Analysis of Oxyhalide Disinfection By-products and
other Anions of Interest in Drinking Water by Ion Chromatography. Jour, of
Chromatog., 602, (1992), 65-74.
8. Standard Methods for the Examination of Water and Wastewater, Method 4500-
C1O2,C "Amperometric Method I" (for the determination of Chlorine Dioxide), 19th
Edition of Standard Methods (1995).
300.1-26
-------
17. TABLES. DIAGRAMS. FLOWCHARTS AND VALIDATION DATA
TABLE 1A. CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION
LIMITS IN REAGENT WATER FOR THE COMMON ANIONS (PART A).
ANALYTE
Fluoride
Chloride
Nitrite-N
Surrogate: DCA
Bromide
Nitrate-N
ortho-Phosphate-P
Sulfate
"
PEAK # (1)
1
- 2
3
4
5
6
7
8
RETENTION TIME
(NUN.)
""' "2.53
4.67
6.01
7.03
8.21
9.84
11.98
13.49
MDL
Fort
Cone,
mg/L
0.020
0.020
0.010
0.040
0.010
0.040
0.040
DETERMINATION
Number
of Replicates
7
7
7
7
7
7
7
DI
MDL
mg/L
0.009
. 0.004
0.001
0.014
0.008
0.019
0.019
Standard Conditions:
Ion Chromatograph:
Columns :
Detector:
Suppressor:
Eluent:
Eluent Flow:
Sample Loop:
Dionex DX500
Dionex AG9-HC / AS9-HC, 2 mm
Suppressed Conductivity Detector, Dionex CD20
ASRS-I, external source electrolytic mode, 100 mA current
9.0mMNa2CO3
0.40 mL/min
lOuL
System Backpressure: 2800 psi
Background Conductivity: 22 uS
Recommended method total analysis time: 25 minutes
(1) See Figure 1
300.1-27
-------
TABLE IB. CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION
LIMITS IN BOTH REAGENT WATER AND HIGH IONIC STRENGTH
WATER FOR THE INORGANIC DISINFECTION BY-PRODUCTS
(PARTB).
MDL DETERMINATION
ANALYTE
Chlorite
Bromate
Surrogate:
DCA
Bromide
Chlorate
PEAK#(1)
1
2
4
5
6
Fort
RETENTION
TIME
(MIN.)
3.63
Cone,
ug/L
2.00
4.19 ! 2.00
7.28
8.48
2.00
9.28 ! 2.00
I
Number
of
Replicates
7
7
7
7
DI
MDL
ug/L
0.89
1.44
.
1.44
1.31
HIW(2)
MDL
ug/L
0.45
1.28
i
i
.2.51 ,!
0.78
Standard Conditions:
Ion Chromatograph:
Columns:
Detector:
Suppressor:
Eluent:
Eluent Flow:
Sample Loop:
DionexDXSOO
Dionex AG9-HC / AS9-HC, 2 mm
Suppressed Conductivity Detector, Dionex CD20
ASRS-I, external source electrolytic mode, 100 mA current
9.0mMNa2CO3
0.40mL/min
50 uL
System Backpressure: 2800 psi
Background Conductivity: 22 uS
Recommended method total analysis time: 25 minutes
(1) See Figure 2 and 3
(2) HIW indicates High Ionic Strength Water which is a simulated drinking water prepared
from reagent water and fortified with chloride at 100 mg/L, carbonate at 100 mg/L, nitrate
at 10.0 mg/L as nitrogen, phosphate at 10.0 mg/L as phosphorous, and sulfate at 100 mg/L.
300.1-28
-------
TABLE 1C. CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION
^ ~? r-flLIMITS IN REAGENT WATER FOR THE INORGANIC
V-l DISINFECTION BY-PRODUCTS USING AN ALTERNATE 4 mm AS9-
ANALYTE ^
Chlorite
Bromate
Surrogate:
DCA
Bromide '
Chlorate '" f '-
• >,,. ',.>.''. ...'.;• „ ;
"',, >, •-,» ' -
. '* f * f •- -
';*•• .--RETENTION:..
' ' • -D'D A "f- -W- " - HPTA JTI~?
. Jr JtiAJv ff . lUVLii •
(MIN.) '
;1 ' 4.43
2 5.10 ••/' ':
4 8.82
•
5 >..•: 10.11
6 ' ; 10.94 •-.->. '
MDL
Fort
Cone,
ug/L
2.00
2.00
2.00
2.00
DETERMINATION
Number
of
Replicates
7
7
7
7
DI
MDL
ug/L
1.44
1.32
0.98
2.55
Standard Conditions:
Ion Chromatograph:
Columns :
Detector:
Suppressor: >
Eluent:
Eluent Flow:
Sample Loop:
DionexDXSOO > : .«
DionexAG9-HC/A"S9-HC,4mm .
Suppressed Conductivity Detector, Dionex CD20
ASRS-Iy external source .electrolytic mode;,! 300 mA current
9.0mMNa2CO3
1.25mL/min .
200 uL
System Backpressure: 1900 psi
Background Conductivity: 21 uS
Recommended method total analysis time: 25 minutes
300.1-29
-------
TABLE 2A. SINGLE-OPERATOR PRECISION AND RECOVERY FOR THE CO!
ANIONS (PART A).
UNFORT FORT #
MATRIX CONG OF MEAN MEAN
ANALYTE MATRIX CONG., mg/L REP mg/L '. %REC SD
mg/L . (n-1)
Fluoride
Chloride
Nitrite-N
Bromide
Nitrate-N
Phosphate-P
Sulfate
Surrogate:
RW
SW
GW
CDW
RW
SW
GW
CDW
RW
SW
GW
CDW
RW
SW
GW
CDW
RW
SW
GW
CDW
RW
SW
GW
CDW
RW
SW
GW
CDW
RW
SW
GW
CDW
-------
TABLE 2B. SINGLE-OPERATOR PRECISION AND RECOVERY FOR THE
INORGANIC DISINFECTION BY-PRODUCTS (PART B).
ANALYTE MATRIX
Chlorite RW
HIW
SW
GW
C1W
CDW
O3W
Bromate RW
HIW
SW
GW
C1W
CDW
O3W
UNFORT
CONC.
ug/L
-------
TABLE 2B. SINGLE-OPERATOR PRECISION ^ND RECOVERY FOR THE
INORGANIC DISINFECTION BY-PRODUCTS (PART B)(contd.).
ANALYTE MATRIX
Bromide RW
mw
•
SW
GW
C1W
CDW
O3W
Chlorate RW
mw
SW
GW
C1W
CDW
O3W
RW = Reagent Water
UNFORT FORT '
GONC. CONC: .,
ug/L ug/L
sbb; •'
'i'bo
!500
100
500
100:
, 500 •
100
500
100
500
100
500
GW
C1W
CDW
O3W
#••?•-"• ''
OF MEA
:'.V:, ;,!-:--y%K-.: '** •,'••"?; i '•"•?,'
REP ,,ug/L
9 20.9
9 ; 107
9 21.8
9 105
9 51.3
9 -140.
9 172
9 265
9 39.3
9 : 125
9 34.4
9 125
9 65.4
9 153
9 98.3
;'9 520
9 86.1
'•'9 502
9 102
, .9 ,. 513,
9 93.5
.9^ ,510
9 ; .136,
9 549
9 223
9 651
9 106
9 523
N "MEAN
%REC
104
107 ;
92.5
102
(2)
109
(2)
__-<2)
115
109;
115
113
_-_(2)
113
98.3
"•'•'-' "104 •'.;';•'•
•y; 86.1
100.
98.3
i 102
.; '";,',' 93.5 ;.';
' .102.''
102
. i . • • .1-
103
__.(2)
106
100
103
SD
(n-l)
0.80 r
0.60
0.79
1.05
0.97
1.88
0.78
2.18
0.64
2.00
0.76
1.24
3.67
1.00
0.80
•'•""' 4.1' 5'
1 47
4 52
1.57
7.11
,2.00
,",'3.84
, ^1.01
sin
3.20
3.50
1.20
2.45
:-3.82
0.56
3.63
1
1.9
1.35
0.45
0.82
1.62
1.6
2.22
0.99
5.61
0.65
' '*•!
;°-82 :;;;'"
: 0.8. '' "
M-7
0.9
1.55
1.39
2.14' "
0.75
0.74
0.57
1.44
0.54
1.13
0.47
= Groundwater
= Chlorinated drinking
= Chlorine
water
dioxide treated drinking water
= Ozonated drinking water
(1)
-------
TABLE 2B. SINGLE-OPERATOR PRECISION AND RECOVERY FOR THE
INORGANIC DISINFECTION BY-PRODUCTS (PART B)(contd.).
ANALYTE
Surrogate: DCA
(see NOTE below)
MATRIX
RW
mw
SW
GW
C1W
CDW
O3W
FORT
CONC
mg/L
5.00
5.00
5.00
5.00
5.00
5.00
5.00
#
OF
REP
. 9
9
9
9
9
9
9
MEAN
mg/L
5.11
4.98
5.00
4.96
4.95
4.99
5.12
5.13
5.15
5.13
5.01
5.04
4.99
5.11
MEAN
%REC
102
99.5
100
99.2
98.9
99.8
102
103
103
103
100
101
99.8
101
SD
(n-i)
0.93
0.69
0.79
1.76
0.70
1.60
0.50
0.50
1.73
1.12
1.02
1.08
0.70
0.53
%RSD
0.91
0.69
0.79
1.78
0.7
1.61
0.49
0.49
1.68
1.09
1.02
1.07
0.7
0.52
RW = Reagent Water
HIW = High Ionic strength Water
[see note (2) in Table IB]
SW- Surface Water
GW- Groundwater
C1W - Chlorinated drinking water
CDW = Chlorine dioxide treated drinking water
O3 W = Qzonated drinking water
NOTE: The surrogate DCA was fortified at 5 mg/L but due to concerns about measuring
trace concentrations of bromide with such high concentration of the neighboring
surrogate peak, the recommended fortified concentration for the surrogate has
been reduced to 1.00 mg/L.
300.1-33
-------
TABLE 3A. STABILITY STUDY RESULTS FOR TfiE COMMON ANIONS (PART A).
ANALYTE Preservative
Fluoride None
Chloride None
Nitrite-N None
Bromide None
Nitrate-N None
Phosphate-P None
Sulfate None
Matrix
RW
SW
GW
CDW
RW
SW
GW
CDW
RW
SW
GW
CDW
RW
SW
GW
CDW
RW
SW
GW
CDW
RW
SW
GW
CDW
RW
SW
GW
CDW
UNFORT
CONC.
mg/L
-------
TABLE 3B STABILITY STUDY RESULTS FOR THE INORGANIC DISINFECTION
BY-PRODUCTS (PART B).
ANALYTE
Chlorite
Chlorite
Bromate
Bromate
Preservative Matrix
None RW
HIW
SW
GW
C1W
CDW
O3W
EDA RW
HIW
SW
GW
C1W.
CDW
O3W
None RW
HIW
SW
GW
C1W
CDW
O3W
EDA RW
HIW
SW
GW
C1W
CDW
O3W
UNFORT
CONC.
ug/L
-------
TABLE 3B. STABILITY STUDY RESULTS FOR THE INORGANIC
DISINFECTION BY-PRODUCTS (PART B)(contd.)
ANALYTE Preservative
Bromide None
Bromide EDA
Chlorate None
Chlorate EDA
Matrix
RW
HIW
SW
GW
C1W
CDW
O3W
RW
HIW
SW
GW
C1W
CDW
O3W
RW
HIW
SW
GW
C1W
CDW
O3W
RW
HIW
SW
GW
C1W
CDW
O3W
UNFORT
CONG.
ug/L
-------
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s
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o
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300.1-37
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DJO
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0
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O
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o
00 Q)
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o
3
ON
%
0)
a
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300.1-38
-------
ra
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CD
300.1-39
-------
— fl) O »_
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300.1-40
-------
ERRATA SHEET TO U.S.EPA METHOD 300.1
April 27,1999
The following were editorial changes which have been incorporated into U.S.EPA Method 300.1.
These minor clarifications are incorporated into the body of this text as follows:
ERRATA #1 -
An additional sentence was added to Section 4.1.1 reiterating the analyst's responsibilities when
incorporating any method change, including modifying eluent strength, or any other method
parameter. The additional sentence states, •
"... The analyst must verify that these changes do not negatively affect performance by
repeating and passing all the QC criteria in Section 9."
On this same theme, section 11.9, was also further clarified and specific precautions were added
as follows,
"...The analysts must verify that this dilution does not negatively affect performance by
repeating and passing all the QC criteria in Section 9. As a specific precaution, upon
dilution of the carbonate eluent, a peak for bicarbonate may be observed within the retention
time window for bromate which will negatively impact the analysis."
ERRATA #2 -
An acronym in Section 9.3.2.2 for Laboratory Fortified Blank (LFB) was incorrectly identified as
LRB. This typographical error was corrected.
ERRATA #3 -
Clarifications and corrections were made to Section 9.4.1.5, 9.4.3.2 and 9.4.3.3. These
clarifications pertain to data reportability for Laboratory Fortified Sample Matrices (LFM) as well
as to analysis continuation when Duplicate Sample QC acceptance criteria are not met.
Section 9.4.1.5 clarifies and now specifies how to report data when the LFM recovery falls outside
the established control criteria by stating,
"...the recovery problem encountered with the LFM is judged to be matrix induced and the
results for that sample and the LFM are reported with a "matrix induced bias " qualifier."
Section 9.4.3.2 required the correction of a typographical reference by removing "%Diff' in the
duplicate sample acceptance criteria and replacing it with the defined RPD, indicating "relative
percent difference".
300.1-41
-------
Section 9.4.3.3, also had a "%Diff reference corrected with RPD and included clarification
regarding continuation of an analysis set when a duplicate analysis fails to meet the acceptance
criteria. This section now reads,
"If the RPD fails to meet these criteria, the samples must be reported with a qualifier
identifying the sample analysis result as yielding a poor duplicate analysis RPD. This should
not be a chronic problem and if it frequently recurs, (>20% of duplicate analysis) it indicates
a problem -with the instrument or individual technique."
300.1-42
-------
METHOD 314.0 DETERMINATION OF PERCHLORATE IN DRINKING WATER
USING ION CHROMATOGRAPHY
Revision 1.0
November 1999
Daniel P. Hautman and David J. Munch, US EPA, Office of Ground Water and Drinking
Water and Andrew D. Eaton and Ali W. Haghani, Montgomery Watson Laboratories
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
314.0-1
-------
METHOD 314.0
DETERMINATION OF PERCHLORATE IN DRINKING WATER USING ION
CHROMATOGRAPHY
1. SCOPE AND APPLICATION
1.1 This method covers the determination of perchlorate in reagent water, surface water,
ground water, and finished drinking water using ion chromatography.
1.2 The single laboratory reagent water Method Detection Limit (MDL, defined in Section
3.16) for the above analyte is listed in Table 1. The MDL for a specific matrix may
differ from those listed, depending upon the nature of the sample and the specific
instrumentation employed.
1.2.1 In order to achieve comparable detection limits, an ion chromatographic system
must utilize suppressed conductivity detection, be properly maintained, and
must be capable of yielding a baseline with no more than 5 nanosiemen (nS)
noise/drift per minute of monitored response over the background conductivity.
1.3 This method is recommended for use only by or under the supervision of analysts
experienced in the use of ion chromatography and in the interpretation of the resulting
ion chromatograms.
1.4 When this method is used to analyze unfamiliar samples for perchlorate, anion
identification should be supported by the use of a laboratory fortified matrix sample.
The fortification procedure is described in Section 9.4.1.
1.5 Users of the method data should identify data quality objectives prior to analysis. Users
of the method must demonstrate the ability to generate acceptable results, using the
procedures described in Section 9.0.
1.6 This method specifies an 1C column and analytical conditions which were determined
to be the most effective for the widest array of sample matrices. Other 1C procedures
have been written which incorporate similar columns and conditions, such as hydroxide
based mobile phases, low hydrophobicity 1C columns, and measurement by suppressed
conductivity detection.1"5 During the development of this method, these other
procedures, as well as the columns and conditions outlined in this method, were
concurrently investigated with comparable results for test matrices with moderate levels
of common inorganic background anions. These findings were consistent with those of
the Inter-Agency Perchlorate Steering Committee, Analytical Subcommittee's Report,6
published in 1998, which reported on the results of an interlaboratory validation of
314.0-2
-------
these other Ion Chromatographic Methods. The columns and conditions identified in
- this method were recommended since they bore the greatest tolerance for the highest
levels of common inorganic anion interference.
2. SUMMARY OF METHOD
2.1 A 1.0 mL volume of sample (see Note), is introduced into an ion chromatograph (1C).
Perchlorate is separated and measured, using a system comprised of an ion
chromatographic pump, sample injection valve, guard column, analytical column,
suppressor device, and conductivity detector.
NOTE: This large sample loop (1.0 mL) can be made using approximately 219 cm (86
inches) of 0.03 inch i.d. PEEK tubing. The exact volume is not critical since
all standards and samples will use the same sample loop. However, the
volume should be verified to be within 5% of this volume by weighing the
sample loop empty, filling the loop with deionized water and re-weighing the
loop. The volume can then be approximated by assuming the density of water
is 1.0 mg/uL.
3. DEFINITIONS
3.1 ANALYSIS BATCH — A sequence of samples, which are analyzed within a 30 hour
period and include no more than 20 field samples. An Analysis Batch must also
include all required QC samples, which do not contribute to the maximum field sample
total of 20. The required QC samples include:
• Instrument Performance Check Standard (DPC)
• Laboratory Reagent Blank (LRB)
• Initial Calibration Check Standard (ICCS)
• Laboratory Fortified Blank (LFB)
• Continuing Calibration Check Standard (CCCS), when the batch contains more than
10 field samples
• End Calibration Check Standard (ECCS)
• Laboratory Fortified Matrix (LFM)
• Either a Field Duplicate, a Laboratory Duplicate or a duplicate of the LFM
• (if pretreated samples are included in batch) Pretreated LRB
• (if pretreated samples are included in batch) Pretreated LFB
• (if pretreated samples are included in batch) Pretreated LFM, for each pretreated
matrix.
NOTE: Every field sample analysis, including both diluted and pretreated field
samples, but excluding any LFM or duplicate field sample analysis which
qualify as QC samples, must be applied to the maximum of 20 total field
samples permitted in an analysis batch.
314.0-3
-------
3.1.1 A field sample(s), included in the analysis batch, can be reanalyzed following
the ECCS provided the 30 hr time limit for the analysis batch has not expired.
The laboratory can reanalyze that sample(s) but must initially conduct a second
ICCS before the reanalysis and an ECCS after the final reanalysis. The ECCS
must be completed within the 30 hr window.
3.2 CALIBRATION STANDARD (CAL) - A solution prepared from the primary dilution
standard solution(s) or stock standard solutions. The CAL solutions are used to
calibrate the instrument response with respect to ahalyte concentration.
3.3 INITIAL CALIBRATION STANDARDS - A series of CAL solutions used to initially
establish instrument calibration and develop calibration curves for individual target
anions (Section 10.2).
3.4 INITIAL CALIBRATION CHECK STANDARD (ICCS)- A CAL solution, which is
analyzed initially, prior to any field sample analyses, which verifies the previously
established calibration curve. The concentration for the initial calibration check
standard MUST be at or below the MRL (Section 3.17) level.
3.5 CONTINUING CALIBRATION CHECK STANDARDS (CCCS) -- A CAL solution
which is analyzed after every tenth field sample analyses, not including QC samples,
which verifies the previously established calibration curve and confirms accurate
analyte quantitation for the previous ten field samples analyzed. The concentration for
the continuing calibration check standards should be either at a middle calibration level
or at the highest calibration level (Section 10.3.2).
3.6 END CALIBRATION CHECK STANDARD (ECCS) - A CAL solution which is
analyzed after the last field sample analyses which verifies the previously established
calibration curve and confirms accurate analyte quantitation for all field samples
analyzed since the last continuing calibration check. The end calibration check standard
should be either the middle or high level continuing calibration check standard (Section
10.3.2).
3.7 FIELD DUPLICATES (FD) ~ 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 field duplicates indicate the precision associated
with sample collection, preservation and storage, as well as with laboratory procedures.
3.8 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) - A solution containing
a specific concentration of perchlorate and other test substances (namely chloride,
sulfate and carbonate) used to evaluate the performance of the instrument system with
respect to a defined set of criteria.
314.0-4
-------
3.9 LABORATORY DUPLICATE (LD) - Two sample aliquots (LD1 and LD2), taken in
the laboratory from a single sample bottle, and analyzed separately with identical
procedures. Analyses of LD1 and LD2 indicate precision associated specifically with
the laboratory procedures by removing variation contributed from sample collection,
preservation and storage procedures.
3.10 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water, or other
blank matrix, to which a known quantity of perchlorate is added in the laboratory. The
LFB is analyzed exactly like a sample, and its purpose is to determine whether the
methodology is in control, and whether the laboratory is capable of making accurate and
precise measurements.
3.11 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) - An aliquot of an
environmental field sample to which a known quantity of perchlorate is 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 result (when compared to
the result for the LFB). The background concentrations of perchlorate, in the sample
matrix, must be initially determined in a separate aliquot and the measured value in the
LFM corrected for this background concentration.
3.12 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or other
blank matrix that is treated exactly as a sample including exposure to all glassware,
equipment, solvents, filtration and reagents that are used with other samples. The LRB
is used to determine if perchlorate or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.13 LINEAR CALIBRATION RANGE (LCR) - The concentration range over which the
instrument response is linear.
3.14 MATERIAL SAFETY DATA SHEET (MSDS) - Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling precautions.
3.15 MATRIX CONDUCTIVITY THRESHOLD (MCT) - The highest permitted
conductance of an unknown sample matrix, measured prior to conducting the analysis,
which is used to determine when sample matrix dilution or pretreatment is required.
The conductance of a sample matrix is proportional to the common anions present in
the matrix (which contribute to the level of total dissolved solids [TDS]) which can
greatly affect the integrity of this analysis. The value for this threshold is dependant on
the conditions, hardware, and state of the hardware employed. Consequently, this
threshold is not method defined and must be determined by the individual analytical
laboratory during the Initial Demonstration of Capability (IDC) and confirmed in each
analysis batch using the Instrument Performance Check (IPC) Solution. Matrix
314.0-5
-------
conductivity is measured in microsiemens/cm (uS/cm) or microMhos/crn (uMhos/cm)
which are considered equivalent terms.
3.16 METHOD DETECTION LIMIT (MDL)- The minimum concentration of an analyte
that can be identified, measured and reported with 99% confidence that the analyte
concentration is greater than zero.7'8
3.17 MINIMUM REPORTING LEVEL (MRL) - The minimum concentration that can be
reported as a quantitated value for a target analyte in a sample following analysis. This
defined concentration can be no lower than the concentration of the lowest calibration
standard and can only be used if acceptable quality control criteria for this standard are
met.
3.18 PEAK AREA TO HEIGHT RATIO (A/H) - The ratio of the peak area divided by the
peak height which is used as a tool to monitor analytical performance. This ratio is
used to establish and monitor the MCT and represents an objective means of assessing
analytical performance when analyzing high conductivity matrices. A gradual
distortion of the baseline is typically observed in the retention time window for
perchlorate as the matrix conductivity increases (consistent with elevated levels of
common anions) which will more significantly influence peak height relative to the
influence on peak area. As the distortion of the baseline increases, this ratio increases,
and the integrity of the measured perchlorate will be compromised.
3.19 PROFICIENCY TESTING (PT) or PERFORMANCE EVALUATION (PE) SAMPLE -
- A certified solution of method analytes whose concentration is unknown to the
analyst. Often, an aliquot of this solution is added to a known volume of reagent water
and analyzed with procedures used for samples. Often, results of these analyses are
used as part of a laboratory certification program to objectively determine the
capabilities of a laboratory to achieve high quality results.
3.20 QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of known
concentrations that is obtained from a source external to the laboratory and different
from the source of calibration standards. It is used to check laboratory performance
with externally prepared test materials.
3.21 STOCK STANDARD SOLUTION (SSS) - A concentrated solution containing
perchlorate which is either prepared in the laboratory using assayed reference materials
or purchased from a reputable commercial source.
3.22 TOTAL DISSOLVED SOLIDS (TDS) - Both organic and inorganic constituent which
are dissolved in a sample matrix and are not removed by particulate filtration.,
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4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in the reagent water, reagents,
glassware, and other .sample processing apparatus that lead to discrete artifacts or
elevated baselines in an ion chromatogram. These interferences can lead to false
positive results for the target analyte as well as reduced detection limits as a
consequence of elevated baseline noise.
4.2 Interferences can be divided into three different categories: direct chromatographic
coelution, where an analyte response is observed at very nearly the same retention time
as the target anion; concentration dependant coelution, which is observed when the
response of higher than typical concentrations of the neighboring peak overlap into the
retention window of the target anion; and, ionic character displacement, where retention
times may significantly shift due to the influence of high ionic strength matrices (high
mineral content or hardness) overloading the exchange sites in the column and
significantly shortening target analyte's retention times.
4.2.1 A direct chromatographic coelution may be solved by changing columns, eluent
strength, modifying the eluent with organic solvents (if compatible with 1C
columns), changing the detection systems, or selective removal of the
interference with pretreatment. Sample dilution will have little to no effect. The
analyst MUST verify that these changes do not induce any negative affects on
method performance by repeating and passing all the QC criteria as described in
Section 9.
4.2.2 Sample dilution may resolve some of the difficulties if the interference is the
result of either concentration dependant coelution or ionic character
displacement, but it must be clarified that sample dilution will alter your
Minimum Reporting Limit (MRL) by a proportion equivalent to that of the
dilution. Therefore, careful consideration of project objectives should be given
prior to performing such a dilution. An alternative to sample dilution, may be
dilution of the eluent as outlined in Section 11.2.6.
4.2.3 Pretreatment cartridges can be effective as a means to eliminate certain matrix
interferences. With any proposed pretreatment, the analyst must verify that the
target analyte is not affected by monitoring recovery after pretreatment
(additional pretreated LFM requirement see Section 11.1.4.6) and that no
background contaminants are introduced by the pretreatment (additional
pretreated LRB requirement see Sections 9.3.1.1 and 11.1.4.2). With advances
in analytical separator column technology which employ higher capacity anion
exchange resins, the need for these cartridges has been greatly reduced.
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4.2.3.1 Extreme caution should be exercised in using these pretreatment
cartridges. Artifacts are known to leach from certain cartridges which
can foul the guard and analytical columns causing loss of column
capacity indicated by shortened retention times and irreproducible
results. Frequently compare your calibration standard chromatograms
to those of the column test chromatogram (received when the column
was purchased) or use calibration chromatograms generated when the
column was initially installed, to insure proper separation and similar
response ratios between the target analytes are observed.
4.2.3.2 If LRB background problems are encountered in the retention time
window for perchlorate when these pretreatment cartridges have been
employed, increase the initial reagent water rinse of the cartridge to
approximately five tunes the .volume specified by the manufacturer.
4.3 Sample matrices with high concentrations of common anions such as chloride, sulfate
and carbonate can make the analysis problematic by destabilizing the baseline in the
retention time window for perchlorate. This is evidenced by observing a protracted
tailing following the initial elution of the more weakly retained anions (chloride,
carbonate, and sulfate) which extends into the perchlorate retention time window.
These common anion levels can be indirectly assessed by monitoring the conductivity
of the matrix. Consequently, all sample matrices must be monitored for conductivity
(Section 11.1.2) prior to analysis. When the laboratory determined Matrix Conductivity
Threshold (MCT, see Section 9.2.8) is exceeded, procedures incorporating sample
dilution and/or pretreatment must be performed as specified in Sections 11.1.3 and
11.1.4, respectively.
4.4 All reagent solutions (eluents, external water for ASRS suppressor, etc...) used by the
instrument must be filtered through no larger than a 0.45 um nominal pore size
membrane or frit to remove particulates and prevent damage to the instrument, columns
and flow systems. Sample filtration must also be employed on every sample prior to
analysis. This applies not only to field samples but also to the laboratory reagent blank
(LRB) and laboratory fortified blank (LFB). The LRB and LFB samples function as
controls and must be filtered to confirm no bias is attributable to the filtration.5 Filter
the samples through a membrane or frit with no larger than a 0.45 um nominal pore
size. Syringe mounted, cartridge type, filters work well. Filters specifically designed
for 1C applications should be used.
4.5 Close attention should be given to the potential for carry over peaks from one analysis
which will effect the proper detection of perchlorate in a second, subsequent analysis.
It is the responsibility of the user to confirm that no late eluting peaks have carried over
into a subsequent analysis thereby compromising the integrity of the analytical results.
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5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method have not been fully
established. Each chemical should be regarded as a potential health hazard and
exposure should be as low as reasonably achievable. Cautions are specifically listed
below in Section 5.3 for hazardous materials.
5.2 Each laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A
reference file of Material Safety Data Sheets (MSDS) should be made available to all
personnel involved in the chemical analysis. The preparation of a formal safety plan is
also advisable. Additional references on laboratory safety are available.9"12
5.3 The following chemicals have the potential to be highly toxic or hazardous, consult
MSDS. ; -
5.3.1 Sodium Hydroxide (NaOH), used in the preparation of the elueht is considered
caustic.
6. EQUIPMENT AND SUPPLIES
6.1 Ion chromatograph (1C) — Analytical system complete with eluent reservoirs, an ion
chromatographic pump, injection valves, both guard and analytical separator columns,
suppressor, conductivity detector, and computer based data acquisition system.
6.1.1 Anion guard column -- Dionex AG16 4 mm (P/N 55377), or equivalent. This
column functions as a protector of the separator column. If omitted from the
system, the retention times will be shorter.
6.1.2 Anion separator column -- Dionex AS16, 4 mm (P/N 55376), or equivalent (see
Sections 6.1.2.1-6.1.2.2). The AS 16, 4 mm column using the conditions
outlined in Table 1 produced the separations shown in Figures 1 through 4.
6.1.2.1 The development of this method included investigations into the
performance of alternate 4 mm 1C guard and analytical separator
columns which have been used for the 1C analysis of perchlorate and
are specified in procedures external to the U.S.EPA.1"5 These alternate
guard /separator columns included the Dionex AG5 / AS 5 and the
Dionex AG11 / AS 11. The AG5 / ASS is currently specified in the
standard operating procedure (SOP) for the 1C analysis of perchlorate
by the State of California, Department of Health Services.1'5 The
AG11 / AS 11 is used by several commercial labs conducting 1C
analysis for perchlorate and is recognized by California as an
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acceptable alternate to the AG5 / AS5. 2'4 A multilab validation study
included both of these analytical columns and indicated comparable
results could be attained.6 In U.S.EPA studies, both the AG5 / ASS
and the AG1 1 / ASH performed well for reagent water and simulated
drinking water samples with low to moderate common anion levels but
as these levels increased, performance began to diminish for both
columns. The AG16 / AS16 columns could tolerate much higher
levels of these common anions and therefore it is recommended in this
method as the column of choice. A summary of the results of
examining these three columns for simulated matrices with various
common anion levels is presented in Table 4.
6. 1 .2.2 Any alternate, equivalent column must be characterized as hydrophilic
or conversely, must be rated as having low to very low
hydrophobicity.4 This is one characteristic that is consistent for the
ASS, ASH and AS 16 analytical separator columns. This requirement
for low hydrophobicity is to allow the efficient, reproducible and
symmetrical band elution of polarizable anions, such as perchlorate.
If the perchlorate analysis is attempted on a hydrophobic column, such
as those typically used for the analysis of common anions,13 poor
performance will result due to very asymmetric, tailing peaks. Using a
middle to high calibration standard, conduct a typical analysis. Any
alternate column must be capable of yielding symmetrical peak elution
for this perchlorate response as demonstrated by yielding a Peak
Gaussian Factor of between 0.80 and 1.15 using the following
equation,
1.83 x WC/2)
PGF = -----------------------
W (V10)
where,
WO/a) is the peak width at half height, and
W (V10) is the peak width at tenth height.
NOTE: Values for WO/a) and W (V10) can be attained through most
data acquisition software.
6. 1 .3 Anion suppressor device — The data presented in this method were generated
using a Dionex Anion Self Regenerating Suppressor (4 mm ASRS, ULTRA,
P/N 53946). An equivalent suppressor device may be utilized provided
comparable conductivity detection limits are achieved and adequate baseline
stability is attained as measured by a combined baseline drift/noise of no more
than 5 nS per minute over the background conductivity. Proper suppressor
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performance is essential to analytical data reproducibility and sensitivity of the
conductivity detector.
6.1.3.1 The ASRS was set to perform electrolytic suppression at a current
setting of 300 mA using the external water mode. External water was
delivered to the suppressor directly from a pressurized source at a flow
rateof5mL/min
6.1.3.2 If pretreated samples (Section 11.1.4), or sample matrices which
contain appreciable concentrations.^!^ansitioiynetal cations (e.g., Fe
or Al) are frequently analyzed, cationic components may bind to the
suppressor membrane and over time effect suppressor performance. If
the instrument begins to have problems with reduced peak response or
asymmetrical perchlorate peaks, the suppressor membranes should be
cleaned. As a quick and easy cleaning step, the manufacturer's ASRS
"Quickstart" procedure for installing a new ASRS should be
followed.14 If this procedure does not correct the problem, follow the
manufacturer's recommended cleaning procedure for removing metal
contaminants.15
6.1.4 Detector — Conductivity cell (Dionex CD20, or equivalent) capable of providing
data as required in Section 9.2.
6.2 Data Acquisition System — The Dionex Peaknet Data Chromatography Software was
used to generate all the data in Tables 1 through 4. Other computer based data systems
may achieve approximately the same performance but the user should demonstrate this
by the procedures outlined in Section 9.
6.3 Conductivity Meter - Used to monitor sample matrix conductance which is directly
related to the common anion levels in a matrix and used to determine if sample
pretreatment is required. At a minimum, this meter should be capable of measuring
matrix conductance over a range of 1 -10,000 uS/cm.
6.4 Analytical balance — Used to accurately weigh target analyte salt for stock standard
preparation (±0.1 mg sensitivity).
6.5 Top loading balance — Used to accurately weigh reagents such as sodium hydroxide
solution in the preparation of eluents (±10 mg sensitivity).
6.6 Weigh boats — Plastic, disposable - for weighing eluent reagents.
6.7 Micro beakers ~ Plastic, disposable - used during sample preparation.
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6.8 Syringes — Plastic, disposable, 10 mL - used during sample preparation.
6.9 Pipets — Pasteur, plastic or glass, disposable, graduated, 5 mL and 10 mL.
6.10 Bottles — High density polyethylene (HDPE) or glass, amber or clear, 30 mL, 125 mL,
250 mL. For sampling and storage of calibration solutions. Stability studies presented
by the Interagency Perchlorate Steering Committee for Analytical Methods 6 and
confirmed at the EPA (see TableS A), indicate perchlorate is neither photoreactive nor
prone to adsorption to the walls of either HDPE plastic or glass bottles.
6.11 Particulate filters — 0.45 micron syringe filters, specifically designed for 1C applications
(Gelman 1C Acrodisc, PN 4485, or equivalent). These cartridges are used to remove
particulates from the sample matrix while loading the sample manually or if the
autosampler employed does not filter the sample during loading.
6.12 Matrix pretreatment cartridges in the barium form — (Dionex OnGuard-Ba cartridges,
PN 046072, or equivalent.) These cartridges are conditioned according to the
manufacturer's directions and are used to reduce the matrix levels of sulfate.
6.13 Matrix pretreatment cartridges in the silver form - (Dionex OnGuard-Ag cartridges
PN 039637, or equivalent.) These cartridges are conditioned according to the
manufacturer's directions and are used to reduce the matrix levels of chloride.
6.14 Matrix pretreatment cartridges in the hydrogen form— Dionex OnGuard-H cartridges
(PN 039596) or equivalent. These cartridges are conditioned according to the
manufacturer's directions and are used to reduce cations in the sample matrix. This
protects the analytical column by removing silver which has leached from the Ag
cartridge and may indirectly minimize the effect of carbonate by removing the cationic
counter ion.
7. REAGENTS AND STANDARDS
7.1 Reagent water -- Distilled or deionized water 17.8 Mohm or better, free of the anions of
interest. Water should contain particles no larger than 0.20 microns.
7.2 Eluent solution - 50 mM sodium hydroxide (NaOH, [CASRN 1310-73-2]), dissolve
8.0 grams of 50% (WAV) sodium hydroxide in reagent water to a final volume of 2.0 L.
NOTE: This eluent solution is specific to the columns listed in Table 1. Any alternate
columns will likely have unique and specific conditions identified by the manufacturer.
7.2.1 Solutions of NaOH are very susceptible to carbonate contamination resulting
from adsorption of carbon dioxide from the atmosphere. This contamination
will result in poor reproducibility of perchlorate retention times, elevated
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instrument background conductivity, and increased baseline noise/drift.
Consequently, exposure to the atmosphere should be minimized by storing these
eluent solutions in sealed reservoirs under low pressure (3 to 5 psi) helium. In
addition, these solutions should be regularly prepared and held for no more than
5 days. When refilling the eluent reservoir, completely replace old eluent
solution by emptying the old eluent, rinsing the reservoir with reagent water,
and refilling with the freshly prepared eluent solution. With this eluent, the
suppressed conductivity detector background signal should be between 2 - 5 uS.
7.2.2 This eluent solution must be purged for 10 minutes with helium prior to use.
This effectively removes dissolved gases which may form micro bubbles in the
1C, compromising system performance and adversely effecting the integrity of
the data. Alternatively, an in-line degas apparatus maybe employed.
7.2.3 A system or apparatus which automatically generates the hydroxide eluent
(Dionex EG40, or equivalent) is an acceptable alternative to physically
preparing this hydroxide eluent.
7.3 Perchlorate stock standard solution, 1000 mg/L (1 mg/mL) - A stock standard solution
may be purchased as a certified solution or prepared from ACS reagent grade, sodium
salt as listed below. (NOTE: Sodium perchlorate represents a molar weight fraction of
81.2 % perchlorate anion)
7.3.1 Perchlorate (C1O40 1000 mg/L - Dissolve 0.1231 g sodium perchlorate
(NaClO4, CASRN [7601-89-0] hi reagent water and dilute to 100 mL in a
volumetric flask.
NOTE: Stability of standards — Perchlorate stock standards, stored at room
temperature, appear to be very stable and may be stable for an extended period
of time. However, specified expiration dates should be marked on each
prepared stock standard as part of any laboratory's quality control program, hi
this regard, it is recommended that stock standards for perchlorate be held for
no more than 12 months and an expiration date should be clearly specified on
the label.
7.4 Mixed Common Anion Stock Solution - containing the anions chloride, sulfate and
carbonate each at 25 mg/mL anion concentration. This solution is used to prepare
simulated common anion samples in the determination of the MCT (Section 9.2.8).
7.4.1 Dissolve the following salts in reagent water to a final volume of 25.0 mL:
1.0 g sodium chloride (NaCl, CASRN [7647-14-5]) = 0.61 g Cl'
0.93 g sodium sulfate (NajSO* CASRN [7757-82-6]) = 0.63 g SO4=
1.1 g sodium carbonate (NajCOj, CASRN [497-19-8]) = 0.62 g CO3=
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7.5 Conductivity Meter Calibration Solution
7.5.1 Potassium Chloride (KC1), 745 mg/L (total salt weight) - Dissolve 0.745 g
potassium chloride (KC1, [CASRN 7447-40-7]) in reagent water and dilute to a
final volume of 1.00 L in a volumetric flask. On a properly functioning and
calibrated conductivity meter, the reference conductance for this solution is
1410uS/cmat25°C.16
8. SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Samples may be collected in plastic or glass bottles. All bottles must be thoroughly
cleaned and rinsed with reagent water. The volume collected should be sufficient to
insure a representative sample, allow for replicate analysis and laboratory fortified
matrix analysis, if required, and minimize waste disposal.
8.2 Samples do not need to be shipped iced or stored cold in a refrigerator but every effort
should be taken to protect the samples from temperature extremes. A thermally
insulated sampling kit, designed to fit sampling bottles securely during shipment,
should be used to protect the samples from these temperature extremes.
8.3 Sample preservation and holding times for the anions are as follows:
Analvte Preservation Holding Time
Perchlorate None required 28 days
NOTE: Perchlorate has been shown to be stable for more than 28 days6 but extended
holding time studies (beyond 35 days) were not conducted by EPA.
Typically, when analytes are believed to be stable, a 28 day holding time is
established as a sufficient time period to permit a laboratory to conduct the
analysis.
9. QUALITY CONTROL
9.1 Each laboratory using this method is required to operate a formal quality control (QC)
program. The requirements of this program consist of an initial demonstration of
laboratory capability, and subsequent analysis in each analysis batch (Section 3.1) of an
Instrument Performance Check Standard (IPC), Laboratory Reagent Blank (LRB),
Initial Calibration Check Standard (ICCS), Laboratory Fortified Blank (LFB),
Continuing and End Calibration Check Standards (CCCS/ECCS), Laboratory Fortified
Sample Matrix (LFM) and either a Field, Laboratory or LFM duplicate sample analysis.
This section details the specific requirements for each of these QC parameters. The QC
criteria discussed in the following sections are summarized in Section 17, Table 5
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and 6. The laboratory is required to maintain performance records that define the
quality of the data that are generated.
9.2 INITIAL DEMONSTRATION OF CAPABILITY
9.2.1 The Initial Demonstration of Capability (IDC) — This is used to characterize
instrument and laboratory performance prior to performing analyses by this
method. The QC requirements for the IDC discussed in the following section
are summarized in Section 17, Table 5.
9.2.2 Initial demonstration of low system background -- See Section 9.3.1.
9.2.3 Initial Demonstration of Accuracy (IDA) — Prepare and analyze 7 replicate
LFBs fortified at 25.0 ug/L. Calculate the mean measured concentration (Cx) of
the replicate values as follows.
n
where,
Cx = Mean recovered concentration of the replicate analysis.
CltC2 •••Cn= Reco vered concentrations of the replicate 1,2...n.
n =7
To pass the IDA, the value derived for Cx must be within ± 10% of the true
value or between 22.5 ug/L and 27.5 ug/L.
9.2.4 Initial Demonstration of Precision (TOP) — Using the data generated for Section
9.2.3, calculate the percent relative standard deviation (%RSD) of the replicate
analysis, as indicated below. To pass the IDP, the %RSD must be less than
10%.
%RSD = —-"-— x 100
where,
Sn_! = sample standard deviation (n-1) of the replicate analyses.
Cx = mean recovered concentration of the replicate analysis.
9.2.5 Quality Control Sample (QCS) - After calibration curves have initially been
established or have been re-established, or as required to meet, data quality
needs, verify both the calibration and acceptable instrument performance with
the preparation and analyses of an external/second source QCS. If the
determined concentrations are not within ± 10% of the stated values,
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performance of the determinative step of the method is unacceptable. The
source of the problem must be identified and corrected before either proceeding
with the IDC or continuing with on-going analyses.
9.2.6 Method Detection Limit (MDL) - An MDL must be established using reagent
water (blank) fortified at a concentration of three to five times the estimated
instrument detection limit.7'8 To determine MDL values, take seven replicate
aliquots of the fortified reagent water and process through the entire analytical
method over a three day period. These seven MDL replicate analyses may be
performed gradually over three days or may represent data that has been
collected, at a consistent MDL estimated concentration, over a series of more
than three days. Perform all calculations defined in the method and report the
concentration values in the appropriate units. Calculate the MDL as follows:
MDL = (t)x(Sn.1)
where,
t = student's t value for a 99% confidence level and a standard deviation
estimate with n-1 degrees of freedom [t = 3.14 for seven replicates]
Sn.! = sample standard deviation (n-1) of the seven replicate analyses.
9.2.6.1 MDLs should be periodically verified, but MUST be initially
determined when a new operator begins work or whenever there is a
significant change in the background, or instrument response.
NOTE: Do not subtract blank values when performing MDL calculations.
9.2.7 Minimum Reporting Level (MRL) - The MRL is the threshold concentration of
an analyte that a laboratory can expect to accurately quantitate in an unknown
sample. The MRL should be established at an analyte concentration either
greater than three times the MDL or at a concentration which would yield a
response greater than a signal to noise ratio of five. Setting the MRL too low
may cause repeated QC failure upon analysis of the ICCS. Although the
lowest calibration standard may be below the MRL, the MRL must never
be established at a concentration lower than the lowest calibration
standard.
9.2.8 Matrix Conductivity Threshold (MCT) - The MCT is an individual laboratory
defined value which must be determined by preparing a series of sequentially
increasing, common anion fortified, reagent water samples each contain a
constant perchlorate concentration. Initially, a reagent water prepared LFB,
containing no common anions, must be analyzed which contains perchlorate at a
suggested concentration of 25 ug/L perchlorate. Next, the series of sequentially
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increasing anionic solutions are prepared, each containing perchlorate at a
suggested concentration of 25 ug/L, which also containing the individual
common anions of chloride, sulfate and carbonate, all included at uniform
increasing concentrations of 200, 300, 400, 500, 600, 800, and 1000 mg/L for
each anion. A concentration of 25 ug/L perchlorate has been suggested
assuming the MRL has been set in the range of 3.0 ug/L to 5.0 ug/L. If a
laboratory's MRL is higher, choose a perchlorate concentration for this exercise
at approximately 5 times that MRL.
9.2.8.1 Prepare the mixed common anion stock solution (see Section 7.4)
containing chloride, sulfate and carbonate, each at 25 mg/mL.
9.2.8.2 Prepare a perchlorate secondary stock dilution standard at 1.00 mg/L
from the 1000 mg/L perchlorate stock standard (Section 7.3) by
diluting 0.50 mL of the stock solution to a final volume of 500 mL.
9.2.8.3 Prepare the LFB at suggested perchlorate concentration of 25 ug/L by
diluting 0.625 mL of the perchlorate secondary stock dilution standard
(Section 9.2.8.2) to a final volume of 25.0 mL.
9.2.8.4 Next, prepare the series of common anion fortified reagent water
samples by adding 0.20 mL, 0.30 mL, 0.40 mL, 0.50 mL, 0.60 mL,
0.80 mL, and 1.00 mL of the mixed common anion stock solution
(Section 7.4) into separate 25 mL volumetric flasks. Next, add 0.625
mL of the perchlorate secondary stock dilution standard (Section
9.2.8.2) to each 25 mL volumetric flask and dilute to volume with
reagent water to yield a final perchlorate concentration of 25.0 ug/L.
9.2.8.5 Measure and record the conductance of each of these prepared
solutions on a calibrated conductivity meter (This meter must be
calibrated as described in Section 10.4 prior to measuring
conductance). To use as a relative reference conductance, the 400
mg/L mixed anion sample, which contains chloride at 400 mg/L,
sulfate at 400 mg/L and carbonate at 400 mg/L, should display a
conductance of between 3200 uS/cm and 3700 uS/cm.
9.2.8.6 Analyze each solution, recording the peak area to height (A/H) ratio
and the quantified concentration of perchlorate. In many data
acquisition and instrument control software, the peak area to height
ratio is a definable parameter which can be specified for printout on
the analysis report.
9.2.8.7 Both the A/H ratio and quantified perchlorate concentration for the
LFB and the 200 mg/L mixed common anion solution should be
reproducibly consistent but as the common anion levels increase, the
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A/H ratio will also begin to increase as the peak height is distorted and
reduced. As the peak is distorted, the area will also eventually begin to
be distorted and the quantitated concentration will be reduced, but this
is typically secondary, with the ratio of peak area to height initially
predicting this pending quantitation problem.
9.2.8.8 Calculate the A/H ratio percent difference (PDA/u) between the average
A/H ratio for the LFB (A/HLFB) and the average A/H ratios for each
mixed common anion solutions (A/H^) using the following equation.
X 100
9.2.8.9 As the conductivity of the matrices increase, the PD^ will increase.
The MCT is the matrix conductance where the PD^ exceeds 20%.
To derive the MCT, perform a linear regression on these data by
plotting PDjvu (as the independent variable, x) versus the matrix
conductance (as the dependent variable, y). The resulting regression
data should yield an lvalue of > 0.95. (See Figure 5) Record the
"constant" (intercept value) and the "X-coefficient" (slope) and
calculate the MCT as follows,
MCT = (20%) x (X-coefficient) + (constant)
NOTE: Be careful to consistently apply percentages as either whole
numbers or as fractional values (20% = 0.20) for both the regression
analysis and the MCT calculation.
9.2.8.10 As an alternate to the regression analysis, the laboratory can choose to
establish their MCT at the conductance level of the highest mixed
anion solution which yielded a PD^ value below the 20 % threshold.
9.2.8.11 As a final procedure, the laboratory should confirm their perchlorate
MRL in a mixed common anion solution which reflects a conductance
near (within +/- 10%) that specified as the MCT. This solution must
contain perchlorate, at the laboratory determined MRL, as well as the
common anions chloride, sulfate and carbonate, prepared consistent
with the instruction for the mixed anion solutions in this section and at
a concentration estimated to generate a conductance near the MCT.
The conductance of this solution must be measured at within ±10% of
the MCT and following the analysis, the recovered perchlorate must be
between 70 -130% of the MRL concentration. If the MRL recovery
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fails this criteria, the MCT should be lowered by 10% and this MRL
verification must be repeated.
9.2.8.12 Prior to conducting any field sample analysis, the conductivity of that
matrix must be determined. When the conductance of a field sample is
above the MCT, sample dilution or pretreatment, as described in
respective Sections 11.1.3 and 11.1.4 must be performed.
9.3 ASSESSING LABORATORY PERFORMANCE - The following items must be
included in every analysis batch (Section 3.1).
9.3.1 Laboratory Reagent Blank (LRB) - An LRB must be prepared and treated
exactly as a typical field sample including exposure to all glassware, equipment,
solvents, filtration and reagents that are used with field samples. Data produced
are used to assess instrument performance of a blank sample and evaluate
contamination from the laboratory environment. Values that exceed 1A the MRL
indicate a laboratory or reagent contamination is present. The source of the
contamination must be determined prior to conducting any sample analysis.
Any sample included in an automated analysis batch which has an invalid LRB,
indicated by a quantitated perchlorate that exceeds 1A the MRL, must be
reanalyzed in a subsequent analysis batch after the contamination problem is
resolved.
9.3.1.1 When sample matrices have been pretreated to reduce the risk of high
common anion interference (Section 11.1.4), a second LRB must be
prepared, pretreated in exactly the same manner, and analyzed to
confirm no background effects from the pretreatment process are
present. If an analysis batch only contains pretreated samples, then
only a pretreated LRB is required.
9.3.2 Instrument Performance Check (IPC) - The MCT, which was determined as
part of the IDC in Section 9.2.8, must be verified through the analysis of an IPC.
The IPC is three tiered and is used to verify the state of the 1C system, over time,
to quantitate perchlorate in highly ionic matrices. This must be conducted with
each analysis batch since over time, column performance can change.
9.3.2.1 Prepare a mixed common anion solution which reflects a conductance
near (within+/-10%) that specified as the MCT. This solution must
be prepared consistent with the instruction in Section 9.2.8, and
containing the common anions chloride, sulfate and carbonate as well
as perchlorate at a suggested concentration of 25 ug/L. This
perchlorate concentration has been specified assuming the MRL has
been set in the range of 3.0 ug/L to 5.0 ug/L. If a laboratory's MRL is
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higher, chose a perchlorate concentration for this exercise at
approximately 5 times that MRL.
9.3.2.2 Confirm the conductance of the IPC and analyze it as the initial sample
hi the analysis batch. If, after several weeks of storage, the measured
conductance of this solution has shifted by more than 10% from the
original measured value, prepare a fresh IPC solution. Following the
analysis, calculate the PD^ (Section 9.2.8.8), by comparing the peak
area to height ratio of this IPC mixed anion standard (A/E^) for this
analysis batch to the value that was derived for the LFB (A/Hug) either
in the original IDC or in the previous analysis batch. As the first tier
criteria, the value for PD^ must be less than 25% before proceeding
with the analysis batch.
9.3.2.3 At the second tier criteria, the measured recovery for perchlorate in this
IPC must fall between 80% and 120 % (20.0 ug/L to 30.0 ug/L for a 25
ug/L fortification).
9.3.2.4 As a third tier and final criteria for the IPC, the laboratory must closely
monitor the perchlorate retention time for this analysis. Small
variations hi retention time can be anticipated when a new solution of
eluent is prepared but if sudden shifts of more than 5% are observed in
the perchlorate retention time, some type of instrument problem may
be present. Potential problems include improperly prepared eluent,
erroneous method parameters programmed such as flow rate or some
other system problem. The observed retention time for perchlorate
should closely replicate the times established when the column was
originally installed. As a column ages, it is normal to see a gradual
shift and shortening of retention times, but if after several years of use,
extensive use over less than a year, or use with harsh samples, this
retention time has noticeably shifted to any less than 80% of the
original recorded value, the column requires cleaning (according to
manufacturer's instructions) or replacement. A laboratory should
retain a historic record of retention times for perchlorate to provide
evidence of an analytical column's continued performance.
9.3.2.5 If any of the conditions defined in Section 9.3.2.2 through 9.3.2.4 are
not met, the MCT must be repeated and revised to a more appropriate
lower matrix conductivity threshold or the source of the problem must
be determined and the IPC reanalyzed.
9.3.3 Laboratory Fortified Blank (LFB) - Prepare a secondary dilution stock using the
same stock solution used to prepare the calibration standards. This separate,
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secondary dilution stock is used as a concentrate to fortify the LFB and the
LFMs (Section 9.4.1). An external source stock or QCS, which is used to verify
the accuracy of the calibration curve when it was initially prepared (Section
10.2.5), should not be used to prepare this secondary dilution stock.
Laboratories are required to analyze a LFB (filtered as if it were a field sample)
with each analysis batch immediately following the ICCS. The LFB must be
prepared with the same solution used to prepare the LFM and should be
prepared at concentrations no greater than ten times the highest concentration
observed in any field sample and should be varied to reflect the range of
concentrations observed in field samples. By analyzing the LFB initially, a
control check is performed on the concentrated solution used to prepare the
LFM. If any deviations in the perchlorate concentration are present, it will be
reflected in the LFB and not exclusively attributed to a matrix upon analysis of
the LFM. Calculate accuracy as percent recovery (Section 9.4.1.3). The
recovery for perchlorate must fall in the range of 85 -115% prior to analyzing
samples. If the LFB recovery for an analysis batch does not meet these recovery
criteria the data are considered invalid, and the source of the problem should be
identified and resolved before continuing analyses.
9.3.3.1 When sample matrices have been pretreated to reduce the risk of high
common anion interference (Section 11.1.4), a second LFB must be
prepared, pretreated in exactly the same manner, and analyzed to
confirm no background effects or recovery bias induced by the
pretreatment are present. If an analysis batch only contains pretreated
samples, then only a pretreated LFB is required.
9.4 ASSESSING ANALYTE RECOVERY AND DATA QUALITY - The following must
be included in every analysis batch (Section 3.1).
9.4.1 Laboratory Fortified Sample Matrix (LFM) - The laboratory must add a known
amount of each target analyte to a minimum of 5% of the collected field samples
or at least one with every analysis batch, whichever is greater. Samples which
exceed the MCT must either be diluted (Section 11.1.3) or pretreated to reduce
the common anion levels (Section 11.1.3). Samples which are pretreated have
additional LFM requirements described in Section 11.1.4.6, and must be
fortified before pretreatment. For a LFM to be valid, the target analyte
concentrations must be greater than the native level and should adhere to the
requirement outlined in Section 9.4.1.2. It is recommended that the solutions
used to fortify the LFM be prepared from the same stocks used to prepare the
calibration standards and not from external source stocks. This will remove the
bias contributed by an externally prepared stock and focus on any potential bias
introduced by the field sample matrix.
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9.4.1.1 The fortified concentration must be equal to or greater than the native
sample concentration. Fortified samples that exceed the calibration
range must be diluted to be within the linear range, hi the event that
the fortified level is less than the observed native level of the
unfortified matrix, the recovery should not be calculated. This is due
to the difficulty in calculating accurate recoveries of the fortified
concentration when the native sample concentration to fortified
concentration ratio is greater than one.
9.4.1.2 For normal drinking waters, the LFM typically should be prepared in
the range of 20 - 50 ug/L. The LFM should not be prepared at
concentration greater than ten times the highest concentration observed
in any field sample and should be varied to reflect the range of
concentrations expected in field samples.
9.4.1.3 Calculate the percent recovery for each target analyte, corrected for
concentrations measured in the unfortified sample. Percent recovery
should be calculated using the following equation:
(Cs - C)
%REC = x 100
s
where,
%REC = percent recovery,
Cs = measured perchlorate in the fortified sample,
C = measured native perchlorate sample concentration, and
s = concentration equivalent of analyte added to sample.
9.4.1.4 Recoveries may exhibit a matrix dependence. If the recovery for
perchlorate falls outside 80 -120%, and the laboratory's performance
for all other QC performance criteria is acceptable, 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 and the LFM must be labeled suspect/matrix to inform the data
user that the result is suspect due to matrix effects. Repeated failure to
meet suggested recovery criteria indicates potential problems with the
procedure and should be investigated.
9.4.2 FIELD, LABORATORY DUPLICATES OR DUPLICATE LFM - The
laboratory must analyze either a field duplicate, a laboratory duplicate, or a
duplicate LFM for a minimum of 5% of the collected field samples or at least
one with every analysis batch, whichever is greater. The sample matrix selected
for this duplicate analysis must contain measurable concentrations of the target
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anions in order to establish the precision of the analysis set and ensure the
quality of the data. Without prior knowledge or strong suspicion that an
unknown sample has measurable perchlorate concentrations, the best alternative
is to analyze a duplicate LFM.
9.4.2.1 Calculate the relative percent difference (RPD) of the initial
quantitated concentration (Ic) and duplicate quantitated concentration
(Dc) using the following formula.
I0c-Dc)|
RPD = X100
(Dc + DJ/2)
9.4.2.2 Duplicate analysis may exhibit a matrix dependance. If the RPD for
the duplicate measurements of perchlorate falls outside ±15% and if
all other QC performance criteria are met, laboratory precision is out
of control for the sample and perhaps the analytical batch. The result
for the sample and duplicate should be labeled as suspect/matrix to
inform the data user that the result is suspect due to a potential matrix
effect, which led to poor precision. This should not be a chronic
problem and if it frequently recurs (>20% of duplicate analyses), it
indicates a problem with the instrument or individual technique that
must be corrected.
9.4.3 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options, such as the use of different columns (which meet the
criteria in Section 6.1.2.2), injection volumes, and/or eluents, to improve the
separations or lower the cost of measurements. Each time such modifications to
the method are made, the analyst is required to repeat the procedure in Section
9.2 and adhere to the condition of conductivity baseline stability found in
Section 1.2.1.
9.4.4 It is recommended that the laboratory adopt additional quality assurance
practices for use with this method. The specific practices that are most
• productive .depend upon the needs of the laboratory and the nature of the
samples. Whenever possible, the laboratory should perform analysis of quality
control check samples and participate in relevant proficiency testing (PT) or
performance evaluation (PE) sample studies.
10. CALIBRATION AND STANDARDIZATION
10.1 Demonstration and documentation of acceptable initial calibration is required prior to
the IDC and before any samples are analyzed, and is required intermittently throughout
sample analysis to meet required QC performance criteria outlined in this method and
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summarized in Table 6. Initial calibration verification is performed using a QCS as
well as with each analysis batch using an initial, continuing (when more than 10 field
samples are analyzed), and end calibration check standards. The procedures for
establishing the initial calibration curve are described in Section 10.2. The procedures
to verify the calibration with each analysis batch is described in Section 10.3.
10.2 INITIAL CALIBRATION CURVE
10.2.1 Establish ion chromatographic operating parameters equivalent to those
indicated in Table 1.
10.2.2 Estimate the Linear Calibration Range (LCR) - The LCR should cover the
expected concentration range of the field samples and should not extend over
more than two orders of magnitude in concentration. The restriction of two
orders of magnitude is prescribed since beyond this it is difficult to maintain
linearity throughout the entire calibration range.
10.2.2.1 If quantification is desired over a larger range, then two separate
calibration curves should be prepared.
10.2.2.2 A minimum of three calibration standards are required for a curve that
extends over a single order of magnitude and a minimum of five
calibration standards are required if the curve covers two orders of
magnitude.
10.2.2.3 Since the anticipated concentration range for perchlorate in actual field
samples is expected to cover two orders of magnitude, the use of at
least five calibration standards in the range 4 - 400 jig/L is
recommended.
10.2.3 Prepare the calibration standards by carefully adding measured volumes of the
stock standard (Section 7.3).to a volumetric flask and diluting to volume with
reagent water.
10.2.4 Inject 1.0 mL of each calibration standard. Tabulate peak area responses against
the perchlorate concentration. The results are used to prepare a calibration
curve. Acceptable calibration is confirmed after reviewing the curve for
linearity (second order fits are also acceptable) and passing the criteria for the
initial calibration check standard in Section 10.3.1. Alternately, if the ratio of
area to concentration (response factor) is constant over the LCR (indicated by <
15% 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.
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10.2.4.1 Peak areas must be used as a measure of response since they have been
found to be more consistent, in terms of quantitation, than peak
heights. Peak height can tend to be suppressed as a result of high
levels of common anions in a given matrix which can compete for
exchange sites leading to peak broadening. Using peak areas, it is the
analyst's responsibility to review all chromatograms to insure accurate
baseline integration of target analyte peaks, since poorly drawn
baselines will significantly influence peak areas.
10.2.5 After establishing or reestablishing calibration curves, the accuracy of this
calibration must be verified through the analysis of a QCS or externally
prepared second source. The QCS should be prepared at a concentration near
the middle of the calibration curve. As specified in Section 9.2.5, determined
concentrations must fall within ± 10% of the stated values.
10.3 CONTINUING CALIBRATION VERIFICATION - Initial calibrations may be stable
for extended periods of time. Once the calibration curve has been established it MUST
be verified for each analysis batch, prior to conducting any field sample analysis using
an Initial Calibration Check Standard. Continuing Calibration Check Standards and
End Calibration Check Standards are also required as described in the sections below.
10.3.1 INITIAL CALIBRATION CHECK STANDARD (ICCS) - For each analysis
batch the calibration must initially be verified prior to analyzing any samples.
The lowest level standard used to prepare the linear calibration curve must be
used, hi cases where the analyst has chosen to set the MRL above the lowest
standard, a standard at a concentration equal to the MRL is acceptable. Percent
recovery for the ICCS must be in the range or 75 - 125% before continuing the
analysis batch and conducting any sample analyses.
10.3.2 CONTINUING CALIBRATION CHECK/END CALIBRATION CHECK
STANDARDS (CCCS/ECCS) - Continuing calibration check standards MUST
be analyzed after every tenth field sample analysis and at the end of the analysis
batch as an end calibration check standard. If more than 10 field samples are
included in an analysis batch, the analyst must alternate between the middle and
high continuing calibration check standard levels.
10.3.2.1 The percent recovery for perchlorate in the CCCS/ECCS must be
between 85-115%.
10,3.2.2 If during the analysis batch, the measured concentration for perchlorate
in the CCCS or ECCS differs by more than the calibration verification
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criteria shown above, or if the perchlorate peak retention time shifts
outside the retention time window (as defined in Section 11.2.4), all
samples analyzed after the last acceptable check standard are
considered invalid and must be reanalyzed. The source of the problem
must be identified and resolved before reanalyzing the samples or
continuing analyses. .
10.3.2.3 In the case where the end calibration fails to meet performance criteria,
but the initial and middle calibration checks are acceptable, the
samples bracketed by the acceptable calibrations may be reported.
However, all field samples between the middle and end calibration
checks MUST be reanalyzed.
10.4 CONDUCTIVITY METER CALIBRATION - Prior to conducting the MCT and
coinciding with each analysis batch, conductivity meter calibration must be verified or
established using a standard KC1 solution (Section 7.5).
10.4.1 Thoroughly rinse the conductivity electrode with reagent water. Place the
electrode in the reagent water, turn on the meter and confirm the conductance of
this blank is < 1 uS/cm.
10.4.2 Pour approximately 15 mL of the standard KC1 solution (Section 7.5) into a
plastic disposable micro beaker (Section 6.7) and place the electrode into the
solution. The reference conductance for this solution is 1410 uS/cm at 25 °C.16
The conductivity meter must yield a conductance between 1380 uS/cm and 1440
uS/cm to be in calibration.
10.4.3 If the conductivity meter fails calibration, recalibrate the unit per manufacture's
instruction and repeat the procedure in 10.4.2 as if the standard solution were an
unknown matrix.
11. PROCEDURE
11.1 SAMPLE PREPARATION
11.1.1 Samples do not need to be refrigerated but if samples are held refrigerated as a
standard practice for sample control, ensure the samples have come to room
temperature prior to conducting sample analysis.
11.1.2 MATRIX CONDUCTANCE VERIFICATION - Prior to conducting the
analysis of a field sample matrix, the conductance of that matrix must be
measured. Matrix conductivity is directly related to the common anion levels
which, at high concentrations, can influence the integrity of the perchlorate
analysis.
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11.1.2.1 Verify conductivity detector calibration by following the procedure
outlined in Section 10.4.
11.1.2.2 Pour approximately 15 mL of sample into a plastic disposable micro
beaker (Section 6.7) and reseal the sample bottle to protect the sample
integrity.
11.1.2.3 Place the electrode into the matrix and measure the conductivity.
11.1.2.4 If the conductance is less than the MCT, continue to Section 11.1.5.
11.1.2.5 If the conductance is greater than the MCT, the matrix requires
dilution or pretreatment prior to analysis. The dilution procedure is
found in Section 11.1.3. Pretreatment is described in Section 11.1.4.
11.1.2.6 Discard this aliquot of sample and be certain to thoroughly rinse the
electrode with reagent water between each matrix conductivity
measurement.
11.1.3 MATRIX DILUTION - If matrix conductivity is less than the MCT, go to
Section 11.1.5.
11.1.3.1 A sample can be analyzed once diluted with reagent water to a
conductance below the MCT. The exact magnitude of this dilution
will adversely increase the MRL by an equivalent proportion.
11.1.3.2 Knowing the matrix conductance exceeds the MCT, estimate the
proportion required for the dilution by dividing the measured matrix
conductance by the MCT. Round up to the next whole number and
dilute the sample by a proportion equivalent to this value. For
example, if the established MCT is 6100 uS/cm and a sample
reflecting a conductance of 8000 uS/cm was measured, dilute the
sample with reagent water by a factor of 2.
11.1.3.3 Measure the conductance of the diluted sample to confirm it is now
below the MCT. Analyze the sample as specified in Section 11.1.5
with the understanding that the MRL has now been elevated by a
proportion equivalent to the dilution.
11.1.3.4 If perchlorate is measured above the elevated MRL, back calculate
actual field sample concentration and report. If no perchlorate is
measured above the elevated MRL and analysis or project objectives
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required monitoring below the concentration of the elevated MRL,
proceed to Section 11.1.4 and pretreat the matrix.
11.1.4 PRETREATMENT FOR MATRICES WHICH EXCEED THE MCT - If
matrix conductivity is less than the MCT, go to Section 11.1.5. If sample
dilution did not yield the required results, sample pretreatment should be
employed. When the MCT is exceeded, it is most often due to a high levels of
common anions (chloride, sulfate, and carbonate) in a particular matrix. If the
analyst were to attempt the 1C analysis of this particular matrix, the common
anions present in the sample would distort the baseline and negatively affect the
accurate quantitation of perchlorate. To effectively reduce a significant amount
of these anions which contribute to the high conductivity reading, a series of
pretreatment cartridges must be employed. For this pretreatment, three
cartridges are attached in series in the following order: Ba, Ag, and H. It is
recommended that all three cartridges be employed unless the analyst has
specific knowledge that a matrix primarily has high levels of a specific common
anion.
11.1.4.1 Individually and thoroughly rinse each pretreatment cartridge with
reagent water in order to insure all residual background contaminants
are removed from the cartridge. Perform this rinse per manufacturer's
instructions.
11.1.4.2 Prior to pretreating any field samples, prepare and pretreat both an
LRB and an LFB. These pretreated quality control samples are
required when an analysis batch contains a matrix which must be
pretreated. This pretreatment is conducted by placing the cartridges in
the following prescribed series (->Ba->Ag->H). The pretreated
LRB and LFB are used to verify that no background interference or
bias is contributed by the pretreatment. If a response is observed in
the pretreated LRB, triple or quadruple the volume of reagent water
rinse suggested by the manufacturer in Section 11.1.4.1 and repeat
until a blank measures no more than 1A the MRL. If this additional
rinsing procedure is required, it must be consistently applied to all the
cartridges prior to conducting any matrix pretreatment.
11.1.4.3 Filter 3 mL of sample through the series of rinsed, stacked cartridges
as an initial sample rinse (Ba, Ag and H) at a flow rate of 1.0 mL/ min
or less (approximately one drop every 3 to 4 seconds). This flow rate
is critical to the pretreatment and must be carefully followed. Discard
this fraction and begin collecting the pretreated sample aliquot of
collected sample.
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11.1.4.4 When sufficient volume has been collected, measure the conductance
of the pretreated sample aliquot being certain the conductivity meter's
probe has been thoroughly rinsed and excess water has been shaken
from the tip. If the conductance is now below the MCT, the sample is
ready for analysis. If the conductance is still above the MCT, the flow
rate through the pretreatment cartridge is likely too fast and the
pretreatment should be repeated with new cartridges. In some
instances, double pretreatment cartridges may need to be applied.
When this pretreatment is performed properly, U.S.EPA has found
70% to 95% reduction in matrix conductance with good recoveries for
perchlorate.
11.1.4.5 Place this aliquot of pretreated sample into an autosampler vial as
described in Section 11.1.3.
11.1.4.6 In order to ensure data quality, all samples which fail the MCT and
have been selected for pretreatment, as described in Section 11.1.4,
must also be used to prepare an LFM. This LFM must be fortified
with perchlorate at concentrations close to, but greater than, the level
determined in the native sample prior to the pretreatment. Initially, the
pretreated sample is analyzed and perchlorate level is determined.
Then, a second aliquot of sample must be fortified with perchlorate,
pretreated to reduce the high common anion levels, and analyzed to
assess perchlorate recovery from that matrix. This additional QC is
required to rule out matrix effects and to confirm that the laboratory
performed the pretreatment step appropriately. If the perchlorate
recovery falls outside the acceptance range of 80 - 120% (Section
9.4.1.4), that particular sample should be reported as
suspect/matrix.
11.1.4.7 The pretreatments prescribed above are effective at reducing the
chloride and sulfate content of a sample matrix but will not reduce
matrix concentrations of other anions such as nitrate or phosphate.
11.1.5 Pour approximately 15 mL of sample into a micro beaker (Section 6.7) and
reseal the sample bottle to protect the sample integrity. Using a Luer lock,
plastic 10 mL syringe, withdraw approximately 10 mL of sample from the
micro beaker and attach a 0.45 um particulate filter (Section 6.11), which has
been demonstrated to be free of ionic contaminants, directly to the syringe.
Filter the sample into an autosampler vial or manually load the injection loop
injecting a fixed amount of filtered, well mixed sample. If using a manually
loaded injection loop, flush the loop thoroughly between sample analysis using
sufficient volumes of each new sample matrix.
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11.1.5.1 If the autosampler vials or vial caps are designed to automatically filter
the sample matrix as the sample is loaded on the 1C system, this
filtration procedure can be omitted and the sample can be directly
transferred to the autosampler vial.
11.2 SAMPLE ANALYSIS
11.2.1 Table 1 summarizes the recommended operating conditions for the ion
chromatograph. Included in this table is the estimated retention time for
perchlorate which has been achieved by this method. Other columns,
chromatographic conditions or detectors maybe used if the requirements of
Sections 1.2.1, 6.1.2.2 and 9.2 are met.
11.2.2 Establish a valid initial calibration and verify this calibration by conducting a
QCS as described in Section 10.2 and complete the IDC (Section 9.2). Initially,
analyze the IPC solution, followed by the LRB. Then confirm the 1C system
calibration by analyzing an ICCS (Section 10.3.1) and, if required, recalibrate as
described in Section 10.2. Lastly, analyze the LFB.
11.2.3 Inject 1.0 mL of each filtered sample. Use the same size loop for standards and
samples. An automated constant volume injection system may also be used.
Record the resulting peak size in area units and retention time for each analyte.
11.2.4 The width of the retention time window used to make identifications should be
based upon measurements of actual retention time variations of standards
measured over several days. Three times the standard deviation of retention
time may be used as a suggested window size but the retention time window
should not extend beyond ± 5% of the retention time for perchlorate. The
experience of the analyst should weigh heavily in the interpretation of these
chromatograms.
11.2.5 If the response of a sample analyte exceeds the calibration range, the sample
must be diluted with an appropriate amount of reagent water and reanalyzed. If
this is not possible then three new calibration concentrations must be employed
to create a separate high concentration calibration curve, one standard near the
estimated concentration and the other two bracketing around an interval
equivalent to approximately ± 25% the estimated concentration. The response
generated by these three new high concentration calibration standards must not
exceed the upper linear range for the conductivity detector. The latter procedure
involves significantly more time than a simple sample dilution therefore, it is
advisable to collect sufficient sample to allow for sample dilution and sample
reanalysis, if required.
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11.2.6 Should more complete resolution be needed between perchlorate and a
coeluting, shoulder peak, the eluent (Section 7.2) may be diluted. This will
spread out the peaks, causing later elution of perchlorate. Analysts are advised
to carefully evaluate any of these eluent dilutions since when these eluent
changes are incorporated, other coelutions may be encountered which were not
initially evident. Additionally, the analyst must verify that this dilution does not
negatively affect performance by repeating and passing all the QC criteria in
Section 9, and by reestablishing a valid initial calibration curve (Section 10.2).
11.2.6.1 Eluent dilution will reduce the overall response of an anion due to
chromatographic band broadening which will be evident by shortened
and broadened peaks. This will adversely effect the MDLs for each
analyte.
11.3 AUTOMATED ANALYSIS WITH METHOD 314.0
11.3.1 Laboratories conducting analyses on large numbers of samples often prepare
large analysis batches that are run in an automated manner. When conducting
automated analyses, careful attention must be paid to ensure sufficient volume
of eluent in the reservoir is available to sustain extended operation. In order to
ensure their data are of acceptable quality, laboratories must ensure that all QC
performance criteria are met throughout the analysis batch through subsequent
careful inspection of the data.
11.3.2 Analysis sequences must be carefully constructed to meet required QC
specifications and frequency (Table 6). To help with this task, an acceptable
sequence for a sample analysis batch, with all the method-required QC, is shown
in Table 7. This schedule is included only as an example of a hypothetical
analysis batch which contains normal sample matrices as well as samples which
have failed the MCT. Within this analysis batch, references to exact
concentrations for the ICCS, CCCS and ECCS are for illustrative purposes only.
11.3.3 Table 7 may be used as a guide when preparing analysis batches. Additional
batches may be added sequentially on to the end of these types of schedules as
long as all QC samples, which define an individual batch (EPC, LRB, ICCS,
LFB, LFM, etc.) are individually reanalyzed with each successive serial batch
and the QC criteria for these analyses are continually met (from the IPC through
ECCS).
12. DATA ANALYSIS AND CALCULATIONS
12.1 Identify perchlorate in the sample chromatogram by comparing the retention time of a
suspect peak within the retention time window to the actual retention time of a known
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analyte peak in a calibration standard. If the perchlorate retention time has slightly
shifted (generally towards shorter times) since the initial calibration, but is still within
acceptance criteria and are reproducible during the analysis batch, the analyst should
use the retention time in the daily calibration check standards to confirm the presence or
absence of perchlorate anion.
12.1.1 If a low concentration of perchlorate is suspected in an unknown sample, but the
retention time has drifted to the edge of the retention time window, a low level
perchlorate LFM, prepared at nearly the same concentration as the suspect peak,
should be prepared from this sample matrix to confirm the matrix induced
retention time shift. If the fortified sample reveals a split or shouldering peak
response, the low concentration in the unfortified sample is likely an interferant
and should not be reported as perchlorate.
12.2 Compute sample concentration using the initial calibration curve generated in Section
10.2.
12.3 Report ONLY those values that fall between the MRL and the highest calibration
standards. Samples with a perchlorate response which exceeds the highest calibration
standard concentration must be diluted and reanalyzed. When this is not possible the
alternate calibration procedures described in Section 11.2.5 must be followed. Samples
with perchlorate identified but quantitated below the concentration established by the
lowest calibration standard, may be reported as "trace present" above the MDL but
below the minimum reporting limit (MRL) and therefore not reported as a quantitated
concentration.
12.4 Report results in u.g/L.
13. METHOD PERFORMANCE
13.1 Table 1 gives the standard conditions, typical retention time, single laboratory MCT and
single laboratory MDL in reagent water, as determined for perchlorate. This retention
time is graphically indicated in the chromatograms in Figures 1 through 4.
13.2 Table 2 shows the precision and accuracy of the perchlorate measurement at two
fortified concentrations, in reagent water, simulated high ionic strength water (HIW),
simulated high organic content water (HOW), ground water, untreated surface water
and treated surface water. The mean perchlorate recovered concentration (accuracy
relative to the fortified level) and the precision (expressed as %RSD of the replicate
analysis) are tabulated. The HIW was designed to simulate a high ionic strength field
sample and the HOW designed to simulate a high organic content field sample. The
HIW was prepared from reagent water which was fortified with the common anions of
chloride at 400 mg/L, carbonate at1600 mg/L, and sulfate at 500 mg/L. The HOW was
prepared from reagent water fortified with 10.0 mg/L fulvic acid.
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-------
13.3 Table 3 shows the stability data for perchlorate held for 35 days and stored under
various conditions. Conditions investigated included sample bottle construction
(HDPE plastic vs. glass), storage condition (refrigerated vs. held at room temperature)
and various matrices including some with a measured perchlorate concentration
assumed to contain microbiological constituents acclimated to the presence of the
anion. Matrices without perchlorate were fortified at 25 ug/L. Each data point in this
table represents the mean percent recovery following triplicate analyses. These data
were used to formulate the holding times shown in Section 8.3.
13.4 Table 4, in conjunction with the chromatograms overlaid in Figure 4 as well as the
linear regression plots in Figure 5, show the results of the single laboratory MCT
determination. The data presented in Table 4 and graphically illustrated in Figure 5,
show results for not only the AS16 but also the AS11 and AS5. The chromatogram
shown in Figure 4 were generated using the AS 16 column.
14. POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity
or toxicity of waste at the point of generation. Numerous opportunities for pollution
prevention exist in laboratory operation. The EPA has established a preferred hierarchy
of environmental management techniques that places pollution prevention as the
management option of first choice. Whenever feasible, laboratory personnel should use
pollution prevention techniques to address their waste generation. When wastes cannot
be feasiblely reduced at the source, the Agency recommends recycling as the next best
option.
* - , .
14.2 Quantity of chemicals purchased should be based on expected usage during its shelf-
life and the disposal cost of unused material. Actual reagent preparation volumes
should reflect anticipated usage and reagent stability.
14.3 For information about pollution prevention that may be applicable to laboratories and
research institutions, consult "Less is Better: Laboratory Chemical Management for
Waste Reduction," available from the American Chemical Society's Department of
Government Regulations and Science Policy, 1155 16th Street N.W., Washington D.C.
20036, (202) 872-4477.
15. WASTE MANAGEMENT
15.1 The Environmental Protection Agency requires that laboratory waste management
practices be conducted consistent with all applicable rules and regulations. Excess
reagents, samples and method process wastes should be characterized and disposed of
in an acceptable manner. The Agency urges laboratories to protect the air, water, and
land by minimizing and controlling all releases from hoods and bench operations,
complying with the letter and spirit of any waste discharge permit and regulations, and
314.0-33
-------
by complying with all solid and hazardous waste regulations, particularly the hazardous
waste identification rules and land disposal restrictions. For further information on
waste management consult the "Waste Management Manual for Laboratory Personnel,"
available from the American Chemical Society at the address listed in Section 14.3.
16. REFERENCES
1. "Determination of Perchlorate by Ion Chromatography." State of California, Department of
Health Services, Sanitation and Radiation Laboratories Branch, Rev. No. 0 (June 3,1997).
2. "Analysis of Low Concentrations of Perchlorate in Drinking Water and Ground Water by
Ion Chromatography." Application Note 121, Dionex Corporation, Sunnyvale, CA (1998).
3. 'Terchlorate by Ion Chromatography, Modified EPA 300.0 Using lonPac ASH." Standard
Operating Procedure, Montgomery Watson Laboratories (March 17, 1998).
4. Jackson, P.E.; Laikhtman, M.; and Rohrer, J.S. "Determination of Trace Level Perchlorate
in Drinking Water and Ground Water by Ion Chromatography," Journal of Chromatography
A, 850 (1999), 131-135.
5. Okamoto, H.S.; Rishi, D.K.; Steeber, W.R.; Baumann, F.J.; and Perera, S.K. "Using Ion
Chromatography to Detect Perchlorate," Journal AWWA. Vol. 91 (October 1999), 73-84.
6. Biter-Agency Perchlorate Steering Committee, Analytical Subcommittee Report (1998).
Report on the interlaboratory validation of 1C methods for perchlorate.
7. Glaser, J.A.; Foerst, D.L.; McKee, G.D..; Quave, S.A. and Budde, W.L. "Trace Analyses
for Wastewater," Environmental Science and Technology. Vol. 15, Number 12, page 1426,
December, 1981.
8. Code of Federal Regulations 40, Pt. 136, Appendix B (July 1, 1998).
9. "OSHA Safety and Health Standards, General Industry," (29CFR1910). Occupational Safety
and health Administration, OSHA 2206, (Revised, Jan. 1976).
10. ASTM Annual Book of Standards, Part E, Volume 11.01, D3370-82, "Standard Practice for
Sampling Water," American Society for Testing and Materials, Philadelphia, PA, 1986.
11. "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.
12. "Safety In Academic Chemistry laboratories," 3rd Edition, American Chemical Society
Publication, Committee on Chemical Safety, Washington, D.C., 1979.
314.0-34
-------
13. U.S. EPA Method 300.1, EPA Document number: EPA/600/R-98/118. NTIS number
PB98-169196 INZ.
14. "Anion Self-Regenerating Suppressor (ASRS) Quickstart Procedure", Document Number
031368-01, Dionex Corporation, Sunnyvale, CA, March,1988.
15. "Installation Instructions and Troubleshooting Guide for the Anion Self-Regenerating
Suppressbr-Ultra", Document Number 031367, Rev. 03, Section 5.1, Dionex Corporation,
Sunnyvale, CA, December, 1988.
16. CRC Handbook of Chemistry and Physics. Standard Solutions for Calibrating Conductivity
Cells, p. D-166, 70th Ed., 1989-1990, CRC Press, Boca Raton, Florida.
314.0-35
-------
?LES. DIAGRAMS. FLOWCHARTS AND VALIDATION DATA
TABLE 1. CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION
LIMITS IN REAGENT WATER FOR PERCHLORATE.
Standard Conditions and Eauinment(a):
Ion Chromatograph:
Sample Loop:
Eluent:
Eluent Flow:
Columns:
Suppressor:
Detectors:
Determined MCT00:
DionexDXSOO
1000 uJL
SOmMNaOH
1.5mL/min
Dionex AG16,4 mm / AS16, 4 mm
Typical System Backpressure: 2600 psi
ASRS ULTRA (P/N 53946), external water mode, 300 mA current
Suppressed Conductivity Detector, Dionex CD20
Background Conductivity: 2 - 3 \iS
6100 uS/cm
Recommended method total analysis time: 15 minutes (may be shortened to 12 minutes)
Analvte Retention Times and Method Detection Limits (MDLs^:
Analyte
Perchlorate
Retention Time (c)
(min.)
10.1 ±0.2
MDL DETERMINATION
Fortified Cone. #of MDL
(|ig/L) Reps. (Hg/t)
2.0 7 0.53
(a) Mention of trade names or commercial products does not necessarily constitute endorsement or
recommendation for use.
(b) This was the single laboratory MCT determined for these conditions listed (See Table 4 and Figure
5 for more detail as well as data pertaining to the AS 11 and ASS).
(c) Reference to chromatograms in Figure 1 through 4.
314.0-36
-------
TABLE 2. SINGLE LABORATORY PRECISION AND RECOVERY FOR
PERCHLORATE IN VARIOUS MATRICES
Matrix Unfortified
Conductivity Cone.
Matrix uS/cm (ng/L)
Reagent Water
Synthetic High
Inorganic Water w
Synthetic High
Organic Water (c)
Ground Water
(highTDS)
Untreated Surface
Water
Chlorinated
Surface Water
~ 1
4200
5.0
710
460
460
-------
TABLE 3. STABILITY STUDY RESULTS FOR PERCHLORATE IN VARIOUS MATRICES
A. Stability when stored in various sampling bottles - All stored at room temperature
Matrix
Bottle type
Unfortified Fortified
Conc.((ig/L) Conc.(ug/L)
DayO
Analyte % Recovery
Day 7 Day 14 Day 28 Day 35
Reagent Water
Reagent Water
Reagent Water
Reagent Water
Clear Glass
Amber Glass
Opaque HOPE Plastic
Translucent HDPE
Plastic
-------
TABLE 4. SINGLE LABORATORY RESULTS FOR THE DETERMINATION OF
MCT - Determination on the AS16, ASH and the ASS.
AS16 Studies - Perchlorate fortified at 25 ug/L
Sample
LFB
MA(50)«
MA(IOO)
MA(200)
MA(400)
MA(600)
MA(800)
MAnnnm
Conductivity
uS/cm
<1
540
932
1770
3570
5010W
6450
7890
RT
min.
10.3
10.3
10.3
10.2
10.2
Measured
C104-, ug/L
25.3
26.0
26.3
26.2
25.2
10.2 j 24.2
10.1
10 2
25.1
24 3
%Rec
101%
104%
105%
105%
101%
97%
100%
97%
Area
20268
20799
21060
20998
20170
19307
20038
15400
Height
1151
1135
1144
1112
1028
954
932
878
A/H ratio
17.6
18.3
18.4
18.9
19.6
20.2
21.5
99 1
PDA/H
0.00%
4.07%
4.54%
7.24%
11.4%
14.9%
22.1%
95 S°/
AS1 l(c) Studies - Perchlorate fortified at 25 ug/L
Sample
LFB
MA(50)(a)
MA(100)
MA(200)
MA(400)
MA(600)
MA(800)
MAnooo^
Conductivity
uS/cm
<1
540
932
1770 *)
3570
5010
6450
789.0
RT
min.
8.9
8.9
9.0
9.0
9.0
9.0
8.9
8 8
Measured
C1O4-, ug/L
25.0
25.2
25.0
24.1
23.6
22.7
19.9
170
%Rec
100%
101%
100%
96%
94%
91%
80%
68%
Area
25213
25445
25192
24340
23855
22922
20243
1J407
Height
1591
1515
1486
1384
1243
1101
870
678
A/H ratio
15.8
16.8
17.0
17.6
19.2
20.8
23.3
95 7
PD^
0.00%
5.98%
6.98%
11.0%
21.1%
31.4%
46.8%
62 0%
AS5(d) Studies - Perchlorate fortified at 25 ug/L
Sample
LFB
MA(50)(a)
MA(IOO)
MA(200)
MA(400)
MA(600)
MA(800)
MAnooo^
Conductivity
uS/cm
<1
540
932
1770 <»
3570
5010
6450
7890
RT
min.
9.7
9.7
9.7
9.7
9.6
9.6
9.6
Q6
Measured
C1O4-, ug/L
22.75
24.89
23.72
22.99
23.51
23.84
21.01
92 95
%Rec
91.0%
99.6%
94.9%
92.0%
94.0%
95.4%
84.0%
91 8%
Area
30348
33505
31776
30704
31474
31948
27792
30650
Height
1780
1751
1721
1591
1478
1441
1214
1 183
A/H ratio
17.0
19.1
18.5
19.3
21.3
22.2
22.9
95 Q
PDA/H
0.00%
12.2%
8.30%
13.2%
24.9%
30.0%
34.3%
59 0%
(a) "MA" indicates mixed common anion solution with each anion (chloride, sulfate and carbonate)
included in the sample matrix at the parenthetical mg/L concentration for each anion.
(b) If the regression analysis is not performed on these data, 5010 uS/cm, 1770 uS/cm and 1770 uS/cm
would be the default MCT for the AS 16, ASH and AS5, respectively, as described in Section 9.2.8.10.
See Figure 5 for a graphical representation of this data, applying a regression analysis of PD^ vs
matrix conductivity for the AS 16, ASH and ASS.
(c) ASH conditions: See reference #2 and #3.
(d) AS5 conditions: See reference #1.
314.0-39
-------
TABLE 5. INITIAL DEMONSTRATION OF CAPABILITY QC REQUIREMENTS.
Requirements prior to beginning any analysis batch
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Sect. 9.2.2
9.3.1
Initial
Demonstration of
Low System
Background
Analyze a method blank (LRB) and
determine that all target analytes are
below l/2 of the proposed MRL prior to
performing the IDC.
The LRB concentration
must be <,1A of the
proposed MRL.
Sect. 9.2.3
Initial
Demonstration of
Accuracy (IDA)
Analyze 7 replicate LFBs fortified with
perchlorate at 25 ug/L. Calculate the
mean recovered concentration
See Equation in Section 9.2.3.
The GX must be ± 10% of
true value.
Sect. 9.2.4
Initial
Demonstration of
Precision (TOP)
Calculate percent relative standard
deviation (%RSD)of IDA replicates.
See Equation in Section 9.2.4.
The %RSD must be '<. 10%
Sect. 9.2.5
Quality Control
Sample (QCS)
Initially, upon reestablishing calibration
or at least quarterly analyze a QCS from
an external/second source.
The QCS must be ± 10%
of the true value.
Sect. 9.2.6
Method
Detection Limit
(MDL)
Determination
Select a fortifying level at 3-5 times the
estimated instrument detection limit.
Analyze 7 replicate LFBs over multiple
days and calculate MDL using equation in
Section 9.2.6 - do not subtract blank
Sect. 9.2.7
Minimum
Reporting Level
(MRL)
An MRL should be established for
perchlorate during the IDC.
The low CAL standard can
be lower than the MRL,
but the MRL MUST be no
lower than the low CAL
standard
Sect. 9.2.8
Sect.
9.2.8.11
Matrix
Conductivity
Threshold (MCT)
MRL verification
Prepare a series of LFB samples, each
containing a suggested perchlorate '
concentration of 25 ug/L, at sequentially
increasing fortified levels of common
anions. Measure sample conductance and
analyze each, calculate average A/H ratios
and PD^VH (using equation in Section
9.2.8.8). Perform linear regression to
calculate MCT (using equation in Section
9.2.8.9) or follow step outlined in Section
9.2.8.10.
Verify the MRL in a solution prepared at
the MCT.
MCT, based upon linear
regression, is point where
equals 20%.
Alternatively, the MCT is
set at the highest measured
conductance observed in
the last fortified MCT
sample to yield a PD^
value below 20%.
Prepared within ±10% of
the MCT.
Perchlorate recovery must
be 70-130% of the MRL.
314.0-40
-------
TABLE 6.
QUALITY CONTROL REQUIREMENTS (SUMMARY).
Requirements specific for each analysis batch
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Sect. 8.3
Sample
Holding Time 7
Preservation /
Storage >
Perchlorate 28 days
No Preservation technique required.
Room Temperature adequate for shipping
and storage.
Holding time must not be
exceeded.
Sect. 10.2
Initial
Calibration
Generate calibration curve. At least 5
calibration standards are recommended.
MRL MUST be no lower
than the lowest calibration
standard
Sect. 9.3.2
Instrument
Performance
Check (IPC)
Designed to verify Matrix Conductivity
Threshold (MCT). Prepare mixed common
anion solution at the MCT (prepared
consistent with procedures in Section
9.2.8). Confirm the sample's conductance
and analyze at the beginning of each
analysis batch.
Prepared within ±10% of
the MCT.
IPC solution conductance
verified to within ± 10%
of original measured value
(when originally prepared)
PDA/H> (when compared to
the A/HLFB) must be <
25%.
Perchlorate quantitated
between 80 -120% of
fortified level.
<5% shift in perchlorate
retention time.
Sect. 10.3.1
Initial
Calibration
Check (ICCS)
With each analysis batch, initially verify
calibration at the MRL by analyzing an
initial low-level continuing calibration
check standard (ICCS).
Recovery must be 75-
125% of the true value.
Sect. 10.3.2
Continuing
Calibration
(CCCS) and
End Calibration
Checks (ECCS)
Alternately analyze separate mid and high
level CCCS/ECCS after every 10 samples
and after the last sample in an analysis
batch.
Recoveries must fall
between 85-115%
Sect. 9.3.1
Laboratory
Reagent Blank
(LRB)
Include LRB with every analysis batch (up
to 20 samples)
Analyze prior to analyzing field samples
Perchlorate must be
< '/2MRL
314.0-41
-------
TABLE 6. QUALITY CONTROL REQUIREMENTS (SUMMARY CONTINUED).
Requirements specific for each analysis batch
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Sect. 9.3.1.1
PRETREATED
Laboratory
Reagent Blank
(LRB)
REQUIRED in any analysis batch
which includes samples which have
exceeded the MCT and have been
pretreated in any way to reduce the
common anion levels.
Perchlorate must be
s '/2 MRL
Sect. 9.3.3
Labortary
Fortified Blank
(LFB)
Laboratory must analyze LFB in each
analysis batch following the ICCS.
Calculate %REC prior to analyzing
samples. The concentration selected for
the LFB in subsequent analysis batches
should be varied throughout the
calibration range.
Recovery for LFB MUST
be 85-115% prior to
analyzing samples.
Sample results from
batches that fail LFB are
invalid.
Sect. 9.3.1:1
PRETREATED
Laboratory
Reagent Blank
(LRB)
REQUIRED in any analysis batch
which includes samples which have
exceeded the MCT and have been
pretreated in any way to reduce the
common anion levels. Fortification
must be made prior to pretreatment.
Recovery for pretreated
LFB MUST be 85-115%
prior to analyzing samples.
Sample results from
batches that fail a
pretreated LFB are invalid.
Sect. 9.4.1
Sect. 11.1.4.6
Laboratory
Fortified Sample
Matrix (LFM)
SPECIAL LFM
for matrices
requiring
pretreatment
Must add known amount of perchlorate
to a minimum of 5% of field samples or
at least one within each analysis batch.
LFM must be fortified above the native
level and at no greater than 10 x the
highest field sample concentration.
Calculate target analyte recovery using
formula (Sect. 9.4.1.3).
When a sample exceeds the MCT and
pretreatment is employed to reduce the
common anion levels, an additional
LFM must be prepared from this matrix
and subsequently pretreated exactly as
the unfortified matrix.
Recovery must be
80 -120%
If fortified sample fails the
recovery criteria, label
both as suspect/matrix.
Same criteria, recoveries
must be 80-120%.
Sect. 9.4.2
Field or
Laboratory
Duplicates or
LFM Duplicate
Analyze either a field, laboratory or
LFM duplicate for a minimum of 5% of
field samples or at least one within each
analysis batch.
Calculate the relative percent difference
(RPD) using formula in Section 9.4.2.1.
RPDmustbe±15%.
Sect. 6.1.2.2
ALTERNATE 1C
analytical column
performance
criteria
If a laboratory chooses an alternate
analytical column for this analysis, it
must be hydrophilic and pass the
criteria for Peak Gaussian Factor (PGF)
using equation (Sect. 6.1.2.2).
PGF must fall between
0.80 and 1.15.
314.0-42
-------
TABLE 7. EXAMPLE SAMPLE ANALYSIS BATCH WITH QUALITY CONTROL
REQUIREMENTS
Injection
#
1..
2
3
4
5
6
• 7
8
9
10
11
12
13
14
15
16
17
18
19
20
Sample
Description
Instrument Performance Check Standard at MCT
Laboratory Reagent Blank (LRB)
ICCS at the MRL (4.0 (j.g/L)
Laboratory Fortified Blank (LFB)
Sample 1
Sample 1 - Laboratory Duplicate (LD) (a)
Sample 2
Sample 2 - Laboratory Fortified Matrix (LFM) (a)
Sample3
Sample 4
Sample 5
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
CCCS (25.0 ug/L)
Sample 1 1 (failed MCT, matrix conductance = 8000 uS/cm)
- Analyzed diluted (Section 11.1.3) by factor of 2 or by
50% with reagent water (diluted matrix conductance =
3800uS/cm).
Sample 12
Sample 13
Acceptance
Criteria
PD^ forBPC<25%
s'/2MRL
3.00 to 5.00 ug/L
Recovery of 85-115%
normal analysis
±15%RPD
normal analysis
Recovery of 80 - 120%
normal analysis
normal analysis
normal analysis
normal analysis
normal analysis
normal analysis
normal analysis
normal analysis
2 1.3 to 28. 8 ug/L
MRL increases from 4 to 8
ug/L, noted in analysis report -
sample found to contain 50 ug/L
(measured at 25 ug/L in diluted
sample)
normal analysis
normal analysis
CONTINUED TO NEXT PAGE
314.0-43
-------
Injection
#
Sample
Description
Acceptance
Criteria
21
Sample 14 (failed MCT, matrix conductance^ 5000 uS/cm)
Analyzed diluted (Section 11.1.3) by a factor of 3 or by
33% with reagent water (Diluted matrix conductance =
4600 uS/cm)
MRL increases from 4 to 12
ug/L, noted in analysis report -
No perchlorate > 12ug/L
measured - project required
monitoring to MRL - sample
pretreatment is therefore
required
22
Ba/Ag/H Pretreated LRB (Section 9.3.1.1)
23
Ba/Ag/H Pretreated LFB (Section 9.3.3.1)
Recovery of 85-115%
24
Sample 14 - Ba/Ag/H pretreated (Section 11.1.4), following
pretreatment the matrix conductance = 230 uS/cm.
normal pretreated analysis
perchlorate < MRL of 4.0 ug/L
25
Sample 14 w - pretreated LFM (Section 11.1.4.6)
Recovery of 80 -120%
26
Sample 15
normal analysis
27
Sample 16
normal analysis
28
Sample 17
normal analysis
29
Sample 18
normal analysis
30
Sample 19W
normal analysis
31
ECCS (100 ug/L)
85.0 to 125 ug/L
W If no analytes are observed above the MRL for a sample, san alternate sample which contains reportable
values should be selected as the laboratory duplicate. Alternately, the LFM can be selected and reanalyzed
as the laboratory duplicate ensuring the collection of QC data for precision.
w Sample #19 (inj #30) was the final field sample permitted in this batch but 20 total field samples were
analyzed. Sample #14 (inj #21 and #24) was analyzed both initially as a diluted sample and subsequently
as a pretreated sample, therefore it accounted for two "field sample analyses" toward the maximum of
twenty in an analysis batch (Section 3.1).
Note: Sample #11 and #14 illustrate examples of proper ways to handle sample matrices which exceed the
MCT.
314.0-44
-------
'dsay PQ puoo
314.0-45
-------
o
o
o
_p
CM
o
o
3
D)
CO
-------
05 2
0)
Sri 'dssy
314.0-47
-------
TO
§
I
LU
o
O
o
p
O
p
-------
FIGURE 5. REGRESSION ANALYSIS OF THE MCT DETERMINATION DATA
8000
£
o
to 6000
8"
£ 4.000
o
•a
£ 2000
0
0
0
AS16
MCT Regression analysis
Repression Data
R-square = 0.986 #pts = 8
y = 32076x-338 6100
600 _-*•"*'***''*""
J^
20.0^-^**"^
. J^—^*
3 ^^f
% 5% 10% 15% 20
PD (A/H)
1000
^**
sMSr
— • Actual Data Points for —
samples
^" Regression Line
A MCTindicated at 610Cr~
— uS/cm —
% 25% 30
%
9000
8000
E 7000
^ 6000
o- 5000
| 4000
| 3000
o 2000
1000
0
0
ASH
MCT Regression analysis
Regression DC
R-square = 0.977 #
y=13136x + 2
' onn J2SSCT
•m^ •
^-^•F
!*a ^*3oo
pts = 8 ^^^"^ u
20 b£^**^
j***8""^^
600 rf^-fl**f"^^^
JL^**^*'^^
"** Regression Line
* MCTindicated at 2850 uS/cm
es
% 10% 20% 30% 40% 50% 60% 70%
PD (A/H)
9000
8000
7000
•S 6000
J- OUUU
3 5000
u
o 4nnn
•§ 3000
3
•D 9nnn
O° 1000
-1000
0
Repression Data
R-square = 0.949 #p
y= 16990X-452
2950
200,^^"*^"^
100^^^*^
I ~**^***"^ H
\^
% 10% 20
ASS
MCT Regression analysis
^*f300
sou _-^^***>*"^'*^
600 _-^-""*"
^'
j$tutt£
,
• Actual Data Poiuls for respective-
mixed aninn samples
•*• Regression Line
A MCTindicated at 2950 uS/cm
' ' ' '-i -' ' i '• • i'
1% 30% 40% 50% 60
PD (A/H)
%
314.0 - 49
-------
-------
METHOD 317.0 DETERMINATION OF INORGANIC OXYHALIDE DISINFECTION
BY-PRODUCTS IN DRINKING WATER USING ION
CHROMATOGRAPHY WITH THE ADDITION OF A
POSTCOLUMN REAGENT FOR TRACE BROMATE ANALYSIS
Revision 1.0
May 2000
Herbert P. Wagner and Barry V. Pepich, IT Corporation and Daniel P. Hautman and
David J. Munch, US EPA, Office of Ground Water and Drinking Water
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
317.0-1
-------
METHOD 317.0
DETERMINATION OF INORGANIC OXYHALIDE DISINFECTION BY-PRODUCTS
IN DRINKING WATER USING ION CHROMATOGRAPHY WITH THE ADDITION
OF A POSTCOLUMN REAGENT FOR TRACE BROMATE ANALYSIS
1. SCOPE AND APPLICATION
1.1 This method covers the determination of inorganic oxyhalide disinfection by-product
anions in reagent water, surface water, ground water, and finished drinking water. In
addition, bromide can be accurately determined in source or raw water and it has been
included due to its critical role as a disinfection by-product precursor. Bromide
concentration in finished water can differ significantly between preserved and
unpreserved samples and should not be attempted due to numerous variables which can
influence the concentration. Since this method, prior to the addition of the postcolumn
reagent (PCR), employs the same hardware as EPA Method 300.11, the analysis of the
common anions (using EPA Method 300.1, Part A1) can be performed using this
instrument setup with the postcolumn hardware attached but "off-line" and with the
appropriate smaller sample loop.
Inorganic Disinfection Bv-products bv Conductivity Detection
Bromate (report values > 15.0 ug/L) Chlorite
Bromide (source or raw water only) Chlorate
Inorganic Disinfection Bv-product bv Postcolumn UV/VTS Absorbance Detection
Bromate (report values > Minimum Reporting Limit (MRL) to 15.0 ug/L)
1.2 The single laboratory reagent water Method Detection Limits (MDL, defined in Section
3.14) for the above analytes are listed in Table 1. The MDL for a specific matrix may
differ from those listed, depending upon the nature of the sample and the specific
instrumentation employed.
1.2.1 In order to achieve comparable detection limits on the conductivity detector, an
ion chromatographic system must utilize suppressed conductivity detection, be
properly maintained and must be capable of yielding a baseline with no more
than 5 nanosiemen (nS) noise/drift per minute of monitored response over the
background conductivity.
1.2.2 In order to achieve acceptable detection limits on the postcolumn absorbance
detector, the postcolumn reagent must be delivered pneumatically and some
form of software signal filtering or smoothing of the absorbance signal from the
absorbance detector must be incorporated.2
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1.3 This method is recommended for use only by or under the supervision of analysts
experienced in the use of ion chromatography and in the interpretation of the resulting
ion chromatograms.
1.4 When this method is used to analyze unfamiliar samples for any of the above anions,
anion identification should be supported by the use of a fortified sample matrix
covering the anions of interest. The fortification procedure is described in Section
9.4.1.
1.5 Users of the method data should state the data quality objectives prior to analysis.
Users of the method must demonstrate the ability to generate acceptable results with
this method, using the procedures described in Section 9.0.
2. SUMMARY OF METHOD
2.1 A volume of sample, approximately 225 uL (see Note), is introduced into an ion
chromatograph (1C). The anions of interest are separated and measured, using a system
comprised of a guard column, analytical column, suppressor device, conductivity
detector, a postcolumn reagent delivery system (pneumatically controlled), a heated
postcolumn reaction coil, and a ultraviolet/visible (UV/VIS) absorbance detector.2'3
NOTE: A 225 uL sample loop can be made using approximately 111 cm (44 inches)
of 0.02 inch i.d. PEEK tubing. Larger injection loops may be employed.4
The volume should be verified to be within 5% by weighing the sample loop
empty, filling the loop with deionized water and re-weighing the loop
assuming the density of water is 1 mg/uL.
3. DEFINITIONS
3.1 ANALYSIS BATCH - A sequence of samples, which are analyzed within a 30 hour
period and include no more than 20 field samples. An analysis batch must also include
all required QC samples, which do not contribute to the maximum field sample total of
20. The required QC samples include:
• Laboratory Reagent Blank (LRB)
•• Initial Calibration Check Standard (ICCS)
• Laboratory Fortified Blank (LFB)
• Continuing Calibration Check Standard (CCCS), when the batch contains more than
10 field samples
• End Calibration Check Standard (ECCS)
• Laboratory Fortified Matrix (LFM)
• Either a Field Duplicate (FD), a Laboratory Duplicate (LD) or a duplicate of the LFM
3.2 CALIBRATION STANDARD (CAL) - A solution prepared from the primary dilution
standard solutions) or stock standard solutions and the surrogate analyte. The CAL
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solutions are used to calibrate the instrument response with respect to analyte
concentration.
3.3 INITIAL CALIBRATION STANDARDS - A series of CAL solutions (either
individual or combined target analytes) used to initially establish instrument calibration
and develop calibration curves for individual target anions (Section 10.2).
3.4 INITIAL CALIBRATION CHECK STANDARD (ICCS) - A CAL solution, (either
individual or combined target analytes) which is analyzed initially, prior to any field
sample analyses, which verifies previously established calibration curves. The
concentration for the initial calibration check standard MUST be at or below the MRL
(Section 3.15) level which is also the level of the lowest calibration standard (Section
10.3.1).
3.5 CONTINUING CALIBRATION CHECK STANDARDS (CCCS) - A CAL solution
(either individual or combined target analytes) which is analyzed after every tenth field
sample analyses which verifies the previously established calibration curves and
confirms accurate analyte quantitation for the previous ten field samples analyzed. The
concentration for the continuing calibration check standards should be either at a
middle calibration level or at the highest calibration level (Section 10.3.2).
3.6 END CALIBRATION CHECK STANDARD (ECCS) - A CAL solution (either
individual or combined target analytes) which is analyzed after the last field sample
analysis which verifies the previously established calibration curves and confirms
accurate analyte quantitation for all field samples analyzed since the last continuing
calibration check. The end calibration check standard should be either the middle or
high level continuing calibration check standard (Section 10.3.2).
3.7 FIELD DUPLICATES (FD) - Two separate field samples collected at the same time
and place under identical circumstances and handled exactly the same throughout field
and laboratory procedures. Analyses of field duplicates indicate the precision
associated with sample collection, preservation and storage, as well as with laboratory
procedures.
3.8 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) - A solution of one or
more method analytes, surrogates, or other test substances used to evaluate the
performance of the instrument system with respect to a defined set of criteria.
3.9 LABORATORY DUPLICATE (LD) - Two sample aliquots, taken in the laboratory
from a single field sample bottle, and analyzed separately with identical procedures.
Analysis of the initial sample (Lj) and the duplicate sample [(Dc) Section 9.4.3.1]
indicate precision associated specifically with the laboratory procedures by removing
variation contributed from sample collection, preservation and storage procedures.
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3.10 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water or other
blank matrix to which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its purpose is to determine
whether the methodology is in control, and whether the laboratory is capable of making
accurate and precise measurements.
3.11 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) - An aliquot of an
environmental field 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 field sample matrix contributes bias to the analytical result. The
background concentrations of the analytes in the field sample matrix must be
determined in a separate, unfortified aliquot and the measured values in the LFM
corrected for background concentrations.
3.12 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or other
blank matrix that is handled exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, and surrogates that are used with other samples. The'
LRB is used to determine if method analytes or other interferences are present in the
laboratory environment, the reagents, or the apparatus.
3.13 MATERIAL SAFETY DATA SHEET (MSDS) - Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling precautions.
3.14 METHOD DETECTION LIMIT (MDL) - The minimum concentration of an analyte
that can be identified, measured and reported with 99% confidence that the analyte
concentration is greater than zero.5
3.15 MINIMUM REPORTING LEVEL (MRL)- The minimum concentration that can be
reported as a quantitated value for a target analyte in a sample following analysis. This
defined concentration can be no lower than the concentration of the lowest calibration
standard and can only be used if acceptable quality control criteria for the ICCS are met.
3.16 PROFICIENCY TESTING (PT) or PERFORMANCE EVALUATION (PE) SAMPLE
- A certified solution of method analytes whose concentration is unknown to the
analyst. Frequently, an aliquot of this solution is added to a known volume of reagent
water and analyzed with procedures used for samples. Often, results of these analyses
are used as part of a laboratory certification program to objectively determine the
capabilities of a laboratory to achieve high quality results.
3.17 QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of known
concentrations that is obtained from a source external to the laboratory and different
from the source of calibration standards. It is used to check laboratory performance
with externally prepared test materials.
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3.18 SURROGATE ANALYTE - An analyte added to all samples, standards, blanks, etc.,
which is unlikely to be found at a significant concentration, and which is added directly
hi a known amount before any sample processing procedures are conducted (except in
the procedure for the removal of chlorite as described is Section 11.1.4). It is measured
with the same procedures used to measure other sample components. The purpose of
the surrogate analyte is to monitor method performance with each sample.
3.19 STOCK STANDARD SOLUTION (SSS) - A concentrated solution containing one or
more method analytes prepared hi the laboratory using assayed reference materials or
purchased from a reputable commercial source.
4. INTERFERENCES
4.1 Interferences can be divided into three different categories: direct chromatographic
coelution, where an analyte response is observed at very nearly the same retention time
as the target anion; concentration dependant coelution, which is observed when the
response of higher than typical concentrations of the neighboring peak overlap into the
retention window of the target anion; and, ionic character displacement, where retention
times may significantly shift due to the influence of high ionic strength matrices (high
mineral content or hardness) overloading the exchange sites on the column and
significantly shortening target analyte's retention tunes.
4.1.1 A direct chromatographic coelution may be solved by changing columns, eluent
strength, modifying the eluent with organic solvents (if compatible with 1C
columns), changing the detection systems, or selective removal of the
interference with pretreatment. Sample dilution will have little to no effect.
The analyst must verify that these changes do not induce any negative affects on
method performance by repeating and passing all the QC criteria as described in
Section 9.
4.1.2 Sample dilution may resolve some of the difficulties if the interference is the
result of either concentration dependant coelution or ionic character
displacement, but it must be clarified that sample dilution will alter your
Minimum Reporting Limit (MRL) by a proportion equivalent to that of the
dilution. Therefore, careful consideration of project objectives should be given
prior to performing such a dilution. An alternative to sample dilution, may be
dilution of the eluent as outlined in Section 11.2.6.
4.1.3 Pretreatment cartridges can be effective as a means to eliminate certain matrix
interferences. With any proposed pretreatment, the analyst must verify that
target analyte(s) are not affected by monitoring recovery after pretreatment.
With advances in analytical separator column technology which employ higher
capacity anion exchange resins, the need for these cartridges has been greatly
reduced.
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4.2 Method interferences may be caused by contaminants in the reagent water, reagents,
glassware, and other sample processing apparatus that lead to discrete artifacts or
elevated baselines in an ion chromatogram. These interferences can lead to false
positive results for target analytes as well as reduced detection limits as a consequence
of elevated baseline noise.
4.3 Samples that contain particles larger than 0.45 microns and reagent solutions that
contain particles larger than 0.20 microns require filtration to prevent damage to
instrument columns and flow systems.
4.4 Close attention should be given to the potential for carry over peaks from one analysis
which will effect the proper detection of analytes of interest in a second or subsequent
analysis. Normally, in this analysis, the elution of sulfate (retention time of 17.5 min.)
indicates the end of a chromatographic run, but, in the ozonated and chlorine dioxide
matrices, which were included as part of the single operator accuracy and bias study, a
small response (200 nS baseline rise) was observed for a very late eluting unknown
peak following the response for sulfate. Consequently, a run time of 25 minutes is
recommended to allow for the proper elution of any potentially interferant late peaks. It
is the responsibility of the user to confirm that no late eluting peaks have carried over
into a subsequent analysis thereby compromising the integrity of the analytical results.
4.5 Any residual chlorine dioxide present in the sample will result in the formation of
additional chlorite prior to analysis. If residual chlorine dioxide is suspected in the
sample, the sample must be purged with an inert gas (helium, argon or nitrogen) for
approximately five minutes. This sparging must be conducted prior to ethylenediamine
preservation and at the time of sample collection.
4.6 The presence of chlorite can interfere with the quantitation of low concentrations of
bromate on the postcolumn UV/VIS absorbance detector. In order to accurately
quantify bromate concentrations in the range 0.5 - 15.0 (ig/L in this postcolumn system,
the excess chlorite must be removed prior to analysis as outlined in Section 11.1.4.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method have not been fully
established although the postcolumn reagent o-dianisidine, is listed as a potential
human carcinogen. Each chemical should be regarded as a potential health hazard and
exposure should be as low as reasonably achievable. Cautions are included for known
extremely hazardous materials or procedures.
5.2 Each laboratory is responsible for maintaining a current awareness file of Occupational
Safety and Health Administration (OSHA) regulations regarding the safe handling of
. the chemicals specified in this method. A reference file of Material Safety Data Sheets
(MSDS) should be made available to all personnel involved in the chemical analysis.
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The preparation of a formal safety plan is also advisable. Additional references on
laboratory safety are available.6"9
5.3 The following chemicals have the potential to be highly toxic or hazardous, consult
MSDS.
5.3.1 Sulfuric acid - used to prepared a 25 mN sulfuric acid regenerant solution for
chemical suppression using a Dionex Anion Micro Membrane Suppressor
(AMMS) and for pretreatment for chlorite removal (Section 11.1.4)
5.3.2 Nitric acid - used to prepare the postcolumn reagent.
5.3.3 o-dianisidine [3, 3'- dimethoxybenzidine dihydrochloride (ODA)] - used as the
postcolumn reagent.
6. EQUIPMENT AND SUPPLIES
6.1 Ion chromatograph - Analytical system complete with ion chromatographic pump and
all required accessories including syringes, analytical columns, compressed gasses,
suppressor, conductivity detector, mixing "tee", postcolumn reagent delivery system,
reaction coil, reaction coil heater, UV/VIS absorbance detector (Figure 1) and a PC
based data acquisition and control system.
NOTE: Because of its acidic nature and high salt content, the PCR MUST be flushed
from the reaction coil upon completion of the final analysis and prevented from
draining through the reaction coil by gravity once the system is shut down. This can be
accomplished either manually or by incorporating a column switching valve in
combination with a flush and close method in the schedule.
6.1.1 Anion guard column - Dionex AG9-HC 4 mm (P/N 51791), or equivalent.
This column functions as a protector of the separator column. If omitted from
the system the retention times will be shorter.
6.1.2 Anion separator column - Dionex AS9-HC column, 4 mm (P/N 51786), or
equivalent (see Note). The AS9-HC, 4 mm column using the conditions outlined
in Table 1 produced the separations shown in Figures 2 and 3.
NOTE: The use of 2 mm columns is not recommended. A 50 uL sample loop
would be required with the 2 mm columns. This reduced injection volume
would decrease the "on-column" bromate and negatively affect PCR reactivity
and the subsequent absorbance response. As well, the 2 mm columns require a
flow rate approximately 4 times less than the 4 mm columns. At the lower flow
rates, band broading may become an issue and it would be difficult, if not
impossible, to accurately maintain the appropriate reduced flow rate for the
PCR.
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6.1.3 Anion suppressor device - The data presented in this method were generated
using a Dionex Anion Self Regenerating Suppressor (4 mm ASRS, P/N 46081).
An equivalent suppressor device may be utilized provided comparable
conductivity detection limits are achieved and adequate baseline stability is
attained as measured by a combined baseline drift/noise of no more than 5 nS
per minute over the background conductivity. The suppressor must be able to
withstand approximately 80 -120 psi back pressure which results from
connecting the postcolumn hardware to the eluent out side of the suppressor.
6.1.3.1 The ASRS was set to perform electrolytic suppression at a current
setting of 100 mA using the external water mode. Insufficient baseline
stability was observed on the conductivity detector using the ASRS in
recycle mode.
6.1.3.2 This method was developed as a multiple component procedure
employing both suppressed conductivity and postcolumn UV/VIS
absorbance detectors in series. If a laboratory is exclusively interested
in monitoring trace bromate using the PCR and the UV/VTS
absorbance detector, the suppressor may not be required. The
performance data presented within this method for the PCR and
UV/VIS absorbance detector, is based upon a suppressed mobile phase
system. A laboratory must generate comparable data as a result of a
complete IDC (Section 9.2) in order to demonstrate comparability of a
non suppressed system.
6.1.4 Detector - Conductivity cell (Dionex CD20, or equivalent) capable of providing
data as required in Section 9.2.
6.1.5 Detector - Absorbance detector (Dionex AD20 or equivalent with 10 mm cell
pathlength, equipped with a tungsten source bulb, or equivalent and capable of
measuring absorbance at 450 nm) capable of providing data as required in
Section 9.2.
6.1.6 Postcolumn reagent delivery system (Dionex PC-10, or equivalent),
pneumatically delivers the postcolumn reagent to mixing tee. The pressure
settings will need to be established on an individual basis for each specific
instrument configuration and at a level which yields the prescribed PCR flow
rates.
6.1.7 Reaction Coil, 500 uL internal volume, knitted, potted or configured to fit
securely in the postcolumn reaction coil heater. (Dionex P/N 39349, or
equivalent).
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6.1.8 Postcolumn Reaction Coil Heater, capable of maintaining 60°C. (Dionex PCH-
2, or equivalent).
6.2 Data System - The Dionex Peaknet Data Chromatography Software was used to
generate all the data in the attached tables. Other computer based data systems may
achieve approximately the same MDLs but the user must demonstrate this by the
procedure outlined in Section 9.2.
6.3 Analytical balance — Used to accurately weigh target analyte salts for stock standard
preparation (±0.1 mg sensitivity).
6.4 Top loading balance - Used to accurately weigh reagents to prepare eluents (±10 mg
sensitivity).
6.5 Weigh boats - Plastic, disposable - for weighing eluent reagents.
6.6 Syringes - Plastic, disposable, 10 mL - used during sample preparation.
6.7 Pipets - Pasteur, plastic or glass, disposable, graduated, 5 mL and 10 mL.
6.8 Bottles - High density polyethylene (HOPE), opaque or glass, amber, 30 mL, 125 mL,
250 mL, used for sample collection and storage of calibration solutions. Opaque or
amber bottles are required due to the photoreactivity of the chlorite anion.
6.9 Micro beakers — Plastic, disposable - used during sample preparation.
6.10 Particulate filters - Gehnan ion chromatography Acrodisc 0.45 micron (PN 4485)
syringe filters or equivalent. These cartridges are used to remove particulates and
[Fe(OH)3(s)] which is formed during the oxidation-reduction reaction between Fe (H)
and C1O2".
6.11 Hydrogen cartridges - Dionex OnGuard-H cartridges (PN 039596) or equivalent.
These cartridges are conditioned according to the manufacturer's directions and are
used to protect the analytical column and the suppressor membrane by removing excess
ferrous iron [Fe (H)]. The ferrous iron is added to field samples to reduce chlorite
levels prior to analysis of chlorine dioxide disinfected water samples.
7. REAGENTS AND STANDARDS
7.1 Reagent water - Distilled or deiom'zed water 18 M Q or better, free of the anions of
interest. Water should contain particles no larger than 0.20 microns.
7.2 Eluent solution - Sodium carbonate (CASRN 497-19-8) 9.0 mM. Dissolve 1.91 g
sodium carbonate (NajC^) in reagent water and dilute to 2 L.
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7.2.1 This eluent solution must be purged for 10 minutes with helium prior to use to
remove dissolved gases which may form micro bubbles in the 1C compromising
system performance and adversely effecting the integrity of the data.
Alternatively, an in-line degas apparatus may be employed.
7.3 Stock standard solutions, 1000 mg/L (1 mg/mL) — Stock standard solutions may be
purchased as certified solutions or prepared from ACS reagent grade, potassium or
sodium salts as listed below, for most analytes. Chlorite requires careful consideration
as outlined below in Section 7.3.4.1.
7.3.1 Bromide (Br ) 1000 mg/L - Dissolve 0.1288 g sodium bromide (NaBr, CASRN
7647-15-6) in reagent water and dilute to 100 mL in a volumetric flask.
7.3.2 Bromate (BrO3~) 1000 mg/L — Dissolve 0.1180 g of sodium bromate (NaBrO3,
CASRN 7789-38-0) in reagent water and dilute to 100 mL in a volumetric flask.
7.3.3 Chlorate (CICy) 1000 mg/L - Dissolve 0.1275 g of sodium chlorate (NaClO3,
CASRN 7775-09-9) in reagent water and dilute to 100 mL in a volumetric flask.
7.3.4 Chlorite (C1O2~) 1000 mg/L - If the amperometric titration of the technical
grade sodium chlorite (NaC 102), specified in 7.3.4.1, had indicated the purity of
the salt to be 80.0 % NaClO2, the analyst would dissolve 0.1676 g of sodium
chlorite (NaClO2, CASRN 7758-19-2) in reagent water and dilute to 100 mL in
a volumetric flask.
7.3.4.1 High purity sodium chlorite (NaCIO 2) is not currently commercially
available due to its potential explosive instability. Recrystallization of
the technical grade (approx. 80%) can be performed but it is labor
intensive and time consuming. The simplest approach is to determine
the exact purity of the NaCIO 2 using the iodometric titration
procedure.10 Following titration, an individual component standard of
chlorite must be analyzed to determine if there is any significant
contamination (greater than 1% of the chlorite weight) from chlorate,
bromate or bromide (as other method target anions) in the technical
grade chlorite standard.
NOTE: Stability of standards - Stock standards (Section 7.3) for most
anions are stable for at least 6 months when refrigerated at <6°C. The
chlorite standard is only stable for two weeks when stored refrigerated
at <6°C and protected from light. Dilute working standards should be
prepared monthly, except those that contain chlorite, which must be
prepared every two weeks or sooner if signs of degradation are
indicated by repeated QC failure.
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7.4 Ethylenediamine (EDA) preservation solution, 100 mg/mL - Dilute 2.8 mL of
ethylenediamine (99%) (CASRN 107-15-3) to 25 mL with reagent water. Prepare fresh
monthly.
7.5 Surrogate Solution, 0.50 mg/mL dichloroacetate (DCA) - Prepare by dissolving 0.065 g
dichloroacetic acid, potassium salt (C12CHCO2K, CASRN 19559-59-2) in reagent water
and diluting to 100 mL in a volumetric flask.
7.5.1 Dichloroacetate is potentially present in treated drinking waters as the acetate
of the organic disinfection byproduct, dichloroacetic acid (DCAA). Typical
concentrations of DCAA rarely exceed 50 jig/L, which, for this worst case
example, would represent only a five percent increase in the observed
response over the fortified concentration of 1.00 mg/L. Consequently, the
criteria for acceptable recovery (90% to 115%) for the surrogate is weighted to
115% to allow for this potential background.
7.5.2 Prepare this solution fresh every 3 months or sooner if signs of degradation are
indicated by the repeated failure of the surrogate QC criteria.
7.5.3 If the analyst is exclusively interested in monitoring trace bromate using the
PCR and the UV/VIS absorbance detector, the surrogate may be omitted since it
only yields a signal on the conductivity detector. If the surrogate is removed,
the laboratory must adhere to the alternate QC requirements found in Section
9.3.3.3 hi order to monitor and demonstrate proper instrument performance.
7.6 Postcolumn reagent - The postcolumn reagent is prepared by adding 40 mL of 70%
redistilled nitric acid (purity as 99.999+%, Aldrich, Cat. No. 22,571-1, Milwaukee, WI,
or equivalent) to approximately 300 mL reagent water in a well rinsed 500 mL
volumetric flask (see Note) and adding 2.5 grams of ACS reagent grade KBr (Sigma,
Cat. No. P-5912, St. Louis, MO, or equivalent). Two-hundred-and-fifty milligrams of
purified grade o-dianisidine, dihydrochloride salt [(ODA), (Sigma, Cat. No. D-3252, or
equivalent)] are dissolved, with stirring, in 100 mL methanol (Spectrophotometric
grade, Sigma, Cat. No. M-3641, St. Louis MO, or equivalent). After dissolution, the o-
dianisidine solution is added to the nitric acid/KBr solution and diluted to volume with
reagent water. The reagent is stable for up to one month.
7.6.1 The purity of all reagents employed in the preparation of the postcolumn reagent
is critical. Some commercial manufacturers/suppliers of laboratory chemicals
sell inferior grades of o-dianisidine dihydrochloride. ONLY the purified grade
of this reagent is acceptable. The purified ODA dihydrochloride salt is a white,
fine crystalline powder.
NOTE: All glassware used to prepare the postcolumn reagent must be
thoroughly rinsed with reagent water prior to use. A champagne or light amber
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coloration of the PCR reagent may be evident when freshly prepared. Over
several hours, this slight coloration will fade. Consequently, the PCR must be
prepared in advance and allowed to sit until it is clear, for a minimum of 4 hours
(preferably overnight) prior to use. Occasionally, no matter how well all the
glassware used to prepare the postcolumn reagent is rinsed, a darkly colored
solution (oxidized ODA) may result. These solutions MUST be discarded. For
this reason, it is recommended that the PCR be made in a series of 500 mL lots
with dedicated glassware. The clear solution should be filtered using a 0.45
micron membrane to remove particulates before use.
7.7 Ferrous iron [1000 mg/L Fe (II)] solution - Dissolve 0.124 g ferrous sulfate
heptahydrate (FeSO4.7H2O, Sigma, F-7002) in approximately 15 mL reagent water
containing 6 uL concentrated nitric acid and dilute to 25 mL with reagent water in a
volumetric flask (final pH ~2). The Fe (IT) solution must be prepared fresh every two
days.
7.8 Sulfuric acid (0.5 N) - Dilute 1.4 mL of concentrated sulfuric acid (Fisher Scientific
Certified ACS Plus, A 300-500) to 100 mL.
8. SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 Samples should be collected in plastic or glass bottles. All bottles must be thoroughly
cleaned and rinsed with reagent water. The volume collected should be sufficient to
insure a representative sample, allow for replicate analysis and laboratory fortified
matrix analysis, if required, and minimize waste disposal.
8.2 Special sampling requirements and precautions for chlorite.
8.2.1 Sample bottles used for chlorite analysis must be opaque or amber to protect the
sample from light.
8.2.2 When preparing the LFM, be aware that chlorite is an oxidant and may react
with the natural organic matter in an untreated drinking water matrix as a result
of oxidative demand. If untreated water is collected for chlorite analysis, and
subsequently used for the LFM, EDA preservation will not control this demand
and reduced chlorite recoveries may be observed.
8.3 Sample preservation and holding times for the anions are as follows:
Analyte Preservation Holding Time
Bromate 50 mg/L EDA, refrigerate at <6°C 28 days
Chlorate 50 mg/L EDA, refrigerate at <6°C 28 days
. Chlorite 50 mg/L EDA, refrigerate at <6°C 14 days
Bromide (source/raw water only) EDA permitted, refrigerate at <6°C 28 days
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NOTE: Samples for chlorite analysis must arrive at the laboratory within 48 hours of
collection and must be received at 10°C or less.
8.4 When collecting a field sample from a treatment plant employing chlorine dioxide, the
field sample must be sparged with an inert gas (helium, argon, nitrogen) prior to
addition of the EDA preservative at time of sample collection.
8.5 All four anions (bromate > 15.0 ug/L) can be analyzed by conductivity, in a sample
matrix which has been preserved with EDA. Add a sufficient volume of the EDA
preservation solution (Section 7.4) such that the final concentration is 50 mg/L in the
sample. This would be equivalent to adding 0.5 mL of the EDA preservation solution
to 1 L of sample.
8.6 Chlorite is susceptible to degradation both through catalytic reactions with dissolved
iron salts and reactivity towards free chlorine which exists as hypochlorous
acid/hypochlorite ion in most drinking water as a residual disinfectant.11 EDA serves a
dual purpose as a preservative for chlorite by chelating iron as well as any other
catalytically destructive metal cations and removing hypochlorous acid/hypochlorite ion
by forming an organochloramine. EDA preservation of chlorite also preserves the
integrity of chlorate which can increase in unpreserved samples as a result of chlorite
degradation. EDA also preserves the integrity of bromate concentrations by binding
with hypobromous acid/hypobromite ion which is an intermediate formed as a by-
product of the reaction of either ozone or hypochlorous acid/hypochlorite ion with
bromide ion. If hypobromous acid/hypobromite ion is not removed from the matrix,
further reactions may form bromate ion.
9. QUALITY CONTROL
9.1 Each laboratory using this method is required to operate a formal quality control (QC)
program. The requirements of this program consist of an initial demonstration of
laboratory capability (IDC), and subsequent analysis in each analysis batch (Section
3.1) of a Laboratory Reagent Blank (LRB), Initial Calibration Check Standard (ICCS),
Laboratory Fortified Blank (LFB), Instrument Performance Check Standard (D?C),
Continuing Calibration Check Standards (CCCS), Laboratory Fortified Sample Matrix
(LFM) and either a Field, Laboratory or LFM duplicate sample analysis. This section
details the specific requirements for each of these QC parameters for both the
conductivity and absorbance detectors used in this application. Although this method
involves both conductivity and absorbance detection, the MDLs and MRLs may differ
but the QC requirements and acceptance criteria are the same for both detectors. The
QC criteria discussed in the following sections are summarized in Section 17, Tables 4
and 5. The laboratory is required to maintain performance records that define the
quality of the data that are generated.
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9.2 INITIAL DEMONSTRATION OF CAPABILITY
9.2.1 The Initial Demonstration of Capability (IDC) - This is used to characterize
instrument and laboratory performance prior to performing analyses by this
method. The QC requirements for the IDC discussed in the following section
are summarized in Section 17, Table 4.
9.2.2 Initial demonstration of low system background-Section 9.3.1.
9.2.3 Initial Demonstration of Precision (TOP) - For the 4 conductivity detector
analytes, prepare 7 replicate LFBs fortified at a recommended concentration of
20 ug/L. For the absorbance detector, prepare 7 replicate LFBs fortified at a
recommended concentration of 2.0 ug/L bromate. The percent relative standard
deviation (RSD) of the results must be less than 20%.
9.2.4 Initial Demonstration of Accuracy (IDA) - Using the data generated for Section
9.2.3, calculate the average recovery. The average recovery of the replicate
values must be within ± 15% of the true value.
9.2.5 Quality Control Sample (QCS) - After calibration curves have initially been ,
established or have been re-established, on a quarterly basis or as required to
meet data quality needs, verify both the calibration and acceptable instrument
performance with the preparation and analyses of an external/second source
QCS. If the determined concentrations are not within ± 20% of the stated
values, performance of the method is unacceptable. The source of the problem
must be identified and corrected before proceeding with the IDC.
9.2.6 Method Detection Limit (MDL) — MDLs must be established for all analytes,
using reagent water (blank) fortified at a concentration of three to five times the
estimated instrument detection limit.4 To determine MDL values, take seven
replicate aliquots of the fortified reagent water and process through the entire
analytical method. The replicates must be prepared and analyzed over three
days. Report the concentration values in the appropriate units. Calculate the
MDL as follows:
MDL = (t) x (S)
where, t = student's t value for a 99% confidence level and a standard
deviation estimate with n-1 degrees of freedom
[t = 3.14 for seven replicates], and
S = standard deviation of the replicate analyses.
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9.2.6.1 MDLs should be periodically verified, but MUST be initially
determined when a new operator begins work or whenever there is a
significant change in the background, or instrument response.
NOTE: Do not subtract blank values when performing MDL
calculations.
9.2.7 Minimum Reporting Level (MRL) - The MRL is the threshold concentration of
an analyte that a laboratory can expect to accurately quantitate in an unknown
sample. The MRL should be established at an analyte concentration either
greater than three times the MDL or at a concentration which would yield a
response greater than a signal to noise ratio of five. Setting the MRL too low
may cause repeated QC failure upon analysis of the ICCS. Although the lowest
calibration standard may be below the MRL, the MRL must never be
established at a concentration lower than the lowest calibration standard.
9.3 ASSESSING LABORATORY PERFORMANCE
9.3.1 Laboratory Reagent Blank (LRB) - The laboratory must analyze at least one
LRB with each analysis batch (Section 3.1). Data produced are used to assess
contamination from the laboratory environment. Values that exceed 1A the MRL
indicate a laboratory or reagent contamination is present. If a method analyte is
observed hi the LRB it must not exceed 1A the MRL. Analytes that exceed this
level will invalidate the analysis batch for that method analyte in all
corresponding field samples.
9.3.1.1 EDA must be added to the LRB at 50 mg/L. By including EDA in the
LRB, any bias as a consequence of the EDA which may be observed in
the field samples, particularly in terms of background contamination,
will be identified.
9.3.1.2 When the PCR method is used for low level bromate analysis on
samples from public water systems (PWSs) which employ chlorine
dioxide disinfection, the matrix must be pretreated to remove the
potentially interferant chlorite anion (Section 11.1.4). When these
types of pretreated samples, or any type of pretreatment is applied to
field samples included as part of an analysis batch, a second LRB must
be prepared, pretreated and analyzed to confirm no background effects
of the pretreatment are present. If the analysis batch contains only
pretreated samples, then only a pretreated LRB is required.
9.3.2 Laboratory Fortified Blank (LFB) - Prepare a secondary dilution stock using the
same stock solutions used to prepare the calibration standards and the LFM
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fortification solution. Since calibration solutions are prepared in large volumes
and can be used over an extended period of time, the integrity of the
concentration of the solution used to fortify the LFM is checked by analyzing
the LFB. The recovery of all analytes must fall in the acceptable recovery range,
as indicated below, prior to analyzing samples. If the LRB recovery for an
analysis batch does not meet these recovery criteria the data are considered
invalid, and the source of the problem must be identified and resolved before
continuing with analyses.
LFB Fortified Concentration range LFB Percent Recovery Limits
MRLtoSxMRL 75-125%
5 x MRL to highest calibration level 85 - 1 1 5 %
9.3.2.1 EDA must be added to the LFB at 50 mg/L. The addition of EDA to
all reagent water prepared calibration and quality control samples is
required not as a preservative but rather as a means to normalize any
bias attributed by the presence of EDA in the field samples.
9.3.3 Instrument Performance Check (IPC) - The Initial Calibration Check Standard
(ICCS) is to be evaluated as the IPC solution in order to confirm proper
instrument performance. As specified in Section 10.3.1, this must be done using
the lowest calibration standard or the standard level established as the MRL.
This analysis confirms the MRL and demonstrates proper chromatographic
performance at the beginning of each analysis batch. Chromatographic
performance is judged by calculating the Peak Gaussian Factor (PGF), which is
a means to measure peak symmetry and monitoring retention time drift in the
surrogate peak over time. If these criteria are not met, corrective action must be
performed prior to analyzing additional samples. Major maintenance like
replacing columns require rerunning the IDC (Section 9.2).
9.3.3.1 Critically evaluate the surrogate peak in the initial calibration check
standard, and calculate the PGF as follows:
1.83 x WO/2)
PGF = -----------------------
W (V10)
where, W(l/z) is the peak width at half height, and
W (V10) is the peak width at tenth height.
NOTE: Values for WO/a) and W (V10) can be attained through most
data acquisition software.
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9.3.3.2 Small variations in retention time can be anticipated when a new
solution of eluent is prepared but if sudden shifts of more than 5% are
observed in the surrogate retention time, some type of instrument
problem is present. Potential problems include improperly prepared
eluent, erroneous method parameters programmed such as flow rate or
some other system problem. The chromatographic profile (elution
order) of the target anions following an ion chromatographic analysis
should closely replicate the profile displayed in the test chromatogram
that was shipped when the column was purchased. As a column ages, it
is normal to see a gradual shift and shortening of retention times, but if
after several years of use, extensive use over less than a year, or use
with harsh samples, this retention tune has noticeably shifted to any
less than 80% of the original recorded value, the column requires
cleaning or replacement; especially if resolution problems are
beginning to become common between previously resolved peaks. A
laboratory should retain a historic record of retention times for the
surrogate and all the target anions to provide evidence of an analytical
columns vitality. . .
9.3.3.3 If a laboratory chooses to monitor exclusively for trace bromate using
PCR and the UV/VIS absorbance detector, and no other analytes are
being monitored on the conductivity detector, the surrogate may be
omitted from the procedure. In this case, no measurement of PGF is
required. However, the laboratory must carefully monitor the bromate
retention time in the ICCS as an alternate to the surrogate retention
time and, in the same manner, adhere to those specifications set forth
in Section 9.3.3.2. During the course of the analysis, bromate retention
times in the CCCS and ECCS must also be closely monitored to be
certain they adhere to the QC requirements set forth hi Section
10.3.2.2.
9.4 ASSESSING ANALYTE RECOVERY AND DATA QUALITY
9.4.1 Laboratory Fortified Sample Matrix (LFM) - The laboratory must add a known
amount of each target analyte to a minimum of 5% of the collected field samples
or at least one with every analysis batch, whichever is greater. Additional LFM
requirements, as described in Section 9.4.1.5, apply when the PCR system is
used for low level bromate in chlorine dioxide disinfected waters. For a LFM to
be valid, the target analyte concentrations must be greater than the native level
and must adhere to the requirement outlined in Section 9.4.1.2. It is
recommended that the solutions used to fortify the LFM be prepared from the
same stocks used to prepare the calibration standards and not from external
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source stocks! This will remove the bias contributed by an externally prepared
stock and focus on any potential bias introduced by the field sample matrix.
9.4. f.1 The fortified concentration must be equal to or greater than the native
concentration. Fortified samples that exceed the calibration range
must be diluted to be within the linear range. In the event that the
v ' fortified level is less than the observed native level of the unfortified
matrix, the recovery should not be calculated. This is due to the
difficulty in calculating accurate recoveries of the fortified
concentration when the native sample concentration to fortified
concentration ratio is greater than one.
9.4.1 .2 The LFM should be prepared at concentrations no greater than ten
times the highest concentration observed in any field sample and
should be varied to reflect the range of concentrations observed in field
samples. If no analytes are observed in any field sample, the LFM
should be fortified near the MRL.
9.4. 1 .3 Calculate the percent recovery for each target analyte, corrected for
" concentrations measured in the unfortified sample. Percent recovery
should be calculated using the following equation:
%REC = — -— — xlOO
where, %REC = percent recovery,
Cs= fortified sample concentration,
C = native sample concentration, and
s = concentration equivalent of analyte added to sample.
9.4. 1 .4 Recoveries may exhibit a matrix dependence. If the recovery of any
analyte falls outside 75 - 125%, and the laboratory's performance for
• all other QC performance criteria are acceptable, 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
and the LFM must be labeled suspect/matrix to inform the data user
that the result is suspect due to matrix effects. Repeated failure to
meet suggested recovery criteria indicates potential problems with the
pjrocedure and should be investigated.
9.4.1.5 When the PCR method is used for low level bromate analysis on field
samples from PWSs which employ chlorine dioxide disinfection and
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consequently contain chlorite, a LFM must be prepared, exclusively
for trace bromate, for each of these field samples. Initially, the field
, sample is analyzed and chlorite, chlorate and bromide levels are
determined. Then, a second aliquot of field sample is pretreated to
remove chlorite, as described in Section 11.1.4, and analyzed to
determine native bromate concentration. A third aliquot of the field
sample then must be fortified with bromate, pretreated as described in
Section 11.1.4 to remove chlorite, and analyzed to assess bromate
recovery from that matrix. This additional QC is required to rule out
matrix effects and to confirm that the laboratory performed the chlorite
removal step (Section 11.1.4.1) appropriately. This LFM should be
fortified with bromate at concentrations close to but greater than the
level determined in the native sample. Recoveries are determined as
described above (Section 9.4.1.3). Samples that fail the LFM percent
recovery criteria of 75 -125% must be reported as suspect/matrix.
9.4.2 SURROGATE RECOVERY - The surrogate is specific to the conductivity
detector and shows no response on the postcolumn absorbance detector.
Calculate the surrogate recovery for the conductivity detector from all analyses
using the following formula:
SRC1
%REC = ---—--- x 100
SFC
where, %REC = percent recovery,
SRC = surrogate recovered concentration, and
SFC = surrogate fortified concentration.
9.4.2.1 Surrogate recoveries must fall between 90-115% for proper instrument
performance and analyst technique to be verified. The recovery range
for the surrogate is extended to 115% to allow for the potential
contribution of trace levels of dichloroacetate as a halogenated organic
disinfection by-product (DBF) of dichloroacetic acid (DCAA).
Background levels of this organic DBF are rarely observed above 50
Hg/L (0.05 mg/L) which constitutes only 5% of the 1.00 mg/L
recommended fortified concentration.
9.4.2.2 If the surrogate recovery falls outside the 90-115% recovery window,
an analysis error is evident and sample reanalysis is required. Poor
recoveries could be the result of imprecise sample injection or analyst
fortification errors. If the second analysis also fails the recovery
criterion, report all data for that sample as suspect.
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9.4.2.3 If a laboratory chooses to monitor exclusively for trace bromate using
PCR and the UV/VIS absorbance detector, and no other analytes are
being monitored on the conductivity detector, the surrogate may be
omitted from the procedure. In this situation, the laboratory MUST
adopt the QC protocol outlined in Section 9.3.3.3.
9.4.3 FIELD OR LABORATORY DUPLICATES - The laboratory must analyze
either a field or a laboratory duplicate for a minimum of 5% of the collected
field samples or at least one with every analysis batch, whichever is greater.
The sample matrix selected for this duplicate analysis must contain measurable
concentrations of the target anions in order to establish the precision of the
analysis set and insure the quality of the data. If none of the samples within an
analysis batch have measurable concentrations, the LFM should be repeated as a
laboratory duplicate.
9.4.3 . 1 Calculate the relative percent difference (RPD) from the mean using
the following formula:
RPD = -------------- x 100
(Pc + Dd/2)
where, RPD = relative percent difference
Ic = the initial quantitated concentration, and
Dc = the duplicate quantitated concentration
9.4.3.2 Duplicate analysis acceptance criteria.
Concentration range RPD Limits
MRL to 5 x MRL ± 20 %
5 x MRL to highest calibration level ± 10 %
9.4.3.3 If the RPD for any target analyte falls outside the acceptance criteria
(Section 9.4.3.2) and if all other QC performance criteria are met for
that analyte, the result for the sample and duplicate should be labeled
as suspect/matrix to inform the data user that the result is suspect due
to a potential matrix effect, which led to poor precision. This should
not be a chronic problem and if it frequently recurs (>20% of duplicate
analyses), it indicates a problem with the instrument or analyst
technique that must be corrected.
9.4.4 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options, such as the use of different columns, injection
volumes, and/or eluents, to improve the separations or lower the cost of
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measurements. Each time such modifications to the method are made, the
analyst is required to repeat the procedure in Section 9.2 and adhere to the
condition of conductivity baseline stability found in Section 1.2.1.
9.4.5 It is recommended that the laboratory adopt additional quality assurance (QA)
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. Whenever possible, the laboratory should perform analysis of quality
control check standards and participate in relevant proficiency testing (PT) or
performance evaluation (PE) sample studies.
10. CALIBRATION AND STANDARDIZATION
10.1 Demonstration and documentation of acceptable initial calibration is required prior to
the IDC and before any samples are analyzed, is required intermittently throughout
sample analysis to meet required QC performance criteria outlined in this method and is
summarized in Tables 4 and 5. Initial calibration verification is performed using a QCS
as well as with each analysis batch using an initial, continuing (when more than 10 field
samples are analyzed), and end calibration standards. The procedures for establishing
the initial calibration curve are described in Section 10.2. The procedures to verify the
calibration with each analysis batch is described in Section 10.3.
10.2 INITIAL CALIBRATION CURVE
10.2.1 Establish ion chromatographic operating parameters equivalent to those
indicated in Table 1 and configured as shown in Figure 1.
10.2.2 Estimate the Linear Calibration Range - The linear concentration range is the
concentration range over which the instrument response is linear. On the
conductivity detector for the four target analytes (chlorite, bromate, bromide and
chlorate) the linear range should cover the expected concentration range of the
field samples and should not extend over more than two orders of magnitude in
concentration. The restriction of two orders of magnitude is prescribed since
beyond this it is difficult to maintain linearity throughout the entire calibration
range.
10.2.2.1 If quantification is desired over a larger range, then two separate
calibration curves must be prepared.
10.2.2.2 For an individual calibration curve, a minimum of three calibration
standards are required for a curve that extends over a single order of
magnitude and a minimum of five calibration standards are required if
the curve covers two orders of magnitude. Because high
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concentrations of chlorite can interfere with the postcolumn analysis of
low levels of bromate, the conductivity and absorbance detectors must
be calibrated separately.
10.2.2.3 Since the concentration ranges in actual field samples by conductivity
detection for chlorite, bromide and chlorate are expected to cover two
orders of magnitude, the use of at least five calibration standards in the
range 5-500 [igTL is recommended. Bromate concentrations are
expected to be significantly lower. It is suggested that the conductivity
detector be calibrated using at least five bromate calibration standard
levels in the range 5-100 u.g/L. Additionally, report values for
bromate by conductivity ONLY when they are measured by the PCR
above 15.0 ug/L. The conductivity detector will observe a response for
bromate at concentration below 15.0 ug/L but concentrations between
5.0 and 15.0 ug/L are within the calibrated range for PCR detection
and will reflect far better precision and accuracy.
10.2.2.4 Although the bromate calibration curve for the absorbance detector
extends over less than two orders of magnitude, the use of five
calibration standards, containing only bromate in the range 0.5 -15.0
|ig/L, is recommended.
10.2.3 Prepare the calibration standards by carefully adding measured volumes of one
or more stock standards (Section 7.3) to a volumetric flask and diluting to
volume with reagent water. Prior to using mixed standards for calibration, it
must be ensured that the individual calibration standards do not contain any
appreciable concentrations of the other target analytes.
10.2.3.1 EDA must be added to the calibration standards at 50 mg/L. The
addition of EDA to all reagent water prepared calibration and quality
control samples is required not as a preservative but rather as a means
to normalize any bias contributed by the addition of EDA to preserve
the field samples.
10.2.3.2 Prepare a 10.0 mL aliquot of surrogate fortified calibration solution
which can be held for direct manual injection or used to fill an
autosampler vial. This is done by adding 20 uL of the surrogate
solution (Section 7.5) to a 20 mL disposable plastic micro beaker.
Next, transfer 10.0 mL of calibration standard into the micro beaker
and mix. These volumes may be adjusted to meet specific laboratory
autosampler volume requirements provided the fortified surrogate
concentration is at the prescribed concentration of 1.0 mg/L. The
calibration standard is now ready for analysis. The same surrogate
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solution that has been employed for the standards should also be used
in Section 11.1 for the field samples.
NOTE: This surrogate fortification procedure may be omitted if a
laboratory chooses to monitor exclusively for trace bromate using PCR
and the UV/VIS absorbance detector, and no other analytes are being
monitored on the conductivity detector. Li this situation, the
laboratory must adopt the QC protocol outlined in Section 9.3.3.3.
10.2.4 Inject 225 \iL of each calibration standard. Increased sensitivity for low level
detection of bromate by PCR can be achieved by increasing the injected sample
volume.4 If the injection volume is increased special operating conditions must
be used to insure proper chromatographic performance.4
10.2.5 Tabulate peak area responses against the concentration for the four target
analytes, the surrogate from the conductivity detector and bromate from the
postcolumn absorbance detector. The results are used to prepare calibration
curves using linear regression analysis for each analyte on the conductivity
detector and using a quadratic regression analysis for bromate on the absorbance
detector.
10.2.5.1 Use of peak areas are strongly recommended since they have been
found to be more consistent, in terms of quantitation, than peak
heights. Peak height can tend to be suppressed as a result of high
levels of common anions in a given matrix which can compete for
exchange sites leading to peak broadening. Using peak areas, it is the
analyst responsibility to review all chromatograms to insure accurate
baseline integration of target analyte peaks, since poorly drawn
baselines will more significantly influence peak areas than peak
heights.
10.2.6 After establishing or reestablishing calibration curves, the accuracy of this
calibration must be verified through the analysis of a QCS or an externally
prepared second source standard. The QCS should be prepared at a
concentration near the middle of the calibration and is best to be analyzed in
triplicate. As specified in Section 9.2.5, determined concentrations must fall
within ± 15% of the stated values.
10.3 CONTINUING CALIBRATION VERIFICATION - Initial calibrations may be stable
for extended periods of time. Once the calibration curves have been established for
both the conductivity and absorbance detectors, they must be verified for each analysis
batch, prior to conducting any field sample analyses using an Initial Calibration Check
Standard. Continuing Calibration Check Standards and End Calibration Check
Standards are also required as described in the sections below.
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10.3.1 INITIAL CALIBRATION CHECK STANDARD (ICCS) - The initial
calibration must be determined to be valid each day prior to analyzing any
samples. Since two detectors are incorporated in this method, this must be
accomplished by using a mixed calibration check standard for the four
conductivity analytes and a separate low level bromate initial calibration check
standard for the absorbance detector, hi both cases, the lowest level standard
used to prepare the calibration curve must be used. In cases where the analyst
has chosen to set the MRL above the lowest standard, a standard at a
concentration equal to or below the MRL is acceptable. Percent recovery for the
ICCS must be in the range or 75 -125% before the analyst is allowed to analyze
samples.
10.3.2 CONTINUING CALIBRATION CHECK/END CALIBRATION CHECK
STANDARDS (CCCS/ECCS) - Continuing calibration check standards must
be analyzed after every tenth field sample analysis and at the end of the analysis
batch as an end calibration check standard. For the reasons noted above, two
separate continuing and end calibration check standards must be incorporated.
If more than 10 field samples are included in an analysis batch, the analyst must
alternate between the middle and high continuing calibration check standard
levels.
10.3.2.1 The percent recovery for the CCCS/ECCS must meet the following
criteria:
Concentration range Percent Recovery Limits
MRL to 5 x MRL 75 - 125 %
5 x MRL to highest calibration level 85 -115 %
10.3.2.2 If during the analysis batch, the measured concentration on either
detector differs by more than the calibration verification criteria shown
above, or the retention times shift more than ± 2% from the last
acceptable initial or continuing calibration check standard for any
analyte, all samples analyzed after the last acceptable calibration check
standard are considered invalid and must be reanalyzed. The source of
the problem must be identified and resolved before reanalyzing the
samples or continuing with the analyses.
10.3.2.3 In the case where the end calibration failed to meet performance
criteria, but the initial and middle calibration check standards were
acceptable, the samples bracketed by the acceptable calibration check
standards may be reported. However, all field samples between the
middle and end calibration check standards must be reanalyzed.
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11. PROCEDURE
11.1 SAMPLE PREPARATION
11.1.1 For refrigerated or field samples arriving to the laboratory cold, ensure the
samples have come to room temperature prior to conducting sample analysis by
allowing the samples to warm on the bench for at least 1 hour.
11.1.2 Prepare a 10.0 mL aliquot of surrogate fortified sample which can be held for
direct manual injection or used to fill an autosampler vial. This is done by
adding 20 \iL of the surrogate solution (Section 7.5) to a 20 mL disposable
plastic micro beaker. Next, place a 10.0 mL aliquot of sample into the micro
beaker and mix. These volumes may be adjusted to meet specific laboratory
autosampler volume requirements provided the fortified surrogate concentration
is at the prescribed concentration of 1.0 mg/L. The sample is now ready for
analysis.
NOTE: The less than 1% dilution error introduced by the addition of the
surrogate is considered insignificant. In addition, this surrogate fortification
procedure maybe omitted if a laboratory chooses to monitor exclusively for
trace bromate using PCR and the UV/VIS absorbance detector, and no other
analytes are being monitored on the conductivity detector. In this situation, the
laboratory must adopt the QC protocol outlined in Section 9.3.3.3.
11.1.3 Using a Luer lock, plastic 10 mL syringe, withdraw the sample from the micro
beaker and attach a 0.45 |im particulate filter (demonstrated to be free of ionic
contaminants) directly to the syringe. Filter the sample into an autosampler vial
(if vial is not designed to automatically filter) or manually load the injection
loop injecting a fixed amount of filtered, well mixed sample. If using a
manually loaded injection loop, flush the loop thoroughly between sample
analysis using sufficient volumes of each new sample matrix.
11.1.4 CHLORINE DIOXIDE - TREATED WATERS CONTAINING CHLORITE -
Treatment plants that use chlorine dioxide as part of their treatment process can
produce high levels of chlorite in samples. Since chlorite can interfere with the
postcolumn quantitation of low levels of bromate as described in Section 4.6,
chlorite must be removed from these samples prior to analysis.12 The oxidation-
reduction reaction between ferrous iron and chlorite13 is used to remove chlorite
without any adverse affects on the bromate concentration.14 The EDA stabilized
sample is acidified to apH of 5-6 (verified using pH test strips), ferrous iron
solution is added and allowed to react for 10 minutes. The sample is then
filtered using a 0.45 micron membrane to remove precipitated ferric hydroxide
and the excess soluble iron is removed by passing the filtered sample through a
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hydrogen cartridge [a solid phase extraction (SPE) clean-up cartridge in the H+
form, (Section 6.11)], prior to analysis. Prior to using any pretreatment, each lot
of cartridges must be QC checked to insure proper analyte recoveries are
maintained and laboratory reagent blanks are free from interferences. In
addition, consistent lots of reagents, pretreatment cartridges, and membrane
cartridges must be used throughout an entire analysis batch to maintain assured
QC uniformity.
11.1.4.1 Place a 10 mL aliquot of sample in a 20 mL micro beaker and add 3 5
uL of 0.5 N sulfuric acid (Section 7.8). After mixing, verify the pH is
between 5 and 6 using pH test strips, add 40 uL of ferrous iron solution
(Section 7.7), mix and allow to react for 10 minutes. Filter the
reaction mixture using a 0.45 micron particulate filter (Section 6.10)
attached to a 10 mL syringe into the barrel of a second syringe to
which a pre-conditioned hydrogen cartridge (Section 6.11) is attached.
Pass the solution through a hydrogen cartridge at a flow rate of
approximately 2 mL per minute. Discard the first 3 mL, and collect an
appropriate volume (depending on autosampler vial size) for analysis.
Add the respective volume of surrogate solution, depending on the
volume collected, and the sample is ready for analysis.
NOTE: Pretreated samples can be held for no more than 30 hours after
initial pretreatment. If this time has expired, the pretreatment steps
must be repeated on a second aliquot of both the field sample matrix
and the respective LFM.
11.1.4.2 In order to ensure data quality, all samples from PWSs which utilize
chlorine dioxide which have been pretreated to remove chlorite,
MUST also be used to prepare a pretreated LFM specific to trace
bromate. This LFM should be fortified with bromate at concentrations
close to but greater than the level determined in the native sample.
Initially, the field sample is analyzed and chlorite, chlorate and
. bromide levels are determined. Then, a second aliquot of field sample
is pretreated to remove chlorite, as described above and analyzed to
determine native bromate concentrations. A third aliquot of the field
sample then must be fortified with bromate, pretreated to remove
chlorite, and analyzed to assess bromate recovery from that matrix.
This additional QC is required to rule out matrix effects and to confirm
that the laboratory performed the chlorite removal step appropriately.
If the bromate recovery falls outside the acceptance range of 75 - 125%
(Section 9.4.1.5), that particular sample should be reported as
suspect/matrix.
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11.1.4.3 Suppressor devices which have had long term exposure to iron cations
may have reduced method performance in other applications, such as
the determination of certain common inorganic anions. If reduced
peak response is observed, particularly for fluoride or phosphate, the
ASRS should be cleaned according to the manufacturer's
recommendations.
11.2 SAMPLE ANALYSIS
11.2.1 Table 1 summarizes the recommended operating conditions for the ion
chromatograph and delivery of the postcolumn reagent. Included in this table is
estimated retention times that can be achieved by this method. Other columns
or chromatographic conditions may be used if the requirements of Section 9.2
are met.
11.2.2 Establish a valid initial calibration as described in Section 10.2 and complete the
IDC (Section 9.2). Check system calibration by analyzing an ICCS (Section
10.3.1) as part of the initial QC for the analysis batch and, if required,
recalibrate as described in Section 10.3.
11.2.3 Inject 225 [iL of each sample. Use the same size loop for standards and
samples. An automated constant volume injection system may also be used.
11.2.3.1 Increased sensitivity for low level detection of bromate by PCR can be
achieved by increasing the inj ected sample volume.4 If the inj ection
volume is increased (Section 10.2.4) special operating conditions must
be used to insure proper chromatographic performance.4
11.2.4 The width of the retention time window used to make identifications should be
based upon measurements of actual retention time variations of standards
measured over several days. Three times the standard deviation of retention
time can be used to calculate a suggested window size for each analyte.
However, the experience of the analyst should weigh heavily in the
interpretation of chromatograms.
11.2.5 If the response of a sample analyte exceeds the calibration range, the sample
must be diluted with an appropriate amount of EDA fortified reagent water and
reanalyzed. If this is not possible then three new calibration concentrations
must be employed to create a separate high concentration calibration curve, one
standard near the estimated concentration and the other two bracketing around
an interval equivalent to approximately ± 25% the estimated concentration. The
latter procedure involves significantly more time than a simple sample dilution
and, therefore, it is advisable to collect sufficient sample to allow for sample
dilution and sample reanalysis, if required.
317.0-28
-------
11.2.6 Should more complete resolution be needed between any two coeluting peaks,
the eluent (Section 7.2) can be diluted. This will spread out the run, however,
and will cause late eluting anions to be retained even longer. The analyst must
verify that this dilution does not negatively affect performance by repeating and
passing all the QC criteria in Section 9, and by reestablishing a valid initial
calibration curve (Section 10.2). As a specific precaution, upon dilution of the
carbonate eluent, a peak for bicarbonate may be observed by conductivity within
the retention time window for bromate which will negatively impact the
analysis.
11.2.6.1 Eluent dilution will reduce the overall response of an anion due to
chromatographic band broadening which will be evident by shortened
and broadened peaks. This will adversely effect the MDLs for each
analyte.
11.3 AUTOMATED ANALYSIS WITH METHOD 317.0
11.3.1 Laboratories conducting analyses on large numbers of samples often prepare
large analysis batches that are run in an automated manner. When conducting
automated analyses, careful attention must be paid to all reservoirs to be certain
sufficient volumes are available to sustain extended operation. Laboratories
must ensure that all QC performance criteria are met as described in preceding
sections to ensure their data are of acceptable quality.
11.3.1.1 Special attention must be made when the PCR reservoir is refilled.
Since this is a pneumatically driven system, the baseline will require a
minimum of ten minutes to restabilize after the reservoir has been
refilled and the bottle repressurized.
11.3.2 Because this method has two detectors that require independent calibration,
analysis sequences must be carefully constructed to meet required QC
specifications and frequency (Table 5). To help with this task, an acceptable
sequence for a sample analysis batch, with all the method-required QC, is shown
in Table 6. This schedule is included only as an example of a hypothetical
analysis batch where the analyst desires to collect data using both detectors.
Within the analysis batch, references to exact concentrations for the ICCS,
CCCS and ECCS are for illustrative purposes only. The analyses for sample
#14 provides an example of the QC requirements for a complete conductivity
and trace bromate PCR analysis of a sample from a PWS employing chlorine
dioxide disinfection.
11.3.3 Table 6 may be used as a guide when preparing analysis batches.
317.0-29
-------
12. DATA ANALYSIS AND CALCULATIONS
12.1 Identify the method analytes in the sample chromatogram by comparing the retention
time of the suspected analyte peak to the retention time of a known analyte peak in a
calibration standard. If analyte retention times have shifted (generally towards shorter
times) since the initial calibration, but are still within acceptance criteria and are
reproducible during the analysis batch, the analyst should use the retention time in the
daily calibrations to confirm the presence or absence of target analytes.
12.2 Compute sample concentration using the initial calibration curve generated in Section
10.2. •-.-•.". V. ,
12.3 Report ONLY those values that fall between the MRL and the highest calibration
standards. Samples with target analyte responses .exceeding the highest standard must
be diluted and reanalyzed. When this is not possible the alternate calibration
procedures described in Section 11.2.5 must be followed. Samples with target analytes
identified but quantitated below the concentration established by the lowest calibration
standard may be reported as present, but below the minimum reporting limit (MRL),
and consequently not quantitated.
12.3.1 Report bromate concentrations using the postcolumn UV/VTS absorbance
detector when they fall between the MRL and 15.0 ug/L. When bromate
concentrations exceed 15.0 ug/L, as detected by UV/VIS absorbance, either
report by conductivity, calibrate the postcolumn UV/VIS absorbance detector to
a higher bromate concentration, or dilute the sample.
12.4 Report results in u.g/L.
12.5 Software filtering of the postcolumn UV/VIS absorbance signal is.required to improve
the precision of peak measurements, minimize non-random noise and improve peak
appearance. Olympic smoothing (25 points, 5 seconds with 1 iteration) was chosen
using peak area for quantitation because it was determined to have minimal effect on
peak height and/or area.2'15 The use of alternate smoothing routines is acceptable
providing all QC criteria are met. . , ,_ „
13. METHOD PERFORMANCE
13.1 Table 1 gives the standard conditions, typical retention times and single laboratory
MDLs in reagent water, as determined for each of the inorganic pxyhah'de DBPs and
bromide. Included in this table is a comparison of the MDLs determined by
conductivity both with and without the postcolumn UV/VIS absorbance system on-line.
These data indicate that the postcolumn UV/VIS detector system has no effect on
conductivity detector performance (careful attention must however be paid to insure
backpressure on the suppressor is kept below 120 psi).
317.0-30
-------
13.2 Table 2 shows the precision and accuracy of the trace bromate measurement, evaluated
on both detectors, at two fortified concentrations, in chlorinated surface water, a
simulated high ionic strength water (HIW) and a simulated high organic (HOW) content
water. The mean bromate recovered concentration (accuracy relative to the fortified
level) and the precision (expressed as %RSD of the replicate analyses) are tabulated.
The HIW was designed to simulate a high ionic strength field sample and the HOW
designed to simulate a high organic content field sample. The HIW was prepared from
reagent water which was fortified with the common anions of chloride at 100 mg/L,
carbonate at 100 mg/L, nitrate at 10.0 mg/L as nitrogen, phosphate at 10.0 mg/L as
phosphorous, and sulfate at 100 mg/L.1 The HOW was prepared from reagent water
fortified with 1.0 mg/L fulvic acid.1
13.3 Table 3 gives the single laboratory standard deviation and precision (% RSD) for each
anion included in the method in a variety of waters for the standard conditions
identified in Table I.1'2
13.4 Table 3A shows the stability data for the inorganic oxyhalide DBFs. Each data point in
these tables represent the mean percent recovery following triplicate analyses. These
data were used to formulate the holding times shown in Section 8.3.1
14. POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity
or toxicity of waste at the point of generation. Numerous opportunities for pollution
prevention exist in laboratory operation. The EPA has established a preferred hierarchy
of environmental management techniques that places pollution prevention as the
management option of first choice. Whenever feasible, laboratory personnel should use
pollution prevention techniques to address their waste generation. When wastes cannot
be feasiblely reduced at the source, the Agency recommends recycling as the next best
option.
14.2 Quantity of the chemicals purchased should be based on expected usage during its
shelf-life and disposal cost of unused material. Actual reagent preparation volumes
should reflect anticipated usage and reagent stability.
14.3 For information about pollution prevention that may be applicable to laboratories and
research institutions, consult "Less is Better: Laboratory Chemical Management for
Waste Reduction," available from the American Chemical Society's Department of
Government Regulations and Science Policy, 115516th Street N.W., Washington D.C.
20036, (202) 872-4477.
317.0-31
-------
15. WASTE MANAGEMENT
15.1 The Environmental Protection Agency requires that laboratory waste management
practices be conducted consistent with,all applicable rules and regulations. Excess
reagents, samples and method process wastes should be characterized and disposed of
in an acceptable manner. The Agency urges laboratories to protect the air, water, and
land by minimizing and controlling all releases from hoods and bench operations,
complying with the letter and spirit of any waste discharge permit and regulations, and
by complying with all solid and hazardous waste regulations, particularly the hazardous
waste identification rules and land disposal restrictions. For further information on
waste management consult the "Waste Management Manual for Laboratory Personnel,"
available from the American Chemical Society at the address listed in Section 14.3.
16. REFERENCES
1. U.S. EPA Method 300.1. EPA Document number: EPA/600/R-98/118. NTIS number
PB98-169196INZ.
2. Wagner, H.P., Pepich, B.V., Hautman, D.P. and Munch, D.J. "Analysis of 500 ng/L Levels
of Bromate in Drinking Water by Direct Injection and Suppressed Ion Chromatography
Coupled with a Single, Pneumatically Delivered Postcolumn Reagent." Journal
ChromatographvA. 850 (1999), 119-129.
3. Wagner, H.P., Alig, A.E., Pepich, B.V., Frebis, C.P., Hautman, D.P. and Munch, D.J. "A
Study of Ion Chromatographic Methods for Trace Level Bromate Analysis in Drinking
Water Comparing the Selective Anion Concentration (SAC) Method, U.S. EPA 300.1 and a
Postcolumn Reagent Procedure." AWWA WOTC Proceedings. 4D-1, San Diego, CA,
(November 1998).
4. Wagner, H.P., Pepich, B.V., Hautman, D.P. and Munch, D.J. "Performance Evaluation of a
Method for the Determination of Bromate in Drinking Water by Ion Chromatography (EPA
317.0) and validation of EPA Method 324.0 " Presented at the International Ion
Chromatography Symposium, San Jose, CA, September, 1999. Accepted for publication
Journal of Chromatographv A, (anticipated) June 2000.
5. Glaser,J.A., Foerst,D.L., McKee, G.D., Quave, S.A., and Budde, W.L. "Trace Analyses
for Wastewater," Environmental Science and Technology. Vol. 15, Number 12, page 1426,
December, 1981.
6. "OSHA Safety and Health Standards, General Industry," (29CFR1910). Occupational Safety
and Health Administration, OSHA 2206, (Revised, Jan. 1976).
317.0-32
-------
I
7. ASTM Annual Book of Standards, Part EL, Volume 11.01, D3370-82, "Standard Practice for
Sampling Water," American Society for Testing and Materials, Philadelphia, PA, 1986.
8. "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.
9. "Safety In Academic Chemistry Laboratories," 3rd Edition, American Chemical Society
Publication, Committee on Chemical Safety, Washington, D.C., 1979.
10. Standard Methods for the Examination of Water and Wastewater, "Method 4500-C1O 2,C
Amperometric Method I (for the determination of Chlorine Dioxide)," 19th Edition of
Standard Methods (1995).
11. Hautman, D.P. & Bolyard, M. "Analysis of Oxyhalide Disinfection By-products and other
Anions of Interest in Drinking Water by Ion Chromatography." Journal of Chromatographv,
602, (1992 ), 65-74.
12. Wagner, H.P., Pepich, B.V., Hautman, D.P. and Munch, DJ. "The Use of EPA Method
300.1 with the Addition of a Postcolumn Reagent for the Analysis of Ultra Trace Levels of
Bromate in Drinking Water and the Analysis of Bromate in Bottled Waters." Presented at
Pittcon 99, Orlando FL, paper 1414, (March 1999).
13. latrou, A. and Knocke, W.R. Removing Chlorite by the Addition of Ferrous Iron. Journal of
the AWWA. Research and Technology, (November, 1992), 63-68.
14. Wagner, H.P., Pepich, B.V., Hautman, D.P. and Munch, DJ. "Eliminating the Chlorite
Interference in US Environmental Protection Agency Method 317.0 Permits the Analysis of
Bromate in all Drinking Water Matrices." Accepted for publication in Journal of
Chromatography A. (anticipated) Fall 2000.
15. Schibler, J.A. American Laboratory. (December, 1997), 63-64.
16. Dixon, WJ. "Processing Data Outliers." Biometrics. BIOMA. 9 (No.l):74-89 (1953).
317.0-33
-------
17. TABLES. DIAGRAMS. FLOWCHARTS AND VALIDATION DATA
TABLE 1. CHROMATOGRAPHIC CONDITIONS AND METHOD DETECTION
LIMITS IN REAGENT WATER FOR THE INORGANIC OXYHALIDE
DISINFECTION BY-PRODUCTS AND BROMIDE.
Standard Conditions and Equipment00:
Ion Chromatograph:
Sample Loop:
Eluent:
Eluent Flow:
Columns:
Typical System Backpressure:
Suppressor:
Detectors:
Postcolumn Reagent Flow:
Dionex DX500
225 uL
9.0mMNa2C03
1.3 mL/min
Dionex AG9-HC / AS9-HC, 4 mm
2300 psi
ASRS-I, external water mode, 100 mA current
Suppressed Conductivity Detector, Dionex CD20
Background Conductivity: 24 |o,S
Absorbance Detector, Dionex AD20 (10 mm cell path)
Set for absorbance at 450 nm (Tungsten lamp)
0.7 mL/min
Postcolumn Reactor Coil: knitted, potted for heater, 500 uL internal volume
Postcolumn Heater: 60° C
Recommended method total analysis time: 25 minutes
Analyte
Chlorite^
Chlorite'*
Bromate(c)
Bromate(d)
Bromate(c)
Surrogate: DCA(d)
Bromide{c)
Bromide(d)
Chlorate(c)
Chlorate®
Retention Time w
(min.)
4.20
4.20
4.85
4.85
5.35
8.50
10.0
10.0
11.0
11.0
MDL
Fortified Cone.
(Hg/L)
2.0
2.0
2.0
2.0
0.50
2.0
2.0
2.0
2.0
DETERMINATION
#of
Reps.
8
8
8
8
7
8
8
8
8
MDL
(Hg/L)
0.45
0.89
0.98
0.71
0.12
0.54
0.69
0.92
0.62
(a) Mention of trade names or commercial products does not constitute endorsement or recommendation
for use.
(b) Reference to chromatograms in Figure 2 and 3.
(c) Method 317.0 conductivity detection without PCR online.
(d) Method 317.0 conductivity detection with PCR online.
(c) Method 317.0 ONLY bromate by postcolumn UV7VIS absorbance detection.
317.0-34
-------
TABLE 2.
SINGLE LABORATORY PRECISION IN VARIOUS MATRICES FOR
BROMATE BY CONDUCTIVITY AND ABSORBANCE DETECTION.
Matrix Detection
Reagent Conductivity
Water
Conductivity
Absorbance
Absorbance
Chlorinated Conductivity
Drinking Water
Conductivity
Absorbance
Absorbance
High Ionic Conductivity
Water
Conductivity
Absorbance
Absorbance
High Conductivity
Organic
Water Conductivity
Absorbance
Absorbance
PRECISION
Fortified
Cone.
(jj,g/L)
0.50
5.0
0.50
5.0
0.50
5.0
0.50
5.0
0.50
5.0
'
0.50
5.0
0.50
5.0
0.50
5.0
#of Reps.
8
8
8
8
8
7(b)
8
8
• 8
8
8
8
8
8
Mean
(|j,g/L)
-------
TABLE 3. SINGLE-LABORATORY PRECISION AND RECOVERY FOR THE INORGANIC
DISINFECTION BY-PRODUCTS AND BROMIDE.1'2
Chlorite
Bromate
by
Conductivity
RW
HIW
SW
GW
C1W
CDW
O3W
RW
HIW
SW
GW
C1W
CDW
O3W
Unfortified
Cone.
(HS/L)
) NC = Not calculated since amount fortified was less than unfortified native matrix concentration (Section
9.4.1.1.).
317.0-36
-------
TABLES. SINGLE-LABORATORY PRECISION AND RECOVERY FOR THE INORGANIC
DISINFECTION BY-PRODUCTS AND BROMIDE (cont.).1'2
Analyte Matrix
Bromide RW
HIW
SW
GW
C1W
CDW
O3W
Chlorate RW
>
HIW
SW •.
GW
C1W
CDW
O3W
Unfortified
Cone.
(Hg/L)
-------
TABLE 3. SINGLE-LABORATORY PRECISION AND RECOVERY FOR THE INORGANIC
DISINFECTION BY-PRODUCTS AND BROMIDE (cont.).1'2
Analyte
Surrogate: DCA
(see Note)
Matrix
RW
HIW
SW
GW ,
C1W
CDW
O3W
Fortified
Cone;
(mg/L)
5.0
5.0
5.0
5.0
5.0
5.0
5.0
#of
Reps.
9
9
9
9
9
9
9
Mean
(mg/L)
5.1
5.0
5.0
5.0
4.9
5.0
5.1
5.1
5.2.
5.1
5.0
5.0
5.0
5.1
Mean
%REC
102
99.5
100.
99.2
98.9
99.8
102
103
103
103
100.
101
99.8
101
SD(n-l)
0.93
0.69
0.79
1.76
0.70
1.60
0.50
0.50
1.73
1.12
1.02
1.08
0.70
0.53
%RSD
0.91
0.69
0.79
1.78
0.7
1.61
0.49
0.49
1.68
1.09
1.02
1.07
0.7
0.52
RW = Reagent Water
HIW = High Ionic Strength Water
SW= Surface Water
GW = Groundwater
C1W = Chlorinated Drinking Water
CDW = Chlorine Dioxide Treated Drinking Water
O3W = Ozonated Drinking Water
NOTE: The surrogate DCA was fortified at 5 mg/L but due to concerns about measuring trace
concentrations of bromide with such high concentration of the neighboring surrogate peak, the
recommended fortified concentration for the surrogate has been reduced to 1.00 mg/L.
317.0-38
-------
TABLE 3A. STABILITY STUDY RESULTS FOR THE INORGANIC DISINFECTION BY-
PRODUCTS AND BROMIDE.1
Analyte
Chlorite
Chlorite
Bromate
Bromate
Preservative Matrix
None RW
HIW
SW
GW
C1W
CDW
O3W
EDA RW
HIW
SW
GW
C1W
CDW
O3W
None RW
HIW
SW .
GW
C1W
CDW
03W
EDA RW
HIW
SW
GW
C1W
CDW
O3W
Unfortified
Cone.
-------
TABLE 3A. STABILITY STUDY RESULTS FOR THE INORGANIC DISINFECTION
BY-PRODUCTS AND BROMIDE (cont.).1'2
Analyte
Bromide
Bromide
Chlorate
Chlorate
iPreservative Matrix
None RW
BOW
SW
GW
C1W
CDW
O3W
EDA RW
fflW
SW
GW
C1W
CDW
O3W
None RW
fflW
SW
GW
C1W
CDW
03W
EDA RW
HIW
SW
GW
C1W
CDW
O3W
Unfortified
Cone.
(W?/L)
-------
TABLE 4. INITIAL DEMONSTRATION OF CAPABILITY QC REQUIREMENTS.
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Sect. 9.2.2
9.3.1
Initial
Demonstration of
Low System
Background
Analyze a method blank (LRB) and determine
that all target analytes are below l/2 of the
proposed MRL prior to performing the IDC
The LRB
concentration must be
<, Vz of the proposed
MRL
Sect. 9.2.3
Initial
Demonstration of
Precision (TOP)
Conductivity: analyze 7 replicate LFBs
recommend fortify at 20 ug/L
Absorbance: analyze 7 replicate LFBs
recommend fortify with bromate at 2.0 ug/L
%RSD must be <;20%
Sect. 9.2.4
Initial
Demonstration of
Accuracy (IDA)
Calculate average recovery of DDP replicates
Mean % recovery
must be ± 15% of true
value.
Sect. 9.2.5
Quality Control
Sample (QCS)
Initially and at least quarterly analyze a QCS
from an external/second source
QCS must be ± 20%
of the true value
Sect. 9.2.6
Method
Detection Limit
(MDL)
Determination
Select a fortifying level at 3-5 times the
estimated instrument detection limit at or lower
than the MRL. Analyze 7 replicate LFBs
Calculate MDL using equation in Section 9.2.6
- do not subtract blank
Sect. 9.2.7
Minimum
Reporting Level
(MRL)
MRLs MUST be established for all analytes
during the IDC.
The low CAL
standard can be lower
than the MRL, but the
MRL MUST be no
lower than the low
CAL standard
317.0-41
-------
TABLES. QUALITY CONTROL REQUIREMENTS (SUMMARY).
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Sect. 8.3
Sample Holding
Time/
Preservation
Bromate 28 days, refrig. at <6°C /
EDA Preservation
Bromide 28 days, EDA Permitted
Chlorate 28 days, refrig. at <6°C / EDA
Preservation
Chlorite 14 days, refrig. at <6°C / EDA
Preservation
Holding time and temperature
must not be exceeded. EDA
added to all samples
Sect.
11.1.4.1
(specific
toPCR)
Pretreated
Sample
(acidified/Fe[IT|
added to remove
chlorite) Holding
Time
ONLY REQUIRED when samples
containing chlorite are pretreated and PCR
is employed to measure trace bromate in
samples.
MAXIMUM PRETREATED SAMPLE
HOLDING TIME: 30 hours
Pretreated sample holding time
must not be exceeded
Sect. 10.2
Initial
Calibration
Conductivity: generate calibration curve
using at least 5 standards
Absorbance: generate calibration curve
using at least 5 bromate standards
MRL MUST be no lower than
the lowest calibration standard
Sect.
10.3.1
Initial
Calibration
Check
Daily, verify calibration of conductivity
detector at the MRL by analyzing an initial
low-level continuing calibration check
standard (ICCS) and a separate low-level
ICCS for the absorbance detector at the
MRL.
Recovery must be 75-125% of
the true value on both detectors
Sect.
10.3.2
Continuing
Calibration and
End Calibration
Checks
Alternately analyze separate mid and high
level CCCS/ECCS after every 10 samples
and after the last sample
MRL to 5 x MRL must have 75
125% recovery on both
detectors
For 5 x MRL to highest CCCS
must have 85-115% recovery
on both detectors
Sect 9.3.1
Laboratory
Reagent Blank
(LRB)
Include LRB with every analysis batch (up
to 20 samples)
Analyze prior to analyzing field samples
All analytes must be
< !/2 MRL
Sect.
9.3.1.2
(specific
to PCR)
PRETREATED
Laboratory
Reagent Blank
(LRB)
REQUIRED in any analysis batch which
includes samples which have been
pretreated to remove chlorite prior to PCR
measurement of trace bromate.
PCR measured bromate
<'/2MRL
Sect. 9.3.2
Laboratory
Fortified Blank
(LFB)
Laboratory must analyze LFB in each
analysis batch following the ICCS.
Calculate %REC prior to analyzing
samples
LFB recovery if fortified at cone.
from MRL to 5X MRL must be
75 - 125%. For 5X MRL to
highest CCCS must be 85 -
115%. Must have acceptable
recoveries prior to analyzing
samples. Sample results from
batches that fail LFB are invalid
317.0-42
-------
TABLE 5. QUALITY CONTROL REQUIREMENTS (SUMMARY CONTINUED).
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Sect. 9.3.3
Instrument
Performance
Check (IPC)
Calculate Peak Gaussian Factor (PGF)
using equation (Sect. 9.3.3.1) and
monitor retention time for surrogate in
Initial Calibration Check Standard
(ICCS) each day
PGF must fall between
0.80 and 1.15
Ret. Time (RT) for
surrogate must remain
80% of initial RT when
column was new
Sect. 9.4.1
Laboratory
Fortified Sample
Matrix (LFM)
Sect.
11.1.4.2
Must add known amount of each target
analyte to a minimum of 5% of field
samples or at least one within each
analysis batch for both detectors
LFM must be fortified above the native
level and at no greater than 5 x the
highest field sample concentration
Calculate target analyte recovery using
formula (Sect. 9.4.1.3)
When field samples from chlorine ,
dioxide plants which contain chlorite
are pretreated prior to the PCR
measurement of trace bromate, an
additional LFM must be prepared for
each pretreated field sample (Sect.
9.4.1.5)
Recovery should be,
75-125%
.If fortified sample fails the
recovery criteria, label
both as suspect/matrix.
Sect. 9.4.2
Surrogate
Dichloroacetate is added to all blanks,
samples and standards (if measuring by
conductivity and absorbance)
Calculate recovery using formula in
Section 9.4.2
Surrogate recovery must'
be 90-115%.
Samples that fail surrogate
recovery must be
reanalyzed. If second
analysis fails label result
as suspect/matrix
Sect. 9.4.3
Field or
Laboratory
Duplicates
Analyze either a field or laboratory
duplicate for a minimum of 5% of field
samples or at least one within each
analysis batch for both detectors
Calculate the relative percent difference
(RPD) using formula in Section 9.4.3.1
The RPD for
concentrations at MRL to
5 x MRL should be ± 20%
on both detectors, and ±
10% on both detectors
for concentrations at 5 x
MRL to highest CCCS. If
this range is exceeded,
label both as •
suspect/matrix
317.0 - 43
-------
TABLE 6. EXAMPLE SAMPLE ANALYSIS BATCH WITH QUALITY CONTROL
REQUIREMENTS
Injection
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Sample
Description
Laboratory reagent blank (LRB)
ICCS conductivity detector (5.0 ug/L)
ICCS absorbance detector (0.5 ug/L)
Laboratory Fortified Blank (LFB) -
conductivity detector
LFB - absorbance detector
Field sample 1
Field sample 1 - Laboratory Duplicate (LD) (a)
Field sample 2
Field sample 2 - Laboratory Fortified Matrix (LFM) (a) at
concentrations specific for conductivity detector
Field sample 2 - LFM specific for trace bromate on the
absorbance detector .
Field sample 3
Field sample 4
Field sample 5
Field sample 6
Field sample 7
Field sample 8
Field sample 9
Field sample 10
CCCS conductivity detector (75.0 ug/L)
CCCS absorbance detector (5.0 ug/L)
Field sample 11
Field sample 12
Acceptance
Criteria
<; 'AMRL
3.75 to 6.25 ug/L
0.375 to 0.625 ug/L
±25% fortified level
±25% fortified level
± 15 % RPD
± 25% fortified level
± 25% fortified level
63.8 to 86.3 ug/L
4.25 to 5.75 ug/L
317.0-44
-------
23
24
25
26
27
28
29
30
31
32
33
34
Field sample 13
Field sample 14 - (finished water from PWS using chlorine
dioxide)
Pretreated LRB (Section 9.3.1.2) using the acid/Fe(IT)
chlorite removal procedure (Section 1 1.1.4)
Field sample 14 w - (finished water from PWS using
chlorine dioxide) pretreated with acid/Fe(H) (Section
11.1.4)
Field sample 14 - (finished water from PWS using chlorine
dioxide) LFM specific for trace bromate on the absorbance
detector, pretreated with acid/Fe(n) (Section 1 1.1.4.2)
Field sample 15
Field sample 16
Field sample 17 ,
Field sample 18
Field sample 19®
ECCS conductivity detector (500.0 ug/L)
ECCS absorbance detector (15.0 ug/L)
< '/2MRL
•
± 25% fortified level
425 to 575 ug/L
12.8 to 17.3 ug/L
(a) If no analytes are observed above the MRL for a sample, an alternate sample which contains
reportable values should be selected as the laboratory duplicate. Alternately, the LFM can be selected
and reanalyzed as the laboratory duplicate ensuring the collection of QC data for precision.
w Field sample #19 was the final field sample permitted in this batch but 20 total field samples were
analyzed.
Field sample #14 was analyzed both initially and as a acid/Fe (IT) pretreated sample, therefore, it
accounted for two "field sample analyses" toward the maximum of twenty in an analysis batch
(Section 3.1).
317.0-45
-------
System Configuration for EPA Method 317.0
Eluent
'.''
utosa''mpler
'1-""- •'
DX -500
Injection loop
AG9-HC
AS9-HC
PC10 PCR
Reservoir
Electrolytic
Suppressor
Conductivity
Detector
Knitted Reaction Coil
PCH-2 Heater (Si 60 °C
Absorbance
Detector
Effluent to waste
Figure 1: Schematic detailing the configuration of postcolumn hardware addition to an ion
chromatograph. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use. If the requirements found in Section 9 are met,
equivalent products or hardware can be employed.
NOTE: In a typical Method 300.1 hardware configuration, a backpressure coil is included after
the conductivity cell as part of the waste stream when this manufacturer's equipment is used.
These backpressure coils are not required when the Method 317.0 instrument configuration is
employed since the additional PCR system components, placed in-line, function in the same
capacity and provide sufficient backpressure.
317.0-46
-------
0.500 -r
0.400- -
0.300- •
0.200- -
0.100--
-0.100- •
-0200
20.00 22.50 25.00
2.00X10*
1.50x10-*- -
1.00X10"8- •
5.00X10-*
-5.00X10"4
bromate
I
chlorite
UV/VTS Detector at 450 nm
-i
2.50 5.00 7.SO
10.00
Minutes
12.50 15.00 17.50 20.00 22.50 25.00
Figure 2: Reagent water fortified with inorganic disinfection by-products and bromide at 10
ug/L.
317.0-47
-------
0.500
0.400-
0.300. .
0200
0.100- •
-0.100 ••
•0,200
Surrogate:
DCA
bromate
2.50
7.50
10.00
Minutes
Conductivity Detector
12.50 15.00 17.50 20.00 22.50 25.00
tOOxlO"3. .
5,00x1 o-'r
•S.OOxlO"4*-
i—I—i
2.60
bromate
UV/VIS Detector at 450 nm
5.00' '/.SO
-H-
-1
10.00
Minutes
12.60 1».'UU 17.50 20.00 22.50 25.00
Figure 3: Chlorinated tap water fortified with bromate at 2.0 ug/L.
317.0-48
-------
METHOD 321.8 DETERMINATION OF BROMATE IN DRINKING WATERS
BY ION CHROMATOGRAPHY INDUCTIVELY COUPLED
PLASMA/MASS SPECTROMETRY
Revision 1.0
December 1997
John T. Creed, Carol A. Brockhoff and Theodore D. Martin, ORD, NERL
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
321.8-1
-------
METHOD 321.8
DETERMINATION OF BROMATE IN DRINKING WATERS
BY ION CHROMATOGRAPHY
INDUCTIVELY COUPLED PLASMA - MASS SPECTROMETRY
1. SCOPE AND APPLICATION
1.1 This method provides a procedure for determination of bromate in drinking water.
Chemical Abstract Services
Analyte Registry Numbers (CASRN)
Bromate (BrO30 15541-45.-4
1.2 For reference where this method is approved for use in compliance monitoring
programs [e.g., Safe Drinking Water Act (SDWA)], consult both the appropriate
sections of the Code of Federal Regulation (Part 141 § 141.23 for drinking water),
and the latest Federal Register announcements.
1.3 This method should be used by analysts experienced in the use of inductively
coupled plasma mass spectrometry (ICP-MS), and the interpretation of spectral and
matrix interferences. A minimum of six months experience with commercial
instrumentation is recommended. It is also recommended that the analyst have
experience in liquid chromatography and the use of ICP-MS as a chromatographic
detector.
1.4 Users of the method data should state the data-quality objectives prior to analysis.
Users of the method must document and have on file the required Initial
Demonstration of Performance data described in Section 9.2 prior to using the
method for analysis.
2. SUMMARY OF METHOD
2.1 An aliquot of a finished drinking water is passed through a preparatory cartridge
capable of removing the trisubstituted haloacetic acids which interfere with the
analysis of bromate. The sample is then injected onto a column which separates the
remaining brominated haloacetic acids and bromide from the bromate. The ICP-MS
is interfaced to the ion chromatograph and both mass 79 and mass 81 are monitored
in time as bromate elutes from the column. The-resulting signal is integrated and a
concentration determined from a calibration curve. Mass 79 is used for quantitation
while mass 81 provides isotope ratio information which can be used to screen for
potential polyatomic interferences.
321.8-2
-------
2.2 Chromatography: The chromatographic separation is based on an anion exchange
resin. The sample is injected on the column and the matrix and analyte partition
themselves between the mobile phase and the stationary phase as they move along
the column. 'Early eluting analytes spend most of their time in the mobile phase
while the late eluting compounds spend a larger percentage of their time interacting
with the stationary phase. The matrix anions can influence retention times by
blocking the interaction of the stationary phase with the analytes producing a shift in
the retention time.
Inductively Coupled Plasma Mass Spectrometer: The detection technique is based
on the use of an ICP-MS for the detection of trace elements[l-3]. The
chromatographic eluent is introduced by pneumatic nebulization into a radio
frequency plasma where energy transfer processes cause desolvation, atomization
and ionization. The ions are extracted from the plasma through a differentially
pumped vacuum interface and separated on the basis of their mass-to-charge ratio by
the mass spectrometer having a minimum resolution capability of 1 amu peak width
at*5% peak height. The ions transmitted through the mass spectrometer are detected
by an electron multiplier or Faraday detector and the ion information is processed by
the data system. Interferences relating to the technique (Sect. 4) must be recognized.
Although ICP-MS is typically used for multi-analyte determinations, it is used in
321.8 for species specific quantification, hi this mode the signal response is
recorded via chromatographic or time resolved software. The use of ion
chromatography in combination with ICP-MS detection has been reported for the
detection b'fbfomate [4-7]. ; -
3. DEFINITIONS
3.1 CALIBRATION BLANK - A volume of reagent water pH adjusted (to 10) with the
same base as in the calibration standards.
3.2 CALIBRATION STANDARD (CAL) - A solution prepared from the dilution of
stock standard solutions. The CAL solutions are used to calibrate the instrument
response with respect to analyte concentration. This solution is pH adjusted to 10.
3.3 INSTRUMENT PERFORMANCE CHECK (IPC) SOLUTION - A solution of
method analytes, used td evaluate the performance of the instrumental system with
respect to defined set of method criteria. Within this method, the IPC is identical to
!-• :" • • the laboratory fortified blank.
3.4 DRIFT STANDARD- A calibration standard added to a post column sample loop
• •">•• - '(via^a second valve) which is transported into the plasma when the sample is
' ! injected; This analyte does not traverse the column and is used to compensate for
instrumental (ICP-MS) drift during the analysis of a set of samples.
321.8-3
-------
3.5 LABORATORY DUPLICATES (LD1 and LD2) - Two aliquots of the same sample
taken in the laboratory and analyzed separately with identical procedures. Analyses
of a number of LD1 and LD2 indicates precision associated with laboratory
procedures, but not with sample collection, preservation, or storage procedures.
3.6 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of LRB to, which known
quantities of the method analyte is added in the laboratory. The LFB is analyzed
exactly like a sample, and its purpose is to determine whether the methodology is in
control and whether the laboratory is capable of making accurate and precise
measurements.
3.7 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) - An aliquot of an
environmental sample to which known quantities of the method analyte is 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 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or other
blank matrices that are treated exactly as a sample including exposure to all
glassware, equipment, solvents, and reagents that are used with other samples. The
LRB is used to determine if method analyte or other interferences are present in the
laboratory environment, reagents, or apparatus.
3.9 LINEAR DYNAMIC RANGE (LDR) - The concentration range over which the
instrument response to an analyte is linear.
3.10 METHOD DETECTION LIMIT (MDL) - The minimum concentration of an analyte
that can be identified, measured, and reported with 99% confidence that the analyte
concentration is greater than zero.
3.11 QUALITY CONTROL SAMPLE (QCS) - A solution of the method analyte of
known concentrations which is used to fortify an aliquot of LRB or sample matrix.
The QCS is obtained from a source external to the laboratory and different from the
source of calibration standards. It is used to check either laboratory or instrument
performance.
3.12 STOCK STANDARD SOLUTION - A concentrated solution containing the method
analytes prepared in the laboratory using assayed reference materials or purchased
from a reputable commercial source.
3.13 TUNING SOLUTION - A solution which is used to determine acceptable instrument
performance prior to calibration and sample analyses.
321.8-4
-------
3.14 WATER SAMPLE - For the purpose of this method, a sample taken from a finished
drinking water supply.
4. INTERFERENCES
4.1 Several interference sources may cause inaccuracies or imprecisions in the
determination of bromate by ICP-MS. These are:
4.1.2 Abundance sensitivity - This is a property defining the degree to which the
"wings" of a mass peak contribute to adjacent masses. The abundance
sensitivity is affected by ion energy and operating pressure. "Wing" overlap
interferences may result when a small ion peak is being measured adjacent
to a large one. The potential for these interferences should be recognized
and the mass spectrometer operating conditions adjusted to minimize the
effect.
This interference is relevant in this method given the large 40Ar40Ar+ dimer
adjacent to mass 79 which is present in conventional ICP-MS. The extent
to which the dimer contributes to the signal on mass 79 can be determined
by scanning over masses 76-83 using 20 points per amu (skipping mass 80)
using a 5rnM HNO3 solution(See Figure 1). The interference signal from the
argon dimer is apparent by examining the background signal at mass 79.4
relative to 76.4, 77.4 and 78.4. With proper mass calibration and adequate
abundance sensitivity, the signal on masses 76.4, 77.4, and 78.4 should be
close to normal photon background. The signal on 79.4 commonly is
elevated relative to the above masses. This elevated signal is caused by the
mass spectrometers insufficient abundance sensitivity.
The signal should decrease as the mass decreases from 79.5 to 79.3 etc. To
determine if the instrument has adequate abundance sensitivity the decrease
(79.5 to 79.3) in this signal should be extrapolated to mass 79.0 at which
point it should be no higher than twice the normal photon background(See
Figure 1). The signal may increase as mass 79.0 is approached depending
on the bromide contamination in the 5mM HNO3 The resolution etc.,
should be adjusted to minimize the dimers contribution to mass 79.0.
Note: If the decrease in the signal from 79.5 to 79.0 does not
have an inflection point, this may indicate that the abundance
sensitivity is insufficient to resolve 79Br+ from 40Ar40Ar+
4.1.3 Isobaric polyatomic ion interferences are caused by ions consisting of more
than one atom which have the same nominal mass-to-charge ratio as the
isotope of interest, and which cannot be resolved by the mass spectrometer
in use[8]. These ions are commonly formed in the plasma or interface
system from support gases or sample components. The two polyatomics
321.8-5
-------
which are inherent to conventional ICP-MS are the '"'Ar^ArH and
40Ar40ArH. 40Ar40ArH contributes to the background signal on mass 81. To
minimize this, it is recommended that the sample flow rate remain on
between injections. This will produce smoother baselines on mass 81 and
thereby produce more reproducible integrations on mass 81 for the early
eluting peaks.
The ICP-MS interferences which apply to the detection of bromate are
listed in Table 1. These spectral interferences are common to the plasma or
produced by matrix anions eluting from the column. These interferences
are outside the retention window for bromate but may cause baseline shifts
which will degrade integration precision.
4.1.4 Physical interferences are associated with the physical processes which
govern the transport of sample into the plasma, sample conversion
processes in the plasma, and the transmission of ions through the plasma-
mass spectrometer interface. These interferences may result in differences
between instrument responses for the sample and the calibration standards.
Samples containing high concentration of chloride (which elutes
immediately after bromate) may cause chromatographic baseline shift
possibly from a small change in the 40Ar40ArH production during the
chloride elution. m addition, the removal of high concentrations of sodium
and potassium which may exist in the drinking water are removed by using
an anion self regenerating suppressor. This minimizes their deposition on
the sampling cone and thus improves long term stability.
Note: The mobile phase in this method was chosen based on its
ability to produce a stable baseline and sensitivity over a multiple
week period. The long term stability of the instrument is
monitored by injecting a post column drift standard with each
chromatogram. The analyte concentration in a sample is
corrected by using this drift standard in the same fashion that an
internal standard is used in Method 200.8[9].
4.1.5 Baseline Drift - This results when a constituent from the sample matrix is
not quantitatively removed from the column leading to a slow column bleed
of the strongly retained species. If this is suspected, the column should be
flushed according to the manufacturer's recommendations. A slowly rising
baseline can be caused by trisubstituted brominated haloacetic acids.
4.1.6 Chromatographic Interferences - The known chromatographic interferences
for the determination of bromate in drinking water via ICP-MS detection
are listed in Table 1 and their approximate retention characteristics are
reported in Figure 2. These known interferences have been
chromatographically resolved using the procedure described in this method.
321.8-6
-------
Given the diversity of environmental samples, the possibility of unidentified
interferences exist. The following section is written to provide the analyst
with some guidance if an interference is identified. The two possible
interferences are chromatographic overlap with a bromine containing
species or a co-eluting polyatomic. In the case of co-elution with a bromine
containing anion it is recommended that the analyst try the following
method modifications in the order presented in an attempt to resolve the co-
elution.
1.) Use weaker mobile phases (i.e. lower NH4NO3
concentrations).
2.) Alternative columns.
3.) Pretreatment cartridge which selectively removes the
interference.
This co-elution should be documented and the changes in the mobile and
stationary phase should produce a method capable of meeting all
requirements in Section 9.
In the case of a co-elution with a polyatomic interference (on mass 79), the
recommendation to the analyst is to try the following method modifications
in the order presented in an attempt to resolve the co-elution.
1.) Use mass 81 for quantitation.
2.) Use weaker mobile phases.
3.) Alternative columns
4.) Pretreatment cartridge to selectively remove the
interference.
This co-elution should be documented and the changes in the mobile and
stationary phase should produce a method capable of meeting all
requirements in Section 9.
4.1.7 Samples that contain particles larger than 0.45 microns and reagent
solutions that contain particles larger than 0.2 micron require filtration to
prevent damage to instrument columns and HPLC pumping system.
4.1.8 The analyst should be aware of the potential for carryover peaks from one
analysis which will effect the proper detection of bromate in the subsequent
analysis. Carryover was not observed in the analysis listed in Table 3 using
the column, mobile phase and flow rate reported in Table 2. However, the
analyst should be aware of the potential for carryover peaks.
321.8-7
-------
4.1.9 Retention time shifts in ion chromatography are possible do to weak eluent
strengths and high ionic strength matrices. These shifts are minimized by
the eluent system reported in table 2. However, the analyst should be aware
of the potential for retention time shifts do to high ionic strength matrices.
These effects can be minimized by dilution of the sample matrix.
5. SAFETY
5.1 The toxiciry or carcinogenicity of bromate and reagents used in this method have not
been fully established. Each chemical should be regarded as a potential health
hazard and exposure to these compounds should be as low as reasonably achievable.
Each laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method
[10,11]. A reference file of material data handling sheets should also be available to
all personnel involved in the chemical analysis. Specifically, concentrated nitric
presents various hazards as it is moderately toxic and extremely irritating to skin and
mucous membranes. Use these reagents in a fume hood whenever possible and if
eye or skin contact occurs, flush with large volumes of water. Always wear safety
glasses or a shield for eye protection, protective clothing, and observe proper mixing
when working with these reagents.
5.2 Analytical plasma sources emit radio frequency radiation, in addition to intense UV
radiation. Suitable precautions should be taken to protect personnel from such
hazards. The inductively coupled plasma should only be viewed with proper eye
protection from UV emissions.
5.3 It is the responsibility of the user of this method to comply with relevant disposal
and waste regulations. For guidance see Sections 14.0 and 15.0.
6. EQUIPMENT AND SUPPLIES
6.1 Inductively coupled plasma mass spectrometer. This instrument must meet the
following requirements:
6.1.1 An instrument capable of scanning the mass range 5-250 amu with a
minimum resolution capability of 0.75 amu peak width at 5% peak height is
required. This instrument may be fitted with a conventional or extended
dynamic range detection system. The abundance sensitivity must be greater
than 1.0 x 106 on the low side of mass 80 or such that the dimer's
(40Ar40Ar+)low mass shoulder does not influence mass 79.
6.1.2 Radio-frequency generator compliant with FCC regulations.
6.1.3 Argon gas supply - High purity grade (99.99%). When analyses are
conducted frequently, liquid argon is more economical.
321.8-8
-------
6.1.4 A variable-speed peristaltic pump may be used to pump the drain of the
spray chamber to waste.
6.1.5 A mass-flow controller on the nebulizer gas supply is required. A water-
cooled spray chamber may be of benefit in reducing the water vapor
entering the plasma and thereby minimizing the 40Ar40ArH+. A double-pass
spray chamber is recommended to increase background stability on mass 81
if 40Ar40ArH+ is present in the spectrum generated while nebulizing the
mobile phase.
6.1.6 If an electron multiplier detector is being used, precautions should be taken,
where necessary, to prevent exposure to high ion flux. Otherwise, changes
in instrument response or damage to the multiplier may result. This may be
true for samples containing bromide in the parts per million range.
6.1.7 A nebulizer with a low dead volume is recommended.
6.2 Ion Chromatograph. This instrument must meet the following specifications:
6.2.1 Eluent Pump - Programmable flow high pressure pumping system capable
of delivering pressures up to 3000 psi and flow rates up to 1.5 ml/min.
6.2.2 Control Valves - Inert double stacked pneumatic operated 4 way valves
capable of withstanding 3000 psi.
6.2.3 Sample Loops- narrow bore, high pressure tefzel® tubing or equivalent.
6.2.4 Tubing- narrow bore high pressure tefzel® tubing or equivalent.
6.2.5 Guard and Analytical Column - Dionex PA-100 or equivalent.
6.2.6 Suppressor - Dionex (ASRS) anion self regenerating suppressor or
equivalent.
6.2.7 Pretreatment Cartridges - Dionex On-Guard-RP or equivalent
6.3 Analytical balance, with capability to measure to 0.1 mg, for use in weighing
samples and preparing standards.
6.4 An air displacement pipette capable of delivering volumes ranging from 50 to 2500
|iL with an assortment of high quality disposable pipet tips. Calibration of the
pipette should be verified frequently by weighing aliquots of distilled deionized
water using the analytical balance to assure precision and accuracy of the pipette.
321.8-9
-------
6.5 Labware - For determination of bromate, plastic labware has been used exclusively
and measurable concentrations of bromate in the blank have not been observed.
6.5.1 Narrow-mouth storage bottles, FEP (fluorinated ethylene propylene) with
ETFE (ethylene tetrafluorethylene) screw closure, 125-mL to 250-mL
capacities.
6.5.2 One-piece stem FEP wash bottle with screw closure, 125-mL capacity.
6.5.3 Syringes- lOcc Becton-Dickinson plastic syringes or equivalent.
7. REAGENTS AND STANDARDS
7.1 Reagents may contain elemental impurities that might affect the integrity of
analytical data. Due to the high sensitivity of ICP-MS, high-purity reagents should
be used whenever possible. All acids used for this method must be of ultra high-
purity grade. The acid used to prepare the 5mM HNO3 in the mobile phase does
contain some bromide background. Care should be taken to minimize this
background intensity.
7.1.1 Nitric acid, concentrated (sp.gr. 1.41).
7.1.2 Nitric acid (1+1) - Add 500 mL cone, nitric acid to 400 mL of reagent grade
water and dilute to 1L.
7.1.3 Ammonium Nitrate - Fisher ACS certified or equivalent.
7.1.4 Sodium Hydroxide - Fisher 50/50 liquid mixture or equivalent.
7.2 Reagent water - All references to reagent grade water in this method refer to ASTM
type I water (ASTM Dl 193)[12]. Suitable water may be prepared by passing
distilled water through a mixed bed of anion and cation exchange resins.
7.3 Standard Stock Solutions - Stock standards may be purchased from a reputable
commercial source or prepared from ultra high-purity grade chemicals. These
standards have extremely low ionic strengths and should be pH adjusted to 10 (with
NaOH)in order to minimize bromate interaction with the plastic sample loop
tubing[6]. Replace stock standards when they can not be verified with QC
standards.
7.3.1 Preparation of calibration standards - a fresh bromate standard should be
prepared once a month or as needed. Dilute the stock bromate standard
solution to levels appropriate to the operating range of the instrument using
reagent water and adjust the pH to 10 using NaOH. The bromate
321.8-10
-------
concentrations in the standards should be sufficiently high to produce good
isotope ratio precision (<2%) and to accurately define the slope of the
response curve. Depending on the sensitivity of the instrument,
concentrations ranging from 10 \igfL to 50 |j,g/L are suggested.
: Note: A blank and one calibration standard or multi-point
calibration can be utilized to calibrate the response of the
instrument.
7.4 Drift standard - The drift standard should be made such that its concentration is
approximately five times higher than the bromate calibration standard. (This will
compensate for the sample loop volume difference between the sample and the drift
standard.) The drift standard concentration should be chosen based on analytical
precision (< 5% rsd of replicate peak integration).
7-5 Blanks - The calibration blank is used to establish and verify the analytical
calibration.
7.5.1 Calibration blank - Consists of reagent grade water which is pH adjusted to
10 with sodium hydroxide.
7.6 Tuning Solution - This solution is used for instrument tuning (lens, argon flows
etc.), mass calibration and abundance sensitivity prior to analysis. The tuning
solution should be approximately twice the bromate calibration standard
concentration (producing greater than 30,000cps) and should be delivered to the
plasma using a peristaltic pump at a flow rate of 1 mL/min. The instrument should
be tuned for maximum signal-to-noise using mass 79. After tuning, the tuning
solution should be analyzed by scanning over masses 76-84 using 20 points per amu
(skipping mass 80). This data can be used to verify the mass calibration (mass shifts
of greater than 0.1 amu should be corrected) and check the instrument sensitivity
(approximately 35,000cps/100ppb bromate given the instrument conditions outlined
in Table 2)., The calibration blank or a 5mM HNO3 solution should then be analyzed
using the same scanning conditions to check the mass analyzer's abundance
sensitivity. This preanalysis routine should be performed daily(see figure 1).
7.7 Quality Control Sample (QCS) - The QCS should be obtained from a source outside
the laboratory. The concentration of the QCS solution analyzed will depend on the
sensitivity of the instrument. To prepare the QCS dilute an appropriate aliquot
bromate to a concentration which is approximately 75% of the concentration of the
highest calibration standard. The QCS should be analyzed as needed to meet data-
quality needs and a fresh solution should be prepared quarterly or more frequently as
needed.
7.8 Laboratory Fortified Blank (LFB) - To an aliquot of Calibration Blank add an aliquot
of the stock standard solution to prepare the LFB. The fortified concentration should
321.8-11
-------
produce percent relative standard deviations of 4-7% on replicate determinations.
The LFB must be carried through the same entire preparation scheme as the samples.
This solution should be pH adjusted to 10 with sodium hydroxide.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 The pH of all aqueous samples should be tested immediately prior to analysis to
ensure the sample has been properly preserved. If the sample requires a pH
adjustment throughly mix the sample after the sodium hydroxide has been added.
The pH of the sample must be adjusted to 10. This pH adjustment should be
performed just prior to analysis. This pH adjustment assures the solubility of
bromate within the sample loop [6].
8.2 If required by the data user, prepare a field blank using reagent water. Use the same
sample containers (see section 6.5.1 for container recommendations)as used in
sample collection.
9. QUALITY CONTROL
9.1 Each laboratory using this method is required to operate a formal quality control
(QC) program. The minimum requirements of this program consist of an initial
demonstration of laboratory capability, and the periodic analysis of laboratory
calibration blanks, fortified blanks and calibration solutions as a continuing check on
performance. The laboratory is required to maintain performance records that define
the quality of the data generated.
NOTE: Because the sample preparation step of this method is limited to pH
adjustment prior to analysis, the number of required solutions needed to
verify data quality has been reduced. In this method the calibration blank
(Section 7.5 and 9.3) is used to establish baseline calibration and is used to
verify the absence of contamination. The laboratory fortified blank
(Sections 7.8 and 9.3) is used to assess both method accuracy and
instrument performance.
9.2 Initial Demonstration of Performance (mandatory)
9.2.1 The initial demonstration of performance is used to characterize instrument
performance (determination of linear calibration range and analysis of
quality control sample) and laboratory performance (determination of
method detection limit) prior to analyses conducted by this method.
9.2.2 Linear dynamic range - Linear dynamic range is detector or
chromatographic resolution limited.
321.8-12
-------
The useable linear range must be determined for the instrument
configuration to be used.
Note: The linear dynamic range maybe limited by the chromatographic
resolution of bromate from bromoacetic acid or other interferences, hi
this situation, the linear calibration range is limited to a concentration of
"' bromate which can be chromatographically resolved from bromoacetic
acid. Given the experimental conditions in Table 2, the linear dynamic
range was limited to 50|ig/L based on chromatographic resolution.
9.2.3 Quality control sample (QCS) - When beginning the use of this method, on
a quarterly basis or as required to meet data-quality needs, verify the
calibration standards and acceptable instrument performance with the
preparation and analyses of a QCS. To verify the calibration standards, the
determined mean concentration from three analyses of the QCS must be
within ± 10% of the stated QCS value. If the calibration standards cannot be
verified, the source of the problem must be identified and corrected before
either proceeding on with the initial determination of method detection
limits or continuing with on-going analyses.
9.2.4 Method detection limit (MDL)- This should be established using reagent
water (blank) fortified at a concentration of two to five times the estimated
detection limit[13]. To determine MDL values, take seven replicate
aliquots of the fortified reagent water and process through the entire
analytical method. Perform all calculations defined in the method and
report the concentration values in the appropriate units. Calculate the MDL
as follows:
MDL = (t)(n.1;1.alpha=0.99) x (S)
where:
(Ooi-w-aipha=o.99) - Student's t value for a 99% confidence level and
a standard deviation estimate with n-1 degrees of freedom (t=3.14
for seven replicates].
n= number of replicates
S = standard deviation of the replicate analyses.
The MDL should be determined annually, when a new operator begins work
or whenever, in the judgement of the analyst, a change in analytical
performance caused by either a change in instrument hardware or operating
conditions would dictate they be redetermined.
The MDL for bromate using the conditions listed in Table 2 is 0.3 |o.g/L.
9.3 Assessing Laboratory Performance (mandatory)
9.3.1 Calibration blank - Within this method a calibration blank and the
321.8-13
-------
laboratory reagent blank are operationally the same. The calibration
blank will be used as the LRB and a fresh calibration blank should be made
daily to verify the lack of contamination.
Analysis of the calibration blank can be used to verify the calibration
baseline and to assess chromatographic carryover interference or
contamination. The calibration blank should be analyzed as a sample. If the
calibration blank produces an integrable signal for bromate, the laboratory
should find the source of this prpblem prior to Analyzing samples. The
laboratory must complete the analysis of one calibration blank with each
batch of 20 samples.
9.3.2 Laboratory fortified blank (LFB) - Within this method the LFB is used to
assess both laboratory and instrument performance. The laboratory
must analyze at least one LFB (Sect, 7.8) immediately after calibration and
after each 10 samples. Calculate accuracy as percent recovery using the
following equation:
LFB-CB
R= xlOO
where: R = percent recovery.
LFB = laboratory fortified blank concentration.
CB = calibration blank concentration.
S = concentration equivalent of analyte
. added to fortify the CB splution..
If the recovery falls outside the required control limits of 85-115%, bromate
is judged out of control, and the source of the prpblem should be identified
and resolved before continuing analyses. .
9.3.3 The laboratory must use LFB analyses data to assess laboratory perfor-
mance against the required control limits of 85-115%. When sufficient
internal performance data become available (usually a minimum of twenty
to thirty analyses), optional control limits can be developed from the mean
percent recovery (x) and the standard deviation (S) of the mean percent
recovery. These data can be used to establish the upper and lower control
limits as follows: ' , .
UPPER CONTROL LIMIT = x + 3S '...•••
LOWER CONTROL LIMIT = x - 3S
The optional control limits must be equal to or better than the required
control limits of 85-115%. After each five to ten new recovery measure-
ments, new control limits can be calculated using only the most recent
321.8-14
-------
twenty to thirty data points. Also, the standard deviation (S) data should be
used to establish an on-going precision statement for the level of
concentratibns included in the LFB.
These data must be kept on file and be available for review.
Note: Using the experimental conditions in Table 2, the average
recovery of the LFB was 99.8% with a three sigma control limit of
10.2%
9.3.4 Instrument performance - For all determinations the laboratory must check
instrument performance and verify that the instrument is properly calibrated
on a continuing basis. This is accomplished via the recovery on the LFB
(85-115%,section 9.3.2) and monitoring the instrument drift standard
injected with each sample.
9.3.5 Instrument Drift Standard - The analyst is expected to monitor the response
from the instrument drift standard(in each sample) throughout the sample
set being analyzed. The absolute response of any one drift standard must
not deviate more than 70-13 0% of the original response associated with the
calibration blank. If deviations greater than these are observed, the reason
for the drift should be investigated. Possible causes of drift may be a
partially blocked sampling cone or a change in the tuning condition of the
instrument.
9.4 Assessing Analyte Recovery and Data Quality
9.4.1 The chemical nature of the sample matrix can affect analyte recovery and
.•'•;•. . .the quality of the data. Taking separate aliquots from the sample for
replicate and fortified analyses can in some cases assess the effect. Unless
otherwise specified by the data user, laboratory or program, the following
laboratory fortified matrix (LFM) procedure is required.
9.4.2 The laboratory must add a known amount of analyte to a minimum of 10%
of the routine samples. In each case the LFM aliquot must be a duplicate of
the aliquot used for sample analysis. The added bromate concentration must
be the same as that used in the laboratory fortified blank. Over time all
routine sample sources should be fortified.
9.4.3 Calculate the percent recovery for bromate, corrected for background
concentrations measured in the unfortified sample, and compare these
values to the designated LFM recovery range of 70-130%. Percent recovery
may be calculated using the following equation:
321.8-15
-------
R =
C.-C
xlOO
where:
R
Cs
C
s
= percent recovery.
= fortified sample concentration.
= sample background concentration.
= concentration equivalent of bromate added to fortify the
sample.
Note: The precision and recovery in six drinking water matrices are
reported in Table 3.
9.4.4 If recovery falls outside the designated range and laboratory performance is
shown to be in control, the recovery problem encountered with the fortified
sample is judged to be matrix related, not system related. The data user
should be informed that the result for the unfortified sample is suspect due
to an uncorrected matrix effect.
10. CALIBRATION AND STANDARDIZATION
10.1 Operating conditions - Because of the diversity of instrument hardware, no detailed
instrument operating conditions are provided. The analyst is advised to follow the
recommended operating conditions provided by the manufacturer. It is the
responsibility of the analyst to verify that the instrument configuration and operating
conditions satisfy the analytical requirements of this method and to maintain quality
control data verifying instrument performance and analytical results. Instrument
operating conditions which were used to generate precision and recovery data for
this method are included in Table 2,
10.2 Precalibration routine - The following precalibration routine must be completed
prior to calibrating the instrument.
10.2.1 Initiate proper operating Configuration of instrument and data system.
Allow a period of not less than 30 min for the instrument to warm up.
Conduct mass calibration and resolution checks using the tuning solution.
The tuning solution should be analyzed by scanning over masses 76-83
using 20 points per amu (skipping mass 80). For good performance, adjust
spectrometer resolution to produce a peak width of approximately 0.75 amu
at 5% peak height. Adjust mass calibration if it has shifted by more than
0.1 amu from unit mass. The abundance sensitivity must be checked by
analyzing the calibration blank(See Figure 1).
10.3 Instrument drift - An instrument drift solution must be injected into the column
effluent at the same time a sample is injected onto the column to verify instrument
321.8-16
-------
drift throughout the analysis of a set of samples. This solution does not traverse the
column. The correction factor is calculated by ratioing the drift standard response in
the calibration solution to the drift standard response in the current sample. This
correction factor is then multiplied by the sample concentration. This is similar to
an internal standard correction used in EPA Method 200.8[9].
Note: The stability of the baseline on mass 81 is strongly influenced by the
1C pump being turned on and off. Therefore it is recommended that this
pump remain on throughout an analysis set.
10.4 Calibration - Prior to initial calibration, set up proper instrument software routines
for the collection of time resolved data. (See Table 2 for the experimental
parameters used to collect the data within the method.) The instrument must be
calibrated using the calibration blank and a calibration standard prepared at one or
more concentration levels. If single point calibration is used, the standard
concentration should be near the determined upper linear range.
10.5 The rinse blank should be used to flush the 1C injection loop and by-pass loops.
This procedure is recommended between injections.
11. PROCEDURE
11.1 Aqueous Sample Preparation - The sample must be adjusted to pH 10 prior to
analysis. The sample must be room temperature and pretreated with an on-guard RP
cartridge prior to analysis. The RP cartridges were used according to the
manufacturer's recommendations. Trisubstituted haloacetic acids (removed by the
RP cartridge) can cause a slowly rising baseline because these trisubstiruted
haloacetic acids are strongly retained on the column.
11.2 The sample is injected onto the column at the same time the instrument drift
standard is injected into the post column mobile phase. For sample and drift
standard sample loop volumes see table 2.
11.3 Samples having concentrations higher than the established linear dynamic range
should be diluted into range and reanalyzed.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Sample data should be reported in units of u.g/L for aqueous samples. Do not report
bromate concentrations below the determined MDL. Drift standard correction
should be applied to all sample concentrations.
12.2 For data values less than ten, two significant figures should be used for reporting
element concentrations. For data values greater than or equal to ten, three significant
figures should be used.
321.8-17
-------
12.3 If additional dilutions were made to any samples, the appropriate dilution factor
should be applied.
12.4 The primary quantitative isotope should be 79 because it has the most stable
background and is less prone to the known interferences. Mass 81 should be
monitored to verify that the bromide isotope ratio within the retention window is
near unity. A ratio of the two isotopes can provide useful information for the analyst
in detecting a possible spectral interference.
12.5 The QC data obtained during the analyses provide an indication of the quality of the
sample data and should be provided with the sample results.
13. METHOD PERFORMANCE
13.1 Instrument operating conditions used for single laboratory testing of the method are
summarized in Table 2.
13.2 Data obtained from single laboratory testing of the method are summarized in Table
3 for six drinking water samples. The average concentrations reported in the second
column are the native bromate concentrations. The percent relative standard
deviations associated with the native concentrations are reported in the second
column. The samples were then fortified with 25ug/L bromate. The average
recovery and precision of this recovery is reported in the 4th and 5th columns
respectively.
14. POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or eliminates the
quantity or toxicity of waste at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The EPA has established a
preferred hierarchy of environmental management techniques that places pollution
prevention as the management option of first choice. Whenever feasible, laboratory
personnel should use pollution prevention techniques to address their waste
generation. When wastes cannot be feasibly reduced at the source, the Agency
recommends recycling as the next best option.
14.2 For information about pollution prevention that may be applicable to laboratories
and research institutions, consult "Less is Better: Laboratory Chemical
Management for Waste Reduction ", available from the American Chemical Society's
Department of Government Relations and Science Policy, 1155 16th Street N.W.,
Washington D.C. 20036, (202)872-4477.
321.8-18
-------
15. WASTE MANAGEMENT
15.1 The Environmental Protection Agency requires that laboratory waste management
practices be conducted consistent with all applicable rules and regulations. The
Agency urges laboratories to protect the air, water, and land by minimizing and
controlling all releases from hoods and bench operations, complying with the letter
and spirit of any sewer discharge permits and regulations, and by complying with all
solid and hazardous waste regulations, particularly the hazardous waste
identification rules and land disposal restrictions. For further information on waste
management consult "The Waste Management Manual for Laboratory Personnel",
available from the American Chemical Society at the address listed in the Section
14.2.
16. REFERENCES
1. Gray A.L. and A. R. Date. Inductively Coupled Plasma Source Mass Spectrometry
Using Continuum Flow Ion Extraction. Analyst 108, 1033-1050, 1983.
2. Houk R.S., V.A. Fassel, G.D. Flesch, HJ. Svec, A.L. Gray, C.E. Taylor. Inductively
Coupled Argon Plasma as an Ion Source for Mass Spectrometric Determination of
Trace Elements. Anal. Chem. 52, 2283-2289, 1980.
3. Houk R.S.. Mass Spectrometry of Inductively Coupled Plasmas. Anal. Chem. 58,
97A-105A, 1986.
4. Heitkemper D.T., L.A. Kaine, D.S. Jackson, K.A. Wolnik. Practical Applications of
Element-Specific Detection by Inductively Coupled Plasma Atomic Emission
Spectroscopy and Inductively Coupled Plasma Mass Spectrometry to Ion
Chromatography of Food. J. Chrom. A. 671,, 101-108, 1994.
5. Creed J.T., M.L. Magnuson, J.D. Pfaff, C.A. Brockhoff. Determination of Bromate
in Drinking Waters by Ion Chromatography With Inductively Coupled Plasma Mass
Spectrometric Detection. /. Chrom. A. 753. 261-67, 1996.
6. Creed J.T., M.L. Magnuson, C.A. Brockhoff. Determination Of Bromate in the
Presence of Brominated Haloacetic Acids by Ion Chromatography With Inductively
Coupled Plasma Mass Spectrometric Detection. ES&T 3_1, 2059-2063,1997.
7. Diemer J., K.G. Heumann. Bromide / Bromate Speciation by NTI-IDMS and ICP-
MS Coupled With Ion Exchange Chromatography. Fres. J. Anal. Chem. 357, 74-79,
1997.
8. Inductively Coupled Plasmas in Analytical Atomic Spectrometry, Second Edition,
copyright 1992 VCH Publishers, edited by Akbar Montaser and D.W. Golightly,
Chapter 12.
321.8-19
-------
9. US EPA Method 200.8, Determination of Trace Elements in Waters and Wastes by
Inductively Coupled Plasma - Mass Spectrometry, Revision 5.4,1994. Available
from the National Technical Information Service (NTIS) as PB-94-184942.
10. Carcinogens - Working With Carcinogens, Department of Health, Education, and
Welfare, Public Health Service, Center for Disease Control, National Institute for
Occupational Safety and Health, Publication No. 77-206, Aug. 1977. Available
from the National Technical Information Service (NTIS) as PB-277256.
11. Safety in Academic Chemistry Laboratories, American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
12. American Society for Testing and Materials. Standard Specification for Reagent
Water, Dl 193-77. Annual Book of ASTM Standards, Vol. 11.01. Philadelphia, PA,
1991.
13. Code of Federal Regulations 40, Ch. 1, Pt. 136 Appendix B.
321.8-20
-------
17. TABLES. DIAGRAMS. FLOWCHARTS AND VALIDATION DATA
TABLE 1. SPECTRAL AND CHROMATOGRAPHIC INTERFERENCES
Spectral Interferences
Interferent
Source
Plasma
Sulfate*
Phosphate**
Potassium
MASS
79
40Ar38Arjr
PO3+
4OAr39K+
MASS
81
40Ar40ArH+
SO,H+
PO3H2+
* Determined in 300 u.g/mL sulfate.
** Determined in 100 ng/mL phosphate.
Potential Chromatographic or Coelution Interferences
Retention Time*
Bromate
Bromoacetic Acid
Dibromoacetic Acid
Bromochloroacetic Acid
Bromide
Phosphate Matrix (P(V)
Sulfate Matrix (SO3H+)
230
170
460
400
570
180
400
*Based on experimental conditions listed in Table 2. Reported in seconds to the leading edge of
the peak using the drift standard as t = 0.
321.8-21
-------
TABLE 2. EXPERIMENTAL CONDITIONS FOR THE DETECTION
OF BROMATE VIA ICP-MS
ICP-MS Experimental Condition and Detection Limit
Instrument Upgraded VG Elemental PQ1 :''
Power . 1.4 KW
Cool Gas • 12.0 L/min
AuxGas 1.2 L/min
Nebulizer Gas 0.957 L/min (Concentric)
m/z Monitored 79 and 81
Analysis Mode Time Resolved or Chromatographic
Time Slice 0.4 seconds
Spray Chamber 5°C
Sensitivity (l,OOng/L BrO3') 35,000cps m/z 79
Background
(5mM HNO3 + 25mM NH4NO3) 100 cps m/z=79; 2500cps m/z=81
Detection Limit 0.3ug/L
Chromatographic Experimental Conditions
Chromatograph Dionex Gradient GPM-2
Column PA100 Guard and Analytical
Flowrate 1 mL/min.
Pretreatment Cartridge On-Guard RP
Mobile Phase 5mM HNO3 + 25mM NH4NO3 (Isocratic)
Sample Loop 580 uL (based on i.d. and length)
Drift Standard Loop 170 uL
321.8-22
-------
TABLE 3. PRECISION AND RECOVERY DATA FOR BROMATE
IN OZONATED DRINKING WATER*
WATER
1
2
3
4
5
6
AVERAGE
CONCENTRATION
ng/mL
22.2
3.0
10.1
2.7
1.3
0.8
%RSD
OfAVG
CONC.
5.5
6.4
3.6
5.1
10.6
15.5
AVG%**
RECOVERY
LFM
97
98
98
96
96
102
%RSD
Of LFM
RECOVERY
3.6
1.4
3.4
3.8
3.0
2.4
* n=5 for all analyses, determined using experimental conditions listed in Table 2.
** Fortified with 25fig/L Bromate
321.8-23
-------
Figure 1: Abundance Sensitivity Considerations
for Bromate Analysis
400 r
300
200
100
A) Borderline abundance sensitivity
Extrapol atioi
Line
V,
75 76 77 78 79 80 81 82 83
300
250
200
150
100
50
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-
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•
•
.
^^U;
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H
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y
Extrapo
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latic
*
n
V/v
rt.
75 76 77
83
321.8-24
-------
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321.8-25
-------
-------
METHOD 515.3 DETERMINATION OF CHLORINATED ACIDS IN DRINKING
WATER BY LIQUID-LIQUID EXTRACTION, DERIVATIZATION
AND GAS CHROMATOGRAPHY WITH ELECTRON CAPTURE
DETECTION.
Revision 1.0
July 1996
R.C. Dressman and J.J. Lichtenberg - EPA 600/4-81-053, Revision 1.0 (1981)
J.W. Hodgeson - Method 515, Revision 2.0 (1986)
T. Engels (Battelle Columbus Laboratory) and D.Munch (U.S.EPA, Office of Water) -
National Pesticide Survey Method 3, Revision 3.0 (1987)
R.L. Graves - Method 515.1, Revision 4.0 (1989)
J.W. Hodgeson - Method 515.2, Revision 1.0 (1992)
Anne M. Pawlecki-Vonderheide, International Consultants, Inc. and David J. Munch
U.S.EPA, Office of Water
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
515.3-1
-------
METHOD 515.3
DETERMINATION OF CHLORINATED 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 (1-12) applicable to the determination
of the listed chlorinated acids in drinking water, ground water, raw source water
and water at any intermediate treatment stage.
Chemical Abstract Services
Analyte Registry Number
Acifluorfen(a) 50594-66-6
Bentazon 25057-89-0
Chloramben ' 133-90-4
2,4-D 94-75-7
Dalapon 75-99-0
2,4-DB 94-82-6
Dacthal acid metabolites^
Dicamba 1918-00-9
3,5-Dichlorobenzoic acid 51-36-5
Diclorprop 120-36-5
Dinoseb 88-85-7
5-Hydroxydicamba 7600-50-2
4-Nitrophenol 100-02-7
Pentachlorophenol 87-86-5
Picloram 1918-02-1
2,4,5-T 93-76-5
2,4,5-TP (Silvex) 93-72-1
00 The herbicide Lactofen will be quantitated as Acifluorfen as their structures
represent different esters of the same carboxylate moiety.
w Dacthal monoacid and diacid metabolites as well as the parent di-ester
included in method scope; Dacthal diacid used for validation studies.
1.2 This method is also applicable to the determination of salts and esters of analyte
acids. The form of each acid is not distinguished by this method. Results are
calculated and reported for each listed analyte as the total free acid.
515.3-2
-------
1.3 Experimentally determined method detection limits (MDLs) (Section 9.4) for the
above listed analytes are provided in Tables 2 and 3. Actual MDLs may vary
according to the particular matrix analyzed and the specific instrumentation
employed.
1.4 This method is designed for analysts skilled in liquid-liquid extractions,
derivatization procedures and the use of GC and interpretation of gas
chromatograms. Each analyst must demonstrate the ability to generate acceptable
results with this method using the procedure described in Section 9.3.
1.5 When this method is used for the analyses of waters from unfamiliar sources, it is
strongly recommended that analyte identifications be confirmed by GC using a
dissimilar column or by GC/MS if concentrations are sufficient.
1.6 When using the diazomethane derivatization procedure, it is recommended that
only qualitative identification be performed for 4-nitrophenol and 5-
hydroxydicamba. Examination of supporting data presented in Tables 2,4, 6, 8,
10 and 12 shows control over precision has not been achieved for these method
analytes, and quantitative identification is therefore not recommended.
1.7 When using the base-promoted methylation procedure, it should be noted that the
esterification efficiences of dinoseb and picloram were found to be less than 50%.
Although the supporting data presented in Tables 3, 5, 7, 9,11 and 13
demonstrates that accurate and precise data can be obtained through the use of
procedural standards, care should be exercised.
1.8 5-Hydroxydicamba was not recovered from chlorinated waters. The exact
interaction between this compound and the free chlorine is not known. The
extremely low recoveries of 5-hydroxydicamba found in Tables 8 and 9 serve to
illustrate this. As noted, the matrix used to obtain these results was local
chlorinated tap water that was first fortified and then dechlorinated. (Note that in
further experiments, 5-hydroxydicamba was recovered in waters that were
dechlorinated prior to fortification.)
2. SUMMARY OF METHOD
2.1 A 40-mL volume of sample is adjusted to pH 12 with 4N sodium hydroxide for
one hour to hydrolyze derivatives. (NOTE: Since many of the analytes contained
in this method are applied as a variety of esters and salts, it is imperative to
hydrolyze them, to the parent acid prior to extraction). The aqueous sample is then
acidified and extracted with 4-mL of methyl-tert-butyl-ether (MtBE). The chlori-
nated acids that have been partitioned into the organic phase are then converted to
their methyl esters by one of two derivatization techniques. The first uses
515.3-3
-------
diazomethane as the methylating agent; the second is a base-promoted
esterification procedure and involves the addition of tetramethylammonium
hydroxide followed by the addition of methyl iodide. The target esters are then
identified and measured by capillary column gas chromatography using an elec-
tron capture detector (GC/ECD). Analytes are quantitated using procedural
standard calibration.
3. DEFINITIONS
3.1 INTERNAL STANDARD (IS) - A pure analyte(s) added to a sample, extract, or
standard solution in known amount(s) and used to measure the relative responses
of other method analytes and surrogates that are components of the same sample
or solution. The internal standard must be an analyte that is not a sample
component.
3.2 SURROGATE ANALYTE (SA) -- A pure analyte(s), which is extremely unlikely
to be found in any sample, and which is added to a sample aliquot in known
amount(s) before extraction or other processing and is measured with the same
procedures used to measure other sample components. The purpose of the SA is
to monitor method performance with each sample.
3.3 LABORATORY DUPLICATES (LD1 AND LD2) ~ Two aliquots of the same
sample designated as such in the laboratory. Each aliquot is extracted, derivatized
and analyzed separately with identical procedures. Analyses of LD1 and LD2
indicate the precision associated with laboratory procedures, but not with sample
collection, preservation, or storage procedures.
3.4 FIELD DUPLICATES (FD1 AND FD2) -- Two separate samples collected at the
same time and place under identical circumstances and treated exactly the same
throughout field and laboratory procedures. Analyses of FD1 and FD2 give a
measure of the precision associated with sample collection, preservation and
storage, as well as with laboratory procedures.
3.5 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or
other blank matrix that are treated exactly as a sample including exposure to all
glassware, equipment, solvents, preservation and other reagents, internal
standards, and surrogates that are used with other samples. The LRB is used to
determine if method analytes or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.6 FIELD REAGENT BLANK (FRB) - An aliquot of reagent water or other blank
matrix that is placed in a sample container in the laboratory and treated as a
sample in all respects, including shipment to the sampling site, exposure to
515.3-4
-------
sampling site conditions, storage, preservation and all analytical procedures. The
purpose of the FRB is to determine if method analytes or other interferences are
present in the field environment.
3.7 LABORATORY FORTIFIED BLANK (LFB) -- An aliquot of reagent water or
other blank matrix to which known quantities of the method analytes are added in
the laboratory. The LFB should be treated like a sample including the addition of
all preservation and other reagents. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise measurements.
3.8 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) - An aliquot of an
environmental sample to which known quantities of the method analytes are
added in the laboratory. The LFM is analyzed exactly like a sample, including the
addition of all preservation and other reagents, and its purpose is to determine
whether the sample matrix contributes bias to the analytical results. The back-
ground 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 (SSS) - A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed reference
materials or purchased from a reputable commercial source.
3.10 PRIMARY DILUTION STANDARD SOLUTION (PDS) - A solution of several
analytes prepared in the laboratory from stock standard solutions and diluted as
needed to prepare calibration solutions and other needed analyte solutions.
3.11 CALIBRATION STANDARD (CAL) - A solution prepared from the primary
dilution standard solution and stock standard solutions of the internal standards
and surrogate analytes. The CAL solutions are used to calibrate the instrument
response with respect to analyte concentration.
3.12 QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of
known concentration which is used to fortify an aliquot of reagent water or sample
matrix. The QCS is obtained from a source external to the laboratory and
different from the source of calibration standards. It is used to check laboratory
performance with externally prepared test materials.
3.13 LABORATORY PERFORMANCE CHECK SOLUTION (LPC) - A solution of
selected method analytes used to evaluate the performance of the instrumental
system with respect to a defined set of method criteria.
515.3-5
-------
3.14 METHOD DETECTION LIMIT (MDL) - The minimum concentration of an
analyte that can be detected, identified, measured and reported with 99% confi-
dence that the analyte concentration is greater than zero.
3.15 MATERIAL SAFETY DATA SHEET (MSDS) - Written information provided
by vendors concerning a chemical's toxicity, health hazards, physical properties,
fire and reactivity data including storage, spill, and handling precautions.
3.16 ESTIMATED DETECTION LIMIT (EDL) - Defined as either the MDL or a level
of a compound in a sample yielding a peak in the final extract with a signal to
noise (S/N) ratio of approximately 5, whichever is greater.
3.17 PROCEDURAL STANDARD QUANTITATION - A quantitation method where
aqueous calibration standards are prepared and processed (e.g. purged, extracted
and/or derivatized) in exactly the same manner as a sample. All steps in the
process from addition of sampling preservatives through instrumental analyses are
included in the calibration. Using procedural standard calibration compensates
for any inefficiencies in the processing procedure.
3.18 CONTINUING CALIBRATION CHECK (CCC) -- A procedural calibration
standard containing the method analytes, which is extracted, derivatized and
analyzed to verify the accuracy of the existing calibration curve or response
factors for those analytes.
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 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 9.5.
Subtracting blank values from sample results is not permitted.
4.1.1 Glassware must be scrupulously cleaned. (1) 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 and reagent water. Drain and heat in an oven or muffle furnace
at 400°C for 1 hr. Do not heat volumetric ware. (Thorough rinsing with
reagent grade acetone may be substituted for the heating. Thermally stable
materials such as PCBs may not be eliminated by this treatment.) After
drying and cooling, store glassware in a clean environment to prevent any
accumulation of dust or other contaminants. Store inverted or capped with
aluminum foil.
515.3-6
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4.1.2 The use of high purity reagents and solvents helps to minimize interfer-
ence 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 (Tables 2 and 3) at the retention times of the
analytes of interest. Purification of solvents by distillation in all-glass sys-
tems may be required.
4.2 Interfering contamination may occur when a sample containing low concentra-
tions of analytes is analyzed immediately following a sample containing relatively
high concentrations of analytes. Routine between-sample rinsing of the sample
syringe and associated equipment with MTBE can minimize sample cross-
contamination. After analysis of a sample containing high concentrations of
analytes, one or more injections of MTBE should be made to ensure that accurate
values are obtained for the next sample.
4.3 Matrix interferences may be caused by contaminants that are coextracted from the
sample. The extent of matrix interferences will vary considerably from source to
source, depending upon the water sampled. Analyte identifications should be
confirmed using the confirmation column specified in Table 1 or another column
that is dissimilar to the primary column or by GC/MS if the concentrations are
sufficient.
4.4 Interferences by phthalate esters can pose a major problem in pesticide analysis
when using an electron-capture detector. These compounds generally appear in
the chromatogram as large peaks. Common flexible plastics contain varying
amounts of phthalates that are easily extracted or leached during laboratory
operations. Cross contamination of clean glassware routinely occurs when
plastics are handled during extraction steps, especially when solvent-wetted
surfaces are handled. Interferences from phthalates can best be minimized by
avoiding the use of plastics in the laboratory. Exhaustive purification of reagents
and glassware may be required to eliminate background phthalate contamination.
(2,3)
4.5 The presence of water may cause incomplete methylation when using the base-
promoted esterification procedure. It is imperative to ensure that all reagents and
glassware are completely free of water.
4.6 5-Hydroxydicamba was not recovered from chlorinated waters. The exact
interaction between this compound and the free chlorine is not known. The
extremely low recoveries of 5-hydroxydicamba found in Tables 8 and 9 serve to
illustrate this. As noted, the matrix used to obtain these results was local
chlorinated tap water that was first fortified and then dechlorinated. (Note that in
515.3-7
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further experiments, 5-hydroxydicamba was recovered in waters that were
dechlorinated prior to fortification.)
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been
precisely defined; however, each chemical compound must be treated as a
potential health hazard. From this viewpoint, exposure to these chemicals must
be minimized. The laboratory is responsible for maintaining a current awareness
file of OSHA regulations regarding the safe handling of the chemicals specified in
this method. A reference file of material safety data sheets should also be made
available to all personnel involved in the chemical analysis. Additional references
to laboratory safety are available and have been identified (4-6) for the
information of the analyst.
5.2 The toxicity of the extraction solvent, MTBE, has not been well defined.
Susceptible individuals may experience adverse affects upon skin contact or
inhalation of vapors. 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.
5.3 Diazomethane is a toxic carcinogen which can explode under certain conditions.
The following precautions must be followed:
5.3.1 Use the diazomethane generator behind a safety shield in a well ventilated
fume hood. Under no circumstances can the generator be heated above
90°C, and all grinding surfaces such as ground glass joints, sleeve bearings
and glass stirrers must be avoided. To minimize safety hazards, the
diazomethane generator apparatus used in the esterification procedure
(Section 11.2) produces micromolar amounts of diazomethane in solution.
If the procedure is followed exactly, no possibility for explosion exists.
5.4 Methyl iodide is a toxic carcinogen. When handling, protective clothing and
gloves should be worn, and this reagent should only be used in a fume hood or
glove box.
6. APPARATUS AND EQUIPMENT
6.1 SAMPLE CONTAINERS - Amber glass bottles, approximately 50 mL, fitted
with teflon-lined screw caps.
6.2 EXTRACTION VIALS ~ 60 mL clear glass vials with teflon-lined screw caps.
515.3-8
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6.3 VIALS — Autosampler, 2.0 mL vials with screw or crimp cap and a teflon-faced
seal.
6.4 STANDARD SOLUTION STORAGE CONTAINERS - 10-20 ml amber glass
vials with teflon-lined screw caps.
6.5 GRADUATED CONICAL CENTRIFUGE TUBES WITH TEFLON-LINED
SCREW CAPS ~ 15-mL with 1 mL graduation markings.
6.6 BLOCK HEATER (or SAND BATH) ~ Capable of holding screw cap conical
centrifuge tubes in Section 6.5.
6.7 PASTEUR PIPETS - Glass, disposable.
6.8 PIPETS ~ 2.0 mL and 4.0 mL, type A, TD, glass.
6.9 VOLUMETRIC FLASKS -- 5 ml, 10 mL, 100 mL.
6.10 - MICRO SYRINGES - 10 \iL, 25 uL, 50 |iL, 100 jxL, 250 [iL, 500 \iL and 1000
jiL.
6.11 BALANCE--analytical, capable of weighing to 0.0001 g.
6.12 DIAZOMETHANE GENERATOR ~ See Figure 1 for a diagram of an all glass
system custom made for these validation studies. A micromolar generator is also
available from the Aldrich Chemical Company.
6.13 GAS CHROMATOGRAPH •-- Analytical system complete with gas chromato-
graph equipped for electron-capture detection, split/splitless capillary or direct
' ~ * 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 electron-capture detector should be vented through the
laboratory fume hood system.
6.14 PRIMARY GC COLUMN ~ DB-1701 [fused silica capillary with chemically
bonded (14% cyanopropylphenyl)-methylpolysiloxane)] or equivalent bonded,
•" . . fused silica column, 30 m x 0.25 mm ID, 0.25 |j.m film thickness.
6.15 CONFIRMATORY GC COLUMN ~ DB-5.625 [fused silica capillary with
chemically bonded (5% phenyi)-methylpolysiloxane)] or equivalent bonded, fused
silica column, 30m x 0.25mm ID, 0.25 um film thickness.
515.3-9
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7. REAGENTS AND STANDARDS
7.1 REAGENT WATER — Reagent water is defined as a water in which an interfer-
ence is not observed z to the MDL of each analyte of interest.
7.1.1 A Millipore Super-Q water system or its equivalent may be used to
generate deionized reagent water. Distilled water that has been passed
through granular charcoal may also be suitable.
7.1.2 Reagent water is monitored through analysis of the laboratory reagent
blank (Section 9.5).
7.2 SOLVENTS
7.2.1 METHYL tert-BUTYL ETHER (MtBE) - High purity, demonstrated to
be free from analytes and interferences, redistilled in glass if necessary.
7.2.2 ACETONE — High purity, demonstrated to be free from analytes and
interferences.
7.2.3 CARBITOL (DIETHYLENE GLYCOL MONOETHYL ETHER) - High
purity, demonstrated to be free from analytes and interferences.
7.2.4 ETHYL ETHER — High purity, unpreserved, demonstrated to be free from
analytes and interferences.
7.3 REAGENTS
7.3.1 SODIUM SULFATE, Na2SO4 - (ACS) granular, anhydrous. If
interferences are observed, it may be necessary to heat the sodium sulfate
in a shallow tray at 400°C for up to 4 hr. to remove phthalates and other
interfering organic substances. Alternatively, it can be extracted with
methylene chloride in a Soxhlet apparatus for 48 hr. Store in a capped
glass bottle rather than a plastic container.
7.3.2 ACIDIFIED SODIUM SULFATE -- Acidify by slurrying 500g of muffled
sodium sulfate with enough ethyl ether to just cover the solid. Add 0.7 mL
concentrated sulfuric acid dropwise while mixing thoroughly. Remove the
ether under vacuum. Mix Ig of the resulting solid with 5 mL of reagent
water and measure the pH of the mixture. The pH must be below pH 4.
Store at 100°C.
515.3-10
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7.3.3 COPPER E SULFATE PENTAHYDRATE, CuSO4 5H2O - ACS
reagent grade.
7.3.4 SODIUM HYDROXIDE, pellets - ACS reagent
grade.
7.3.5 POTASSIUM HYDROXIDE, pellets - ACS reagent grade
7.3.6 SODIUM THIOSULFATE, Na^A - ACS reagent grade, used as
a dechlorinating agent in this method.
7.3.7 DIAZALD - ACS reagent grade.
7.3.8 SULFURIC ACID, CONCENTRATED - ACS reagent grade
7.3.9 METHYL IODIDE - ACS reagent grade
7.3.10 TETRABUTYLAMMONIUM HYDROXIDE - ACS reagent grade.
This reagent can be purchased as a l.OM solution in methanol. It is impor-
tant that it contain no water as moisture may result in incomplete methyla-
tion.
7.3.11 SILICA GEL — ACS reagent grade. If interferences are observed, it may
be necessary to heat this reagent at 100°C for 1 hour.
7.3.12 FLORISEL - 60-100/PR mesh. Activate by heating in a shallow container
at 150°C for at least 24 hours and not more than 48 hours.
7.4 SOLUTIONS
7.4.1 4N NaOH SOLUTION - Dissolve 16g NaOH pellets in reagent water and
dilute to 100 mL.
7.4.2 37% (w/v) KOH SOLUTION - Dissolve 37g KOH pellets in
reagent water and dilute to 100 mL.
7.4.3 DIAZALD SOLUTION - Prepare a solution containing 5g Diazald in 50
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
bottle with a Teflon-lined screw cap.
515.3-11
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7.5 STANDARDS
7.5.1 4,4'-DffiROMOOCTAFLUOROBIPHENYL, 99+% -- For use as the
internal standard. Prepare a stock internal standard solution of 4,4'-
Dibromooctafluorobiphenyl by accurately weighing approximately 0.0200
g of neat material. Dissolve the neat material in MtBE and dilute to
volume in a 10 mL volumetric flask. Transfer the solution to an amber
glass vial with a teflon-lined screw cap and store at 4°C. The resulting
concentration of the stock internal standard solution will be approximately
2.0 mg/mL. Prepare a primary dilution standard at approximately 2.5
lig/mL by the addition of 12.5 \iL of the stock standard to 10 mL of MtBE.
Transfer the primary dilution to an amber glass vial with a teflon-lined
screw cap and store at 4°C. Addition of 10 uL of the primary dilution
standard to the final 1 mL extract results in a final internal standard
concentration of 25 ng/mL. The solution should be replaced when
ongoing QC indicates a problem. This compound has been shown to be an
effective internal standard for the method analytes, but other compounds
may be used if the QC requirements in Section 9 are met.
7.5.2 2,4-DICHLOROPHENYLACETIC ACID, 99+% ~ For use as a surrogate
compound. Prepare a surrogate stock standard solution of 2,4-
Dichlorophenylacetic acid by accurately weighing approximately 0.0100 g
of neat material. Dissolve the neat material in acetone and dilute to
volume in a 10 mL volumetric flask. Transfer the solution to an amber
glass vial with a teflon-lined screw cap and store at 4°C. The resulting
concentration of the stock surrogate solution will be approximately 1.0
mg/mL. Prepare a primary dilution standard at approximately 100 [ig/mL
by the addition of 1 mL of the stock standard to 10 mL of acetone.
Transfer the primary dilution to an amber glass vial with a teflon-lined
screw cap and store at 4°C. Addition of 10 |iL of the primary dilution
standard to the 40 mL aqueous sample results in a surrogate concentration
of 25 \ig/L. The solution should be replaced when ongoing QC indicates a
problem. This compound has been shown to be an effective surrogate for
the method analytes, but other compounds may be used if the QC require-
ments in Section 9 are met.
7.5.3 STOCK STANDARD SOLUTION (SSS) - Prepare separate stock
standard solutions for each analyte of interest at a concentration of 1-5
mg/mL in acetone. Method analytes may be obtained as neat materials or
ampulized solutions (> 99% purity) from a number of commercial suppli-
ers but ampulized solutions should not be used if the solvent is methanol.
(7) These stock standard solutions should be stored at 4°C. They are
515.3-12
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stable for at least one month but should be checked frequently for signs of
evaporation.
7.5.3.1 For analytes which are solids in their pure form, prepare stock
standard solutions by accurately weighing approximately 0.01
to 0.05 grams of pure material in a 10.0 mL volumetric flask.
Dilute to volume with acetone. Each compound's purity must
be assayed to be 96% or greater.
7.5.3.2 Stock standard solutions for analytes which are liquid in their
pure form at room temperature can be accurately prepared in
the following manner.
7.5.3.3 Place about 9.8 mL of acetone into a 10.0 mL volumetric flask.
Allow the flask to stand, unstoppered, for about 10 minutes to
allow solvent film to evaporate from the inner walls of the
volumetric, and weigh to the nearest 0.1 mg.
7.5.3.4 Use a 10 uL syringe and immediately add 10.0 |j,L of standard
material to the flask by keeping the syringe needle just above
the surface of the acetone. Be sure that the standard material
falls dropwise directly into the acetone without contacting the
inner wall of the volumetric.
7.5.3.5 Reweigh, dilute to volume, stopper, then mix by inverting the
flask several times. Calculate the concentration in milligrams
per milliliter from the net gain in weight.
7.5.4 PRIMARY DILUTION STANDARD (PDS) - Prepare the primary
dilution standard solution by combining and diluting stock standard
solutions with acetone. This primary dilution standard solution should be
stored at 4°C. It is stable for at least one month but should be checked
before use for signs of evaporation. As a guideline to the analyst, the
primary dilution standard solution used in the validation of this method is
described below.
515.3-13
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Concentration. \i g/mL
Acifluorfen 5.0
Bentazon 10.
Chloramben 5.0
2,4-D 10.
Dalapon 10.
2,4-DB 10.
Dacthal acid metabolites 5.0
Dicamba 5.0
3,5-Dichlorobenzoic acid 5.0
Diclorprop 10.
Dinoseb 10.
5-Hydroxydicamba 5.0
4-Nitrophenol 10.
Pentachlorophenol 1.0
Picloram 10.
2,4,5-T 2.5
2,4,5-TP (Silvex) 2.5
This primary dilution standard is used to prepare calibration standards,
which comprise five concentration levels of each analyte with the lowest
standard being at or near the MDL of each analyte. The concentrations of
the other standards should define a range containing the expected sample
concentrations or the working range of the detector.
7.5.5 CALIBRATION STANDARDS (CAL) - A five-point calibration curve is
to be prepared by diluting the primary dilution standard into acetone at the
appropriate levels. A designated amount of each acetone calibration
standard is then spiked into separate 40 mL aliquots of reagent water to
produce a calibration curve ranging from near the detection limit to
approximately 10-20 times the lowest calibration level. These aqueous
calibration standards should be treated like samples and therefore require
the addition of all preservation and other reagents. They are extracted by
the procedure set forth in Section 11. (The calibration standard solutions
in acetone should be stored at 4°C. They are stable for at least one month
but should be checked frequently for signs of evaporation.)
7.5.6 LABORATORY PERFORMANCE CHECK STANDARD (LPC) - The
low level standard of the calibration curve can serve as the LPC standard.
(Section 9.2)
515.3-14
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SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 SAMPLE VIAL PREPARATION
8.1.1 Grab samples must be collected in accordance with conventional sampling
practices (8) using amber glass containers with TFE-lined screw-caps and
capacities of at least 50 ml.
8.1.2 If residual chlorine is present, add 4 mg of sodium thiosulfate per 50 mL
of sample to the sample bottle prior to collecting the sample.
8.2. SAMPLE COLLECTION
8.2.1 Fill sample bottles but take care not to flush out the sodium thiosulate.
Because the target analytes of this method are not volatile, it is not
necessary to ensure that the sample bottles are completely headspace free.
8.2.2 When sampling from a water tap, open the tap and allow the system to
flush until the water temperature has stabilized (usually about 3-5
minutes). Remove the aerator so that no air bubbles can be visibly
detected and collect samples from the flowing system.
8.2.3 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 sample vials from the container.
8.2.4 After collecting the sample in the bottle containing the sodium thiosulfate,
seal the bottle and agitate by hand for 15 seconds.
8.2.5 Because of the several pH adjustments made to the samples in the course
of this method, the addition of hydrochloric acid to the samples to retard
biological activity has been omitted. However, the analyst should be
aware of the potential for the biological degradation of the analytes.
8.3 SAMPLE STORAGE/HOLDING TIMES
8.3.1 Samples must be iced or refrigerated at 4°C and maintained at these
conditions away from light until extraction. Synthetic ice (i.e. blue ice) is
not recommended. Holding studies performed to date have shown that, in
samples preserved with sodium thiosulfate, the analytes are stable for up to
14 days. (See Tables 16-19) Thus, once extracted, samples must be
analyzed within 14 days. Since stability may be matrix dependent, the
515.3-15
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analyst should verify that the prescribed preservation technique is suitable
for the samples under study.
8.3.2 Extracts (Sections 11.2.7 and 11.3.11) must be stored at 4°C or less away
from light hi glass vials with Teflon-lined caps. Holding time studies
indicate that the analytes are stable for up to 14 days in the extracts.
(Tables 20-23)
9. QUALITY CONTROL
9.1 Each laboratory that uses this method is required to operate a formal quality
control (QC) program. Minimum quality control requirements are monitoring the
laboratory performance check standard, initial demonstration of laboratory
capability, performance of the method detection limit study, analysis of laboratory
reagent blanks and laboratory fortified sample matrices, determination of
surrogate compound recoveries in each sample and blank, monitoring internal
standard peak area or height in each sample, blank and CCC, and analysis of QC
samples. Additional QC practices may be added.
9.2 LABORATORY PERFORMANCE CHECK STANDARD (LPC) - At the
beginning of an analysis batch, prior to any calibration standard or sample analysis
and after an initial solvent blank, a laboratory performance check standard must
be analyzed. It is not necessary that a new check standard be extracted each day.
This check standard ensures proper performance of the GC by evaluation of the
instrument parameters of detector sensitivity, peak symmetry, and peak resolution.
It also demonstrates that instrument sensitivity has not changed drastically since
the analysis of the MDL study. Inability to demonstrate acceptable instrument
performance indicates the need for re-evaluation of the instrument system.
Criteria are listed in Table 14.
9.2.1 The sensitivity requirement is based on the EDLs published in this
method. If laboratory EDLs differ from those listed in Tables 2 and 3,
concentrations of the LPC standard maybe adjusted to be compatible with
the laboratory EDLs.
9.2.2 The compounds listed in Table 15 for the LPC may not be included in the
analyses of a particular laboratory. Therefore, other analytes may be
chosen as long as each of the parameters (detector sensitivity, peak
symmetry and peak resolution) can be sufficiently evaluated.
9.2.3 If column or chromatographic performance cannot be met, a new column
may need to be installed, column flows corrected or modifications made to
the oven temperature program.
515.3-16
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9.3. INITIAL DEMONSTRATION OF CAPABILITY (IDC)
9.3.1 Select a representative fortification concentration for each of the target
analytes. Concentrations near those in Tables 6 and 7 are recommended.
Prepare 4-7 replicate laboratory fortified blanks by adding an appropriate
aliquot of the primary dilution standard or another certified quality control
sample to reagent water. Analyze the LFBs according to the method
beginning in Section 11.
9.3.2 Calculate the mean percent recovery and the standard deviation of the
recoveries. For each analyte, the mean recovery value, expressed as a
percentage of the true value, must fall in the range of 80-120% and the
relative standard deviation should be less than 20%. For those compounds
that meet these criteria, performance is considered acceptable and sample
analysis may begin. For those compounds that fail these criteria, this pro-
cedure must be repeated using 4-7 fresh samples until satisfactory
performance has been demonstrated. Maintain these data on file to
demonstrate initial capabilities.
9.3.3 Furthermore, before processing any samples, the analyst must analyze at
least one laboratory reagent blank to demonstrate that all glassware and
reagent interferences are under control. ••
9.3.4 The initial demonstration of capability is used primarily to preclude a
laboratory from analyzing unknown samples via a new, unfamiliar method
prior to obtaining some experience with it. As laboratory personnel gain
experience with this method, the quality of data should improve beyond
those required here.
9.3.5 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 Section 9.3.1 through
Section 9.3.4.
9.4 METHOD DETECTION LIMIT STUDY (MDL)
9.4.1 Prior to the analysis of any field samples, the method detection limits must
be determined. Initially, estimate the concentration of an analyte which
would yield a peak equal to 5 times the baseline noise and drift. Prepare
seven replicate laboratory fortified blanks at this estimated concentration.
Analyze the LFB's according to the method beginning in Section 11.
515.3-17
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9.4.2 Calculate the mean recovery and the standard deviation for each analyte.
Multiply the Student's t value at 99% confidence and n-1 degrees of
freedom (3.143 for seven replicates) by this standard deviation to yield a
statistical estimate of the detection limit. This estimate is the MDL.
9.4.3 MDL's should be recalculated after major changes in the chromatographic
temperature program or stationary phase or after a change in instrument or
detector.
9.5 LABORATORY REAGENT BLANKS (LRB) - Each time a set of samples is
extracted or reagents are changed, a LRB must be analyzed. The LRB must
contain the preservation and other reagents added to the sample. If the LRB
produces an interferant peak within the retention tune window (Section 12.3) of
any analyte that would prevent the determination of that analyte or a peak of
concentration greater than the MDL for that analyte, the analyst must determine
the source of contamination and eliminate the interference before processing
samples. For the analyte(s) that failed to meet this criteria, concentrations in field
samples are considered suspect.
9.6 LABORATORY FORTIFIED BLANK (LFB) - Since this method utilizes proce-
dural calibration standards, which are fortified reagent water, there is no
difference between the LFB and the continuing calibration check standard.
Consequently, the analysis of an LFB is not required (Section 10.2).
9.7 LABORATORY FORTIFIED SAMPLE MATRIX (LFM)
9.7.1 The concentrations of the analytes in a given sample may be equal to or
greater than the fortified concentrations. Subsequently, relatively poor
accuracy and precision may be anticipated when a large background must
be subtracted. For many samples, the concentrations may be so high that
fortification may lead to a final extract with instrumental responses
exceeding the working range of the electron capture detector. If this
occurs, the extract must be diluted. In spite of these problems, sample
sources should be fortified and analyzed as described below. By fortifying
sample matrices and calculating analyte recoveries, any matrix induced
analyte bias is evaluated.
9.7.2 The laboratory must add known concentrations of analytes to one sample
per extraction set or a minimum of 10% of the samples, whichever is
greater. The concentrations should be equal to or greater than the back-
ground concentrations in the sample selected for fortification. If the
fortification level is less than the background concentration, recoveries are
515.3-18
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not reported. Over time, samples from all routine sample sources should
be fortified.
9.7.3 Calculate the mean percent recovery, R, of the concentration for each
analyte, after correcting the total mean measured concentration, A, from
the fortified sample for the back-ground concentration, B, measured in the
unfortified sample, i.e..•
R=100(A-B)/C,
where C is the fortifying concentration. In order for the recoveries to be
considered acceptable, they must fall between 70% and 130% for all the
target analytes.
9.7.4 If a recovery falls outside of this acceptance range, a matrix induced bias
can be assumed for the respective analyte and the data for'that analyte
must be reported to the data user as suspect due to matrix effects.
9.8 ASSESSING SURROGATE RECOVERY - The surrogate standard is fortified
into the aqueous portion of all calibration standards, samples, QC samples and
laboratory reagent blanks. The surrogate is a means of assessing method perfor-
mance from extraction to final chromatographic performance.'
9.8.1 When surrogate recovery from a sample, blank, QC sample or CCC is <
70% or > 130%, check (1) calculations to locate possible errors, (2)
standard solutions for degradation, (3) contamination, and (4) instrument
performance. If those steps do not reveal the cause of the problem,
reanalyze the extract.
9.8.2 If the extract reanalysis meets the surrogate recovery criterion, report only
data for the reanalyzed extract.
9.8.3 If the extract reanalysis fails the 70-130% recovery criterion, the analyst
should check the calibration by analyzing the most recently acceptable
continuing calibration check standard. If the CCC fails the criteria of
Section 10.2.1, recalibration is in order per Section 10.1. If the CCC is
acceptable, it may be necessary to extract another aliquot of sample. If the
sample re-extract also fails the recovery criterion, report all data for that
sample as suspect.
515.3-19
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9.9 ASSESSING THE INTERNAL STANDARD
9.9.1 The analyst must monitor the IS response (peak area or peak height) of all
injections during each analysis day. A mean IS response should be
determined from the five point calibration curve. The IS response for any
run should not deviate from this mean IS response by more than 30%.
9.9.2 If a greater deviation than this occurs with an individual extract, optimize
instrument performance and inject a second aliquot of that extract.
9.9.2.1 If the reinjected aliquot produces an acceptable internal
standard response, report results for that aliquot.
9.9.2.2 If a deviation of greater tiian 30% is obtained for the reinjected
extract, the analyst should check the calibration by analyzing
the most recently acceptable CCC. If the CCC fails the criteria
of Section 10.2.1, recalibration is in order per Section 10.1. If
the CCC is acceptable, analysis of the sample should be
repeated beginning with Section 11, provided the sample is still
available. Otherwise, report results obtained from the reinject-
ed extract, but annotate as suspect.
9.10 QUALITY CONTROL SAMPLE (QCS) -- At least quarterly, analyze a QCS
from an external source. If measured analyte concentrations are not of acceptable
accuracy, check the entire analytical procedure to locate and correct the problem
source.
9.11 The laboratory may adapt additional QC practices for use with this method. The
specific practices that are most productive depend upon the needs of the laborato-
ry 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 maybe used to assess contamination of samples under site
conditions, transportation and storage.
10. CALIBRATION AND STANDARDIZATION
10.1 INITIAL CALIBRATION CURVE
10.1.1 Establish GC operating parameters equivalent to the suggested specifi-
cations in Table 1. The GC system must be calibrated using the internal
standard (IS) technique. Other columns or conditions may be used if
equivalent or better performance can be demonstrated.
515.3-20
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10.1.2 Calibration standards at five concentrations are required. The lowest
should contain the analytes at a concentration near to but greater than the
EDL (Tables 2 and 3) for each compound. The others should be evenly
distributed throughout the concentration range expected in the samples.
10. 1 .3 Inject 2 uL of each calibration standard extract and tabulate peak height or
area response and concentration for each analyte and the internal standard.
10.1 .4 Generate a calibration curve by plotting the area ratios (Aa/AjS) against the
concentration ratios (Ca/Cis) of the five calibration standards where
Aa is the peak area of the analyte.
AJS is the peak area of the internal standard.
Ca is the concentration of the analyte.
Cis is the concentration of the internal standard.
This curve can be defined as either first or second order; the correlation
coefficients must be greater than 0.95. Also, the working calibration curve
must be verified daily by measurement of one or more calibration stan-
dards (Section 10.2). If the response for any analyte falls outside the pre-
dicted response by more than 30%, the calibration check must be repeated
using a freshly prepared calibration standard. Should the retest fail, a new
calibration curve must be generated.
10.1.5 Alternately, an average relative response factor can be calculated and used
for quantitation. Relative response factors are calculated for each analyte
at the five concentration levels using the equation below:
(Aa)(Cis)
RRF = ----- -—
If the RRF value over the working range is constant (<20% RSD), the
RRF can be assumed to be invariant and the average RRF used for
calculations. Also, the average RRF must be verified daily by
measurement of one or more calibration standards (Section 10.2). If the
RRF for the continuing calibration standard deviates from the average
RRF by more than 30%, the calibration check must be repeated using a
freshly prepared calibration standard. Should the retest fail, a new
calibration curve must be generated.
515.3-21
-------
10.1.6 A data system maybe used to collect the chromatographic data, calculate
relative response factors, or calculate linear or second order calibration
curves.
10.2 CONTINUING CALIBRATION CHECK (CCC)
10.2.1 At least one CCC must be extracted with each set of samples. A CCC
must be analyzed at the beginning of each analysis set, after every tenth
sample analysis and after the final sample analysis, to ensure that the
instrument is still within calibration. These checks should be at two
different concentration levels. Calculate analyte recoveries for all target
analytes. In order for the calibration check to be considered valid and
subsequently for the preceding ten samples to be considered acceptable
with respect to calibration, recoveries must fall between 70% and 130%
for all the target analytes. Additionally, the internal standard area must be
within 30% of the mean IS response. (Section 9.9.1)
NOTE: Continuing calibration check standards need not all be different
extracts but can be injections from the same extract as long as the holding
tune requirements for extracts (Sect. 8.3.2) are met. However, at least one
must be extracted with each batch of samples.
10.2.2 If this criterion cannot be met, the continuing calibration check standard
extract is re-injected in order to determine if the response deviations
observed from the initial analysis are repeated. If this criterion still cannot
be met, a CCC that has already been analyzed and has been found to be
acceptable should be run. If this second CCC fails, then the instrument is
considered out of calibration and needs to be recalibrated. Should all
CCC's associated with a particular set of samples fail, the set of samples
must be re-extracted.
11. PROCEDURE
11.1 SAMPLE EXTRACTION AND HYDROLYSIS
11.1.1 Remove the samples from storage (Sect. 8.3.1) and allow them to
equilibrate to room temperature.
11.1.2 Place 40 mL of the water sample into a precleaned 60 mL glass vial with a
teflon-lined screw cap using a graduated cylinder.
11.1.3 Add 10 uL of surrogate standard (100 ng/mL 2,4-Dichlorophenylacetic
acid in acetone per Section 7.5.2) to the aqueous sample.
515.3-22
-------
NOTE: After injection, cap the sample and invert once. This ensures that
the fortification volume is mixed well with the water.
11.1.4 Add 1 mL of the 4N NaOH solution prepared in Section 7.4.1 to each
glass vial. Check the pH of the sample with pH paper or a pH meter; if the
sample does not have a pH greater than or equal to 12, adjust the pH by
adding more 4N NaOH solution. Let the sample sit at room temperature
for 1 hour, shaking the contents periodically.
NOTE: Since many of the herbicides contained in this method are applied
as a variety of esters and salts, it is vital to hydrolyze them to the parent
acid prior to extraction. This step must be included in the analysis of all
extracted field samples, LRBs, LFMs and calibration standards.
11.1.5 Adjust the pH to less than 0.5 by adding at least 2 mL of concentrated
sulfuric acid. Cap, shake and then check the pH with a pH meter or
narrow range pH paper.
11.1.6 Quickly add approximately 2 g of copper n sulfate pentahydrate and shake
until dissolved. This colors the aqueous phase blue and therefore allows
the analyst to better distinguish between the aqueous phase and the organic
phase in this micro extraction.
11.1.7 Quickly add approximately 16 g of muffled sodium sulfate and shake for 3
to 5 minutes until almost all is dissolved. Sodium sulfate is added to
increase the ionic strength of the aqueous phase and thus further drive the
chlorophenoxy acids into the organic phase. The addition of this salt and
the copper n sulfate pentahydrate should be done quickly so that the heat
generated from the addition of the acid (Section 11.1.5) will help dissolve
the salts.
11.1.8 Add exactly 4.0 mL MtBE and place on the mechanical shaker for 30
minutes. (If hand-shaken, two minutes is sufficient if performed
vigorously).
11.1.9 Allow the phases to separate for approximately 5 minutes.
11.2 METHYLATION - DIAZOMETHANE
NOTE: It is not recommended that this method of derivatization be used if 4-
nitrophenol and 5-hydroxydicamba are included in the target list.
11.2.1 Generation of Diazomethane.
515.3-23
-------
11.2.1.1 Assemble the diazomethane generator (Figure 1) in a hood.
The collection vessel is a 10 or 15-mL glass vial equipped with
a teflon-lined screw cap and maintained at 0-5 °C.
11.2.1.2 Add a sufficient amount of ethyl ether (approximately 7 ml) to
tube 1 to cover the first impinger. Add 5 ml of MtBE to the
collection vial. Set the nitrogen flow at 5-10 mL/min. Add 4
mL Diazald solution (Section 7.4.3) and 3 mL of 37% KOH
solution (Section 7.4.2) to the second impinger. Connect the
tubing as shown and allow the nitrogen flow to purge the
diazomethane from the reaction vessel into the collection vial
for 30 minutes. Cap the vial when collection is complete and
maintain at 0-5°C. When stored at 0-5°C, this diazomethane
solution may be used over a period of 48 hours.
11.2.2 Using a Pasteur pipet, transfer the sample extract (upper MtBE layer) to
a 10 mL screw cap vial. Add 0.1 g acidified sodium sulfate and shake.
This step is included to ensure the MtBE extract contains no water.
11.2.3 Using a Pasteur pipet, transfer exactly 3 mL of the dried MtBE extract to
a 15 mL graduated conical centrifuge tube.
11.2.4 Add 250 uL of the diazomethane solution prepared in Section 11.2.1.2.
to each centrifuge tube. The contents of the centrifuge tube should
remain slightly yellow in color. If this is not the case, more
diazomethane solution may be added, making sure to add the exact addi-
tional amount to every calibration standard, blank, QC sample and field
sample. Let the esterification reaction proceed for 30 minutes.
11.2.5 Remove any unreacted diazomethane by the addition of 0.1 g silica gel.
Effervescence (evolution of nitrogen) is an indication that excess
diazomethane was present. Allow the extracts to sit for 0.5 hour.
11.2.6 Place a small plug of glass wool into a disposable Pasteur pipet. Fill the
pipet with approximately 2 inches of florisil. (Section 7.3.12) (This step
is the preparation of clean-up columns for the methylated extracts. One
column should be prepared for each extract.)
11.2.7 Apply the methylated extract to the prepared clean-up column and
collect the eluate in a 5 mL vial.
11.2.8 Transfer exactly 1.0 ml of the MtBE extract to an autosampler vial. A
duplicate vial should be filled using the excess extract.
515.3-24
-------
11.2.9 Add 10 uL of internal standard to the vial to be analyzed. (2.5
4,4'-Dibromooctafluorobiphenyl in MtBE per Section 7.5.1). Internal
standard should be added to the duplicate vial before analysis.
11.2.10 Analyze the sample extracts as soon as possible. The sample extract
may be stored up to 14 days if kept at 4°C or less. Keep the extracts
away from light in amber glass vials with Teflon-lined caps.
11.3. METHYLATION - BASE-PROMOTED ESTERIFICATION
11.3.1 Using a Pasteur pipet, transfer approximately 3 mL of the sample extract
(upper MtBE layer) to a 10 mL screw cap vial. Add 0.1 g acidified
sodium sulfate and shake. This step is included to ensure that the MtBE
extract contains no water.
11.3.2 Using a Pasteur pipet, transfer exactly 3 mL of the dried MtBE extract to
a 15 mL graduated conical centrifuge tube.
11.3.3 Add 80 pL of the l.OM solution of tetrabutylammonium hydroxide.
11.3.4 Add 40 |j,L of methyl iodide.
11.3.5 Cap the centrifuge tubes and place in the heating block (or sand bath) at
50°C and maintain for 1.5 hr. The vials must fit snugly into the heating
block to ensure proper heat transfer. At this stage, methylation of the
method analytes is attained and the tetrabutylammonium iodide by-
product may be viewed as a precipitate.
11.3.6 Remove the centrifuge tubes from the heating block (or sand bath) and
allow them to cool before removing the caps.
11.3.7 Place a small plug of glass wool into a disposable Pasteur pipet. Fill the
pipet with approximately 2 inches of florisil. (Section 7.3.12) (This step
is the preparation of clean-up columns for the methylated extracts. One
column should be prepared for each extract.)
11.3.8 Apply the methylated extract to the prepared clean-up column and
collect the eluate in a 5 mL vial.
11.3.9 Transfer exactly 1.0 ml of the MtBE extract to an autosampler vial. A
duplicate vial should be filled using the excess extract.
515.3-25
-------
1 1.3.10 Add 10 nL of internal standard to the vial to be analyzed. (2.5 |ig/mL
4,4'-Dibromooctafluorobiphenyl in MTBE per Section 7.5. 1). Internal
standard should be added to the duplicate vial before analysis.
11.3.11 Analyze the samples as soon as possible. The sample extract may be
stored up to 14 days if kept at 4°C or less. Keep the extracts away from
light in amber glass vials with Teflon-lined caps.
11.4 GAS CHROMATOGRAPHY
1 1 .4. 1 Table 1 summarizes recommended GC operating conditions and reten-
tion times observed using this method. Figure 2 illustrates the perfor-
mance of the recommended primary column with the method analytes.
Figure 3 illustrates the performance of the recommended confirmation
column with the method analytes. Other GC columns or chromato-
graphic conditions may be used if the requirements of Section 9 are met.
1 1 .4.2 Calibrate the system daily by either the analysis of a calibration curve
(Section 1 0. 1) or a continuing calibration check as described in Section
10.2.
1 1.4.3 Inject 2 jxL of the sample extract. Record the resulting peak sizes in area
or height units.
1 1 .4.4 If the response for the peak exceeds the working range of the system,
dilute the extract, add an appropriate additional amount of internal
standard and reanalyze. The analyst must not extrapolate above or
below the calibration range established.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Identify sample components by comparison of retention times to retention data
from the calibration standard analysis. If the retention time of an unknown peak
corresponds, within limits (Section 12.3), to the retention time of a standard
compound, then the identification is considered positive. Calculate analyte
concentrations hi the samples and reagent blanks from the calibration curves
generated in Section 10.1.
12.2 If an average relative response factor has been calculated, analyte concentrations
in the samples and reagent blanks are calculated using the following equation:
(Aa)(Qs)
515.3-26
-------
12.3 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 chromato-
gram.
13. METHOD PERFORMANCE
13.1 In a single laboratory, accuracy and precision data were obtained at three
concentrations in reagent water (Tables 5-8). The MDL and EDL data are given
in Tables 2 and 3. In addition, recovery and precision data were obtained at a
medium concentration for dechlorinated tap water (Tables 9 and 10), high ionic
strength ground water (Tables 11 and 12) and high humectant reagent water
(Tables 13 and 14).
14. POLLUTION PREVENTION
14.1 This method utilizes a micro-extraction procedure which requires the use of very
small quantities of organic solvents. This feature reduces the hazards involved
with the use of large volumes of potentially harmful organic solvents needed for
conventional liquid-liquid extractions.
14.2 For information about pollution prevention that may be applicable to laboratory
operations consult "Less is Better: Laboratory Chemical Management for Waste
Reduction" available from the American Chemical Society's Department of
Government Relations and Science Policy, 1155 16th Street N.W., Washington
D.C. 20036.
15. WASTE MANAGEMENT .
15.1 Due to the nature of this method there is little need for waste management. No
large volumes of solvents or hazardous chemicals are used. The matrices of
concern are finished drinking water or source water. However, the Agency
requires that laboratory waste management practices be conducted consistent with
all applicable rules and regulations, and that laboratories protect the air, water, and
land by minimizing and controlling all releases from fume hoods and bench
operations. Also compliance is required with any sewage discharge permits and
regulations, particularly the hazardous waste identification rules and land disposal
restrictions. For further information on waste management, consult "The Waste
Management Manual for Laboratory Personnel" also available from the American
Chemical Society at the address in Sect. 14.2.
515.3-27
-------
16. REFERENCES
1. , ASTM Annual Book of Standards, Part JJ, Volume 11,02, D3694-82, "Standard
Practice for Preparation of Sample Containers and for Preservation," American
Society for Testing and Materials, Philadelphia, PA, p.86,1986.
2. Giam, C.S., Chan, H.S., and Nef, G.S., "Sensitive Method for Determination of
Phthalate Ester Plasticizers in Open-Ocean Biota Samples", Analytical Chemistry,
47, 2225 (1975).
3. 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, 701-708,1976.
4. ''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.
5. "OSHA Safety and Health Standards, General Industry", (29CFR1910), OSHA
2206, Occupational Safety and Health Administration, Washington, D.C. Revised
January 1976.
6. "Safety In Academic Chemistry Laboratories", 3rd Edition, American Chemical
Society Publication, Committee on Chemical Safety, Washington, D.C., 1979.
7. Xie, Yuefeng, Reckhow, David A. and Rajan, R.V., "Spontaneous Methylation of
Haloacetic Acids in Methanolic Stock Solutions", Environ. Sci. Technol., Vol.27,
No.6,1993, pp!232-1234,
8. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sam-
pling Water", American Society for Testing and Materials, Philadelphia, PA, p.
76,1980.
9. Frei, R.W. and Lawrence, J.F., Chemical Derivatization in Analytical Chemistry,
Plenum Press, New York, 1981.
10. Blau, K. and King, G.S., Handbook of Derivatives for Chromatographv, Heyden
and Son, Ltd., London, 1978.
11. Knapp, D.R., Handbook of Analytical Derivatization Reactions, John Wiley and
Sons, New York, 1979.
12. Drozd, J., Chemical Derivatization in Gas Chromatographv. Elsevier Scientific
Publishing Company, New York, 1981.
515.3-28
-------
17. TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. RETENTION DATA AND CHROMATOGRAPHIC CONDITIONS OF
ANALYTE METHYL DERIVATIVES
Retention Time, min.
Analyte Primary column Confirmatory column
Dalapon
3,5-Dichlorobenzoic Acid
4-Nitrophenol
2,4-Dichlorophenylacetic Acid^
Dicamba
Dichlorprop
4,4'Dibromooctafluorobiphenyl(a)
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Dacthal acid metabolites
Picloram
Acifluorfen
15.43
34.62
38.45
40.62
,41.88
47.67
49.13
49.83
50.18
53.58
55.32
55.60
57.17
57.38
59.68
61.55
62.78
65.77
72.57
19.73
39.98
40.31
48.50
49.21
53.59
55.88
54.60
58.13
59.13
59.59
60.21
59.59
62.77
62.61
63.64
67.82
66.70
75.60
Primary DB-1701, 30 m x 0.25 mm i.d., 0.25 |im film thickness, Injector Temp. =
column: 200°C, Detector Temp. = 290°C, Helium Linear Velocity = 24 Cm/sec at 35°C,
Splitless injection with 30 s delay
Program: Hold at 35°C for 10 min, ramp to 150°C at 5C°/min. and hold 10 min., ramp to
222°C at 4C°/min. and hold 5 min, ramp to 260°C at 5C°/min. and hold 6 min.
Confirmatory DB-5.625, 30 m x 0.25 mm i.d., 0.25 u,m film thickness, Injector Temp. =
column: 200°C, Detector Temp. = 290°C, Linear Helium Velocity = 25 cm/sec at 35°C,
splitless injection with 30 s delay.
Program: Hold at 35°C for 10 min, ramp to 150°C at 5C°/min. and hold 10 min., ramp to
222°C at 4C°/min. and hold 5 min, ramp to 260°C at 5C°/min. and hold 6 min.
(a) Internal Standard
^ Surrogate Compound
515.3-29
-------
TABLE 2. ANALYTE ACCURACY AND PRECISION DATA
AND METHOD DETECTION LIMITS"
DERIVATIZATION BY DIAZOMETHANE
LEVEL 1 IN REAGENT WATER
Analyte
Dalapon
3,5-Dichlorobenzoic
Acid
4-Nitrophenold
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicambad
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid
Metabolites
Fortified
Cone., \LgfL
1.25
0.625
1.25
0.625
1.25
1.25
0.125
0.314
0.625
0.314
0.625
1.25
1.25
1.25
1.25
0.625
1.25
Mean
Measured
Cone., ng/L
1.60
0.804
1.24
0.844
1.66
1.71
0.176
0.409
0.822
0.307
0.857
1.61
1.56
1.46
1.31
0.720
1.47
Std.
Dev.,
Ug/L
0.31
0.060
0.29
0.095
0.16
0.11
0.027
0.046
0.19
0.064
0.079
0.21
0.26
0.28
0.33
0.15
0.20
Relative
Std. Dev.,
%
19
7.5
23
11
9.6
6.4
15
11
23
21
9.2
13
17
19
25
21
14
Method
Detection
Limitb, |J,g/L
0.97
0.19
0.91
0.30
0.51
0.35
0.085
0.14
0.60
0.20
0.25
0.66
0.82
0.88
1.0
0.47
0.63
Estimated
Detection
Limit0, ng/L
1.25
0.625
1.25
0.30
0.51
0.35
0.085
0.14
0.625
0.20
0.25
1.25
0.82
1.25
1.0
0.47
0.63
a Produced by analysis of seven aliquots of fortified reagent water.
b The MDL is a statistical estimate of the detection limit. To determine the MDL for each analyte, the standard deviation
of the mean concentration of the seven replicates is calculated. This standard deviation is then multiplied by the Student's
t-value at 99% confidence and n-1 degrees of freedom (3.143 for seven replicates). The result is the MDL.
c The EDL is defined as either the MDL or a level of a compound in a sample yielding a peak in the final extract with a
signal to noise (S/N) ratio of approximately 5, whichever is greater.
d Quantisation not recommended due to poor precision.
515.3-30
-------
TABLE 3. ANALYTE ACCURACY AND PRECISION DATA
AND METHOD DETECTION LIMITS3
DERTVATIZATION BY TETRABUTYLAlVmOMUM HYDROXTOE AND METHYL IODIDE
LEVEL 1 IN REAGENT WATER
Analyte
Dalapon
3,5-Dichlorobenzoic
Acid
4-Nitrophenol
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid
Metabolites
Fortified
Cone., (ig/L
1.25
0.625
1.25
0.625
1.25
1.25
0.125
0.314
0.625
0.314
0.625
1.25
1.25
1.25
1.25
0.625
1.25
Mean
Measured
Cone., p,g/L
1.37
0.781
1.07
0.689
1.55
1.39
0.108
0.326
0.708
0.255
0.644
1.47
1.06
0.927
1.12
0.639
1.52
Std.
Dev.,
Hg/L
0.17
0.041
0.21
0.065
0.13
0.11
0.0068
0.023
0.068
0.052
0.044
0.19
0.24
0.16
0.15
0.12
0.12
Relative
Std. Dev.,
%
12
5.2
20
9.4
8.4
7.9
6.3
7.1
9.6
20
6.8
13
. 23
17
13
21
7.9
Method
Detection
Limitb, \igfL
0.53
0.13
0.66
0.20
0.41
0.36
0.021
0.072
0.21
0.16
0.14
0.60
0.75
0.50
0.47
0.38
0.38
Estimated
Detection
Limit0 , ng/L
1.25
0.625
1.25
0.20
0.41
0.36
0.021
0.072
0.21
0.16
0.14
0.60
1.25
1.25
0.47
0.38
0.38
a Produced by analysis of seven aliquots of fortified reagent water.
b The MDL is a statistical estimate of the detection limit. To determine the MDL for each analyte, the standard deviation
of the mean concentration of the seven replicates is calculated. This standard deviation is then multiplied by the Student's
t-value at 99% confidence and n-1 degrees of freedom (3.143 for seven replicates). The result is the MDL.
c The EDL is defined as either the MDL or a level of a compound in a sample yielding a peak in the final extract with a
signal to noise (S/N) ratio of approximately 5, whichever is greater.
515.3-31
-------
TABLE 4. ANALYTE ACCURACY AND PRECISION DATA"
DERIVATIZATION BY DIAZOMETHANE
LEVEL 2 IN REAGENT WATER
Analyte
Dalapon
3,5-Dichlorobenzoic Acid
4-Nitrophenolb
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicambab
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid Metabolites
Fortified
Cone., [ig/L
5.00
2.50
5.00
2.50
5.00
5.00
0.500
1.25
2.50
1.25
2.50
5.00
5.00
5.00
5.00
2.50
5.00
Mean
Measured
Cone., [ig/L
5.26
3.04
5.69
2.62
5.97
6.34
0.524
1.26
2.32
1.30
2.51
5.88
5.55
5.09
5.12
2.82
5.53
Std.
Dev.,
Hg/L
0.34
0.27
0.96
0.068
0.18
0.20
0.012
0.084
0.45
0.065
0.13
0.37
0.21
0.18
0.35
0.24
0.38
Relative
Std.
Dev., %
6.5
8.9
17
2.6
3.0
3.2
2.3
6.7
19
5.0
5.2
6.3
3.8
3.6
6.8
8.3
6.8
Percent
Recovery, %
105
122
114
105
119
127
105
101
93
104
100
118
111
102
102
113
111
* Produced by the analysis of seven aliquots of fortified reagent water.
b Quantitation not recommended due to poor precision.
515.3-32
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TABLE 5. ANALYTE ACCURACY AND PRECISION DATA3
DERIVATIZATION BY TETRABUTYLAMMONIUM HYDROXIDE
AND METHYL IODIDE
LEVEL 2 IN REAGENT WATER
Analyte
Dalapon
3,5-Dichlorobenzoic Acid
4-Nitrophenol
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid Metabolites
Fortified
Cone., |ig/L
5.00
2.50
5.00
2.50
5.00
5.00
0.500
1.25
2.50
1.25
2.5.0
5.00
5.00
5.00
5.00
2.50
5.00
Mean
Measured
Cone., ng/L
6.37
3.26
5.24
. 2.75
5.70
5.59
0.520
1.31
2.75
1.20
2.34
5.46
5.42
4.54
4.28
2.58
5.16
StcL
Dev.,
[igfL
0.67
0.18
0.29
0.22
0.29
0.33
0.065
0.081
0.17
0.19
0.25
0.24
0.69
0.33
0.52
0.50
0.66
Relative
Std.
Dev., %
10
5.4
5.5
8.2
5.1
5.9
12
6.2
6.0
16
11
4.4
13
7.3
12
19
13
Percent
Recovery, %
127
131,
105
110
114
112
104
104
110
96
94
109
108
91
86
103
"ids"
1 Produced by the analysis of seven aliquots of fortified reagent water.
515.3-33
-------
TABLE 6. ANALYTE ACCURACY AND PRECISION DATA3
DERIVATIZATION BY DIAZOMETHANE
LEVEL 4 IN REAGENT WATER
Analyte Fortified
Cone., (ig/L
Dalapon
3,5-Dichlorobenzoic Acid
4-Nitrophenolb
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicambab
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid Metabolites
10.0
5.00
10.0
5.00
10.0
10.0
1.00
2.50
5.00
2.50
5.00
10.0
10.6
10.0
10.0
5.00
10.0
Mean .
Measured
Cone., \igfL
10.3
4.63
14.3
4.80
9.26
9.67
0.967
2.43
6.70
2.46
4.95
9.67
9.78
9.78
10.2
5.15
8.85
Std. Relative
Dev., Std. Dev.,
Ug/L %
0.27
0.24
3.1
0.083
0.28
0.19
0.015
0.032
2.4.
0.088
0.081
0.22
0.19
0.19
0.25
0.40
0.31
2.6
5.2
22..
1.7
3.1
1.9
1.5
1.3
36.
3.6
1.6
2.3
2.0
1.9
2.4
7.8
3.5
Percent
Recovery, %
103
93
143
96
93
97
97
97
134
98
99
97
98
98
102
103
88
a Produced by the analysis of seven aliquots of fortified reagent water.
b Quantisation not recommended due to poor precision.
515.3-34
-------
TABLE 7. ANALYTE ACCURACY AND PRECISION DATAa
DERIVATIZATION BY TETRABUTYLAMMONIUM HYDROXIDE
AND METHYL IODIDE
LEVEL 4 IN REAGENT WATER
Analyte
Dalapon
3,5-Dichlorobenzoic Acid
4-Nitrophenol
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid Metabolites
Fortified
Cone.,
Hg/L
10.0
5.00
10.0
5.00
10.0
10.0
1.00
2.50
5.00
2.50
5.00
10.0
10.0
10.0
10.0
5.00
10.0
Mean
Measured
Cone.,
Hg/L
8.37
4.55
9.52
4.55
8.87
9.09
0.870
2.29
. 4.23
2.26
4.76
10.2
10.7
9.54
9.60
5.27
8.17
Std.
Dev.,
Hg/L
0.88
0.16
i.o
0.14
0.18
0.38
0.16
0.050
0.30
0.12
0.34
0.34
3.1
0.84
0.44
0.46
1.2
Relative
Std. Dev.,
%
10
3.5
11
3.0
2.0
4.2
19
2.2
7.1
5.2
7.2
3.3
29
8.8
4.6
8.7
15
Percent
Recovery, %
84
91
95
91
89
91
87
92
85
90
95
102
107
95
96
105
82
1 Produced by the analysis of seven aliquots of fortified reagent water.
515.3-35
-------
TABLE 8. ANALYTE ACCURACY AND PRECISION DATA3
DERIVATIZATION BY DIAZOMETHANE
LEVEL 3 IN DECHLORINATED TAP WATER"
Analyte
Dalapon
3,5-Dichlorobenzoic
Acid
4-Nitrophenolc
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicambac
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid
Metabolites
Background
Cone., [xg/L
<1.25
O.625
<1.25
<0.30
<0.51
<0.35
<0.085
<0.14
<0.625
<0.20
<0.25
<1.25
<0.82
<1.25
<1.0
O.47
<0.63
Fortified
Cone.,
Hg/L
7.50
3.75
7.50
3.75
7.50
7.50
0.750
1.87
3.75
1.87
3.75
7.50
7.50
7.50
7.50
3.75
7.50
Mean
Measured
Cone., |ig/L
8.18
4.07
5.76
3.91
7.29
7.00
0.754
1.70
0.233
1.66
3.93
7.51
8.02
7.64
7.91
3.97
7.87
Std.
Dev.,
Hg/L
0.93
0.38
1.0
0.16
0.40
0.38
0.016
0.077
0.12
0.046
0.25
0.70
0.32
0.44
1.0
0.38
0.52
Relative
Std.
Dev., %
11
9.3
18
4.0
5.4
5.4
2.2
4.5
51
2.8
6.4
9.3
4.0
5.8
13
9.7
6.6
Percent
Recovery,
%
109
108
77
104
97
93
101
90
6d
88
105
100
107
102
105
106
105
* Produced by the analysis of seven aliquots of fortified reagent water.
b Chlorinated surface water from a local utility to which sodium thiosulfate was added as the
dechlorinating agent.
0 Quantitation not recommended due to poor precision.
d As noted in Section 4.6, 5-Hydroxydicamba cannot be recovered from chlorinated waters.
515.3-36
-------
TABLE 9. ANALYTE ACCURACY AND PRECISION DATA"
DERIVATIZATION BY TETRABUTYLAMMONIUM HYDROXIDE
AND METHYL IODIDE
LEVEL 3 IN DECHLORINATED TAP WATER"
Analyte
Dalapon
3 ,5-Dichlorobenzoic
Acid
4-Nitrophenol
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid
Metabolites
Background
Cone., jig/L
<1.25
<0.625
<1.25
<0.20
0.41
<0.36
<0.021
O.072
<0.21
<0.16
<0.14
<0.60
<1.25
<1.25
<0.47
<0.38
<0.38
Fortified
Cone., |ig/L
7.50
3.75
7.50
3.75
7.50
7.50
0.750
1.87
3.75
1.87
3.75
7.50
7.50
7.50
7.50
3.75
7.50
Mean
Measured
Cone.,
[ig/L
8.06
4.18
6.63
3.69
6.70
6.85
0.771
1.69
0.180
1.55
3.22
7.31
8.81
6.98
7.11
4.08
6.71
Std.
Dev.,
u.g/L
0.39
0.54
0.78
0.14
0.24
0.55
0.071
0.13
0.048
0.19
0.26
0.48
1.8
0.58
0.74
0.63
0.70
Relative
Std.
Dev., %
4.8
13
12
3.6
3.6
8.0
9.2
7.6
26
12
8.1
6.6
20
8.3
10
15
10
Percent
Recovery,
%
108
111
88
98
89
91
103
90
5C
83
86
97
117
93
95
109
90
a Produced by the analysis of seven aliquots of fortified reagent water.
b Chlorinated surface water from a local utility to which sodium thiosulfate was added as the
dechlorinating agent.
0 As noted in section 4.6, 5-Hydroxydicamba cannot be recovered from chlorinated waters.
515.3-37
-------
TABLE 10. ANALYTE ACCURACY AND PRECISION DATA3
DERIVATIZATION BY DIAZOMETHANE
LEVEL 3 IN HIGH IONIC STRENGTH WATER"
Analyte
Dalapon
3,5-Dichlorobenzoic
Acid
4-Nitrophenolc
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicambac
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid
Metabolites
Background
Cone., u.g/L
<1.25
<0.625
<1.25
<0.30
<0.51
<0.35
<0.085
<0.14
<0.625
<0.20
<0.25
<1.25
<0.82
<1.25
<1.0
<0.47
<0.63
Fortified
Cone., |ig/L
7.50
3.75
7.50
3.75
7.50
7.50
0.750
1.87
3.75
1.87
3.75
7.50
7.50
7.50
7.50
3.75
7.50
Mean
Measured
Cone.,
|xg/L
5.98
3.86
10.44
3.80
7.12
6.98
0.787
1.76
5.43
1.72
4.16
8.84
. 7.74
7.83
7.29
3.82
7.73
Std.
Dev.,
Hg/L
0.47
0.14
2.1
0.098
0.16
0.14
0.053
0.066
2.0
0.044
0.24
0.38
0.21
0.44
0.39
0.27
0.28
Relative
Std.
Dev., %
7.8
3.7
20
2.6
2.2
2.0
6.7
3.7
36
2.6
5.8
4.3
2.7
5.6
5.4
7.2
3.7
Percent
Recovery,
%
80
103
139
101
95
93
105
94
145
92
111
118
103
104
97
102
103
8 Produced by the analysis of seven aliquots of fortified reagent water.
b Chlorinated ground water from a water source displaying a hardness of 460 mg/L as CaCO3.
c Quantitation not recommended due to poor precision.
515.3-38
-------
TABLE 11. ANALYTE ACCURACY AND PRECISION DATA3
DERIVATIZATION BY TETRABUTYLAMMONIUM HYDROXIDE
AND METHYL IODIDE
LEVEL 3 IN HIGH IONIC STRENGTH WATER"
Analyte
Dalapon
3 ,5-Dichlorobenzoic
Acid
4-Nitrophenol
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid
Metabolites
Background
Cone., |ig/L
<1.25
<0.625
<1.25
<0.20
O.41
<0.36
O.021
<0.072
<0.21
<0.16
<0.14
<0.60
<1.25
<1.25
0.47
0.38
O.38
Fortified
Cone., \ig/L
7.50
3.75
7.50
3.75
7.50
7.50
0.750
1.87
3.75
1.87
3.75
7.50
7.50
7.50
7.50
3.75
7.50
Mean
Measured
Cone.,
u-g/L
7.33
4.25
7.90
3.96
7.11
6.67
1.00
1.80
3.62
1.65
3.84
7.74
7.45
7.88
6.15
4.17
6.82
Std.
Dev.,
u.g/L
0.65
0.29
0.51
0.12
0.74
0.17
0.068
0.081
0.12
0.074
0.30
0.54
0.72
0.46
0.63
0.46
0.39
Relative
Std.
Dev,%
8.9
6.8
6.4
3.0
10
2.6
6.8
4.5
3.4
4.5
7.8
7.0
9.7
5.8
10
11
5.7
Percent
Recovery,
%
98
113
105
106
95
89
134
96
96
88
102
103
99
105
82
111
91
a Produced by the analysis of seven aliquots of fortified reagent water.
b Chlorinated ground water from a water source displaying a hardness of 460 mg/L as CaCO3.
515.3-39
-------
TABLE 12. ANALYTE ACCURACY AND PRECISION DATA3
DERTVATIZATION BY DIAZOMETHANE
LEVEL 3 IN HIGH HUMIC CONTENT WATERb
Analyte Background Fortified Mean Std. Relative Percent
Cone., ng/L Cone., [ig/L Measured Dev., Std. Recovery,
Cone., M-g/L |ig/L Dev., % %
Dalapon
3,5-Dichlorobenzoic
Acid
4-Nitrophenolc
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba°
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid
Metabolites
<1.25
<0.625
<1.25
<0.30
<0.51
<0.35
<0.085
<0.14
<0.625
<0.20
<0.25
<1.25
0.82
<1.25
<1.0
<0.47
<0.63
7.50
3.75
7.50
3.75
7.50
7.50
0.750
1.87
3.75
1.87
3.75
7.50
7.50
7.50
7.50
3.75
7.50
8.41
4.46
6.21
4.36
9.22
9.28
0.797
1.96
2.52
2.06
3.86
9.10
8.66
7.89
6.79
3.67
9.23
1.92
0.38
2.4
0.19
0.69
0.77
0.020
0.048
1.4
0.19
0.29
0.36
1.0
0.30
1.9
0.53
1.0
23
8.6
39
4.4
7.5
8.3
2.5
2.5
55
9.1
7.6
3.9
12
3.7
29
14
11
112
119
83
116
123
124
106
104
67
110
103
121
115
105
91
98
123
* Produced by the analysis of seven aliquots of fortified reagent water.
b Reagent water fortified at 1.0 mg/L with fulvic acid extracted from Ohio River water. Sample
simulates high TOC matrix.
c Quantisation not recommended due to poor precision.
515.3-40
-------
TABLE 13. ANALYTE ACCURACY AND PRECISION DATA3
DERIVATIZATION BY TETRABUTYLAMMONIUM HYDROXIDE
AND METHYL IODIDE
LEVEL 3 IN HIGH HUMIC CONTENT STRENGTH WATER"
Analyte Background Fortified Mean Std. Relative Percent
Cone., u.g/L Cone., jxg/L Measured Dev., Std. Recovery,
Cone., ng/L [ig/L Dev., % %
Dalapon
3 ,5-Dichlorobenzoic
Acid
4-Nitrophenol
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid
Metabolites
<1.25
<0.625
<1.25
<0.20
<0.41
<0.36
<0.021
<0.072
O.21
<0.16
O.14
<0.60
<1.25
<1.25
<0.47
O.38
<0.38
7.50
3.75
7.50
3.75
7.50
7.50
0.750
1.87
3.75
1.87
3.75
7.50
7.50
7.50
7.50
3.75
7.50
6.82
3.86
7,04
3.67
7.24
6.37
0.736
^1.77
3.17
1.49
3.68
8.01
7.08
7.50
5.66
3.89
6.93
1.1
0.33
0.49
0.13
0.52
0.93
0.12
0.19
0.34
0.29
0.24
0.42
0.81
0.44
1-0
0.45
0.53
16
8.5
7.0
3.4
7.2
15
16
11
11
20
6.6
5.2
11
5.9
18
12
7.6
91
103
94
98
97
85
98
95
84
79
98
107
94
100
. ^ . ' ' '. .(• •
75
104
92 •'-'.; ^
a Produced by the analysis of seven aliquots of fortified reagent water.
b Reagent water fortified at 1.0 mg/L with fulvic acid extracted from Ohio River water. Sample r ->: •'
simulates high TOC matrix.
515.3-41
-------
TABLE 14; LABORATORY PERFORMANCE CHECK SOLUTION
PARAMETER
ANALYTE
CONC., u.g/LIN ACCEPTANCE
WATER CRITERIA
SAMPLE
INSTRUMENT
SENSITIVITY
CHROMATOGRAPffl
C PERFORMANCE
COLUMN
PERFORMANCE
DINOSEB
4-NITROPHENOL
CHLORAMBEN
2,4-DB
2.50
2.50
1.25
2.50
DETECTION OF
ANALYTE; S/N>3a
PGF BETWEEN
0.80 AND 1.15b
RESOLUTION^ . :
0.50C '• ..,
a S/N, a ratio of peak signal to baseline noise.
peak signal - measured as height of peak.
baseline noise - measured as maximum deviation in baseline (in units of height) over a width equal
to the width of the base of the peak.
b PGF = Peak Gaussian Factor
1.83xW1/2 t. " ^ -: ' ' ' -.':.,V
PGF= - :
W1/10 ! / ^ :-.._.
where W1C = the peak width at half height.
W1/10 = the peak width at one-tenth height • . '",
This is a measure of the symmetry of the peak,
c Resolution between two peaks is defined by the equation:
t
R =
W
YV ave
where t = the difference in elution tunes between the two peaks.
Wave = the average peak width of the two peaks (measurements taken at
... . baseline). ...... . , . .- .. ., -....•
This a measure of the degree of separation of two peaks under specific chromatographic conditions.
515.3-42
-------
FIGURE 1. APPARATUS FOR GENERATION OF DIAZOMETHANE
Nitrogen
Ethyl
ether
Diazald solution
and KOH solution
Methyl terf-butyl ether in
_ collection vial
Ice bath
515.3-43
-------
FIGURE 2. CHROMATOGRAM OF CHLOROPHENOXY HERBICIDES ON DB-1701.
DERIVATIZATION BY DIAZOMETHANE (LEVEL 4 CALIBRATION)
-5
-10
-15
-20
-25
-30
-35
-40
-45
-50
-55
-60
-65
-70
12
-14
— 13
-ib
15
19
,17
515.3-44
-------
FIGURE 3. CHROMATOGRAM OF CHLOROPHENOXY HERBICIDES ON DB-5.625.
BASE-PROMOTED DERIVATIZATION (LEVEL 2 CALIBRATION)
-5
-10
15
20
25
:30
:35
-40
745
-50
-55
-60
10
12
JS
£-1~816
-70
-75
19
11,13
17
515.3-45
-------
TABLE 15. KEY FOR PEAK NUMBERS DISPLAYED IN FIGURES 2 AND 3
Peak Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Method 515.3 compound
Dalapon
3,5-Dichlorobenzoic Acid
4-Nitrophenol
2,4-Dichlorophenylacetic Acid (Surrogate)
Dicamba
Dichlorprop
4,4l-Dibromooctafluorobiphenyl (Internal Standard)
2,4-D
Pentachlorophenol
Silvex (2,4,5-TP)
5-Hydroxydicamba
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Dacthal Acid Metabolites
Picloram
Acifluorfen
515.3-46
-------
TABLE 16. HOLDING TIME STUDY FOR AQUEOUS SAMPLES
DERIVATIZATION BY TETRABUTYLAMMONIUM HYDROXIDE
AND METHYL IODIDE
DAYO
Analyte
Dalapon
3,5-Dichlorobenzoic Acid
4-Nitrophenol
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid Metabolites
Fortified
Cone.,
[LgfL
10.0
5.00
10.0
5.00
10.0
10.0
1.00
2.50
5.00
2.50
5.00
10.0
10.0
10.0
10.0
5.00
10.0
Mean
Measured
Cone.,
Hg/L
8.37
4.55
9.52
4.55
8.87
9.09
0.870
2.29
4.23
2.26
4.76
10.2
10.7
9.54
9.60
5.27
8.17
Std.
Dev.,
Hg/L
0.88
0.16
1.0
0.14
0.18
0.38
0.16
0.050
0.30
0.12
0.34
0.34
3.1
0.84
0.44
0.46
1.2
Relative
Std. Dev.,
%
10
3.5
11
3.0
2.0
4.2
19
2.2
7.1
5.2
7.2
3.3
29
8.8
4.6
8.7
15
Percent
Recovery, %
84
91
95
91
89
91
87
92
85
90
95
102
107
95
96
105
82
1 Produced by the analysis of seven aliquots of fortified reagent water.
515.3-47
-------
TABLE 17. HOLDING TIME STUDY FOR AQUEOUS SAMPLES
DERTVATIZATION BY TETRABUTYLAMMONlUM HYDROXIDE
AND METHYL IODIDE
DAY?
Analyte
Dalapon
3,5-Dichlorobenzoic Acid
4-Nitrophenol
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid Metabolites
Fortified
Cone.,
Hg/L
10.0
5.00
10.0
5.00
10.0
10.0
1.00
2.50
5.00
2.50
5.00
10.0
10.0
10.0
10.0
5.00
10.0
Mean
Measured
Cone.,
[ig/L
9.48
4.98
9.65
4.99
9.86
9.76
1.10
2.62
5.07
2.75
5.32
10.3
9.50
10.9
11.5
5.92
10.0
Std. Relative
Dev., Std. Dev.,
Hg/L %
1.4
0.19
0.38
0.052
0.16
0.19
0.15
0.057
0.19
0.20
0.16
0.22
2.4
0.35
2.4
0.38
0.57
15
3.8
4.0
1.0
1.6
2.0
14
2.2
3.8
7.2
3.1
2.1
26
3.2
21
6.4
5.7
Percent
Recovery, %
95
100
97
100
99
98
110
105
101
110
106
103
95
109
115
118
100
a Produced by the analysis of seven aliquots of fortified reagent water.
515.3-48
-------
TABLE 18. HOLDING TIME STUDY FOR AQUEOUS SAMPLES
DERIVATIZATION BY TETRABUTYLAMMONIUM HYDROXIDE
AND METHYL IODIDE
DAY 14
Analyte
Dalapon
3,5-Dichlorobenzoic Acid
4-Nitrophenol
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid Metabolites
Fortified
Cone.,
Hg/L
10.0
5.00
10.0
5.00
10.0
10.0
1.00
2.50
5.00
2.50
5.00
10.0
10.0
10.0
10.0
5.00
10.0
Mean
Measured
Cone.,
VLS/L
9.53
4.85
9.18
4.60
8.98
8.73
1.22
2.42
4.26
2.28
4.65
9.30
10.1
9.92
9.43
4.89
8.23
Std.
Dev.,
Hg/L
0.27
0.16
0.32
0.15
0.23
0.20
0.15
0.066
0.066
0.12
0.12
0.20
1.2
0.33
2.2
0.28
0.67
Relative
Std. Dev.,
%
2.8
3.4
3.5
3.3
2.5
2.3
12
2.7
1.6
5.2
2.7
2.1
12
3.3
23
5.6
8.1
Percent
Recovery, %
"95
97
92
92
90
87
122
97
85
91
93
93
101
99
94
98
82
1 Produced by the analysis of seven aliquots of fortified reagent water.
515.3-49
-------
TABLE 19. HOLDING TIME STUDY FOR AQUEOUS SAMPLES
DERIVATIZATION BY TETRABUTYLAMMONIUM HYDROXIDE
AND METHYL IODIDE
DAY 21
Analyte
Dalapon
3,5-Dichlorobenzoic Acid
4-Nitrophenol
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid Metabolites
Fortified
Cone., |ig/L
10.0
5.00
10.0
5.00
10.0
10.0
1.00
2.50
5.00
2.50
5.00
10.0
10.0
10.0
10.0
5.00
10.0
Mean Std. Relative
Measured Dev., Std. Dev.,
Cone., ng/L M-g/L %
9.65
5.32
9.32
4.64
9.34
8.35
1.10
2.10
3.76
1.97
3.74
8.32
9.66
8.63
11.4
4.20
8.22
1.3
0.49
0.81
0.27
0.46
0.13
0.071
0.13
0.19
0.24
0.46
0.38
1.8
0.81
2.9
0.43
0.78
13
9.2
8.7
5.8
4.9
1.6
6.5
6.2
5.0
12
12
4.6
19
9.4
25
10
9.5
Percent
Recovery, %
97
106
93
93
93
84
110
84
75
79
75
83
97
86
114
84
82 .
a Produced by the analysis of seven aliquots of fortified reagent water.
515.3-50
-------
TABLE 20. HOLDING TIME STUDY FOR MTBE EXTRACTS
DERIVATIZATION BY TETRABUTYLAMMONIUM HYDROXIDE
AND METHYL IODIDE
DAYO
Analyte
Dalapon
3,5-Dichlorobenzoic Acid
4-Nitrophenol
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthai Acid Metabolites
Fortified
Cone.,
. M'g/L
10.0
5.00
10.0
5.00
10.0
10.0
1.00
2.50
5.00
2.50
5.00
10.0
10.0
10.0
10.0
5.00
:io.o ;
Mean
Measured
Cone.,
Hg/L
8.37
4.55
9.52
4.55
8.87
9.09
0.870
2.29
4.23
2.26
4.76
10.2
10.7
9.54
9.60
5.27
8.17
Std.
Dev.,
\Lg/L
0.88
0.16
1.0
0.14
0.18
0.38
0.16
0.050
0.30
0.12
0.34
0.34
3.1
0.84
0.44
0.46
1.2 "-
Relative
Std. Dev.,
%
10
3.5
11
3.0
2.0
4.2
19
2.2
7.1
5.2
7.2
3.3
29
8.8
4.6
8.7
15-
Percent
Recovery, %
84
91
95
91
89
91
87
92
85
90
95
102
107
95
96
,105
" 82 ":::"
1 Produced by the analysis of seven aliquots of fortified reagent water.
515.3-51
-------
TABLE 21. HOLDING TIME STUDY FOR MTBE EXTRACTS
DERIVATIZATION BY TETRABUTYLAMMONIUM HYDROXIDE
AND METHYL IODIDE
DAY?
Analyte
Dalapon
3,5-Dichlorobenzoic Acid
4-Nitrophenol
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid Metabolites
Fortified
Cone.,
Rg/L
10.0
5.00
10.0
5.00
10.0
10.0
1.00
2.50
5.00
2.50
5.00
10.0
10.0
10.0
10.0
5.00
10.0
Mean
Measured
Cone.,
Hg/L
9.67
5.50
10.7
5.31
10.4
10.4
1.21
2.64
5.32
2.58
5.75
11.0
9.60
11.4
13.8
6.27
10.8
Std. \
Dev.,
Hg/L
1.6
0.60
1.0
0.18
0.50
0.66
0.23
0.17
0.38
0.32
0.60
0.24
0.91
0.98
1.3
0.88
1.1
Relative Percent
\ Std. Dev., Recovery, %
\ %
\
• 17
11
9.7
\
3.4
4.8
6.3
\
19
6.5
7.1
12
10
2.1
9.5
8.6
9.6
14
10
97
no
107
106
104
104
121
106
106
103
115
110
96.
114
138
125
108
a Produced by the analysis of seven aliquots of fortified reagent water.
515.3-52
-------
TABLE 22. HOLDING TIME STUDY FOR MTBE EXTRACTS
DERIVATIZATION BY TETRABUTYLAMMONIUM HYDROXIDE
AND METHYL IODIDE
DAY 14
Analyte
Dalapon
3,5-Dichlorobenzoic Acid
4-Nitrophenol
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba
2-,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid Metabolites
Fortified
Cone.,
Hg/L
10.0
5.00
10.0
5.00
10.0
10.0
1.00
2.50
5.00
2.50
5.00
10.0
10.0
10.0
10.0
5.00
10.0
Mean
Measured
Cone.,
[ig/L
10.1
5.55
10.8
5.40
10.1
10.1
1.07
2.57
5.13
2.43
5.40
10.5
9.68
10.8
13.3
5.17
10.1
Std.
Dev.,
ug/L
0.72
0.35
1.1
0.30
0.42
0.50
0.12
0.16
0.40
0.21
0.68
0.74
2.0
1.2
2.6
0.79
1.3
Relative
Std. Dev.,
%
7.2
6.3
9.8
5.5
4.1
5.0
11
6.2
7.8
8.6
12
7.0
21
11
19
15
13
Percent
Recovery, %
101
111
108
108
101
101
107
103
103
97
108
105
97
108
133
103
101
1 Produced by the analysis of seven aliquots of fortified reagent water.
515.3-53
-------
TABLE 23. HOLDING TIME STUDY FOR MTBE EXTRACTS
DERIVATIZATION BY TETRABUTYLAMMONIUM HYDROXIDE
AND METHYL IODIDE
DAY 21
Analyte
Dalapon
3,5-Dichlorobenzoic Acid
4-Nitrophenol
Dicamba
Dichlorprop
2,4-D
Pentachlorophenol
Silvex
5-Hydroxydicamba
2,4,5-T
Chloramben
2,4-DB
Dinoseb
Bentazon
Picloram
Acifluorfen
Dacthal Acid Metabolites
Fortified
Cone.,
Hg/L
10.0
5.00
10.0
5.00
10.0
10.0
1.00
2.50
5.00
2.50
5.00
10.0
10.0
10.0
10.0
5.00
10.0
Mean
Measured
Cone.,
Hg/L
10.8
5.72
10.1
5.13
10.4
9.51
1.06
2.65
5.20
2.50
5.36
11.1
8.92
10.2
12.6
5.67
14.0
Std.
Dev.,
Hg/L
1.8
0.88
1.7
0.50
0.71
0.85
0.27
0.28
0.82
0.27
0.58
1.2
1.6
0.74
1.0
0.71
1.1
Relative
Std. Dev.,
%
16
15
16
9.7
6.8
9.0
26
10
16
11
11
11
18
7.3
8.1
12
7.9
Percent
Recovery, %
108
114
101
103
104
95
106
106
104
100
107
111
89
102
126
113
140
1 Produced by the analysis of seven aliquots of fortified reagent water.
515.3-54
-------
METHOD 526. DETERMINATION OF SELECTED SEMIVOLATILE ORGANIC
COMPOUNDS IN DRINKING WATER BY SOLID PHASE
EXTRACTION AND CAPILLARY COLUMN GAS
CHROMATOGRAPHY/ MASS SPECTROMETRY (GC/MS)
Revision 1.0
June 2000
S.D. Winslow, B. Prakash, M.M. Domino, and B.V. Pepich, IT Corporation and D. J.
Munch USEPA, Office of Ground Water and Drinking Water
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
526-1
-------
METHOD 526
DETERMINATION OF SELECTED SEMIVOLATILE ORGANIC COMPOUNDS IN
DRINKING WATER BY SOLID PHASE EXTRACTION AND CAPILLARY COLUMN
GAS CHROMATOGRAPHY/ MASS SPECTROMETRY (GC/MS)
1.
SCOPE AND APPLICATION
1.1
1.2
This is a gas chromatography/mass spectrometry (GC/MS) method for the
determination of selected semivolatile organic compounds in raw and finished
drinking waters. This method is applicable to the organic compounds listed
below, which are efficiently extracted from water using a polystyrene
divinylbenzene solid phase sorbent, and are sufficiently volatile and thermally
stable for gas chromatography. Accuracy, precision, and method detection limit
(MDL) data have been generated in reagent water, finished ground and surface
water for the following compounds:
Analyte
Acetochlor
Cyanazine
Diazinon
2,4-Dichlorophenol
1,2-Diphenylhydrazine
Disulfoton
Fonofos
Nitrobenzene
Prometon
Terbufos
2,4,6-Trichlorophenol
Chemical Abstract Services
Registry Number
34256-82-1
21725-46-2
61790-53-2
120-83-2
122-66-7
298-04-4
944-22-9
98-95-3
1610-18-0
13071-79-9
88-06-2
MDLs are compound, instrument, and matrix dependent. The MDL is defined as
the statistically calculated minimum concentration that can be measured with 99%
confidence that the reported value is greater than zero.{1) Experimentally
determined MDLs for the above listed analytes are provided in Section 17, Table
3. The MDL differs from, and is lower than, the minimum reporting limit (MRL)
526-2
-------
(Sect. 3.17). Precision and accuracy were evaluated at 0.5 and 20 ug/L. Precision
and accuracy data and sample holding time data are presented Section 17, Tables
4 through 8. Analyte retention times are in Section 17, Table 2.
1.3 This method is restricted to use by or under the supervision of analysts skilled in
solid-phase extractions (SPE) and GC/MS analysis.
2. SUMMARY OF METHOD
2.1 A 1 liter water sample is passed through a SPE disk or cartridge containing
polystyrenedivinylbenzene (SDVB) to extract the target analytes and surrogate
compounds. The extract is dried by passing through a column of anhydrous
sodium sulfate and is concentrated by blowdown with nitrogen to a volume of
about 0.7 mL. Internal standards are added and the extract is diluted to a final
volume of 1 mL. Components are separated chromatographically by injecting an
aliquot of the extract onto a gas chromatograph equipped with a high resolution
fused silica capillary column. The analytes pass from the capillary column into a
mass spectrometer where the they are identified by comparing their measured
mass spectra and retention times to reference spectra and retention times collected
for the same compounds. Instrument specific reference spectra and retention
times for analytes are obtained by the analyses of calibration standards under the
same GC/MS conditions used for samples. The concentration of each identified
component is measured by relating the MS response of the compound's
quantitation ion to the internal standard's quantitation ion MS response.
3. DEFINITIONS
3.1 EXTRACTION BATCH - A set of up to 20 field samples (not including QC
samples) extracted together by the same person(s) during a work day using the
same lot of solid phase extraction devices, solvents, surrogate solution, and
fortifying solutions. Required QC samples include Laboratory Reagent Blank,
Laboratory Fortified Blank, Laboratory Fortified Matrix, and either a Field
Duplicate or Laboratory Fortified Matrix Duplicate.
3.2 ANALYSIS BATCH - A set of samples that is analyzed on the same instrument
during a 24-hour period that begins and ends with the analysis of the appropriate
Continuing Calibration Check standards (CCC). Additional CCCs may be
required depending on the length of the analysis batch and/or the number of Field
Samples.
3.3 INTERNAL STANDARD (IS)-A pure analyte added to an extract or standard
solution in a known amount and used to measure the relative responses of other
method analytes and surrogates. The internal standard must be an analyte that is
not a sample component.
526-3
-------
3.4 SURROGATE ANALYTE (SUR) - A pure analyte, which is extremely unlikely
to be found in any sample, and which is added to a sample aliquot in a known
amount before extraction or other processing, and is measured with the same
procedures used to measure other sample components. The purpose of the SUR is
to monitor method performance with each sample.
3.5 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or
other blank matrix that is treated exactly as a sample including exposure to all
glassware, equipment, solvents, reagents, sample preservatives, internal standards,
and surrogates that are used in the extraction batch. The LRB is analyzed is used
to determine if method analytes or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.6 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water or
other blank matrix to which known quantities of the method analytes and all the
preservation compounds are added. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise measurements.
3.7 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) - An aliquot of an
environmental sample to which known quantities of the method analytes and all
the preservation compounds 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 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFMD) - A
second aliquot of the Field Sample used to prepare the LFM which is fortified,
extracted and analyzed identically. The LFMD is used instead of the Field
Duplicate to access method precision and accuracy when the occurrence of target
analytes is Ipw.
3.9 LABORATORY DUPLICATES (LD1 and LD2) - Two aliquots of the same
sample taken in the laboratory and analyzed separately with identical procedures.
Analyses of LD1 and LD2 indicate precision associated with laboratory
procedures, but not with sample collection, preservation, and storage procedures.
3.10 FffiLD 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.
526-4
-------
3.11 STOCK STANDARD SOLUTION (SSS) - A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed reference
materials or purchased from a reputable commercial source.
3.12 PRIMARY DILUTION STANDARD SOLUTION (PDS) - A solution containing
method analytes prepared in the laboratory from stock standard solutions and
diluted as needed to prepare calibration solutions and other needed analyte
solutions.
3.13 CALIBRATION STANDARD (CAL) - A solution prepared from the primary
dilution standard solution or stock standard solutions and the internal standards
and surrogate analytes. The CAL solutions are used to calibrate the instrument
response with respect to analyte concentration.
3.14 CONTINUING CALIBRATION CHECK (CCC) - A calibration standard
containing one or more method analytes, which is analyzed periodically to verify
the accuracy of the existing calibration for those analytes.
3.15 QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of
known concentrations, that is obtained from a source external to the laboratory and
different from the source of calibration standards. It is used to check standard
integrity.
3.16 METHOD DETECTION LIMIT (MDL) - The minimum concentration of an
analyte that can be identified, measured and reported with 99% confidence that
the analyte concentration is greater than zero (Section 9.2.4). This is a statistical
determination of precision. Accurate quantitation is not expected at this level.(1)
3.17 MINIMUM REPORTING LEVEL (MRL) - The minimum concentration that can
be reported as a quantitated value for a target analyte in a sample following
analysis. This defined concentration can be no lower than the concentration of the
lowest continuing calibration check standard for that analyte, and can only be used
if acceptable quality control criteria for this standard are met.
3.18 MATERIAL SAFETY DATA SHEET (MSDS) - Written information provided
by vendors concerning a chemical's toxicity, health hazards, physical properties,
fire, and reactivity data including storage, spill, and handling precautions.
4. INTERFERENCES
4.1. All glassware must be meticulously cleaned. Wash glassware with detergent and
tap water, rinse with tap water, followed by reagent water. Non-volumetric
526-5
-------
glassware can be heated in a muffle furnace at 400 °C for 2 hours as a substitute
for a solvent rinse. Volumetric glassware should not be heated in an oven above
120 °C.
4.2 Method interferences may be caused by contaminants in solvents, reagents
(including reagent water), sample bottles and caps, and other sample processing
hardware that lead to discrete artifacts and/or elevated baselines in the chromato-
grams. All items such as these must be routinely demonstrated to be free from
interferences (less than V3 the MRL for each target analyte) under the conditions
of the analysis by analyzing laboratory reagent blanks as described in Section 9.4.
Subtracting blank values from sample results is not permitted.
4.3 Matrix interferences may be caused by contaminants that are co-extracted from
the sample. The extent of matrix interferences will vary considerably from source
to source, depending upon the nature of the water. Water samples high in total
organic carbon may have elevated baseline or interfering peaks.
4.4 Relatively large quantities of the buffer and preservatives (Sect. 8.1) are added to
sample bottles. The potential for trace-level organic contaminants in these
reagents exist. Interferences from these sources should be monitored by analysis
of laboratory reagent blanks, particularly when new lots of reagents are acquired.
4.5 Benzophenone was an interferent recovered at low level from one manufacturer's
lot of tris(hydroxymethyl)aminomethane hydrochloride. The spectra of
benzophenone and 1,2-diphenylhydrazine are very similar, sharing the major m/z
ions of 51, 77, 105, 152, and 182. At first glance, the benzophenone may appear
to be a contaminant in the LRB. Given that the two compounds share common
ions, their chromatographic peaks must be resolved.
4.6 During method development, one lot of anhydrous sodium sulfate was found to
add an agent to the extract that, when injected, caused complete loss of prometon
recovery and caused severe chromatographic tailing of the phenols. It is very
important to check a new sodium sulfate lot before general use. When only one or
two samples with the interfering agent were injected, recovery of good
chromatographic performance was possible by removing the first meter of the
capillary column and replacing the deactivated glass inlet liner.
4.7 Solid phase cartridges and disks and their associated extraction devices have been
observed to be a source of interferences. The analysis of field and laboratory
reagent blanks can provide important information regarding the presence or
absence of such interferences. Brands and lots of solid phase extraction devices
should be tested to ensure that contamination will not preclude analyte
identification and quantitation.
526-6
-------
4.8 Analyte carryover may occur when a relatively "clean" sample is analyzed
immediately after a sample containing relatively high concentrations of
compounds. Syringes and splitless injection port liners must be cleaned carefully
or replaced as needed. After analysis of a sample, containing high concentrations
of compounds, a laboratory reagent blank should be analyzed to ensure that
accurate values are obtained for the next sample.
4.9 Silicone compounds may be leached from punctured autosampler vial septa,
particularly when particles of the septa sit in the vial. These silicone compounds
should have no effect on the analysis, but the analyst should be aware of this
potential problem.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent 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 an awareness of OSHA regulations regarding safe
handling of chemicals used in this method. A reference file of MSDSs should be
made available to all personnel involved in the chemical analysis. Additional
references to laboratory safety are available/2^
5.2 Pure standard materials and stock standard solutions of these compounds should
be handled with suitable protection to skin and eyes, and care should be taken not
to breath the vapors or ingest the materials.
6. EQUIPMENT AND SUPPLIES (All specifications are suggested. Catalog numbers are
included for illustration only.)
6.1 SAMPLE CONTAINERS - 1 liter glass bottles fitted with polytetrafluoroethylene
(PTFE) lined screw caps.
6.2 VIALS - Various sizes of amber glass vials with PTFE lined screw caps for
storing standard solutions and extracts. Crimp-top glass autosampler vials, 2 mL,
with PTFE faced septa.
6.3 VOLUMETRIC FLASKS - Class A, suggested sizes include 1, 5, and 10 mL, for
preparation of standards and dilution of extract to final volume.
6.4 GRADUATED CYLINDERS - Suggested sizes include 5, 10, and 250 mL.
6.5 MICRO SYRINGES - Suggested sizes include 25, 50, 100, 250, 500, and 1000
mL.
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6.6 DRYING COLUMN - The drying column must be able to contain 5 to 7 g of
anhydrous sodium sulfate. The drying column should not leach interfering
compounds or irreversibly adsorb target analytes. Any small column may be used,
such as a glass pipet with glass wool plug.
6.7 CONICAL CENTRIFUGE TUBES - 15 mL, or other glassware suitable for
collection of the eluate from the cartridge or disk after extraction.
6.8 COLLECTION TUBES OR VIALS - 25 mL or larger, or other glassware
suitable for collecting extract from drying tube. Conical centrifuge tubes, 50 mL,
with graduations (VWR #: 21049-063) were used to develop this method.
6.9 ANALYTICAL BALANCE - Capable of weighing to the nearest 0.0001 g.
6.10 SOLID PHASE EXTRACTION (SPE) APPARATUS USING CARTRIDGES
6.10.1 SOLID PHASE EXTRACTION CARTRIDGES - 6 mL, packed with 500
mg (125 um dp) of polystyrene divinylbenzene (SDVB) sorbent phase
(Varian Bond Elut ENV phase; cat.#: 1225-5011 or equivalent).
6.10.2 SAMPLE RESERVOIR AND TRANSFER TUBE- Sample reservoirs
(VWR cat.#: JT7120-3 or equivalent) with a volume of about 75 mL are
attached to the cartridges to hold the water sample. An alternative method
is using transfer tubes (Supelco "Visiprep"; cat.#: 57275 or equivalent)
which transfer the sample directly from the sample container to the SPE
cartridge.
6.10.3 VACUUM EXTRACTION MANIFOLD - With flow/vacuum control
(Supelco cat.#: 57044 or equivalent). The use of replaceable needles or
valve liners may be used to prevent cross contamination.
6.10.4 REMOTE VACUUM GAUGE/BLEED VALVE ASSEMBLY - To
monitor and adjust vacuum pressure delivered to the vacuum manifold
(Supelco cat.#: 57161-U or equivalent)
6.10.5 An automatic or robotic system, designed for use with SPE cartridges, may
be used as long as all quality control requirements discussed in Section 9
are met. Automated systems may use either vacuum or positive pressure
to process samples and solvents through the cartridge. All extraction and
elution steps must be the same as in the manual procedure. Extraction or
elution steps may not be changed or omitted to accommodate the use of an
automated system.
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6.11 SOLID PHASE EXTRACTION (SPE) APPARATUS USING DISKS
6.11.1 SOLID PHASE EXTRACTION DISKS-47 mm diameter and 0.5 mm
thick, manufactured with a polystyrene divinylbenzene (SDVB) sorbent
phase (Varian cat. #: 1214-5010 or equivalent). Larger disks may be used
as long as the QC performance criteria outlined in Section 9 are met.
6.11.2 SPE DISK EXTRACTION GLASSWARE - funnel, PTFE coated support
screen, PTFE gasket, base, and clamp used to support SPE disks and
contain samples during extraction. May be purchased as a set (Fisher cat.
#:K971100-0047 or equivalent) or separately.
6.11.3 VACUUM EXTRACTION MANIFOLD - Designed to accommodate
extraction glassware and disks (Varian cat.#: 1214-6001 or equivalent).
6.11.4 An automatic robotic system for disks as described in Section 6.10.5.
6.12 EXTRACT CONCENTRATION SYSTEM - Extracts are concentrated by
blowdown with nitrogen using water bath set to 40°C (Meyer N-Evap, Model 111,
Organomation Associates, Inc. or equivalent).
6.13 LABORATORY OR ASPIRATOR VACUUM SYSTEM - Sufficient capacity to
maintain a vacuum of about 10 inches of mercury for cartridges. A greater
vacuum of 15 to 25 inches of mercury may be used with disks.
6.14 GAS CHROMATOGRAPH/MASS SPECTROMETER (GC/MS)
INSTRUMENTATION
6.14.1 FUSED SILICA CAPILLARY GC COLUMN - 30 m x 0.25 mm i.d.
fused silica capillary column coated with a 0.25 urn bonded film of
poly(dimethylsiloxy)poly(l,4-bis(dimethylsiloxy)phenylene)siloxane
(J&W DB-5MS or equivalent). Any capillary column that provides
adequate resolution, capacity, accuracy, and precision as summarized in
Section 17 may be used. A nonpolar, low-bleed column is recommended
for use with this method to provide adequate chromatography and
minimize column bleed.
6.14.2 GC INJECTOR AND OVEN - Capable of temperature programming and
equipped for split/splitless injection. Targetcompounds included in this
method are subject to thermal breakdown in the injector port, which
increases when the injector is not properly deactivated or at excessive
temperatures. The injection system must not allow analytes to contact hot
stainless steel or other metal surfaces that promote decomposition. The
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performance data in Section 17 was obtained by hot, splitless injection
using a 4 mm i.d. glass, deactivated liner (Restek cat.#: 20772). Other
injection techniques such as temperature programmed injections, cold on-
column injections and large volume injections may be used if the QC
criteria in Sections 9 and 10 are met. Equipment designed appropriately
for these alternate types of injections must be used if these options are
selected.
6.14.3 GC/MS INTERFACE - Interface should allow the capillary column or
transfer line exit to be placed within a few mm of the ion source. Other
interfaces, like jet separators, are acceptable as long as the system has
adequate sensitivity and QC performance criteria are met.
6.14.4 MASS SPECTROMETER- The MS must be capable of electron
ionization at a nominal electron energy of 70 eV to produce positive ions.
The spectrometer must be capable of scanning at a minimum from 45 to
450 amu with a complete scan cycle time (including scan overhead) of 1.0
second or less. (Scan cycle time = total MS data acquisition time in
seconds divided by number of scans in the chromatogram). The
spectrometer must produce a mass spectrum that meets all criteria in Table
1 when a solution containing approximately 5 ng of DFTPP is injected into
the GC/MS. Use a single spectrum at the apex of the DFTPP peak, an
average spectrum of the three highest points of the peak, or an average
spectrum across the entire peak to evaluate the performance of the system.
The scan tune should be set so that all analytes have a minimum of five
scans across the chromatographic peak. Seven to ten scans across
chromatographic peaks are preferred.
6.14.5 DATA SYSTEM — An interfaced data system is required to acquire, store,
and output mass spectral data. The computer software should have the
capability of processing stored GC/MS data by recognizing a GC peak
within a given retention time window. The software must allow
integration of the ion abundance of any specific ion between specified time
or scan number limits. The software must be able to calculate relative
response factors, construct a linear regression or quadratic calibration
curve, and calculate analyte concentrations.
7. REAGENTS AND STANDARDS
7.1 REAGENTS AND SOLVENTS - Reagent grade or better chemicals should be
used in all tests. Unless otherwise indicated, it is intended that all reagents will
conform to the specifications of the Committee on Analytical Reagents of the
American Chemical Society (ACS), where such specifications are available.
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Other grades may be used, provided it is first determined that the reagent is of
sufficiently high purity to permit its use without lessening the quality of the
determination.
7.1.1 HELIUM - 99.999% or better, GC carrier gas.
7.1.2 REAGENT WATER - Purified water which does not contain any
measurable quantities of any target analytes or interfering compounds at or
above 1/3 the MRL for each compound of interest.
7.1.3 METHANOL (CAS# 67^56-1) - High purity, demonstrated to be free of
analytes and interferences (B&J Brand GC2®, Capillary GC/GC-MS Grade
or equivalent).
7.1.4 ETHYL ACETATE (CAS# 141-78-6)- High purity, demonstrated to be
free of analytes and interferences (B&J Brand GC2®, Capillary GC/GC-MS
Grade or equivalent).
7.1.5 METHYLENE CHLORIDE (CAS# 75-09-02) - High purity,
demonstrated to be free of analytes and interferences (B&J Brand GC2®,
Capillary GC/GC-MS Grade or equivalent).
7.1.6 SODIUM SULFATE, ANHYDROUS (CAS# 7757-82-6) - Soxhlet
extracted with methylene chloride for a minimum of four hours or heated
to 400°C for two hours in a muffle furnace. One lot of "ACS grade"
anhydrous sodium sulfate had a contaminant that degraded the capillary
column so that analyte recoveries were unacceptable. An "ACS grade,
suitable for pesticide residue analysis," or equivalent, of anhydrous sodium
sulfate is recommended.
7.1.7 SAMPLE PRESERVATION REAGENTS - These preservatives are
solids at room temperature and may be added to the sample bottle before
shipment to the field.
7.1.7.1 BUFFER SALT MIX, pH 7 - The sample must be buffered to pH
7 with two components: 1) tris(hydroxymethyl)aminomethane, also
called Tris, 0.47 g (CAS# 77-86-1, ACS Reagent Grade or ,
equivalent); and 2) tris(hydroxymethyl)aminomethane
hydrochloride, also called Tris HC1, 7.28 g (CAS# 1185-53-1, ACS
Reagent Grade or equivalent). Alternately, 7.75 g of a commercial
buffer crystal mixture, that is blended in proportion to the amounts
given above, can be used.
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7.2
7.1.7.2 L-ASCORBIC ACID (CAS# 50-81-7) - Ascorbic acid reduces
"free chlorine" at the time of sample collection (ACS Reagent
Grade or equivalent).
7.1.7.3 ETHYLENEDIAMINETETRAACETIC ACID, TRISODIUM
SALT (Trisodium EDTA, CAS# 10378-22-0) - Trisodium EDTA
is added to inhibit metal-catalyzed hydrolysis of analytes. The
trisodium salt is used instead of the disodium salt because the
trisodium salt solution pH is closer to the desired pH of 7 (Sigma
cat.#: ED3SS or equivalent).
7.1.7.4 DIAZOLIDINYL UREA (DZU) (CAS# 78491-02-8) - DZU is
added to inhibit microbial growth. DZU (Sigma cat.#: D-5146) is
commonly used as a preservative in cosmetics such as skin lotion.
STANDARD SOLUTIONS - When a compound purity is assayed to be 96% or
greater, the weight can be used without correction to calculate the concentration of
the stock standard. Solution concentrations listed in this section were used to
develop this method and are included as an example. Standards for sample
fortification generally should be prepared in the smallest volume that can be
accurately measured to minimize the addition of excess organic solvent to
aqueous samples. Even though stability times for standard solutions are
suggested in the following sections, laboratories should used standard QC
practices to determine when their standards need to be replaced.
7.2.1 INTERNAL STANDARD SOLUTIONS - This method uses three
internal standard compounds listed in the table below.
Internal Standards
acenaphthene-c?10
phenanthrene-c?10
chrysene-f/12
CAS#'^-.:;:;:
15067-26-2
1517-22-2
1719-03-5
FW; '.-.•
164.3
188.3
240.4
7.2.1.1 INTERNAL STANDARD PRIMARY DILUTION STANDARD
(500 ug/mL) - Prepare or purchase commercially the Internal
Standard Primary Dilution Standard (PDS) at a concentration of
500 ug/mL. If prepared from neat or solid standards, this solution
requires the preparation of a more concentrated stock standard
similar to the procedure followed for the analyte stock (Sect.
7.2.3.1). The solvent for the Internal Standard PDS may be
acetone or ethyl acetate. The Internal Standard PDS has been
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7.2.2
shown to be stable for 1 year in amber glass screw cap vials when
stored at - 10°C or less.
7.2.1.2 INTERNAL STANDARD EXTRACT FORTIFICATION
SOLUTION (50 ug/mL) - Dilute a portion of the Internal
Standard PDS (500 ug/mL) (Sect. 7.2.1.1) to a concentration of 50
ug/mL in ethyl acetate and use this solution to fortify the final 1
mL extracts (Sect. 11.6). The Internal Extract Fortification
Solution has been shown to be stable in amber glass screw cap
vials for 6 months when stored at - 10°C or less.
SURROGATE (SUR) ANALYTE STANDARD SOLUTIONS - The two
surrogates for this method are listed in the table below.
Surrogates
1 ,3-dimethyl-2-nitrobenzene
triphenylphosphate
CAS#
81-20-9
115-86-6
FW
151.2
326.3
7.2.2.1 SUR STOCK SOLUTION (~ 4 to 10 mg/mL) - Surrogate Stock
Solutions may be purchased commercially or prepared from neat
materials. The solvent for the SUR Stock Solution may be acetone
or ethyl acetate. These solutions have been shown to be stable for
one year when stored in amber glass containers at -10°C or less.
7.2.2.2 SUR PRIMARY DILUTION STANDARD (SUR PDS) (500
ug/mL) - The 500 ug/mL SUR PDS may be purchased
commercially or prepared by volumetric dilution of the SUR Stock
Solutions (Sect. 7.2.2.1) in acetone or ethyl acetate The PDS has
been shown to be stable for one year when stored in amber glass
screw cap vials at- 10°C or less. This solution is used to make the
50 ug/mL solution for sample fortification and also to prepare
calibration solutions.
7.2.2.3 SUR SAMPLE FORTIFICATION SOLUTION (50 ug/mL) -
Dilute the 500 ug/mL SUR PDS in methanol to make a 50 ug/mL
sample fortification solution. Add 100 uL of this 50 ug/mL
solution to each 1 liter water sample before extraction to give a
concentration of 5 ug/L of each surrogate. This solution has been
shown to be stable for .six months when stored in amber glass
screw cap vials at - 10°C or less.
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7.2.3 ANALYTE STANDARD SOLUTIONS - Obtain the analytes listed in the
table in Section 1.1 as neat or solid standards or as commercially prepared
ampulized solutions from a reputable standard manufacturer. Prepare the
Analyte Stock and Primary Dilution Standards as described below.
7.2.3.1 ANALYTE STOCK STANDARD SOLUTIONS (1 to 10 mg/mL) -
Analyte standards may be purchased commercially as ampulized
solutions or prepared from neat materials. Stock standards have
been shown to be stable for one year when stored in amber glass
screw cap vials at - 10°C or less.
7.2.3.2 ANALYTE PRIMARY DILUTION STANDARDS (200 ug/mL and
20 ug/mL) - Prepare the 200 ug/mL Analyte PDS by volumetric
dilution of the Analyte Stock Standard Solution (Sect. 7.2.3.1) in
ethyl acetate to make a 200 ug/mL solution. The 20 ug/mL Analyte
PDS can be made by a volumetric dilution of the 200 ug/mL
Analyte PDS in ethyl acetate. The Analyte PDSs are used to
prepare calibration and fortification standards. They have been
shown to be stable for six months when stored in an amber glass
screw cap vial at - 10°C or less. Check frequently for signs of
evaporation, especially before preparing calibration solutions.
7.2.4 CALIBRATION SOLUTIONS - Prepare a calibration curve of at least 5
CAL levels over the concentration range of interest from dilutions of the
Analyte PDSs in ethyl acetate. All calibration solutions should contain at
least 80% ethyl acetate so that gas chromatographic performance is not
compromised. The lowest concentration of calibration standard must be at
or below the MRL. The level of the MRL will depend on system
sensitivity. A constant concentration of each internal standard and
surrogate (in the range of 2 to 5 ng/uL) is added to each CAL. For instance,
for method development work, 50 uL of the 500 ug/mL Internal Standard
PDS and 50 uL of the 500 ug/mL SUR PDS were added to each CAL level
standard for final concentrations of 5 ug/mL. The calibration solutions
have been shown to be stable for six months when stored in an amber glass
screw cap vial at-10°C or less.
7.2.5 ANALYTE FORTIFICATION SOLUTIONS (0.05 to 5.0 ug/mL) - The
Analyte Fortification Solutions contain all method analytes of interest in
methanol. They are prepared by dilution of the Analyte PDSs (200 ug/mL
or 20 ug/mL). These solutions are used to fortify the LFBs, the LFMs and
LFMDs with method analytes. It is recommended that three concentrations
be prepared so that the fortification levels can be rotated. The Analyte
Fortification Solutions have been shown to be stable for six months when
stored in an amber glass screw cap vial at - 10°C or less.
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7.2.6 GC/MS TUNE CHECK SOLUTION (5 ug/mL) - Prepare a
Decafluorotriphenylphosphine (DFTPP) solution in methylene chloride.
DFTPP is more stable in methylene chloride than in acetone or ethyl
acetate. Store this solution in an amber glass screw cap vial at - 10°C or
less.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 SAMPLE BOTTLE PREPARATION
8.1.1 Grab samples must be collected in accordance with conventional sampling
practices(5) using a 1 liter or 1 quart amber or clear glass bottle fitted with
PTFE-lined screw-caps.
8.1.2 Preservation reagents, listed in the table below, are added to each sample
bottle prior to shipment to the field.
Compound
L- Ascorbic Acid
Ethylenediaminetetraacetic acid
trisodium salt
Diazolidinyl Uriea
*Tris(hydroxymethyl)aminomethane
*Tris(hydroxymethyl)ammometIiane
hydrochloride
Amount
0.10 g/L
0.35 g/L
1.0 g/L
0.47 g/L
7.28 g/L
Purpose •"•••'-.
Dechlorination
Inhibit metal-catalyzed
hydrolysis of targets
Microbial inhibitor
First component of pH 7
buffer mixture
Second component of pH
7 buffer mixture
*Alternately, 7.75 g of a commercial buffer crystal mixture, that is blended
in the proportions given in the table, canbe used (Sect. 7.1.7.1).
8.1.2.1 Residual chlorine must be reduced at the time of sample collection
with 100 mg of ascorbic acid per liter. Sodium thiosulfate and
sodium sulfite cannot be used because they were found to degrade
target analytes. In addition, while ammonium chloride is effective
in converting free chlorine to chloramines, the chloramines also
caused target compound loss.
8.1.2.2 Ethylenediaminetetraacetic acid, trisodium salt (trisodium EDTA)
(0.35 g) must be added to inhibit metal-catalyzed hydrolysis of the
target analytes, principally terbufos, disulfpton, diazinon, fonofos,
and cyanazine.
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8.1.2.3 Diazolidinyl urea (1.0 g) is added to inhibit microbial degradation
of analytes. Diazolidinyl urea is used in cosmetics such as skin
lotion. The antimicrobial activity of diazolidinyl urea has been
proposed as due to protein alkylation of sulfhydryl groups and the
ability to release formaldehyde.(6) Plate count studies conducted
during method development indicated that it was effective in
inhibiting microbial degradation for extended periods.
8.1.2.4 The sample must be buffered to pH 7 to reduce the acid and base
catalyzed hydrolysis of target analytes. The pH buffer has two
components: tris(hydroxymethyl)aminomethane (0.47 g) and
tris(hydroxymethyl)aminomethane hydrochloride (7.28 g). A
commercially prepared combination of these two compounds can
be purchased as pre-mixed crystals. When using the pH 7 pre-
mixed crystals, add 7.75 g per liter of water sample.
8.2 SAMPLE COLLECTION
8.2.1 When sampling from a cold water tap, remove the aerator so that no air
bubbles will be trapped in the sample. Open the tap and allow the system
to flush until the water temperature has stabilized (usually about 3-5
minutes). Collect samples from the flowing system.
8.2.2 When sampling from an open body of water, fill a 1 quart wide-mouth
bottle or 1 L beaker with water sampled from a representative area, and
carefully fill sample bottles from the container. Sampling equipment,
including automatic samplers, must be free of plastic tubing, gaskets, and
other parts that may leach interfering analytes into the water sample.
8.2.3 Fill sample bottles, taking care not to flush out the sample preservation
reagents. Samples do not need to be collected headspace free.
8.2.4 After collecting the sample, cap the bottle and agitate by hand until
preservatives are dissolved. Keep the sample sealed from time of
collection until extraction.
8.3 SAMPLE SHIPMENT AND STORAGE - Samples must be chilled during
shipment and must not exceed 10°C during the first 48 hours after collection.
Samples should be confirmed to be at or below 10°C when they are received at the
laboratory. Samples stored in the lab must be held at or below 6°C until
extraction, but should not be frozen.
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8.4 SAMPLE AND EXTRACT HOLDING TIMES-Results of the sample storage
stability study (Sect. 17, Table 7) indicated that most compounds listed in this
method have adequate stability for 14 days when collected, dechlorinated,
preserved, shipped and stored as described in Sections 8.1, 8.2, and 8.3. At 14
days, even the most unstable compound, terbufos, was recovered above 60 %.
Water samples should be extracted as soon as possible, but must be extracted
within 14 days. The extract storage stability study must be stored at 0°C or less
and analyzed within 28 days after extraction. The extract storage stability study
data are presented in Section 17, Table 8.
9. QUALITY CONTROL
9.1 Quality control (QC) requirements include the Initial Demonstration of Capability
(Sect. 17, Table 9), the determination of the MDL, and subsequent analysis in
each analysis batch of a Laboratory Reagent Blank (LRB), Continuing Calibration
Check Standards (CCC), a Laboratory Fortified Blank (LFB), a Laboratory
Fortified Sample Matrix (LFM), and either a Laboratory Fortified Sample Matrix
Duplicate (LFMD) or a Field Duplicate Sample. This section details the specific
requirements for each QC parameter. The QC criteria discussed in the following
sections are summarized in Section 17, Tables 9 and 10. These criteria are
considered the minimum acceptable QC criteria, and laboratories are encouraged
to institute additional QC practices to meet their specific needs.
9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) - Requirements for the
Initial Demonstration of Capability are described in the following sections and
summarized in Section 17, Table 9.
9.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND -
Any tune a new lot of solid phase extraction (SPE) cartridges or disks is
used, it must be demonstrated that a laboratory reagent blank (Sect. 9.4) is
reasonably free of contamination and that the criteria in Section 9.4 are
met.
9.2.2 INITIAL DEMONSTRATION OF PRECISION - Prepare, extract, and
analyze 4-7 replicate LFBs fortified near the midrange of the initial
calibration curve according to the procedure described in Section 11.
Sample preservatives as described in Section 8.1 must be added to these
samples. The relative standard deviation (RSD) of the results of the
replicate analyses must be less than 20%.
9.2.3 INITIAL DEMONSTRATION OF ACCURACY - Using the same set of
replicate data generated for Section 9.2.2, calculate average recovery. The
average recovery of the replicate values must be within ±30% of the true
value.
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9.2.4 MDL DETERMINATION - Prepare, extract and analyze at least seven
replicate LFBs at a concentration estimated to be near the MDL, over a
period of at least three days (both extraction and analysis should be
conducted over at least three days) using the procedure described in
Section 11. The fortification level may be estimated by selecting a
concentration with a signal of 2 to 5 times the noise level. The appropriate
concentration will be dependent upon the sensitivity of the GC/MS system
being used. Sample preservatives as described in Section 8.1 must be
added to these samples. Calculate the MDL using the equation
MDL = St(n. ^ i. alpha=o 99)
where
V1, i -alpha=0.99)= Students t value for the 99% confidence level
with n-1 degrees of freedom,
n = number of replicates, and
S = standard deviation of replicate analyses.
NOTE: Do not subtract blank values when performing MDL calculations.
The MDL is a statistical determination of precision only.(1) If the MDL
replicates are fortified at a low enough concentration, it is likely that they
will not meet precision and accuracy criteria.
9.2.5 METHOD MODIFICATIONS - The analyst is permitted to modify GC
columns, GC conditions, evaporation techniques, internal standards or
surrogate standards, but each time such method modifications are made,
the analyst must repeat the procedures of the IDC (Sect. 9.2).
9.3 MINIMUM REPORTING LEVEL (MRL) - The MRL is a threshold
concentration of an analyte that a laboratory can expect to accurately quantitate in
an unknown sample. The MRL should be established at an analyte concentration
that is either greater than three times the MDL or at an analyte concentration
which would yield a response greater than a signal-to-noise ratio of five.
Although the lowest calibration standard for an analyte may be below the
MRL, the MRL must never be established at a concentration lower than the
lowest calibration standard.
9.4 LABORATORY REAGENT BLANK (LRB) - An LRB is required with each
extraction batch (Sect. 3.1) of samples to determine the background system
contamination. If the LRB produces a peak within the retention time window of
any analyte that would prevent the determination of that analyte, determine the
source of contamination and eliminate the interference before processing samples.
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Background contamination must be reduced to an acceptable level before
proceeding. Background from method analytes or contaminants that interfere
with the measurement of method analytes must be below 1/3 of the MRL. If the
target analytes are detected in the LRB at concentrations equal to or greater than
this level, then all data for the problem analyte(s) must be considered invalid for
all samples in the extraction batch.
9.5 CONTINUING CALIBRATION CHECK (CCC) - A CCC is a standard prepared
with all compounds of interest which is analyzed during the analysis batch to
ensure the stability of the instrument initial calibration. This calibration check is
required at the beginning of each day that samples are analyzed, after every ten
injections, and at the end of any group of sample analyses. See Section 10.3 for
concentration requirements and acceptance criteria.
9.6 LABORATORY FORTIFIED BLANK (LFB) - An LFB is required with each
extraction batch (Sect. 3.6). The fortified concentration of the LFB should be
rotated between low, medium, and high concentrations from day to day. The low
concentration LFB must be as near as practical to, but no more than two times the
MRL. Similarly, the high concentration LFB should be near the high end of the
calibration range established during the initial calibration (Sect. 10.2). Results of
the low-level LFB analyses must be 50-160% of the true value. Results of the
medium and high-level LFB analyses must be 70-130% of the true value. If the
LFB results do not meet these criteria for target analytes, then all data for the
problem analyte(s) must be considered invalid for all samples in the extraction
batch.
9.7 MS TUNE CHECK - A complete description of the MS tune check is in Section
10.2.1. This check must be performed each time a major change is made to the
mass spectrometer, and prior to establishing and/or re-establishing an initial
calibration (Sect. 10.2). In this method daily DFTPP analysis is not required.
9.8 INTERNAL STANDARDS (IS) - The analyst must monitor the peak area of each
internal standard in all injections during each analysis day. The IS response (as
indicated by peak area) for any chromatographic run must not deviate by more
than ±50% from the average area measured during the initial calibration for that
IS. A poor injection could cause the IS area to exceed these criteria. Inject a
second aliquot of the suspect extract to determine whether the failure is due to
poor injection or instrument response drift.
9.8.1 If the reinjected aliquot produces an acceptable internal standard response,
report results for that aliquot.
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9.8.2 If the internal standard area for the reinjected extract deviates greater than
50% from the initial calibration average, the analyst should check the
continuing calibration check standards that ran before and after the sample.
If the continuing calibration check fails the criteria of Section 9.5 and 10.3,
recalibration is in order per Section 10. If the calibration standard is
acceptable, extraction of the sample should be repeated provided the
sample is still within holding time. Otherwise, report results obtained
from the reinjected extract, but annotate as suspect.
9.9 SURROGATE RECOVERY - The surrogate standards are fortified into the
aqueous portion of all samples, duplicates, LRBs, LFMs and LFMDs prior to
extraction. Surrogates are also added to the calibration curve and calibration
check standards. The surrogate is a means of assessing method performance from
extraction to final chromatographic measurement.
9.9.1 When surrogate recovery from a sample, blank, or CCC is less than 70%
or greater than 130%, check (1) calculations to locate possible errors, (2)
standard solutions for degradation, (3) contamination, and (4) instrument
performance. If those steps do not reveal the cause of the problem,
reanalyze the extract.
9.9.2 If the extract reanalysis meets the surrogate recovery criterion, report only
data for the reanalyzed extract.
9.9.3 If the extract reanalysis fails the 70-130% surrogate recovery criterion, the
analyst should check the surrogate calibration by analyzing the most
recently acceptable calibration standard. If the calibration standard fails
the 70-130% surrogate recovery criteria of Section 9.9.1, recalibration is in
order. If the surrogate recovery of the calibration standard is acceptable,
extraction of the sample should be repeated, provided the sample is still
within the holding time. If the sample re-extract also fails the recovery
criterion, report all data for that sample as suspect/surrogate recovery.
9.9.4 The surrogate, l,3-dimethyl-2-nitrobenzene, is used to track the recovery
of nitrobenzene. The other surrogate, triphenylphosphate, monitors the
recovery of all the other target analytes. If nitrobenzene is not included on
the target analyte list, then the first surrogate, l,3-dimethyl-2-nitrobenzene,
does not need to be analyzed.
9.10 LABORATORY FORTIFIED SAMPLE MATRIX AND DUPLICATE (LFM
AND LFMD) - Analyses of LFMs (Sect. 3.7) are required in each extraction
batch and are used to determine that the sample matrix does not adversely affect
method accuracy. If the occurrence of target analytes in the samples is infrequent,
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or if historical trends are unavailable, a second LFM, or LMFD, must be prepared,
extracted, and analyzed from a duplicate field sample used to prepare the LFM to
assess method precision. Extraction batches that contain LFMDs will not require
the analysis of a Field Duplicate (Sect. 9.11). If a variety of different sample
matrices are analyzed regularly, for example, drinking water from groundwater
and surface water sources, method performance should be established for each.
Over time, LFM data should be documented for all routine sample sources for the
laboratory.
9.10.1 Within each extraction batch, a minimum of one field sample is fortified
as an LFM for every 20 samples extracted. The LFM is prepared by
spiking a sample with an appropriate amount of Analyte PDS (Sect. 7.2.5).
Select a spiking concentration that is at least twice the matrix background
concentration, if known. Use historical data or rotate through the
designated concentrations to select a fortifying concentration. Selecting a
duplicate bottle of a sample that has already been analyzed aids in the
selection of appropriate spiking levels.
9.10.2 Calculate the percent recovery (R) for each analyte using the equation
R= " *100
Vx
where: A = measured concentration in the fortified sample,
B = measured concentration in the unfortified sample, and
C = fortification concentration.
9.10.3 Analyte recoveries may exhibit matrix bias. For samples fortified at or
above their native concentration, recoveries should range between 70 and
130%, except for low-level fortification near or at the MRL where 50 to
160% recoveries are acceptable. If the accuracy of any analyte falls
outside the designated range, and the laboratory performance for that
analyte is shown to be in control in the LFB, the recovery is judged to be
matrix biased. The result for that analyte in the unfortified sample is
labeled suspect/matrix to inform the data user that the results are suspect
due to matrix effects.
9.10.4 If an LFMD is analyzed instead of a Field Duplicate (Sect. 9.11), calculate
DDr. LFM-LFMD 1AA
RPD = * 100
(LFM+LFMD~)/2
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the relative percent difference (RPD) for duplicate LFMs (LFM and
LFMD) using the equation RPDs for duplicate LFMs should fall in the
range of ±30% for samples fortified at or above their native concentration.
Greater variability may be observed when LFMs are spiked near the MRL.
At the MRL, RPDs should fall in the range of ±50% for samples fortified
at or above their native concentration. If the accuracy of any analyte falls
outside the designated range, and the laboratory performance for that
analyte is shown to be in control in the LFB, the recovery is judged to be
matrix biased. The result for that analyte in the unfortified sample is
labeled suspect/matrix to inform the data user that the results are suspect
due to matrix effects.
9.11 FIELD DUPLICATES (FD 1 AND FD2) - Within each extraction batch, a
minimum of one Field Duplicate (FD) or LFMD (Sect. 9.10) must be analyzed.
FDs check the precision associated with sample collection, preservation, storage,
and laboratory procedures. If target analytes are not routinely observed in field
samples, a LFMD (Sect. 9.10) should be analyzed to substitute for this
requirement. Extraction batches that contain LFMDs (Section 9.10) will not
require the analysis of a Field Duplicate.
9. 1 1 . 1 Calculate the relative percent difference (RPD) for duplicate
measurements (FD1 and FD2) using the equation
100 m
9.11.2 RPDs for duplicates should be in the range of ±30%. Greater variability
may be observed when analyte concentrations are near the MRL. At the
MRL, RPDs should fall in the range of ±50%. If the accuracy of any
analyte falls outside the designated range, and the laboratory performance
for that analyte is shown to be in control in the LFB, the recovery is judged
to be matrix biased. The result for that analyte in the unfortified sample is
labeled suspect/matrix to inform the data user that the results are suspect
due to matrix effects.
9.12 QUALITY CONTROL SAMPLES (QCS) - Each time that new standards are
prepared or a new calibration curve is run, analyze a QCS from a source different
than the source of the calibration standards. The QCS may be injected as a
calibration standard or fortified into reagent water and analyzed as a LFB. If the
QCS is analyzed as a continuing calibration, then the acceptance criteria are the
same as for the CCC. If the QCS is analyzed as a LFB, then the acceptance
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criteria are the same as for an LFB. If measured analyte concentrations are not of
acceptable accuracy, check the entire analytical procedure to locate and correct the
problem source.
10. CALIBRATION AND STANDARDIZATION
10.1 Demonstration and documentation of acceptable mass spectrometer tune and
initial calibration is required before any samples are analyzed. After the initial
calibration is successful, a continuing calibration check is required at the
beginning and end of each period in which analyses are performed, and after every
tenth sample. Verification of mass spectrometer tune must be repeated each time
a major instrument modification is made or maintenance is performed, and prior
to analyte calibration.
10:2 INITIAL CALIBRATION
10.2.1 MS TUNE/MS TUNE CHECK- Calibrate the mass and abundance scales
, of the MS with calibration compounds and procedures prescribed by the
manufacturer with any modifications necessary to meet tuning
requirements. Inject 5 ng or less of the DFTPP solution (Sect. 7.2.6) into
the GC/MS system. Acquire a mass spectrum that includes data for m/z
45-450. Use a single spectrum at the apex of the DFTPP peak, an average
spectrum of the three highest points of the peak, or an average spectrum
across the entire peak to evaluate the performance of the system. If the
DFTPP mass spectrum does not meet all criteria in Table 1, the MS must
be retuned and adjusted to meet all criteria before proceeding with the
initial calibration.
10.2.2 INSTRUMENT CONDITIONS - Operating conditions are described
below. Conditions different from those described may be used if QC
criteria in Section 9 are met. Different conditions include alternate GC
columns, temperature programs, and injection techniques, such as cold on-
column and large volume injections. Equipment designed for alternate
types of injections must be used if these options are selected.
10.2.2.1 Inject 1 uL into a hot, splitless injection port held at 210°C with a
split delay of 1 min. The temperature program is as follows:
initially hold at 55°C for one minute, then ramp at 8°C/ min to
320°C. Total run time is approximately 33 min. Begin data
acquisition at 3.5 minutes.
Note: The GC was operated in a constant flow rate mode at a rate
of 1.4 mL per minute and an initial head-pressure of 12.2 psi.
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10.2.2.2 Many of the target compounds exhibit decreased sensitivity for
low-level injections due to degradation or irreversible adsorption
in the injector port. Deactivated glass or quartz inlet liners are
recommended.
10.2.2.3 MS Detection and Sensitivity - Acquire and store data from m/z
45 to 450 with a total cycle time (including scan overhead time)
of 1.0 second or less. Adjust the cycle time to measure at least
five or more spectra during the elution of each GC peak. Seven
to ten scans across each GC peak are recommended. The
GC/MS/DS peak identification software must be able to
recognize a GC peak in the appropriate retention time window
for each of the compounds in the calibration solution, and make
correct qualitative identifications.
10.2.3 CALIBRATION SOLUTIONS - Prepare a set of at least 5 calibration
standards as described in Section 7.2.4. The lowest concentration of the
calibration standard must be at or below the MRL, which will depend on
system sensitivity. Acceptable calibration over a large dynamic range,
greater than about 50 fold range, may require multiple calibration curves.
10.2.4 CALIBRATION - The system is calibrated using the internal standard
technique. Concentrations maybe calculated through the use of average
relative response factor (RRF) or through the use of a calibration curve.
Calculate the RRFs using the equation
where: A,, = integrated abundance (peak area) of the quantitation
ion of the analyte,
AJS = integrated abundance (peak area) of the quantitation
ion internal standard,
Qx = quantity of analyte inj ected in ng or concentration
units, and
Qis = quantity of internal standard inj ected in ng or
concentration units.
Average RRF calibrations may only be used if the RRF values over the
calibration range are relatively constant (<30% RSD). Average RRF is
determined by calculating the mean RRF of a minimum of five calibration
concentrations.
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10.2.5 As an alternative to calculating average RRFs and applying the RSD test,
use the GC/MS data system software to generate a linear regression or
quadratic calibration curve. The analyst may choose whether or not to
force zero to obtain a curve that best fits the data. Examples of common
GC/MS system calibration curve options are: 1) A^ /Ajs vs Qx /Qis and 2)
RRFVSA./A;,
10.2.6 Acceptance criteria for the calibration of each analyte is determined by
calculating the concentration of each analyte and surrogate in each of the
analyses used to generate the calibration curve or average RRF. Each
calibration point, except the lowest point, for each analyte must calculate
to be 70-130% of its true value. The lowest point must calculate to be 50-
150% of its true value. If this criteria cannot be met, reanalyze the
calibration standards, restrict the range of calibration, or select an alternate
method of calibration. The data presented in this method were obtained
using quadratic fit (RRF vs. amount). Quadratic fit calibrations should be
used with caution, because the non-linear area of the curve may not be
reproducible.
10.3 CONTINUING CALIBRATION CHECK (CCC) - The CCC verifies the initial
calibration at the beginning and end of each group of analyses, and after every 10th
sample during analyses, hi this context, a "sample" is considered to be a field
sample. LRBs, LFBs, LFMs, LFMDs and CCCs are not counted as samples. The
beginning CCC for each analysis batch must be at or below the MRL in order to
verify instrument sensitivity prior to any analyses. If standards have been
prepared such that all low CAL points are not in the same CAL solution, it may be
necessary to analyze two CAL solutions to meet this requirement. Subsequent
CCCs should alternate between a medium and high concentration.
10.3.1 Inject an aliquot of the appropriate concentration calibration solution and
analyze with the same conditions used during the initial calibration.
10.3.2 Determine that the absolute areas of the quantitation ions of the internal
standards have not changed by more than ± 50% from the areas measured
during initial calibration. If any IS area has changed by more this amount,
remedial action must be taken (Sect. 10.3.4). Control charts are useful
aids in documenting system sensitivity changes.
10.3.3 Calculate the concentration of each analyte and surrogate in the check
standard. The calculated amount for each analyte for medium and high
level CCCs must be ±30% of the true value. The calculated amount for
the lowest calibration level for each analyte must be within ±50% of the
true value. If these conditions do not exist, then all data for the problem
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analyte must be considered invalid, and remedial action (Sect. 10.3.4)
should be taken which may require recalibration. Any field sample
extracts that have been analyzed since the last acceptable calibration
verification should be reanalyzed after adequate calibration has been
restored, with the following exception. If the continuing calibration fails
because the calculated concentration is greater than 130% (150% for the
low-level CCC) for a particular target compound, and field sample extracts
show no detection for that target compound, non-detects may be reported
without re-analysis.
10.3.4 REMEDIAL ACTION - Failure to meet CCC QC performance criteria
may require remedial action. Major maintenance such as cleaning an ion
source, cleaning quadrapole rods, replacing filament assemblies, etc.,
require returning to the initial calibration step (Sect. 10.2).
11. PROCEDURE
11.1 Important aspects of this analytical procedure include proper preparation of
laboratory glassware and sample containers (Sect. 4.1), and sample collection and
storage (Sect. 8). This section describes the procedures for sample preparation,
solid phase extraction (SPE) using cartridges or disks, and extract analysis.
11.2 SAMPLE BOTTLE PREPARATION
11.2.1 Samples are preserved, collected and stored as presented in Section 8. All
field and QC samples must contain the preservatives listed in Section
8.1.2, including the LRB and LFB. Before extraction, mark the level of
the sample on the outside of the sample bottle for later sample volume
determination. If using weight to determine volume (Sect. 11.3.7), weigh
the bottle with collected sample before extraction.
11.2.2 Add an aliquot of the surrogate fortification solution to each sample to be
extracted. For method development work, a 100 uL aliquot of the 50
ug/mL SUR Sample Fortification Solution (Sect. 7.2.2.3) was added to 1 L
for a final concentration of 5.0 ug/L.
11.2.3 If the sample is an LFB, LFM, or LFMD, add the necessary amount of
analyte fortification solution. Swirl each sample to ensure all components
are mixed.
11.2.4 Proceed with sample extraction using either SPE cartridges (Sect. 11.3) or
disks (Sect. 11.4).
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11.3 CARTRIDGE SPE PROCEDURE - The cartridge extraction procedure is carried
out in a manual mode or by using a robotic or automatic sample preparation
device. This section describes a SPE manual procedure using the equipment
outlined in Section 6.10. The manual mode of sample addition to cartridges is
performed with a large reservoir attached to the cartridge or with a transfer tube
from the sample bottle to the cartridge. Cartridge extraction data in Section 17
was collected using the transfer tube option described below.
11.3.1 CARTRIDGE CLEANUP - Attach the extraction cartridges to the vacuum
manifold. Rinse each cartridge with a 5-mL aliquot of ethyl acetate,
allowing the sorbent to soak in the ethyl acetate for about 1 minute by
turning off the vacuum temporarily. Repeat with a 5-mL aliquot of
methylene chloride (MeCl2). Let the cartridge vacuum dry after each
flush.
11.3.2 CARTRIDGE CONDITIONING - This conditioning step is critical for
recovery of analytes and can have a marked effect on method precision
and accuracy. If the cartridge goes dry during the conditioning phase, the
conditioning must be started over. Once the conditioning has begun, the
cartridge must not go dry until the last portion of the sample passes
because analyte and surrogate recoveries may be affected. The analyst
should note premature drying of the solid phase, because the sample may
require re-extraction due to low surrogate recoveries.
11.3.2.1 CONDITIONING WITH METHANOL-Rinse each cartridge
with a 5-mL aliquot of methanol (MeOH)? allowing the sorbent
to soak for about 30 seconds by turning off the vacuum
temporarily. From this point on, do not allow the cartridge to go
dry until after extraction is complete. Drain most of the MeOH
without going below the top of the cartridge packing and rinse
again with a 5-mL aliquot of MeOH.
11.3.2.2 CONDITIONING WITH REAGENT WATER-Drain most of
the MeOH and rinse the cartridge with two consecutive 5-mL
aliquots of reagent water, being careful not to allow the water
level to go below the cartridge packing. Turn off the vacuum.
Fill the cartridge to the top with reagent water and attach a
reservoir or a transfer tube (Sect. 6.10.2).
11.3.3 CARTRIDGE EXTRACTION - Prepare samples, including the QC
samples, as specified in Section 11.2. The sample may be added to the
cartridge using either a large reservoir attached to the cartridge or using a
transfer tube from the sample bottle to the cartridge.
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11.3.3.1 SAMPLE ADDITION USING RESERVOIRS - Attach a
reservoir to the conditioned cartridge from Section 11.3.2. Fill
the reservoir with sample and turn on the vacuum adding
additional aliquots of sample until the entire 1 L sample is
processed. Adjust the vacuum so that the flow rate is about 20
mL/min. Do not let the cartridge packing go dry before all the
sample has been extracted. After all of the sample has passed
through the SPE cartridge, draw air through the cartridge for 10
minutes at full vacuum (minus 10 to 15 inches Hg). If the
cartridge is dried for period much longer than 10 minutes, there
may be a loss of recovery for nitrobenzene and the surrogate 1,3-
dimethyl-2-nitrobenzene. After drying, turn off and release
vacuum.
11.3.3.2 SAMPLE ADDITION USING TRANSFER TUBES -Fit the
PTFE transfer tube adapter securely to the conditioned cartridge.
The screw on the adapter must be finger-tight, otherwise, air can
leak and the cartridge may go dry. Place the weighted end of the
transfer tube inside on the bottom of the sample bottle. Adjust
the flow rate to about 20 mL/min. If the adapter is securely
attached, the water level in the cartridge should drop only as
much as the volume of the transfer tube and no more. Do not let
the SPE sorbent go dry before all the sample has been extracted.
After all the sample has passed through the SPE cartridge, draw
air through the cartridge for 10 minutes at full vacuum (minus 10
to 15 inches Hg). If the cartridge is dried for a much longer
period than 10 minutes, there may be a loss of recovery for
nitrobenzene and the surrogate l,3-dimethyl-2-nitrobenzene.
After drying, turn off and release vacuum.
11.3.4 CARTRIDGE ELUTION - Keep reservoirs or transfer tubes attached.
Lift the manifold top, place collection tubes into the extraction tank, and
insert valve liners into the collection tubes for extract collection. Add 5
mL of ethyl acetate (EtAc) to the empty sample bottle and rotate the bottle
on its side, rinsing the inside of the bottle. If using the cartridge reservoir
method, pour the EtAc from the bottle into the cartridge reservoir and
draw enough of the EtAc through the cartridge to soak the sorbent. If
using the transfer tubes method, pull the EtAc through the PTFE transfer
tubes and draw enough of the EtAc through the tubes into the cartridge to
soak the sorbent. Turn off the vacuum and vent the system and allow the
sorbent to soak in EtAc for approximately 30 seconds. Start a low vacuum
(minus 2-4 in Hg) and pull the ethyl acetate through in a dropwise fashion
into the collection tube. Repeat rinse with 5 mL MeCl2. Take off
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reservoirs and transfer tubes and rinse the cartridge body with 2 to 3 mLs
of 1:1 mixture of MeCl2 and EtAc (1:1 MeCl2/EtAc).
11.3.5 DRYING OF THE EXTRACT - Small amounts of residual water from
the sample container and solid phase may form an immiscible layer with
the solvent in the extract. Set up a drying column (Sect. 6.6) packed with
about 5 grams of anhydrous sodium sulfate. Pre-rinse the sodium sulfate
column with about 2 mL of 1:1 EtAc/MeCl2. Place a clean collection tube
that can hold at least 20 mL beneath the drying column. Add the extract to
the column and follow with two, 3 mL aliquots of 1:1 EtAc/MeCl2.
11.3.6 EXTRACT CONCENTRATION - Concentrate the extract to about 0.7
mL under a gentle stream of nitrogen in a warm water bath (at ~ 40°C).
Do not blow down samples to less than 0.5 mL, because the most volatile
compounds will show diminished recovery. Transfer the extract to a 1 mL
volumetric flask and add the internal standard (method development used
100 uL of 50 ug/mL internal standard solution for an extract concentration
of5ug/mL). Rinse the collection tube that held the dried extract with
small amounts of EtAc and add to the volumetric flask to bring the volume
up to the 1 mL mark. Transfer to autosampler vial.
11.3.7 SAMPLE VOLUME OR WEIGHT DETERMINATION - Use a
graduated cylinder to measure the volume of water required to fill the
original sample bottle to the mark made prior to extraction (Sect. 11.2.1).
Determine volume to the nearest 10 mL for use in the final calculations of
analyte concentration (Sect. 12.2). If using weight to determine volume,
reweigh empty sample bottle. From the weight of the original sample
bottle measured in Section 11,2.1, subtract the empty bottle weight. Use
this value for analyte concentration calculations in Section 12.2.
11.4 DISK SPE PROCEDURE - The disk extraction procedure may be carried out in a
manual mode or by using a robotic or automatic sample preparation device. This
section describes the disk SPE procedure using the equipment outlined in Section
6.10 in its simplest, least expensive mode without the use of a robotics systems.
The manual mode described below was used to collect data presented hi Section
17.
11.4.1 SAMPLE PREPARATION - Prepare the sample as given in Section 11.2.
11.4.2 DISK CLEANUP - Assemble the extraction glassware onto the vacuum
manifold, placing disks on a support screen between the funnel and base.
Add a 5 mL aliquot of 1:1 mixture of ethyl acetate (EtAc) and methylene
chloride (MeCl2) (1:1 MeCl2/EtAc), drawing about half through the disk,
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and allowing the solvent to soak the disk for about a minute. Draw the
remaining solvent through the disk to waste until the disk is dry of solvent.
11.4.3 DISK CONDITIONING - The conditioning step is critical for recovery of
analytes and can have a marked effect on method precision and accuracy.
If the disk goes dry during the conditioning phase, the conditioning must
be started over. Once the conditioning has begun, the disk must not go dry
until the last portion of the sample passes, because analyte and surrogate
recoveries may be affected. The analyst should note premature drying of
the solid phase, because the sample may require re-extraction due to low
surrogate recoveries. During conditioning, it is not unusual for the middle
of the solid phase disk to form a wrinkle. This typically does not
adversely affect extraction.
11.4.3.1 CONDITIONING WITH METHANOL- Add approximately 10
mL methanol to each disk. Pull about 1 mL of MeOH through
the disk and turn off the vacuum temporarily to let the disk soak
for about one minute. Draw most of the remaining MeOH
through the disk, but leave a layer of MeOH on the surface of the
disk. The disk must not be allowed to go dry from this point
until the end of the sample extraction.
11.4.3.2 CONDITIONING WITH WATER - Follow the MeOH rinse
with two, 10 mL aliquots of reagent water, being careful to keep
the water level above the disk surface. Turn off the vacuum.
11.4.4 DISK EXTRACTION - Add sample to the extraction funnel containing
the conditioned disk and turn on the vacuum. Do not let the disk go dry
before all sample has been extracted. Drain as much water from the
sample container as possible. After all of the sample has passed, draw air
through the disk by maintaining full vacuum (minus 10-15 in Hg) for 10
minutes. If the disk is dried for a period much longer than 10 minutes,
there will be a loss of recovery for nitrobenzene and the surrogate 1,3-
dimethyl-2-nitrobenzene. After drying, turn off and release the vacuum.
11.4.5 DISK ELUTION - Detach the glassware base from the manifold without
disassembling the funnel from the base. Dry the underside of the base.
Insert collection tubes into the manifold to catch the extracts as they are
eluted from the disk. The collection tube must fit around the drip tip of
the base to ensure collection of all the eluent. Reattach the base to the
manifold. Add 5 mL of ethyl acetate to the empty sample bottle and rinse
the inside of the bottle. Transfer the ethyl acetate to the disk and, with
vacuum, pull enough ethyl acetate into the disk to soak the sorbent, and
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allow the solvent to soak the disk for about one minute. Pull the
remaining solvent slowly through the disk into the collection tube. Repeat
the rinse with 5 mL MeCl2. Rinse the SPE funnel surface once with a 2-3
mL aliquot of 1:1 EtAc/MeCl2. Repeat this last rinse of the SPE funnel.
Detach glassware from manifold and remove collection tube from the
manifold.
11.4.6 DRYING OF THE EXTRACT - Proceed with drying the extract, Section
11.3.5.
11.4.7 EXTRACT CONCENTRATION - Proceed with extract concentration,
Section 11.3.6.
11.4.8 SAMPLE VOLUME OR WEIGHT DETERMINATION - Proceed with
sample volume or weight determination, Section 11.3.7.
11.5 ANALYSIS OF SAMPLE EXTRACTS
11.5.1 Establish operating conditions as described in Section 10.2.2. Confirm
that retention times, compound separation and resolution are similar to
those summarized in Table 2 and Figure 1.
11.5.2 Establish a valid initial calibration following the procedures outlined in
Section 10.2 or confirm that the calibration is still valid by running a CCC
as described in Section 10.3. If establishing an initial calibration for the
first time, complete the IDC as described in Section 9.2.
11.5.3 Analyze aliquots of field and QC samples at appropriate frequencies (Sect.
9) with the GC/MS system using the conditions used for the initial and
continuing calibrations. At the conclusion of data acquisition, use the
same software that was used in the calibration procedure to tentatively
identify peaks in predetermined retention time windows of interest. Use
the data system software to examine the ion abundances of components of
the chromatogram.
11.5.4 COMPOUND IDENTIFICATION - Identify a sample component by
comparison of its mass spectrum (after background subtraction) to a
reference spectrum in the user-created data base.
11.5.4.1 Establish an appropriate retention time window for each target
analyte, internal standard and surrogate standard to identify them
in QC and Field Samples chromatograms. Ideally, the retention
time window should be based on measurements of actual
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retention time variation for each compound in standard solutions
collected on each GC/MS over the course of time. The suggested
variation is plus or minus three times the standard deviation of
the retention time for each compound for a series of injections.
The injections from the initial calibration and from the Initial
Demonstration of Capability may be used to calculate a
suggested window size. However, the experience of the analyst
should weigh heavily on the determination of an appropriate
retention window size.
11.5.4.2 In general, all ions that are present above 10% relative abundance
in the mass spectrum of the standard should be present in the
mass spectrum of the sample component and should agree within
an absolute 20%. For example, if an ion has a relative abundance
of 30% in the standard spectrum, its abundance in the sample
spectrum should be in the range of 10 to 50%. Some ions,
particularly the molecular ion, are of special importance, and
should be evaluated even if they are below 10% relative
abundance.
11.5.5 EXCEEDING CALIBRATION RANGE - An analyst must not
extrapolate beyond the established calibration range. If an analyte result
exceeds the range of the initial calibration curve, the extract may be
diluted with ethyl acetate, with the appropriate amount of internal standard
added to match the original level, and the diluted extract injected.
Acceptable surrogate performance (Sect. 9.9) should be determined from
the undiluted sample extract. Incorporate the dilution factor into final
concentration calculations. The dilution will also affect analyte MRLs.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Identify method analytes present in the field and QC samples as described in
Section 11.8.4. Complete chromatographic resolution is not necessary for
accurate and precise measurements of analyte concentrations if unique ions with
adequate intensities are available for quantitation.
12.1.1 Identification is hampered when sample components are not resolved
chromatographically and produce mass spectra containing ions contributed
by more than one analyte. When GC peaks obviously represent more than
one sample component (i.e., broadened peak with shoulder(s) or valley
between two or more maxima), appropriate analyte spectra and
background spectra can be selected by examining plots of characteristic
ions. When analytes coelute (i.e., only one GC peak is apparent), the
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identification criteria can be met but each analyte spectrum will contain
extraneous ions contributed by the coeluting compound.
12.1.2 In validating this method, concentrations were calculated by measuring the
characteristic ions listed in Table 2. Other ions may be selected at the
discretion of the analyst.
12.2 Calculate analyte and surrogate concentrations, using the multipoint calibration
established in Section 10.2. Do not use daily continuing calibration check data to
quantitate analytes in samples. Adjust the final analyte concentrations to reflect
the actual sample volume or weight determined in Section 11.3.7 or 11.4.8. Field
Sample extracts that require dilution should be treated as described in Section
11.5.5.
12.3 Calculations should utilize all available digits of precision, but final reported
concentrations should be rounded to an appropriate number of significant figures.
13. METHOD PERFORMANCE
13.1 PRECISION, ACCURACY, AND MDLs - Method performance data are
summarized in Section 17. Method detection limits (MDLs) are presented in
Table 3 and were calculated using the formula in Section 9.2.4. Single laboratory
precision and accuracy data are presented for reagent water (Sect. 17, Tables 4A
and 4B), chlorinated "finished" ground water (Sect. 17, Tables 5A and 5B), and
chlorinated "finished" surface water (Sect. 17, Tables 6A and 6B).
13.2 POTENTIAL PROBLEM COMPOUNDS
13.2.1 MATRIX ENHANCED SENSITIVITY - Cyanazine, and to a lesser
extent 2,4,6-trichlorophenol and prometon, tend to exhibit "matrix-
induced chromatographic response enhancement."^1!) Compounds that
exhibit this phenomenon often give analytical results that exceed 100 %
recovery in fortified extracts at low concentration and in continuing
calibration checks. More frequent recalibration is required. It has been
proposed that these compounds are susceptible to GC inlet absorption or
thermal degradation so that analytes degrade more when injected in a
"cleaner" matrix. The injection of a "dirty" sample extract coats surfaces
with matrix components and "protects" the problem compounds from
decomposition or adsorption. As a result, a relatively greater response is
observed for analytes in sample extracts than in calibration solutions. This
effect is minimized by using deactivated injection liners (Sect. 10.2.2.2).
The analyst may also choose to condition the injection port after
maintenance by injecting a few aliquots of a field sample extract prior to
526-33
-------
establishing an initial calibration. Preparation of calibration standards in
clean extracts is not allowed.
13.2.2 COMPOUND DEGRADATION - Method development work indicated
that several of the target compounds were unstable when stored in water
without preservation. There were various modes of loss. Hydrolysis of
1,2-diphenylhydrazine, terbufos, diazinon, disulfoton and cyanazine was
accelerated at low and high pH. hi addition, transition metal ions further
catalyzed hydrolysis of terbufos, fonofos, and diazinon. Free chlorine and
chloramines degraded 2,4-dichlorophenol, terbufos, fonofos, diazinon, and
disulfoton. When water samples were not properly preserved, after three
days, there was more than 80% loss of some targets, initially fortified at 5
ppb. Sample preservation conditions (Sect. 8) have been carefully chosen
to rninimize analyte degradation to acceptable levels during the 14 day
sample holding tune.
13.2.3 Inlet liners and/or capillary GC columns that develop active sites can cause
a complete loss of prometon and excessive tailing of 2,4-dichlorophenol
and 2,4,6-trichlorophenol peaks in the chromatogram.
13.3 ANALYTE STABILITY STUDIES
13.3.1 FIELD SAMPLES - Chlorinated surface water samples, fortified with
method analytes at 5.0 ug/L, were preserved and stored as required in
Section 8. The average of triplicate analyses, conducted on days 0, 3, 7,
and 14, are presented in Section 17, Table 7. These data document the 14-
day sample holding time. It is advisable to extract as soon as possible
because some compounds exhibit significant losses by 7 days.
13.3.2 EXTRACTS - Extracts from the day 0 extract holding time study
described above were stored below 0 °C and analyzed on days 0, 6,13, 20,
and 32. The data presented in Section 17, Table 8, document the 28-day
extract holding time.
14. POLLUTION PREVENTION
14.1 This method utilizes solid phase extraction technology to remove the analytes
from water. It requires the use of very small volumes of organic solvent and very
small quantities of pure analytes, thereby minimizing the potential hazards to both
the analyst and the environment involved with the use of large volumes of organic
solvents in conventional liquid-liquid extractions.
526-34
-------
14.2 For information about pollution prevention that may be applicable to laboratory
operations, consult "Less Is Better: Laboratory Chemical Management for Waste
Reduction" available form the American Chemical Society's Department of
Government Relations and Science Policy, 1155 16th Street N.W., Washington,
D.C. 20036.
15. WASTE MANAGEMENT
15.1 The analytical procedures described in this method generate relatively small
amounts of waste since only small amounts of reagents and solvents are used.
The matrices of concern are finished drinking water or source water. However,
the Agency requires that laboratory waste management practices be conducted
consistent with all applicable rules and regulations, and that laboratories protect
the air, water, and land by minimizing and controlling all releases from fume
hoods and bench operations. Also, compliance is required with any sewage
discharge permits and regulations, particularly the hazardous waste identification
rules and land disposal restrictions. For further information on waste
management, consult "The Waste Management Manual for Laboratory Personnel"
also available from the American Chemical Society at the address in Section 14.2.
16 REFERENCES
1. Glaser, J.A., Foerst, D.L., McKee, G.D., Quave, S.A., and Budde, W.L., "Trace Analyses
for Wastewaters." Environ. Sci. Technol.. 15 (1981) 1426-1435.
2. "Carcinogens - Working With Carcinogens," Department of Health, Education, and
Welfare, Public Health Service, Center for Disease Control, National Institute for
Occupational Safety and Health, Publication No. 77-206, Aug. 1977.
3. "OSHA Safety and Health Standards, General Industry," (29CFR1910), Occupational
Safety and Health Administration, OSHA 2206, (Revised, January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part E, Volume 11.01, D3370-82, "Standard Practice
for Sampling Water," American Society for Testing and Materials, Philadelphia, PA,
1986.
6. Llabres, C.M., Ahearn, D.G. "Antimicrobial Activities of N-Chloramines and
Diazolidinyl Urea," Applied and Environmental Microbiology, 49, (1985), 370-373.
526-35
-------
7. Erney, D.R., Gillespie, A.M., Gilvydis, D.M., and Poole, C.F., "Explanation of the
Matrix-Induced Chromatographic Response Enhancement of Organophosphorous
Pesticides During Open Tubular Column Gas Chromatography with Splitless or Hot On-
Column Injection and Flame Photometric Detection." J. Chromatogr.. 638 (1993) 57-63.
8. Mol, H.G.J., Althuizen, M., Janssen, H., and Cramers, C.A., Brinkman, U.A.Th.,
"Environmental Applications of Large Volume Injection in Capillary GC Using PTV
Injectors," J. High Resol. Chromatogr., 19 (1996) 69-79.
9. Emey, D.R., Pawlowski, T.M., Poole, C.F., "Matrix Induced Peak Enhancement of
Pesticides in Gas Chromatography," J. High Resol. Chromatogr.. 20 (1997) 375-378.
10. Hajslova, J., Holadova, K., Kocourek, V., Poustka, J., Godula, M., Cuhra, P., Kempny,
M., "Matrix Induced Effects: A Critical Point in the Gas Chromatographic Analysis of
Pesticide Residues," J. Chromatogr.. 800 (1998) 283-295.
11. Wylie, P., Uchiyama, M., "Improved Gas Chromatographic Analysis of
Organophosphorous Pesticides with Pulsed Splitless Injection," J. AOAC International.
79,2, (1996) 571-577.
526-36
-------
17. TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. ION ABUNDANCE CRITERIA FOR
BIS(PERFLUOROPHENYL)PHENYL PHOSPHINE,
(DECAFLUOROTRIPHENYL PHOSPHINE, DFTPP)
Mass
(M/z)
51
68
70
127
197
198
199
275
365
441
442
443
Relative Abundance Criteria
10-80% of the base peak
<2% of Mass 69
<2% of Mass 69
10-80% of the base peak
<2% of Mass 98
Base peak or >50% of Mass 442
5-9% of Mass 198
10-60% of the base peak
>1% of the base peak
Present and 50% of Mass 198
15-24% of Mass 442
Purpose of Checkpoint1
Low-mass sensitivity
Low-mass resolution
Low-mass resolution
Low- to mid-mass resolution
Mid-mass resolution
Mid-mass resolution and sensitivity
Mid-mass resolution and isotope ratio
Mid- to high-mass sensitivity
Baseline threshold
High-mass resolution
High-mass resolution and sensitivity
High-mass resolution and isotope ratio
'All ions are used primarily to check the mass measuring accuracy of the mass spectrometer and
data system, and this is the most important part of the performance test. The three resolution
checks, which include natural abundance isotope ratios, constitute the next most important part
of the performance test. The correct setting of the baseline threshold, as indicated by the
presence of low intensity ions, is the next most important part of the performance test. Finally,
the ion abundance ranges are designed to encourage some standardization to fragmentation
patterns.
526-37
-------
TABLE 2. RETENTION TIMES CRTs), SUGGESTED QUANTITATION IONS (QIs),
AND INTERNAL STANDARD REFERENCE
Peak
#a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Analyte
Nitrobenzene
2,4-Dichlorophenol
2,4,6-Trichlorophenol
1 ,2-Diphenylhydrazine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
Acenaphthene-c?10 (IS#1)
Phenanthrene-c?10 (IS#2)
Chrysene-^12 (IS#3)
l,3-Dimethyl-2-Nitrobenzene (SURR)
Triphenylphosphate (SURR)
Peak Label in
Figure #1
1
2
4
6
7
8
9
11
12
13
14
5
10
16
3
15
RTb
(rain)
6.33
7.70
10.81
15.08
16.64
17.08
17.14
17.29
17.51
18.39
19.73
12.88
17.20
24.98
7.98
24.29
Quanti-
tation
Ion
77
162
196
182
225
231
246
179
88
146
225
164
188
240
151
326
IS#
Ref.
1
1
1
2
2
2
2
2
2
2
2
-
-
1
3
a- Number refers to peak number in Figure 1.
b- Column: 30 m X 0.25 mm i.d. DBS-MS (J&W), 0.25 urn film thickness.
526-38
-------
TABLE 3. METHOD DETECTION LIMITS IN REAGENT WATER FOR SDVB
DISK AND CARTRIDGE EXTRACTION PROCEDURES
Compound
Nitrobenzene
2,4-Dichlorophenol
2,4,6-TrichIorophenol
1,2-DiphenyIhydrazine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
Disk Extraction
Spiking Cone.
(ug/L)
0.05
0.05
0.05
0.20
0.10
0.05
0.05
0.10
0.10
0.05
0.05
MDLa
(ug/L)
0.015
0.012
0.012
0.028
0.035
0.017
0.022
0.015
0.024
0.015
0.025
Cartridge Extraction
Spiking Cone.
(ug/L)
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
MDLa
(«g/L)
0.09
0.04
0.14
0.10
0.14
0.05
0.06
0.03
0.05
0.10
0.09
"Method detection limits samples were extracted and analyzed over 3 days for 7 replicates following the
procedure outlined in Section 9.2.4.
526-39
-------
TABLE 4A. PRECISION, ACCURACY AND SENSITIVITY DATA FOR METHOD
ANALYTES FORTIFIED AT 0.5 AND 20 UG/L IN REAGENT WATER
EXTRACTED WITH SDVB DISKS
Compound
Nitrobenzene
2,4-DichlorophenoI
2,4,6-Trichlorophenol
1,2-Diphenylhydrazine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
l,3-Dimethyl-2-Nitrobenzene
(SUR)C
Triphenylphosphate (SUR)C
Concentration= 0.5 ug/L,
n=7
Mean %
Recovery
106
114
136
121
138
111
104
101
105
124
153
86.2
106
%RSDa
3.8
1.9
2.3
2.5
2.3
2.7
2.0
2.5
2.4
2.9
2.5
2.6
2.6
S/N
Ratio6
159
71
134
25
42
71
138
14
103
67
10
NC
NC
Concentration = 20
ug/L,n=7
Mean%
Recovery
81.5
97.6
104
103
101
91.4
106
98.3
95.0
98.9
104
84.2
105
%RSDa
5.6
4.3
3.4
3.7
3.8
4.0
4.4
4.7
4.8
4.0
4.6
4.6
5.2
"Relative Standard Deviation = (Standard Deviation/Recovery)* 100.
bSignal-to-noise ratios were calculated for each peak by dividing the peak height for each compound by
the peak-to-peak noise, which was determined for each component from the method blank over a period
of time equal to the full peak width in the target analyte's retention time window.
"Surrogate fortification concentration of all samples was 5 ug/L.
526-40
-------
TABLE 4B. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES
FORTIFIED AT 0.5 AND 20 UG/L IN REAGENT WATER EXTRACTED
WITH SDVB CARTRIDGES
- . , - . "
Compound
Nitrobenzene
2,4-Dichlorophenol
2,4,6-Trichlorophenol
1,2-Diphenylhydrazine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
l,3-Dimethyl-2-Nitrobenzene (SUR)b
Triphenylphosphate (SUR)b
Concentration = 0;5 ug/L,
••" n=7
Mean%
Recovery
86.3
104
129
96.9
148
115
101
104
106
125
147
81.7
107
%RSDa
6.9
7.3
3.8
16
3.2
2.8
3.8
3.4
3.0
3.0
5.6
4.1
7.5
Concentration = 20 ug/L,
'-.'.: n=7 . -'
Mean %
Recovery
73.4
83.8
92.9
123
100
85.0
90.1
91.1
85.5
91.3
101
80.2
108
%RSDa
3.7
3.3
3.0
3.2
0.8
1.8
1.9
1.4
1.7
1.2
1.2
3.0
3.0
"Relative Standard Deviation = (Standard Deviation/Recovery)* 100.
bSurrogate fortification concentration of all samples was 5 ug/L.
526-41
-------
TABLE 5A. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES
FORTIFIED AT 0.5, AND 20 UG/L IN GROUND WATER EXTRACTED
WITH SDVB DISKS
Compound
Nitrobenzene
2,4-Dichlorophenol
2,4,6-Trichlorophenol
1,2-Diphenylhydrazine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
l,3-Dimethyl-2-Nitrobenzene
(SUR)b
Triphenylphosphate (SUR)b
Concentration = 0.5 ug/L,
n = 7 ".
Mean%
Recovery
110
113
148
134
152
119
105
110
113
130
163
86.3
105
%RSDa
4.7
3.0
2.0
3.8
2.4
1.6
2.4
1.5
2.5
2.1
2.3
4.8
2.9
Concentration - 20 ttg/L,
7- ... " ,
. .• • ;;<:- ' , .'.'•'.'
Mean%
.Recovery .•';.'"•••'•;
83.5
96.5
103
102
101
93.4
105
97.6
95.1
98.3
104
83.8
106
:%RSDa;
5.3
5.0
4.1
4.0
3,8
3.5
3.7
3.7
4.2
3.7
3.8
5.5
4.0
"Relative Standard Deviation = (Standard Deviation/Recovery)*100.
'Surrogate fortification concentration of all samples was 5 ug/L.
526-42
-------
TABLE SB. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES
FORTIFIED AT 0.5, 5.0 AND 20 UG/L IN GROUND WATER
EXTRACTED WITH SDVB CARTRIDGES
Compound
Nitrobenzene
2,4-DichIorophenol
2,4,6-Trichlorophenol
1,2-DiphenyIhydrazine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
l,3-Dimethyl-2-
Nitrobenzene (SUR)b
Triphenylphosphate
(SUR)b
Concentration =
0.5 ug/L, n = 7
Mean %
Reeiovery
114
110
136
122
144
120
107
105
108
129
160
86.7
119
%RSDa
5.4
3.4
' 4.1
4.3
3.3
3.0
3.0
2.4
3.8
4.5
3.1
6.3
.4.3
Concentration =
5;0 ug/L, n = 7
Mean %
Recovery
87.7
89.8
102
84.8
103
87.1
87.9
88.7
93.3
103
106
86.3
123
%RSD"
3.9
3.9
3.8
3.2
2.9
4.2
3.5
3.1
3.7
3.7
3.6
3.3
3.4
Concentration =
20 ug/L, n = 7
Mean %
Recovery
84.1
89.1
98.3
90.4
100
84.9
90.8
91.6
88.6
94.2
98.6
84.1
111
%RSDa
1.9
2.2
1.4
3.7
2.3
2.4
2.4
2.3
3.1
1.1
3.6
4.6
14
"Relative Standard Deviation = (Standard Deviation/Recovery)* 100.
bSurrogate fortification concentration of all samples was 5 ug/L.
526-43
-------
TABLE 6A. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES
FORTIFIED AT 0.5,5.0 AND 20 UG/L IN SURFACE WATER
EXTRACTED WITH SDVB DISKS
Compound
Nitrobenzene
2,4-Dichlorophenol
2,4,6-Trichlorophenol
1,2-Diphenylhydrazine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
l,3-DimethyI-2-Nitrobenzene
(SUR)b
Triphenylphosphate (SUR)b
Concentration =
0.5 ug/L, n = 7
Mean %
Recovery
105
111
143
118
136
109
98.9
102
105
124
151
82.9
101
%RSDa
3.8
4.0
2.8
2.4
3.6
3.0
3.8
3.6
3.4
3.4
3.2
4.5
4.5
Concentration =
5.0 ug/L, n = 7
Mean %
Recovery
72.4
81.2
90.8
105
102
85.7
83.0
83.7
81.2
89.2
105
77.4
98.2
%RSD"
4.4
4.0
4.2
4.5
3.1
4.6
4.2
3.5
4.2
3.6
3.9
4.4
3.6
Concentration =
20 ug/L, n - 7
Mean%
Recovery
76.7
89.9
92.4
88.3
89.5
80.2
89.1
86.1
86.6
89.4
93.6
83.9
98.8
%RSDa
4.1
4.8
4.0
3.9
3.9
3.4
4.0
4.1
3.7
3.4
3.9
3.9
4.3
•Relative Standard Deviation = (Standard Deviation/Recovery)* 100.
bSurrogate fortification concentration of all samples was 5 ug/L.
526-44
-------
TABLE 6B. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES
FORTIFIED AT 0.5 AND 20 UG/L IN SURFACE WATER EXTRACTED
WITH SDVB CARTRIDGES
Compound
1 i ,11, **
Nitrobenzene
2,4-DichlorophenoI
2,4,6-Trichlorophenol
1,2-Diphenylhydrazine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
l,3-Dimethyl-2-Nitrobenzene (SUR)b
Triphenylphosphate (SUR)b
Concentration = 0.5 ug/L,
n = 7
Mean %
Recovery
91.7
113
135
107
158
128
109
112
109
132
158
83.0
104
%RSDa
6.6
4.9
4.0
9.9
3.3
3.0
2.3
3.7
3.0
2.8
1.7
5.3
1.9
Concentration = 20 ug/L,
n = 7
Mean %
Recovery
84.9
89.7
97.2
93.7
101
89.0
91.6
92.8
90.0
94.6
99.9
85.1
99.1
%RSDa
2.9
2.2
1.8
2.3
1.3
2.1
1.6
1.5
1.6
1.4
1.0
3.8
1.60
"Relative Standard Deviation = (Standard Deviation/Recovery)* 100.
bSurrogate fortification concentration of all samples was 5 ug/L.
526-45
-------
TABLE 7. SAMPLE HOLDING TIME DATA3 FOR SAMPLES FROM A
CHLORINATED SURFACE WATER, FORTIFIED WITH METHOD
ANALYTES AT 5 UG/L, AND PRESERVED ACCORDING TO
SECTION 8.
Compound
Nitrobenzene
2,4-DichIorophenoI
2,4,6-Trichlorophenol
1,2-Diphenylhydrazine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
DayO
%Rec.
83.5 •
97.2
110
98.8
104
96.7
94.1
93.0
90.8
100
126
Day3
%Rec.
82.4
92.6
102
93.9
97.7
79.3
88.0
86.7
84.7
93.3
119
Day?
% Rec.
76.7
86.9
97.3
87.5
93.1
69.1
84.8
83.4
81.5
90.5
112
Day 14
%Rec.
88.1
98.1
108
97.2
102
65.4
93.1
91.3
89.1
97.6
125
"Storage stability is expressed as a percent recovery value. Each percent recovery value represents the
mean of 3 replicate analyses. Relative Standard Deviations ([Standard Deviation/Recovery]* 100) for
replicate analyses were all less than 13.8 %.
526-46
-------
TABLE 8. EXTRACT HOLDING TIME DATA" FOR SAMPLES FROM A
CHLORINATED SURFACE WATER, FORTIFIED WITH METHOD
ANALYTES AT 5 UG/L, AND PRESERVED ACCORDING TO
SECTIONS.
Compound
Nitrobenzene
2,4-Dichlorophenol
2,4,6-Trichlorophenol
1,2-Diphenylhydrazine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
l,3-DimethyI-2-Nitrobenzene (STJR)b
Triphenylphosphate (SUR)b
DayO
%Rec.
83.5
97.2
110
98.8
104
96.7
94.1
93.0
90.8
100
126
79.3
97.9
Day 6
% Rec.
84.7 .
97.5
110
97.9
104
97.5
95.0
93.0
91.9
100
125
78.5
96.6
Day 13
% Rec.
85.2
96.2
108
97.9
103
97.9
94.3
93.6
92.9
99.8
124
78.7
96.7
Day 20
%Rec.
85.5
96.7
107.
97.0
103
98.6 ,
95.3
94.3
94.6
99.8
122 ,
78.5
... 97.8 -:;
Day 32
% Rec.,
80.5
95.9
112
98.3
101
89.5
97.3
93.0
93.5
,98.7
123
77.1
93.4
"Extracts were stored at less than 0 °C and reinjected periodically. Each table value represents the mean
of 3 replicate analyses. Relative Standard Deviations ([Standard Deviation/Recovery]* 100) for replicate
analyses were all less than 5.7 %.
526-47
-------
TABLE 9. INITIAL DEMONSTRATION OF CAPABILITY (IDC) REQUIREMENTS
Method
Reference
Section
9.2.1
Section
9.2.2
Section
9.2.3
Section
9.2.4
Requirement
Initial
Demonstration
of Low Method
Background
Initial
Demonstration
of Precision
(TOP)
Initial
Demonstration
of Accuracy
Method Detection
Limit (MDL)
Determination
Specification and
Frequency
Analyze LPJ3 prior to any
other IDC steps
Analyze 4-7 replicate LFBs
fortified at midrange
concentration.
Calculate average recovery
for replicates used in IDP
Over a period of three days,
prepare a minimum of 7
replicate LFBs fortified at a
concentration estimated to be
near the MDL. Analyze the
replicates.through all steps of
the analysis. Calculate the
MDL using the equation in
Section 9.2.4.
Acceptance Criteria
Demonstrate that all target
analytes are below V3 the
reporting limit or lowest CAL
standard, and that possible
interference from extraction
media do not prevent the
identification and
quantification of method
analytes.
%RSD must be <20%
Mean recovery 70-130% of true
value.
Note: Data from MDL replicates
are not required to meet method
precision and accuracy criteria.
If the MDL replicates are
fortified at a low enough
concentration, it is likely that
they will not meet precision and
accuracy criteria.
526-48
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TABLE 10. QUALITY CONTROL REQUIREMENTS (SUMMARY)
Method
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Section
10.2.1
MS Tune Check
Analyze DFTPP to verify MS
tune each time the instrument is
calibrated.
Criteria are given in Table 1.
Section
10.2
Initial Calibration
Use internal standard calibration
technique to generate an average
RRF or first or second order
calibration curve. Use at least 5
standard concentrations.
When each calibration
standard is calculated as an
unknown using the
calibration curve, the result
must be 70-130% of the true
value for all except the lowest
standard, which must be 50-
150% of the true value.
Section
9.4
Laboratory Reagent
Blank (LRB)
Daily, or with each extraction
batch of up to 20 samples,
whichever is more frequent.
Demonstrate that all target
analytes are below V3 the
method reporting limit or
lowest CAL standard, and
that possible interferences do
not prevent quantification of
method analytes.
If targets exceed V3 the MRL,
results for all subject analytes
in extraction batch are
invalid.
Section
10.3
Continuing
Calibration Check
(CCC)
Verify initial calibration by
analyzing a calibration standard
at the beginning of each analysis
batch prior to analyzing
samples, after every 10 samples,
and after the last sample.
Low CCC-near MRL
Mid CCC - near midpoint in
initial calibration curve
High CCC - near highest
calibration standard
1) The result for each analyte
must be 70-130% of the true
value for all but the lowest
standard. The lowest
standard must be 50-150% of
the true value.
2) The peak area of internal
standards must be 50-150%
of the average peak area
calculated during the initial
calibration.
Results for analytes that do
not meet IS criteria or are not
bracketed by acceptable
CCCs are invalid.
526-49
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Method
Reference
Requirement
Specification and frequency
Acceptance Criteria
Section
9.6
Laboratory Fortified
Blank (LFB)
One LFB is required daily or for
each extraction batch of up to 20
field samples. Rotate the
fortified concentration between
low, medium, and high amounts.
Results of LFB analyses at
medium and high fortification
must be 70-130% of the true
value for each analyte and
surrogate. LFB Results of the
low level LFB must be 50-
160% of the true value.
Section
9.8
Internal Standard
Acenaphthene-J10
phenanthrene-
-------
Method
Reference
Section
9.11
Section
9.12
Section
8.4
Section
8.4
Requirement
Field Duplicates
(FD)
Quality Control
Sample (QCS)
Sample Holding
Time
Extract Holding
Time
Specification and Frequency
Extract and analyze at least one
FD with each extraction batch
(20 samples or less). ALFMD
may be substituted for a FD
when the frequency of detects
for target analytes is low.
Analyzed QCS quarterly.
14 days with appropriate
preservation and storage
28 days with appropriate storage
Acceptance Criteria
Target analyte RPDs for FD
should be ±30% at mid and
high levels of fortification
and ±50% near MRL.
Results must be 70-130% of
the expected value.
Sample results are valid only
if samples are extracted
within sample hold time.
Sample results are valid only
if extracts are analyzed within
extract hold time.
526-51
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^igure 1 : Total ion chromatogram of surface water sample extract with target compounds, internal standards, and surrogate standards fortified at 5 ppm
evel in extract. Peaks with asterisk (*) are interference associated with the use of diazolidinyl urea.
526-52
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METHOD 528 DETERMINATION OF PHENOLS IN DRINKING WATER BY
SOLID PHASE EXTRACTION AND CAPILLARY COLUMN GAS
CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS)
Revision 1.0
April 2000
Jean W. Munch, USEPA, ORD, NERL
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
528-1
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METHOD 528
DETERMINATION OF PHENOLS IN DRINKING WATER
BY SOLID PHASE EXTRACTION AND CAPILLARY COLUMN
GAS CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS)
1. SCOPE AND APPLICATION
1.1 This method provides procedures for the determination of phenols in finished
drinking water. The method may be applicable to untreated source waters and other
types of water samples, but it has not been evaluated for these uses. The method is
applicable to a variety of phenols that are efficiently partitioned from the water
sample onto a modified polystyrene divinylbenzene solid phase sorbent, and
sufficiently volatile and thermally stable for gas chromatography. The method
includes the following compounds:
ANALYTE
phenol
2-chlorophenol
2-methylphenol (o-cresol)
2-nitrophenol
2,4-dimethylphenol
2,4-dichlorophenol
4-chloro-3 -methylphenol
2,4,6-trichlorophenol
2,4-dinitrophenol
4-nitrophenol
2-methyl-4,6-dinitrophenol
pentachlorophenol
CAS NUMBER
108-95-2
95-57-8
95-48-7
88-75-5
105-67-9
120-83-2
59-50-7
88-06-2
51-28-5
93951-79-2
534-52-1
87-86-5
528-2
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1.2 Method detection limit (MDL) is defined as the statistically calculated minimum
concentration that can be measured with 99% confidence that the reported value is
greater than zero (1). The MDL is compound dependent and is particularly depend-
ent on extraction efficiency, sample matrix, and instrument performance. MDLs for
method analytes range from 0.02-0.58 jig/L, and are listed in Table 1. The concen-
tration calibration range demonstrated by this method is 0.1 u.g/L to 15 |j,g/L for
most analytes, and approximately 1.0 jig/L to 15 |ig/L for 2,4-dinitrophenol, 4-
nitrophenol, 2-methyl-4,6-dinitrophenol, and pentachlorophenol.
1.3 This method should be performed only by analysts with experience in solid phase
extractions and GC/MS analyses.
2. SUMMARY OF METHOD
Analytes and surrogates are extracted by passing a 1 L water sample through a solid phase
extraction (SPE) cartridge containing 0.5 g of a modified polystyrene divinyl benzene
copolymer. The organic compounds are eluted from the solid phase with a small quantity
of methylene chloride. The sample components are separated, identified, and measured by
injecting an aliquot of the concentrated extract into a high resolution fused silica capillary
column of a GC/MS system. Compounds eluting from the GC column are identified by
comparing their measured mass spectra and retention times to reference spectra and
retention times in a data base. Reference spectra and retention times for analytes are
obtained by the measurement of calibration standards under the same conditions used for
samples. The concentration of each identified component is measured by relating the MS
response of the quantitation ion(s) produced by that compound to the MS response of the
quantitation ion(s) produced by a compound that is used as an internal standard. Surrogate
analytes, whose concentrations are known in every sample, are measured with the same
internal standard calibration procedure.
3. DEFINITIONS
3.1 ANALYSIS BATCH — A set of samples analyzed on the same instrument during a
24 hour period that begins and ends with the analysis of the appropriate Continuing
Calibration Check (CCC) standards. Additional CCCs may be required depending
on the length of the analysis batch and/or the number of Field Samples
3.2 CALIBRATION STANDARD (CAL) - A solution prepared from the primary
dilution standard solution or stock standard solutions and the internal standards and
surrogate analytes. The CAL solutions are used to calibrate the instrument response
with respect to analyte concentration.
3.3 CONTINUING CALIBRATION CHECK (CCC) - A calibration standard contain-
ing one or more method analytes, which is analyzed periodically to verify the
accuracy of the existing calibration for those analytes.
528-3
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3.4 EXTRACTION BATCH-A set of up to 20 field samples (not including QC
samples) extracted together by the same person(s) during a work day using the same
lot of solid phase extraction devices and solvents, surrogate solution, and fortifying
solutions. Required QC samples for each extraction batch include: Laboratory
Reagent Blank, Laboratory Fortified Blank, Laboratory Fortified Matrix, and either a
Field Duplicate or Laboratory Fortified Matrix Duplicate.
3.5 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.
3.6 INTERNAL STANDARD (IS) -- A pure analyte(s) added to a sample, extract, or
standard solution in known amount(s) and used to measure the relative responses of
other method analytes and surrogates that are components of the same solution. The
internal standard must be an analyte that is not a sample component.
3.7 LABORATORY FORTIFIED BLANK (LFB) -- An aliquot of reagent water or other
blank matrix to which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, including the use of sample
preservatives, and its purpose is to determine whether the methodology is in control,
and whether the laboratory is capable of making accurate and precise measurements.
3.8 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) -- An aliquot of an
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 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFMD) -- A
second aliquot of the Field Sample, or duplicate Field Sample, that is used to prepare
the LFM. The LFMD is fortified, extracted and analyzed identically to the LFM.
The LFMD is used instead of the Laboratory Duplicate to assess method precision
when the occurrence of target analytes are low.
3.10 LABORATORY REAGENT BLANK (LRB) -- An aliquot of reagent water or other
blank matrix that is treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates, and sample
preservatives that are used with other samples. The LRB is used to determine if
528-4
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method analytes or other interferences are present in the laboratory environment, the
reagents, or the apparatus.
3.11 MATERIAL SAFETY DATA SHEET (MSDS) - Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical properties, fire,
and reactivity data including storage, spill, and handling precautions.
3.12 METHOD DETECTION LIMIT (MDL) - The minimum concentration of an
analyte that can be identified, measured and reported with 99% confidence that the
analyte concentration is greater than zero. This is a statistical determination (Section
9.2.4), and accurate quantitation is not expected at this level.(1)
3.13 MINIMUM REPORTING LEVEL (MRL) - The minimum concentration that can
be reported as a quantitated value for a target analyte in a sample following analysis.
This defined concentration can be no lower than the concentration of the lowest
calibration standard for that analyte, and can only be used if acceptable quality
control criteria for the analyte at this concentration are met.
3.14 PEAK TAILING FACTOR (PTF) - A calculated value that indicates the amount of
peak tailing exhibited by a chromatographic peak. The value is calculated by
dividing the peak width of the back half of the peak (at 10% peak height), by the
peak width of the front half of the peak (at 10% peak height). The calculation is
demonstrated in Figure 4.
3.15 PRIMARY DILUTION STANDARD SOLUTION (PDS) - A solution of several
analytes prepared in the laboratory from stock standard solutions and diluted as
needed to prepare calibration solutions and other needed analyte solutions.
3.16 QUALITY CONTROL SAMPLE (QCS) -- A solution of method analytes of known
concentrations that is obtained from a source external to the laboratory and different
from the source of calibration standards. It is used to check laboratory performance
with externally prepared test materials.
3.17 STOCK STANDARD SOLUTION (SSS) - A concentrated solution containing one
or more method analytes prepared in the laboratory using assayed reference materials
or purchased from a reputable commercial source.
3.18 SURROGATE ANALYTE (SUR) -- A pure analyte, which is extremely unlikely to
be found in any sample, and which is added to a sample aliquot in a known amount
before extraction or other processing, and is measured with the same procedures
used to measure other sample components. The purpose of the SUR is to monitor
method performance with each sample.
528-5
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4. INTERFERENCES
4.1 During analysis, major contaminant sources are reagents and SPE devices. Analyses
of laboratory reagent blanks provide information about the presence of
contaminants. Solid phase extraction devices described in this method have two ,
potential sources of contamination, both the solid phase sorbent and the
polypropylene cartridge that it is packed in. Brands and manufacturers lot numbers
of these devices should be monitored and tracked to ensure that contamination will
not preclude analyte identification and quantitation.
4.2 Interfering contamination may occur when a sample containing low concentrations
of compounds is analyzed immediately after a sample containing relatively high
concentrations of compounds. Syringes and splitless injection port liners must be
cleaned carefully or replaced as needed. After analysis of a sample containing high
concentrations of compounds, a laboratory reagent blank should be analyzed to
ensure that accurate values are obtained for the next sample.
4.3 Silicone compounds may be leached from autosampler vial septa by methylene
chloride. This contamination of the extract will be enhanced if particles of the septa
are introduced into standards and sample extracts by the needle used for injection.
These silicone compounds should, in most cases, have no effect on the analysis.
However, the analyst should be aware of this potential problem.
4.4 Airborne phenol may be a source of phenol contamination in samples and sample
extracts. Samples should not be stored or extracted in areas where phenol is used for
other laboratory operations.
4.5 2,3,4,5-Tetrachlorophenol is used as one of the internal standards for the quantitation
of reactive and thermally labile phenols. Tetrachlorophenol isomers may be present
at low levels (less than 4% total tetrachlorophenol) in pentachlorophenol used as a
pesticide and wood preservative. However, occurrence of pentachlorophenol in U.S.
drinking waters is rare, and measured concentrations are typically 1 u-g/L or less. If a
matrix interference with the internal standard is suspected, an alternate internal
standard may be selected.
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 mamtaining awareness of OSHA regulations regarding safe handling of
chemicals used in this method. Each laboratory should maintain a file of applicable
MSDSs. Additional references to laboratory safety are cited (2-4).
528-6
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5.2 Some method analytes and solvents, including 2,4,6-trichlorophenol, pentachloro-
phenol, and methylene chloride have been classified as known or suspected human
or mammalian carcinogens. Pure standard materials and stock standard solutions of
these compounds should be handled with suitable protection to skin, eyes, etc.
6- EQUIPMENT AND SUPPLIES (All specifications are suggested. References to specific
brands or catalog numbers are included for illustration only.)
6.1 GLASSWARE - All glassware must be meticulously cleaned. This may be
accomplished by washing with detergent and water, rinsing with water, distilled
water, or solvents, air-drying, and heating (where appropriate) in a muffle furnace.
Volumetric glassware should never be heated to the temperatures obtained in a
muffle furnace.
6.2 SAMPLE CONTAINERS - 1 L or 1 qt amber glass bottles fitted with
polytetrafluoroethylene (PTFE) lined polypropylene screw caps. Amber bottles are
highly recommended since some of the method analytes are sensitive to light and
may degrade upon exposure. Clear glass bottles may be used if they are wrapped in
foil, or samples are stored in boxes that prevent exposure to light. Although specific
contamination problems from bottle caps were not observed during method
development, phenolic resin bottle caps should be avoided.
6.3 VOLUMETRIC FLASKS - various sizes.
6.4 LABORATORY OR ASPIRATOR VACUUM SYSTEM - Sufficient capacity to
maintain a vacuum of approximately 25 cm (10 in.) of mercury.
6.5 MICRO SYRINGES - various sizes.
6.6 VIALS - Various sizes of amber vials with PTFE lined screw caps for storing
standard solutions and extracts.
6.7 DRYING COLUMN - The drying tube should contain about 5 to 7 grams of
anhydrous sodium sulfate to remove residual water from the extract. Any small tube
may be used, such as a syringe barrel, a glass dropper, etc. as long as no particulate
sodium sulfate passes through the column into the extract.
6,8 ANALYTICAL BALANCE -- Capable of weighing 0.0001 g accurately.
6.9 FUSED SILICA CAPILLARY GAS CHROMATOGRAPHY COLUMN - Any
capillary column that provides adequate resolution, capacity, accuracy, and precision
can be used. Medium polarity, low bleed columns are recommended for use with
this method to provide adequate chromatography and minimize column bleed.
528-7
-------
Deactivated injection port liners are highly recommended. During the course of the
development of this method, two columns were used. Although these are both
polyphenylmethylsilicone columns, the exact phase is slightly different, ^formation
on the exact composition of each phase is available from the manufacturers. Most of
the work was performed with column 1. Any column which provides analyte
separations equivalent to or better than these columns may be used. Example
chromatograms are shown in Figs 1-3. Retention times are presented in Table 2.
6.9.1. Column 1- 30 m x 0.25 mm id fused silica capillary column coated with a
0.25 nm bonded film of polyphenylmethylsilicone, (J&W DB-5ms).
6.9.2 Column 2- 30 m x 0.25 mm id fused silica capillary column coated with a
0.25 (o-m bonded film of polyphenylmethylsilicone, (SGE BPX5).
6.10 GAS CHROMATOGRAPH/MASS SPECTROMETER/DATA SYSTEM
(GC/MS/DS)--
6.10.1 The GC must be capable of temperature programming and should be
equipped for split/splitless injection. The injection system must not allow
the analytes to contact hot stainless steel or other metal surfaces that
promote decomposition. Other injection techniques such as temperature
programmed injections, cold on-column injections and large volume
injections maybe used if QC criteria in Section 9 and 10 are met. If an
alternate injection technique is performed, the analyst will need to select an
instrument configuration which has been specifically designed for that
application. Performance data in Section 17 include data obtained both by
hot, splitless injection and temperature programmed splitless injection.
6.10.2 The GC/MS interface should allow the capillary column or transfer line exit
to be placed within a few mm of the ion source. Other interfaces, for
example the open split interface, are acceptable if the system has adequate
sensitivity.
6.10.3 The mass spectrometer must be capable of electron ionization at a nominal
electron energy of 70 eV to produce positive ions. The spectrometer must
be capable of scanning at a minimum from 45 to 450 amu with a complete
scan cycle time (including scan overhead) of 1.0 sec or less. (Scan cycle
time = total MS data acquisition time in sec divided by number of scans in
the chromatogram). The spectrometer must produce a mass spectrum that
meets all criteria in Table 3 when an injection of approximately 5 ng of
DFTPP is introduced into the GC. A single spectrum at the apex of the
chromatographic peak, or an average of the three spectra at the apex of the
peak, or an average spectrum across the entire GC peak may be used to
528-8
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„••' , evaluate the performance of the system. Background subtraction is
,,:. . permitted. The scan time must be set so that all analytes have a minimum
of 5 scans across the chromatographic peak. Seven to ten scans across
;..'-•• chromatographic peaks are recommended.
6.10.4 An interfaced data system is required to acquire, store, reduce, and output
,.,-,_ mass spectral data. The computer software should have the capability of
processing stored GC/MS data by recognizing a GC peak within any given
. retention time window. The software must also allow integration of the ion
abundance of any specific ion between specified time or scan number
limits, calculation of response factors as defined in Sect. 10.2.5 or
construction of a linear regression calibration curve, and calculation of
analyte concentrations.
6.11 VACUUM MANIFOLD - A vacuum manifold (Supelco # 57030 and #57275) is
required for processing samples through the extraction/elution procedure. An
automatic or robotic sample preparation system designed for use with solid phase
extraction cartridges may be utilized in this method if all quality control
requirements discussed in Sect. 9 are met. Automated systems may use either
vacuum or positive pressure to process samples and solvents through the cartridge.
All extraction and elution steps must be the same as in the manual procedure.
Extraction and/or elution steps may not be changed or omitted to accommodate the
use of an automated system.
7. REAGENTS AND STANDARDS
7.1 HELIUM - carrier gas, purity as recommended by the GC/MS manufacturer.
7.2 SOLID PHASE EXTRACTION CARTRIDGES - Varian Bond Elut PPL or
equivalent. Cartridges are inert non-leaching plastic, for example polypropylene, or
glass, and must not contain plasticizers that leach into the methylene chloride eluant
and prevent the identification and quantitation of method analytes. The
polypropylene cartridges (6 mL volume) are packed with 0.5 g highly cross-linked,
,, and chemically modified styrene divinyl benzene copolymer. The packing must
have a narrow size distribution and must not leach interfering organic compounds
_ , into the eluting solvent.
7.3 SOLVENTS ~
, , 7.3.1 Methylene chloride, acetone, and methanol. High purity pesticide quality or
equivalent.
528-9
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7.3.2 Reagent water. Water in which an interference is not observed at >l/3 the
MRL of any of the compounds of interest. Prepare reagent water by passing
tap water through a filter bed containing about 0.5 kg of activated carbon or
by using a water purification system. Store in clean, narrow-mouth bottles
with PTFE lined septa and screw caps.
7.4 HYDROCHLORIC ACID - 6 N and 0.05 N.
7.5 SODIUM SULFATE, ANHYDROUS - (Soxhlet extracted with methylene chloride
for a minimum of 4 h or heated to 400°C for 2 h in a muffle furnace.)
7.6 STOCK STANDARD SOLUTIONS -- Individual solutions of surrogates, internal
standards, and analytes, or mixtures of analytes, may be purchased from commercial
suppliers or prepared from pure materials. To prepare stocks from neat materials,
add 10 mg (weighed on an analytical balance to within 0.1 mg) of the pure material
to 1.9 mL of methanol, methylene chloride, or acetone in a 2 mL volumetric flask,
dilute to the mark, and transfer the solution to an amber glass vial. The solvent to be
used is dependent upon the final use of the standard, hi general, calibration
standards and internal standards are prepared in methylene chloride, sample
fortification solutions are prepared in methanol or acetone. Follow any specific
instructions for each standard or standard mixture. If compound purity is confirmed
by the supplier at >96%, the weighed amount can be used without correction to
calculate the concentration of the solution (5 iig/jiL). Store the amber vials at 0°C or
less.
7.7 PRIMARY DILUTION STANDARD SOLUTION - The stock standard solutions
are used to prepare a primary dilution standard solution that contains multiple
method analytes in methylene chloride. Aliquots of each of the stock standard
solutions are combined to produce the primary dilution in which the concentration of
the analytes is at least equal to the concentration of the most concentrated calibration
solution, that is, 15 ng/uL. Store the primary dilution standard solution in an amber
vial at 0°C or less, and check regularly for signs of degradation or evaporation,
especially just before preparing calibration solutions. Mixtures of method analytes
to be used as primary dilution standards may also be purchased from commercial
suppliers.
7.8 CALIBRATION SOLUTIONS (CAL1 through CAL7) -- Prepare a series of seven
calibration solutions in methylene chloride which contain analytes of interest at
suggested concentrations of 15,10, 5, 2, 1, 0.5, and 0.1 ng/uL, with a constant
concentration of each internal standard in each CAL solution (2-5 ng/pL is
recommended). Surrogate analytes are also added to each CAL solution, and maybe
added at a constant concentration or varied concentrations (similar to those for
method analytes), at the discretion of the analyst. CAL1 through CAL7 are prepared
528-10
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by combining appropriate aliquots of a primary dilution standard solution (Sect. 7.7)
and the fortification solution of internal standards and surrogates (Sect. 7.10). All
calibration solutions should contain at least 80% methylene chloride to avoid gas
chromatographic problems due to mixed solvents. Store these solutions in amber
vials at 0°C or less. Check these solutions regularly for signs of evaporation and/or
degradation.
NOTE: Because the MS sensitivity to analytes 9-12 (Table 2) is significantly less
than compounds 1-8, it may be more convenient to prepare calibration solutions in
which the concentrations of analytes 9-12 (Table 2) are higher than the
concentrations of analytes 1-8. Use of this option is at the discretion of the analyst.
Calibration requirements are specified in Sect. 10.
7.9 INTERNAL STANDARD SOLUTION(S) - This method uses two internal
standards: l,2-dimethyl-3-nitrobenzene (IS#1) and 2,3,4,5-tetrachlorophenol (IS#2).
The first internal standard, l,2-dimethyl-3 -nitrobenzene is used to monitor
instrument sensitivity and is used to quantify analytes 1-8 in Table 2. The second
internal standard, 2,3,4,5-tetrachlorophenol is used to quantify analytes 9-12 (Table
2). IS#2 was selected for its chemical similarity to these compounds which are
susceptible to adsorption and/or thermal decomposition in the GC inlet. A full
explanation of the use of 2,3,4,5-tetrachlorophenol to quantify these compounds is
given in Section 13. If cold, on-column or temperature programmed injection
techniques are used, acceptable performance may be obtained using only one
internal standard (IS#1).
7.9.1 1 ,2-Dimethyl-3-nitrobenzene (Aldrich) - 1 00 ng/mL in methylene chloride.
Use 25 uL of this solution per 1 mL of sample extract for a final
concentration of 2.5 |j,g/mL.
7.9.2 2,3,4,5-Tetrachlorophenol (Chem Service Inc.) - 200 |xg/mL in methylene
chloride. Use 25 uL of this solution per 1 mL of sample extract for a final
concentration of 5
7.9.3 The internal standard solutions listed above can be made individually or
together in one solution.
7.10 SAMPLE FORTIFICATION SOLUTIONS --
7.10.1 Surrogate fortification solutions -
7.10.1.1. 2-Chlorophenol-3,4,5,6-d4 (Chem Service Inc.) - 1 00 ng/mL in
methanol. Use 20 uL of this solution per 1 L of water sample
for a final concentration of 2 \ig/L.
528-11
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7.10.1.2 2,4-Dimethylphenol-3,5,6-d3 (CDN Isotopes) - 100 ng/mL in
acetone. Use 20 (iL of this solution per 1 L of water sample for
a final concentration of 2 |ig/L.
7.10.1.3 2,4,6-Tribromophenol - 200 u.g/mL in methanol. Use 25 ui, of
this solution per 1 L water sample for a final concentration of 5
[ig/L.
7.10.2 Analyte fortification solution(s). This solution contains all method analytes
of interest in methanol. These solutions are used to fortify LFBs and LFMs
with method analytes. It is recommended that more than one concentration
of this solution be prepared. During the method development, two solutions
were used. One containing 100 u-g/mL of each analyte, was used for higher
concentration fortifications, and the other containinglO jig/mL of each
analyte in methanol was used for lower level fortifications.
NOTE: Because the MS sensitivity to analytes 9-12 (Table 2) is
significantly less than analytes 1-8, it maybe more convenient to prepare
analyte fortification solutions in which the concentrations of analytes 9-12
are higher than the concentrations of analytes 1-8. Use of this option is at
the discretion of the analyst.
7 11 GC/MS TUNE CHECK SOLUTION - Decafluorotriphenylphosphine (DFTPP), 5
u-g/mL in methylene chloride. Store this solution in an amber vial at 0°C or less.
7.12 SODIUM SULFITE, ANHYDROUS - Reducing agent used to reduce residual
chlorine at the time of sample collection.
8. SAMPLE COT.T.ECTION. PKESERVATTON AND STORAGE
8 1 SAMPLE COLLECTION ~ When sampling from a water tap, open the tap and
allow the system to flush until the water temperature has stabilized (usually about 2
min) Adjust the flow to about 500 mL/min and collect samples from the flowing
stream The sample should nearly fill the 1 L or 1 qt bottle, but does not need to be
headspace free. Keep samples sealed from collection time until analysis. When
sampling from an open body of water, fill the sample container with water from a
representative area. Sampling equipment, including automatic samplers, must be
free of plastic tubing, gaskets, and other parts that may leach interfering analytes into
the water sample.
8 2 SAMPLE DECHLORINATION AND PRESERVATION - All samples must be
dechlorinated and acidified at the time of collection. Residual chlorine is reduced
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by addition of 40-50 mg of sodium sulfite. It may be added as a solid to the sample
bottles before the bottles are transported to the field. It is very important that the
sample be dechlorinated prior to acidification. Wait until sodium sulfite is dissolved
before acidification. Adding sodium sulfite and HC1 (together) to the sample bottles
prior to shipping bottles to the sampling site is not permitted. After dechlorination,
samples are acidified to less than pH 2 with 6 N hydrochloric acid. The acid serves
as a chemical and biological preservative. This pH is the same that is used in the
sample extraction, and is required to support the recovery of several method
analytes.
8.3 SAMPLE TRANSPORT AND STORAGE - All samples should be iced during
shipment and must not exceed 10° C during the first 48 hours. Samples should be
confirmed to be at or below 10° C when they are received at the laboratory. Samples
stored in the lab must be held at or below 6° C until extraction, but should not be
frozen.
8.4 HOLDING TIME - Results of holding time studies of all method analytes showed
that all compounds are stable for 14 days in water samples when the samples are
dechlorinated, preserved, and stored as described in Sect. 8.2 and 8.3. Therefore,
samples must be extracted within 14 days of collection. Sample extracts may be
stored at 0°C or less for up to 30 days after sample extraction. Data from holding
time studies are shown in Tables 7 and 8.
9. QUALITY CONTROL
9.1 Quality control (QC) requirements include: the initial demonstration of laboratory
capability (summarized in Table 9) followed by regular analyses of continuing
calibration checks, laboratory performance check standards, laboratory reagent
blanks, laboratory fortified blanks, and laboratory fortified matrix samples. An
MDL must be determined for each analyte of interest. These criteria are considered
the minimum acceptable QC criteria, and laboratories are encouraged to institute
additional QC practices to meet their specific needs. The laboratory must maintain
records to document the quality of the data generated. A complete summary of QC
requirements is summarized in Table 10.
9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) - Requirements for the
initial demonstration of laboratory capability are described in the following sections
and summarized in Table 9.
9.2.1 INITIAL DEMONSTRATION OF LOW CARTRIDGE EXTRACTION
BACKGROUND AND SYSTEM BACKGROUND - Before any samples
are analyzed, or any time a new supply of solid phase extraction cartridges
is received from a supplier, it must be demonstrated that a laboratory
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reagent blank (LRB) is reasonably free of any contamination that would
prevent the determination of any analyte of concern.
9.2.1.1 A source of potential contamination is the solid phase extraction
cartridge which may contain phthalate esters, silicon
compounds, and other contaminants that could interfere with the
determination of method analytes. Although extraction
cartridges are generally made of inert materials, they may still
contain extractable organic material. If the background
contamination is sufficient to prevent accurate and precise
measurements, the condition must be corrected before
proceeding with the initial demonstration.
9.2.1.2 Other sources of background contamination are solvents,
reagents, and glassware. Background contamination must be
reduced to an acceptable level before proceeding with the next
section. Background from method analytes and interferences
should be < 1/3 the MRL.
9.2.2 INITIAL DEMONSTRATION OF PRECISION (IDP) - Prepare 4-7
replicate LFBs fortified at 5-10 \igl~L, or other mid-range concentration.
Sample preservatives described in Sect. 8.2 must be added to these samples.
Extract and analyze these replicates according to the procedure described in
Section 11. The relative standard deviation (RSD) of the results of the
replicate analyses must be less than or equal to 20% for all method analytes
with the exception of phenol. The RSD for replicate analyses for phenol
must be less than or equal to 30%.
9.2.3 INITIAL DEMONSTRATION OF ACCURACY (IDA) -- Using the same
set of replicate data generated for Section 9.2.2, calculate average recovery.
The average recovery of the replicate values must be within 70-130% of the
true value, except for phenol. Phenol will typically be recovered less
effectively than other method analytes. Because of its higher water
solubility some breakthrough from the extraction cartridge does occur. The
recovery limits for phenol are 50-150%.
9.2.4 MDL DETERMINATION ~ Replicate analyses for this procedure should
be done over at least 3 days (both the sample extraction and the GC
analyses should be done over at least 3 days). Prepare at least 7 replicate
LFBs at a concentration estimated to be near the MDL. This concentration
may be estimated by selecting a concentration at 2-5 times the noise level.
Concentrations shown in the example data in Table 1 may be used as a
guide, however the appropriate concentration will be dependent upon the
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injection technique and the sensitivity of the GC/MS system used. Sample
preservatives described in Sect. 8.2 must be added to these samples.
Analyze the seven replicates through all steps of Section 11. Calculate the
MDL using the following equation:
MDL = St^ n _ 1( j . alpha = 0
where:
*( n - 1,1 - alpha = 0.99) = Student's t value for the 99% confidence level with
n-1 degrees of freedom
n = number of replicates
S = standard deviation of replicate analyses.
NOTE: Do not subtract blank values when performing MDL
calculations.
9.2.5 The analyst is permitted to modify GC columns, GC conditions, extract
evaporation techniques, internal standards or surrogate compounds. Each
time such method modifications are made, the analyst must repeat the
procedures in Sect. 9.2.1 through 9.2.4.
9.3 MINIMUM REPORTING LEVEL (MRL) -- The MRL is the threshold
concentration of an analyte that a laboratory can expect to accurately quantitate in an
unknown sample. The MRL should be established at an analyte concentration either
greater than three times the MDL or at a concentration which would yield a response
greater than a signal-to-noise ratio of five. Although the lowest calibration
standard for an analyte may be below the MRL, the MRL for an analyte must
never be established at a concentration lower than the lowest calibration
standard for that analyte.
9.4 LABORATORY REAGENT BLANKS (LRB) - With each extraction batch,
analyze a laboratory reagent blank to determine the background system
contamination. If, within the retention time window of any analyte, the LRB
produces a peak that would prevent the determination of that analyte, determine the
source of contamination and eliminate the interference before processing samples.
Background contamination must be reduced to an acceptable level before
proceeding. Background from method analytes or contaminants that interfere with
the measurement of method analyses should be ^ 1/3 the MRL. Any time a new
batch of SPE cartridges is received, or new supplies of other reagents are used,
repeat the demonstration of low background described in Sect. 9.2.1.
9.5 CONTINUING CALIBRATION CHECK (CCC) - This calibration check is
required at the beginning of each day that samples are analyzed, after every ten field
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samples, and at the end of any group of sample analyses. See Sect. 10.3 for
concentration requirements and acceptance criteria.
9.6 MS TUNE CHECK -- This performance check consists of verifying the MS tune
using the mass spectrum of DFTPP. A complete description of the check is in Sect.
10.2.1. This check must be performed each time a major change is made to the mass
spectrometer, and each time analyte calibration is performed (i.e. average RFs are
calculated, or first or second order calibration curves are developed).
9.7 PEAK TAILING FACTOR (PTF) -- This check consists of calculating the PTF as
described in Sect. 10.2.3.1. and in Figure 4. This check must be performed once
every 24 hr of instrument operation.
9.8 LABORATORY FORTIFIED BLANK (LFB) - With each extraction batch, extract
and analyze an LFB containing each analyte of concern. If more than 20 field
samples are included in a batch, analyze a LFB for every 20 samples. The fortified
concentration of the LFB should be rotated between low, medium, and high
concentrations from day to day. The low concentration LFB must be as near as
practical to the MRL. Results of LFB analyses corresponding to the lowest CAL
point for an analyte must be 50-150% of the true value for all analytes. Results of
LFB analysis from medium and high level concentrations must be 70-130% of the
true value for all analytes except phenol. The acceptance limit for phenol is 50-
150% of the true value.
9.9 INTERNAL STANDARD (IS) -The analyst must monitor the peak area of the 1,2-
dimethyl-3-nitrobenzene (IS#1) in all injections during each analysis day. The IS#1
response (peak area) in any chromatographic run should not deviate from the
response in the most recent CCC by more than 30%, and must not deviate by more
than 50% from the area measured during initial analyte calibration. If the IS#1 area
in a chromatographic run does not meet these criteria inject a second aliquot of that
extract.
NOTE: The peak area of 2,3,4,5-tetrachlorophenol may not be consistent. It may
vary depending upon the composition of the extract or standard being analyzed. See
Section 13.2.1 for a detailed explanation.
9.9.1 If the reinjected aliquot produces an acceptable internal standard response,
report results for that aliquot.
9.9.2 If a deviation of greater than 30% is obtained for the reinjected extract,
when compared to the most recent CCC, the analyst should check the
calibration by reanalyzing the most recently acceptable calibration standard.
If the calibration standard fails the criteria of Section 10.3.3, recalibration is
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in order per Section 10. If the calibration standard is acceptable, extraction
of the sample may need to be repeated provided the sample is still within
the holding time. Otherwise, report results obtained from the reinjected
extract, but annotate as suspect.
9.10 SURROGATE RECOVERY - The surrogate standards are fortified into all
calibration standards, samples, LFBs, LFMs, FDs, FRBs and LRBs. The surrogate is
a means of assessing method performance from extraction to final chromatographic
measurement.
9.10.1 Surrogate recovery criteria are 70-130% of the fortified amount for 2-
chlorophenol-3,4,5,6-d4 and 2,4-dimethylphenol-3,5,6-d3. The criteria for
2,4,6-tribromophenol is 60-130% of the fortified amount. When surrogate
recovery from a sample, blank, or CCC does not meet these criteria, check
(1) calculations to locate possible errors, (2) standard solutions for
degradation, (3) contamination, and (4) instrument performance. Correct
any problems that are identified. If these steps do not reveal the cause of
the problem, reanalyze the extract.
9.10.2 If the extract reanalysis meets the surrogate recovery criterion, report only
data for the reanalyzed extract.
9.10.3 If the extract reanalysis fails the recovery criterion, the analyst should check
the calibration by reanalyzing the most recently acceptable calibration
standard. If the calibration standard fails the criteria of Section 10.3.3,
recalibration is in order per Section 10. If the calibration standard is '
acceptable, it may be necessary to extract another aliquot of sample if
sample holding time has not been exceeded. If the sample reextract also
fails the recovery criterion, report all data for that sample as suspect.
9.11 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) - Determine that the
sample matrix does not contain materials that adversely affect method performance.
This is accomplished by analyzing replicates of laboratory fortified matrix samples
and ascertaining that the precision, accuracy, and method detection limits of analytes
are in the same range as obtained with laboratory fortified blanks. If a variety of
different sample matrices are analyzed regularly, for example, drinking water from
groundwater and surface water sources, matrix independence should be established
for each. Over time, LFM data should be documented for all routine sample sources
for the laboratory. A laboratory fortified sample matrix should be extracted and
analyzed for each extraction batch. If more than 20 samples are processed in a
batch, extract and analyze a LFM for every 20 samples. If the recovery data for an
LFM does not meet the recovery criteria in Sect. 9.8, and LFBs show the laboratory
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to be in control , then the samples from that matrix (sample location) are
documented as suspect due to matrix effects.
9.11.1 Within each extraction batch, a minimum of one field sample is fortified as
a LFM for every 20 samples analyzed. The LFM is prepared by spiking a
sample with an appropriate amount of the fortification solution. The
concentrations 5, 10, and 15 [ig/L are suggested spiking concentrations.
Select the spiking concentration that is closest to, and at least twice the
matrix background concentration. Use historical data or rotate through the
designated concentrations to select a fortifying concentration. Selecting a
duplicate bottle of a sample that has already been analyzed, aids in the
selection of appropriate spiking levels.
9. 1 1 .2 Calculate the percent recovery (R) for each analyte, after correcting the
measured fortified sample concentration, A, for the background
concentration, B, measured in the unfortified sample, i.e.,
R = Z „ 100
where C is the fortified concentration. Compare these values to
control limits for LFBs (Sect. 9.8).
9.11.3 Recoveries may exhibit a matrix dependence. For samples fortified at or
above their native concentration, recoveries should range between 70 and
130%, for all method analytes except phenol which should be recovered at
50-150%. If the accuracy of any analyte falls outside the designated range,
and the laboratory performance for that analyte is shown to be in control,
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.
NOTE: Matrix effects are expected to be more likely with compounds 9-12
(Table 2) than other method analytes.
9.12 FIELD DUPLICATES (FD) - Within each extraction batch, a minimum of one field
sample should be analyzed in duplicate. Duplicate sample analyses serve as a check
on sampling and laboratory precision. If analytes are not routinely observed in field
samples, duplicate LFMDs should be analyzed to substitute for this requirement.
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9. 12. 1 Calculate the relative percent difference (RPD) for duplicate measurements
(FD1 and FD2) as shown below.
RPD, FD1-FD2
FD2)I2
9. 12.2 Relative percent differences for laboratory duplicates and LFMDs should
fall in the range of ± 30 %.
NOTE: Greater variability may be observed for target analytes with
concentrations at the low end of the calibration range.
9.13 QUALITY CONTROL SAMPLE (QCS) - Each time that new standards are
prepared, analyze a QCS from an external source. If standards are prepared
infrequently, analyze a QCS at least quarterly. The QCS may be injected as a
calibration standard, or fortified into reagent water and analyzed as an LFB. If the
QCS is analyzed as a calibration check standard, then the acceptance criteria are the
same as for the CCC (Sect. 10.3.3). If the QCS is analyzed as a LFB, then the
acceptance criteria are the same as for an LFB (Sect. 9.8). If measured analyte
concentrations are not of acceptable accuracy, check the entire analytical procedure
to locate and correct the problem.
10. CALIBRATION AND STANDARDIZATION
10.1 Demonstration and documentation of acceptable mass spectrometer tune and initial
calibration is required before any samples are analyzed. After initial calibration is
successful, a continuing calibration check is required at the beginning and end of
each period in which analyses are performed, and after every tenth sample.
Verification of mass spectrometer tune must be repeated each time a major
instrument modification or maintenance is performed, and prior to analyte
calibration. A peak tailing factor check is required every day that samples are
analyzed, hi periods of continuous operation, the peak tailing factor must be
performed every 24 hr.
10.2 Initial calibration
10.2.1 MS TUNE - Calibrate the mass and abundance scales of the MS with
calibration compounds and procedures prescribed by the manufacturer with
any modifications necessary to meet tuning requirements. Inject 5 ng or less
of DFTPP solution into the GC/MS system. Acquire a mass spectrum that
includes data for m/z 45-450. If the DFTPP mass spectrum does not meet
all criteria in Table 3, the MS must be retuned and adjusted to meet all
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criteria before proceeding with calibration. A single spectrum at the apex of
the chromatographic peak, or an average of the three spectra at the apex of
the peak, or an average spectrum across the entire GC peak may be used to
evaluate the performance of the system. Background subtraction is
permitted. The tune check may be performed as a separate analysis, or for
routine MS tune verification, DFTPP may be added to one or more of the
CAL standards used for calibration verification, so that the tune check and
calibration verification can be performed in a single analysis. DFTPP elutes
shortly after pentachlorophenol on both of the columns cited in Sect.6.9.
10.2.2 ANALYTE CALIBRATION - Inj ect an aliquot of a medium concentration
calibration solution. For example, 2-10 jig/mL, and acquire and store data
from m/z 45-350 with a total cycle time (including scan overhead time) of
1.0 sec or less. Cycle time must be adjusted to measure at least five or more
scans during the elution of each GC peak. Seven to ten scans across each
GC peak are recommended.
Chromatographic conditions used during method development are outlined
below. These conditions were found to work well on the instrumentation
used. Since some of the method analytes are vulnerable to active sites and
thermal decomposition, optimum chromatographic conditions may vary
with individual instrument design. Although the following conditions are
recommended, GC conditions maybe modified, if all performance criteria
in Sections 9 and 10 are met.
10.2.2.1 The following parameters are suggested GC conditions for hot,
splitless injection: injector temperature 200° C, carrier gas head
pressure 12-15 psi. Inject at an oven temperature of 35° C and
hold in splitless mode for 0.2 min. After 6 min, temperature
program the GC oven at 8° C per min to 250° C. Start data
acquisition at approximately 10 min. Example chromatograms
are shown in Figures 1 and 2.
10.2.2.2 The following parameters are suggested injection conditions for
temperature programmed splitless injection. Inject with the
injector temperature at 25° C, program the injector at 200° C per
min to 200° C. Hold in the splitless mode for 1.0 min. Use the
same column temperature program as listed in Sect. 10.2.2.1.
An example chromatogram is shown in Figure 3.
10.2.3 Performance criteria for the calibration standards. Examine the stored
GC/MS data with the data system software.
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.10.2.3.1 PEAK TAILING FACTOR (PTF) - Peak tailing can be a
problem associated with phenols. The phenols most likely to tail
are those with low acidity constants: 2,4-dinitrophenol, 4-nitro-
phenol, pentachlorophenol and 2-methyl-4,6-dinitrophenol.
These compounds must exhibit a peak tailing factor of 5 or less
at a concentration equivalent to 5-10 |ig/L in a water sample (5-
10 (xg/mL in an extract or calibration standard). For example
peak tailing factor calculations, see Fig.4. Peak tailing factors
must be evaluated for the four analytes listed above each day
that samples are analyzed. In periods of continuous instrument
operation, verify acceptable PTFs every 24 hr. Peak tailing
factors may be evaluated in either a CAL standard, LFB or LFM.
10.2.3.2 The.GC/MS/DS peak identification software should be able to
recognize a GC peak in the appropriate retention time window
for each of the compounds in the calibration solution, and make
correct identifications (Sect. 11.5).
10.2.4 If all performance criteria are met, inject an aliquot of an appropriate
volume (usually 1-2 \iL unless a large volume injector is used) of each of
the other CAL solutions using the same GC/MS conditions.
10.2.4.1 Some GC/MS systems may not be sensitive enough to detect
some of the analytes in the two lowest concentration CAL
solutions (0.1 and 0.5 |J.g/mL). If this is the case, it is acceptable
to calibrate using the remaining (higher concentration) points, as
long as a minimum of 5 calibration points are used to generate
the calibration curve or average response factor (RF) for each
analyte. In addition, some GC/MS systems might reach signal
saturation at the highest calibration concentration. If this is the
case, it is acceptable to drop the highest point and calibrate on
the remaining points, as long as at least 5 calibration
concentrations are used to generate the calibration curve or
average RF for each analyte. Points in the middle of the
calibration range may not be dropped. Data outside of the
established calibration range should never be reported.
10.2.5 Concentrations may be calculated through the use of average response
factor (RF) or through the use of a calibration curve. Average RF
calibrations may only be used if the RF values over the calibration range are
relatively constant (<30% RSD).
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Average RF is determined by calculating the mean RF of each calibration
point, with a minimum of five calibration concentrations.
"
where:
AX = integrated abundance (peak area) of the quantisation ion
of the analyte.
AJS = integrated abundance (peak area) of the quantitation ion
internal standard.
Qx = quantity of analyte injected in ng or concentration units.
Qis = quantity of internal standard inj ected in ng or
concentration units.
10.2.6 As an alternative to calculating average RFs and applying the RSD test, use
the GC/MS data system software to generate a linear regression or quadratic
calibration curve. The analyst may choose whether or not to force zero, to
obtain a curve that best fits the data. Examples of common GC/MS system
calibration curve options are: 1) A,, /Aj? vs Qx /Qis and 2) RF vs A^ /AjS.
10.2.7 Acceptance criteria for the calibration of each analyte is determined by
calculating the concentration of each analyte and surrogate in each of the
analyses used to generate the calibration curve or average RF. Each
calibration point, except the lowest point, for each analyte must calculate to
be 70-130 % of its true value. The lowest point must calculate to be 50-
150% of its true value. If this criteria cannot be met, reanalyze the
calibration standards, or select an alternate method of calibration. The data
presented in this method were obtained using linear regression (RF vs
AX /AjS). Quadratic fit calibrations should be used with caution, because the
non-linear area of the curve may not be reproducible.
10.3 CONTINUING CALIBRATION CHECK (CCC) ~ The minimum daily calibration
verification is as follows. Verify the initial calibration at the beginning and end of
each group of analyses, and after every tenth sample during analyses. (In this
context, a "sample" is considered to be a field sample. LRBs, LFMs, LFBs and
CCCs are not counted as samples.) The beginning CCC each day should be at or
near the MRL in order to verify instrument sensitivity prior to any analyses. If
standards have been prepared such that all low CAL points are not in the same CAL
solution, it may be necessary to analyze two CAL solutions to meet this requirement.
Subsequent CCCs can alternate between a medium and high concentration standard.
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10.3.1 Inject an aliquot of the appropriate concentration calibration solution and
analyze with the same conditions used during the initial calibration.
10.3.2 Determine that the absolute areas of the quantitation ions of the internal
standard l,3-dimethyl-2-nitrobenzene has not changed by more than 30%
from the areas measured in the most recent continuing calibration check, or
by more than 50% from the areas measured during initial calibration. If this
area has changed by more than these amounts, adjustments must be made to
restore system sensitivity. These adjustments may include cleaning of the
MS ion source, or other maintenance as indicated in Sect. 10.3.4. Major
instrument maintenance requires recalibration. Control charts are useful
aids in documenting system sensitivity changes.
10.3.3 Calculate the concentration of each analyte and surrogate in the check
standard. The calculated amount for each analyte for medium and high level
CCCs must be within 70-130% of the true value. The calculated amount
for the lowest calibration point for each analyte must be within 50-150% of
the true value. If these conditions do not exist, remedial action should be
taken which may require recalibration. Any field sample extracts that have
been analyzed since the last acceptable calibration verification should be
reanalyzed after adequate calibration has been restored, with the following
exception. If the continuing calibration check in the middle or at the
end of an analysis batch fails because the calculated concentration is
>130% of the true value, and field sample extracts showed no detection
of method analytes, non-detects may be reported without re-analysis.
10.3.4 Some possible remedial actions are listed below. This list is not meant to
be all inclusive. Major maintenance such as cleaning an ion source,
cleaning quadrupole rods, replacing filament assemblies, etc. require
returning to the initial calibration step (Sect. 10.2).
10.3.4.1 Check and adjust GC and/or MS operating conditions; check the
MS resolution, and calibrate the mass scale.
10.3.4.2 Clean or replace the splitless injection liner; silanize a new
injection liner.
10.3.4.3 Flush the GC column with solvent according to manufacturer's
instructions.
10.3.4.4 Break off a short portion (about 1 meter) of the column from the
end near the injector, or replace GC column. This action will
cause a change in retention times.
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10.3.4.5 Prepare fresh CAL solutions, and repeat the initial calibration
step.
10.3.4.6 Clean the MS ion source and rods (if a quadrupole).
10.3.4.7 Replace any components that allow analytes to come into
contact with hot metal surfaces.
10.3.4.8 Replace the MS electron multiplier, or any other faulty
components.
11. PROCEDURE
11.1 CARTRIDGE EXTRACTION
11.1.1 This procedure may be performed manually or in an automated mode (Sect.
6.11) using a robotic or automatic sample preparation device. If an
automatic system is used to prepare samples, follow the manufacturer's
operating instructions, but all extraction and elution steps must be the same
as in the manual procedure. Extraction and/or elution steps may not be
changed or omitted to accommodate the use of an automated system.
11.1.2 Mark the level of the sample on the outside of the sample bottle for later
sample volume determination (Sect. 11.2). Verify that the sample is at pH 2
or less and is free of residual chlorine. If the sample is a LRB or LFB, add
sodium sulfite and acidify following procedures in Sect.8.2. Add an aliquot
of the surrogate fortification solution(s), and mix immediately until
homogeneous. The resulting concentration of these compounds in the water
should be 2-5 (ig/L. If the sample is a LFB or LFM, add the desired amount
of analyte fortification solution.
11.1.3 CARTRIDGE CLEAN-UP AND CONDITIONING - Rinse each cartridge
with three, 3 mL aliquots of methylene chloride. Let the cartridge drain dry
after each flush. Then rinse the cartridge with three, 3mL aliquots of
methanol, but DO NOT allow the methanol to elute below the top of the
cartridge packing. From this point, do not allow the cartridge packing to go
dry. Rinse with three, 3mL aliquots of 0.05 N hydrochloric acid, but before
the dilute acid level drops below the top edge of the packing, turn off the
vacuum. Add approximately 3 mL additional 0.05 N hydrochloric acid to
the cartridge, attach the transfer tube, and turn on the vacuum, and begin
adding sample to the cartridge.
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11.1.4 Adjust the vacuum so that the approximate flow rate is 20 mL/min (50 min
for a 1 L sample). After all of the sample has passed through the SPE
cartridge, draw air or nitrogen through the cartridge for 15-30 min at high
vacuum (10-15 in Hg). The cartridge packing should appear dry (light tan
color) before continuing with the elution steps. It is important that the
cartridge packing be dry, in order to obtain good recoveries. The drying
time may vary, depending upon the strength of the vacuum source, and the
number of cartridges being processed simultaneously. The color and
appearance of the packing is the most reliable indicator of dryness. During
the method development, drying for more than 60 minutes was not observed
to have any negative effect upon the sample data.
NOTE: Samples with a high level of hardness and/or high TOC may exhibit
a lower flow rate than "cleaner" samples at the same vacuum setting. This
may be due to partial plugging of the solid phase. Fortified sample matrices
of these types showed no loss of method performance.
11.1.5 Rinse the inside of each sample bottle with 8-10 mL methylene chloride and
use vacuum to pull the solvent through the transfer tube and through the
cartridge, collecting the solvent in a collection tube. Remove the transfer
tubing from the top of the cartridge. Add 2-3 mL methylene chloride to the
top of the cartridge with a disposable pipette. Pull this solvent through the
cartridge at low vacuum, such that the solvent exits the cartridge in a
dropwise fashion. Small amounts of residual water from the sample
container and the SPE cartridge may form an immiscible layer with the
eluate. Pass the eluate through the drying column (Sect. 6.7), which is
packed with approximately 5 to 7 grams of anhydrous sodium sulfate, and
collect in a clean collection tube. Wash the sodium sulfate with at least 2
mL methylene chloride and collect in the same tube. Concentrate the
extract to approximately 0.9 mL in a warm (40°C) water bath under a
gentle stream of nitrogen. Do not concentrate the extract to less than 0.5
mL, as this will result in losses of analytes. Add the internal standards (Sect
7.9). Adjust final volume to 1 mL. Make any volume adjustments with
methylene chloride.
11.2 Fill the sample bottle to the volume mark noted in Sect. 11.1.2. with tap water.
Transfer the tap water to a 1000 mL graduated cylinder, and measure the sample
volume to the nearest 10 mL. Record this volume for later analyte concentration
calculations. As an alternative to this process, the sample volume may be
determined by the difference in weight between the full bottle (before extraction)
and the empty bottle (after extraction). Assume a sample density of 1.0.
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11.3 Analyze an aliquot of the sample extract with the GC/MS system under the same
conditions used for the initial and continuing calibrations (Sect. 10.2.2 and 10.3).
11.4 At the conclusion of data acquisition; use the same software that was used in the
calibration procedure to identify peaks in predetermined retention time windows of
interest. Use the data system software to examine the ion abundances of
components of the chromatogram.
11.5 Identification of analytes. Identify a sample component by comparison of its mass
spectrum (after background subtraction) to a reference spectrum in the user-created
database. The GC retention time of the sample component should be within 1-2 sec
of the retention time observed for that same compound in the most recently analyzed
continuing calibration check standard. Ideally, the width of the retention time
window 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 the
chromatogram.
11.5.1 In general, all ions that are present above 10% relative abundance in the
mass spectrum of the .standard should be present in the mass spectrum of
the sample component and should agree within absolute 20%. For example,
if an ion has a relative abundance of 30% in the standard spectrum, its
abundance in the sample spectrum should be in the range of 10 to 50%.
Some ions, particularly the molecular ion, are of special importance, and
should be evaluated even if they are below 10% relative abundance.
ANALYSIS AND CALCULATIONS
12.1 Complete chromatographic resolution is not necessary for accurate and precise
measurements of analyte concentrations if unique ions with adequate intensities are
available for quantitation. Identification is hampered when sample components are
not resolved chromatographically and produce mass spectra containing the same ions
contributed by more than one analyte. When GC peaks obviously represent more
than one sample component (i.e., broadened peak with shoulder(s) or valley between
two or more maxima), appropriate analyte spectra and background spectra can be
selected by examining plots of characteristic ions for each tentatively identified
component. When analytes coelute (i.e., only one GC peak is apparent), the
identification criteria can be met but each analyte spectrum will contain extraneous
ions contributed by the coeluting compound. In validating this method,
concentrations were calculated by measuring the characteristic ions listed in Table 2.
Other ions maybe selected at the discretion of the analyst. If the response of any
analyte exceeds the calibration range established in Section 10, dilute the extract,
528-26
-------
add additional internal standard, and reanalyze. The resulting data should be
documented as a dilution, with an increased MRL.
12.1.1 Calculate analyte and surrogate concentrations, using the multipoint
calibration established in Sect. 10. Do not use daily calibration verification
data to quantitate analytes in samples. Adjust final analyte concentrations
to reflect the actual sample volume determined in Section 11.2.
12.1.2 Calculations should utilize all available digits of precision, but final
reported concentrations should be rounded to an appropriate number of
significant figures (one digit of uncertainty). Experience indicates that three
significant figures maybe used for concentrations above 99 p,g/L, two
significant figures for concentrations between 1.0-9.9 (ig/L, and one
significant figure for lower concentrations.
13. METHOD PERFORMANCE
13.1 PRECISION, ACCURACY AND MDLs- Single laboratory accuracy and precision
data from both fortified reagent water and fortified matrices using hot, splitless
injection are presented in Tables 4 and 5. Table 6 includes data from 2 matrices
using temperature programmed splitless injection. Method detection limits (MDLs)
are presented in Table 1 for both types of injectors used. MDLs were calculated
using the formula in Section 9.2.4. Although the calculated MDLs using the two
different types are not dramatically different, for compounds 9-12 (Table 2) the peak
shapes are significantly better using temperature programmed injection, and the peak
heights and areas are greater.
13.2 POTENTIAL PROBLEM COMPOUNDS -
13.2.1 2,4-Dinitrophenol, 4-nitrophenol, 2-memyl-4,6-dinitrophenol,
pentachlorophenol arid 2,4,6-tribromophenol have a tendency to exhibit a
chromatographic phenomenon known as "matrix-induced chromatographic
response enhancement" (5-8). Compounds that exhibit this phenomenon
often give analytical results that exceed 100% recovery. The theory behind
this phenomenon is that these compounds are susceptible to adsorption
and/or thermal degradation in the GC inlet. The "cleaner" the matrix they
are injected in, e.g. clean solvent, the more they degrade. When they are
injected in a sample extract, matrix components in the sample extract
"protect" these compounds from decomposition and a relatively greater
response is observed. While most of the literature references to this
phenomenon refer to organophosphate pesticides in river water and food
samples, the effect seen during development of this method suggests the
same type of problem occurs with these acidic phenols.
528-27
-------
This method uses 2,3,4,5-tetrachlorophenol as the internal standard for
quantifying these analytes. The chromatographic behavior of 2,3,4,5-
tetrachlorophenol mimics these particular method analytes. Therefore its
use as an internal standard helps maintain accurate measurement of these
analytes. It should be noted however that these particular analytes will
probably not be measured with the same level of precision and accuracy as
other method analytes, but the precision and accuracy requirements should
still be achievable.
13.2.2 The same compounds listed in sect. 13.2.1. also have a tendency to tail. QC
criteria for peak tailing factors have been given in Sect. 10.2.3.1. During
method development, significantly less peak tailing was observed using
temperature programmed injection. Other measures shown to minimize
peak tailing and improve peak shape are pressure pulsed injection, and
increasing the GC oven temperature program rate. Pulsed injection is
recommended on GCs which have that option available. A faster GC oven
temperature program is recommended if there are no interferences, and if
the minimum number of scans across all chromatographic peaks can be
obtained. This is a function of how fast the MS can scan.
13.2.3 Phenol is very water soluble compared to other method analytes.
Breakthrough experiments performed during method development indicate
that some breakthrough from the SPE cartridge can be expected.
Breakthrough can be minimized by monitoring the flow of the sample
through the cartridge. In general, slower flow rates will minimize
breakthrough. Precision and accuracy requirements in Sect. 10 should be
achievable.
13.3 HOLDING TIME STUDY RESULTS -
13.3.1 Holding time studies for aqueous samples were conducted for a period of 3 5
days. Chlorinated surface water samples fortified with method analytes and
preserved and stored according to requirements in Section 8, were analyzed
on days 0, 7, 10, 15, 23, 28, and 35. Small, but statistically significant
losses of 2-chlorophenol, o-cresol, and 2,4-dimethylphenol were observed
beginning between day 15 and 23. Therefore the aqueous holding time was
determined to be 14 days. Data from these studies are in Table 7.
13.3.2 Holding time studies for sample extracts were conducted for a period of 35
days. A single set of extracts were stored at 0°C, and analyzed on days 0,
14,23, and 35. No significant losses were observed within this time frame.
Therefore the extract holding time was established at 30 days. Data from
these studies are in Table 8.
528-28
-------
14. POLLUTION PREVENTION
14.1 This method utilizes SPE technology to remove the analytes from water. It requires
the use of very small volumes of organic solvent and very small quantities of pure
analytes, thereby minimizing the potential hazards to both the analyst and the
environment when compared with the use of large volumes of organic solvents in
conventional liquid-liquid extractions.
14.2 For information about pollution prevention that may be applicable to laboratory
operations, consult "Less Is Better: Laboratory Chemical Management for Waste
Reduction" available from the American Chemical Society's Department of
Government Relations and Science Policy, 1155 16th Street N.W., Washington,
D.C., 20036.
15. WASTE MANAGEMENT
15.1 The analytical procedures described in this method generate relatively small amounts
of waste since only small amounts of reagents and solvents are used. The matrices
of concern are finished drinking water or source water. However, the Agency
requires that laboratory waste management practices be conducted consistent with all
applicable rules and regulations, and that laboratories protect the air, water, and land
by minimizing and controlling all releases from fume hoods and bench operations.
Also, compliance is required with any sewage discharge permits and regulations,
particularly the hazardous waste identification rules and land disposal restrictions.
For further information on waste management, consult "The Waste Management
Manual for Laboratory Personnel" also available from the American Chemical
Society at the address in Section 14.2.
16. REFERENCES
1. Glaser, J. A., D. L. Foerst, G. D. McKee, S. A. Quave, and W. L. Budde, "Trace Analyses
for Wastewaters," Environ. Sci. Technol.. 15 (1981)1426-1435.
2. "Carcinogens - Working With Carcinogens," Department of Health, Education, and
Welfare, Public Health Service, Center for Disease Control, National Institute for
Occupational Safety and Health, Publication No. 77-206, Aug. 1977.
3. "OSHA Safety and Health Standards, General Industry," (29CFR1910), Occupational
Safety and Health Administration, OSHA 2206, (Revised, January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
528-29
-------
5. Erney, D.R., A.M. Gillespie, DM. Gilvydis, and C.F. Poole, "Explanation of the Matrix-
Induced Chromatographic Response Enhancement of Ofganophosphorous Pesticides
During Open Tubular Column Gas Chromatography with Splitless or Hot On-column
Injection and Flame Photometric Detection." J. Chromatogr.. 638 (1993)57-63.
6. Mol, H.G.J., M. Althuizen, H. Janssen, and C.A. Cramers, "Environmental Applications of
Large Volume Injection in Capillary GC Using PTV Injectors," J. High Resol.
Chromatogr.. 19 (1996)69-79.
7. Emey, D.R., T.M. Pawlowski, C.F. Poole, "Matrix Induced Peak Enhancement of
Pesticides in Gas Chromatography," J. High Resol. Chromatogr.. 20 (1997) 375-378.
8. Hajslova, J., k. Holadova, V. Kocourek, J. Poustka, M. Gbdula, P. Cuhra, M. Kempny,
"Matrix Induced Effects:A Critical Point in the Gas Chromatographic Analysis of Pesticide
Residues." J. Chromatogr.. 800 (1998)283-295.
528-30
-------
17. TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. METHOD DETECTION LIMITS a
Analyte
phenol
2-chlorophenol
2-methylphenol (o-cresol)
2-nitrophenol
2,4-dimethylphenol
2,4-dichlorophenol
4-chloro-3 -methylphenol
2,4,6-trichlorophenol
2,4-dinitrophenol
4-nitrophenol
2-methyl-4,6-dinitrophenol
pentachlorophenol
Hot Splitless Injection b
Spiking
Cone. (ng/L)
1.0
0.1
0.1
0.1
0.1
0.1
0.1
0.1
1.0
1.0
1.0
1.0
MDL
(Hg/L)
0.58
0.020
0.026
0.026
0.026
0.027
0.036
0.046
0.31
0.42
0.26
0.25
Temperature Programmed
SpUtless Injection c
Spiking
Cone. (|ig/L)
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.5
0.5
0.5
0.5
MDL
(Hg/L)
0.025
0.041
0.028
0.044
0.034
0.046
0.042
0.024
0.22
0.18
0.092
0.081
a- data obtained using Column 1
b- n=7
c-n=8
528-31
-------
TABLE 2. RETENTION TIMES (RTs) AND SUGGESTED QUANTITATION IONS (QIs)
Cmpd
#a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Analyte
phenol
2-chlorophenol
2-methylphenol (o-cresol)
2-nitrophenol
2,4-dimethylphenol
2,4-dichlorophenol
4-chloro-3 -methylphenol
2,4,6-trichlorophenol
2,4-dinitrophenol
4-nitrophenol
2-methyl-4,6-dinitrophenol
pentachlorophenol
l,2-dimethyl-3-nitrobenzene (IS#1)
2,3,4,5-tetrachlorophenol (IS#2)
2-chlorophenol-3,4,5,6-d4 (SURR)
2,4-dimethylphenol-3,5,6-d3 (SURR)
2,4,6-tribromophenol (SURR)
RTs (min)
column 1
11:00
11:07
12:52
14:36
15:00
15:22
17:46
18:56
21:30
21:53
23:09
25:14
17:43
22:09
11:04
14:59
23:37
RTs (min)
column 2 c
12:38
12:50
14:27
16:22
16:35
17:04
19:27
20:42
23:30
23:44
25:09
27:12
19:29
24:02
12:47
16:34
25:37
QIs
(m/z)
94
128
107
139
107
162
142
97
154,184d
139
121,198d
266
134
232
132
110
330
IS
Ref
1
1 ;
1
1 r
1
1
1
1
2
2
2
2
1
1
2
a- Number refers to peak number in Figures 1-3.
b- Column 1- 30 m * 0.25 mm id DB-5ms (J&W), 0.25 (im film thickness.
c- Column 2- 30 m x 0.25mm id BPX5 (SGE), 0.25 urn film thickness.
d- Because the MS response to these compounds is low, and both the listed ions are near 100%
ion abundance, the signal from both ions may be added together to increase sensitivity.
528-32
-------
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Analyze LRB prior to any other
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528-39
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Acceptance Criteria
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528-40
-------
Results of LFB analyses must be 70-130%
of the true value (except phenol) for each
analyte and surrogate for all fortified
concentrations greater than the lowest CAL
point. Results of LFBs corresponding to the
lowest CAL point must be 50-150% of the
true value.
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for each extraction batch of up to
20 field samples. Rotate the
fortified concentration between
low, medium and high amounts.
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compounds listed in the referenced
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FIGURE 4. PEAK TAILING FACTOR (PTF) CALCULATION.
1.83X
266
2168
25.19
Peak Tailing Factor =
BC
AB
BD = 10 % peak height
Note: the PTF should be calculated from the single ion chromatogram of the quantitation ion.
This example is for the pentachlorophenol peak. The PTF = 2.5.
528-46
-------
METHOD 532. DETERMINATION OF PHENYLUREA COMPOUNDS IN
DRINKING WATER BY SOLID PHASE EXTRACTION AND
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH
UV DETECTION
Revision 1.0
June 2000
M. V. Bassett, S.C. Wendelken, T.A. Dattilio, and B.V. Pepich, IT Corporation and
D. J. Munch, US EPA, Office of Ground Water and Drinking Water
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
532-1
-------
METHOD 532
DETERMINATION OF PHENYLUREA COMPOUNDS IN DRINKING WATER BY
SOLID PHASE EXTRACTION AND HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY WITH UV DETECTION
1. SCOPE AND APPLICATION
1.1 This is a high performance liquid chromatographic (HPLC) method for the
determination of phenylurea pesticides in drinking waters. This method is
applicable to phenylurea compounds that are efficiently extracted from the water
using a Cls solid phase cartridge or disk. Accuracy, precision, and method
detection limit (MDL) data have been generated for the following compounds in
reagent water and finished ground and surface waters:
Chemical Abstracts Service
Analvte Registry Number
Diflubenzuron 35367-38-5
Diuron 330-54-1
Fluometuron 2164-17-2
Linuron 330-55-2
Propanil 709-98-8
Siduron 1982-49-6
Tebuthiuron 34014-18-1
Thidiazuron 51707-55-2
1.2 MDLs are compound, instrument, and matrix dependent. The MDL is defined as
the statistically calculated minimum concentration that can be measured with 99%
confidence that the reported value is greater than zero.(I) Experimentally
determined MDLs for the above listed analytes are provided in Section 17, Table
3. The MDL differs from, and is usually lower than (but never above), the
minimum reporting limit (MRL) (Sect. 3.16). The concentration range for target
analytes hi this method was evaluated between 1.0 ug/L and 30 ug/L for a 500 mL
sample. Precision and accuracy data and sample holding time data are presented
in Section 17, Tables 4 - 9.
532-2
-------
1.3 This method is restricted to use by or under the supervision of analysts skilled in
solid phase extraction (SPE), and HPLC analysis.
2. SUMMARY OF METHOD
2.1 A 500 mL water sample is passed through a SPE cartridge or disk containing a
chemically bonded C18 organic phase to extract the phenylurea pesticides and
surrogate compounds. The analytes and surrogates are eluted from the solid phase
with methanol, and the extract is concentrated to a final volume of 1 mL.
Components are then chromatographically separated by injecting an aliquot of the
extract into an HPLC system equipped with a C]g column and detected using a
UV/Vis detector. Identification of target and surrogate analytes and quantitation
is accomplished by comparison of retention times and analyte responses using
external standard procedures. Sample extracts with positive results are solvent
exchanged and confirmed using a second, dissimilar HPLC column that is also
calibrated using external standard procedures.
3. DEFINITIONS
3.1 EXTRACTION BATCH - A set of up to 20 field samples (not including QC
samples) extracted together by the same person(s) during a work day using the
same lot of solid phase extraction devices and solvents, surrogate solution, and
fortifying solutions. Required QC samples include: Laboratory Reagent Blank,
Laboratory Fortified Blank, Laboratory Fortified Matrix, and either a Field
Duplicate or Laboratory Fortified Matrix Duplicate.
3.2 ANALYSIS BATCH - A set of samples that is analyzed on the same instrument
during a 24-hour period that begins and ends with the analysis of the appropriate
Continuing Calibration Check standards (CCC). Additional CCCs may be
required depending on the length of the analysis batch and/or the number of Field
Samples.
3.3 SURROGATE ANALYTE (SUR) - A pure analyte, which is extremely unlikely
to be found in any sample, and which is added to a sample aliquot in known
amount(s) before extraction or other processing and is measured with the same
procedures used to measure other sample components. The purpose of the SUR is
to monitor method performance with each sample.
3.4 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or
other blank matrix that is treated exactly as a sample including exposure to all
glassware, equipment, solvents, reagents, sample preservatives, and surrogates
that are used in the extraction batch. The LRB is used to determine if method
analytes or other interferences are present in the laboratory environment, the
reagents, or the apparatus.
532-3
-------
3.5 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water or
other blank matrix to which known quantities of the method analytes and all the
preservation compounds are added. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise measurements.
3.6 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) - An aliquot of an
environmental sample to which known quantities of the method analytes and all
the preservation compounds 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 hi the sample matrix must be determined in a separate aliquot and the
measured values in the LFM corrected for background concentrations.
3.7 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFMD) - A
second aliquot of the Field Sample used to prepare the LFM which is fortified,
extracted, and analyzed identically. The LFMD is used instead of the Field
Duplicate to access method precision and accuracy when the occurrence of target
analytes is low.
3.8 LABORATORY DUPLICATES (LD1 and LD2) - Two aliquots of the same
sample taken in the laboratory and analyzed separately with identical procedures.
Analyses of LD1 and LD2 indicate precision associated with laboratory
procedures, but not with sample collection, preservation, or storage procedures.
3.9 FIELD DUPLICATES (FD1 and FD2) - Two separate samples collected at the
same tune 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.
3.10 STOCK STANDARD SOLUTION (S S S) - A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed reference
materials or purchased from a reputable commercial source.
3.11 PRIMARY DILUTION STANDARD SOLUTION (PDS) - A solution containing
method analytes prepared in the laboratory from stock standard solutions and
diluted as needed to prepare calibration solutions and other needed analyte
solutions.
3.12 CALIBRATION STANDARD (CAL) - A solution prepared from the primary
dilution standard solution or stock standard solution and the surrogate analytes.
The CAL solutions are used to calibrate the instrument response with respect to
analyte concentration.
532-4
-------
3.13 CONTINUING CALIBRATION CHECK (CCC) - A calibration standard
containing one or more of the method analytes, which is analyzed periodically to
verify the accuracy of the existing calibration for those analytes.
3.14 QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of
known concentrations that is obtained from a source external to the laboratory and
different from the source of calibration standards. It is used to check standard
integrity.
3.15 METHOD DETECTION LIMIT (MDL) - The minimum concentration of an
analyte that can be identified, measured and reported with 99% confidence that
the analyte concentration is greater than zero. This is a statistical determination of
precision (Sect. 9.2.4). Accurate quantitation is not expected at this level.(1)
3.16 MINIMUM REPORTING LEVEL (MRL) - The minimum concentration that can
be reported as a quantitated value for a target analyte in a sample following
analysis. This defined concentration can be no lower than the concentration of the
lowest continuing calibration standard for that analyte, and can only be used if
acceptable quality control criteria for this standard are met.
3.17 MATERIAL SAFETY DATA SHEET (MSDS) - Written information provided
by vendors concerning a chemical's toxicity, health hazards, physical properties,
fire, and reactivity data including storage, spill, and handling precautions.
3.18 PEAK GAUSSIAN FACTOR (PGF)-Thepeak gaussian factor is calculated
using the equation in Section 10.2.3.1. It provides a quantitative measure of peak
asymmetry. A perfectly symmetric peak would have a PGF of 1. Poor peak
symmetry can result in imprecise quantitation, degraded resolution and poor
retention reproducibiliry. For this reason, columns and conditions that produce
symmetric peaks are required.
4. INTERFERENCES
4.1 All glassware must be meticulously cleaned. Wash glassware with detergent and
tap water, rinse with tap water, followed by reagent water. A final rinse with
solvents may be needed. In place of a solvent rinse, non-volumetric glassware can
be heated in a muffle furnace at 400 °C for 2 hours. Volumetric glassware should
not be heated above 120 °C.
4.2 Method interferences may also be caused by contaminants in solvents, reagents
(including reagent water), sample bottles and caps, and other sample processing
hardware that lead to discrete artifacts and/or elevated baselines in the chromato-
grams. All items such as these must be routinely demonstrated to be free from
interferences (less than V3 the MRL for each analyte) under the conditions of the
. 532-5
-------
analysis by analyzing laboratory reagent blanks as described in Section 9.3.
Subtracting blank values from sample results is not permitted.
4.2.1 An extraneous peak was noted that elutes very near fluometuron on the
confirmation column that can cause problems with quantitation if the
chromatography is not fully optimized. No interferences were observed
for the primary column analysis.
4.3 Matrix interferences may be caused by contaminants that are co-extracted from
the sample. The extent of matrix interferences will vary considerably from source
to source, depending upon the nature and diversity of the matrix being sampled.
Water samples high in total organic carbon may have an elevated baseline and/or
interfering peaks.
4.4 Solid phase cartridges and disks and their associated extraction devices have been
observed to be a source of interferences in other EPA organic methods. The
analysis of field and laboratory reagent blanks can provide important information
regarding the presence or absence of such interferences. Brands and lots of solid
phase extraction devices should be monitored to ensure that contamination will
not preclude analyte identification or quantitation.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been
precisely defined. Each chemical compound should be treated as a potential
health hazard, and exposure to these chemicals should be minimized. The
laboratory is responsible for maintaining an awareness of OSHA regulations
regarding the safe handling of the chemicals used in this method. A reference file
of MSDSs should also be made available to all personnel involved in the chemical
analysis. Additional references to laboratory safety are available.(2"4)
5.2 Pure standard materials and stock standards of these compounds should be
handled with suitable protection to skin and eyes, and care should be taken not to
breath the vapors or ingest the materials.
6. EQUIPMENT AND SUPPLIES (All specifications are suggested. Brand names and/or
catalog numbers are included for illustration only.)
6.1 SAMPLE CONTAINERS - 500 mL amber or clear glass bottles fitted with PTFE
(polytetrafluoroethylene) lined screw caps.
6.2 VIALS - Screw cap or crimp top glass autosampler vials with PTFE faced septa,
amber or clear.
532-6
-------
6.3 VOLUMETRIC FLASKS - Class A, suggested sizes include 1,5,and 10 mL.
6.4 GRADUATED CYLINDERS -Suggested sizes include 5,10, and 500 mL.
6.5 MICRO SYRINGES - Various sizes.
6.6 ANALYTICAL BALANCE - Capable of accurately weighing to the nearest
0.0001 g.
6.7 DISPOSABLE SYRINGES - 1 mL (B-D catJ: 309602 or equivalent) size, used
to filter sample extracts before analysis.
6.8 FILTERS - Disposable filters to filter sample extracts before analysis (Gelman
0.45 urn Nylon Acrodisk cat.#: 4426 or equivalent).
6.9 SOLID PHASE EXTRACTION (SPE) APPARATUS USING CARTRIDGES
6.9.1 EXTRACTION CARTRIDGES - 6 mL, packed with 500 mg (40 urn dp)
silica bonded with CI8 (Varian catJ: 1210-2052 or equivalent).
6.9.2 SAMPLE RESERVOIRS - (VWR catJ: JT7120-3 or equivalent) These
are attached to the cartridges and water samples are poured into them,
although they hold only 75 mL at one time. An alternative is a transfer
tube system (Supelco "Visiprep"; cat. #: 57275 or equivalent) which
transfers the sample directly from the sample container to the SPE
cartridge.
6.9.3 VACUUM EXTRACTION MANIFOLD - With flow/vacuum control
(Supelco cat.#: 57044 or equivalent). The use of replaceable needles or
valve liners may be used to prevent cross contamination.
6.9.4 REMOTE VACUUM GAUGE/BLEED ASSEMBLY - To monitor and
adjust vacuum pressure delivered to the vacuum manifold (Supelco catJ:
57161-U or equivalent).
6.9.5 CONICAL CENTRIFUGE TUBES - 15 mL, or other glassware suitable
for elution of the sample from the cartridge after extraction.
6.9.6 An automatic or robotic system designed for use with SPE cartridges may
be used if all quality control requirements discussed in Section 9 are met.
Automated systems may use either vacuum or positive pressure to process
samples and solvents through the cartridge. All extraction and elution
steps must be the same as the manual procedure. Extraction or elution
steps may not be changed or omitted to accommodate the use of the
automated system.
532-7
-------
6.10 SOLID PHASE EXTRACTION (SPE) APPARATUS USING DISKS
6.10.1 EXTRACTION DISKS - 47 mm diameter, manufactured with a C18
bonded sorbent phase (Varian cat.#: 1214-5004 or equivalent). Larger
disks may be used as long as the QC performance criteria outlined in
Section 9 are met.
6.10.2 SPE DISK EXTRACTION GLASSWARE - Funnel, PTFE coated support
screen, PTFE gasket, base, and clamp used to support SPE disks and
contain samples during extraction. May be purchased as a set (Fisher cat#
K971100-0047 or equivalent) or separately.
6.10.2 VACUUM EXTRACTION MANIFOLD - Designed to accommodate
extraction glassware (Varian cat. #: 1214-6001 or equivalent).
6.10.3 CONICAL CENTRIFUGE TUBES - 15 mL, or other glassware suitable
for collection of the eluent that drips from the disk extraction base.
6.10.4 An automated or robotic system may be used as specified in Section 6.9.6.
6.11 EXTRACT CONCENTRATION SYSTEM - To concentrate extracts in 15 mL
conical tubes, the bottoms of which are submersed in a 40°C water bath, under a
steady steam of nitrogen to the desired volume (Meyer N-Evap, Model III,
Organomation Associates, Inc. or equivalent).
6.12 LABORATORY OR ASPIRATOR VACUUM SYSTEM - Sufficient capacity to
maintain a minimum vacuum of approximately 25 cm (10 in.) of mercury for
cartridges. A greater vacuum of approximately 66 cm (26 in.) of mercury may be
used with disks.
6.13 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
INSTRUMENTATION
6.13.1 HPLC SYSTEM - Capable of reproducibly injecting 20 or 10 uL
aliquots, and performing binary linear gradients at a constant flow rate
near 1.5 mL/min.
6.13.2 HPLC DETECTOR - A UV detector capable of collecting data at 240-
245 nm. For the development of this method, a photodiode array detector
was used. A LC/MS system may also be used.
6.13.3 PRIMARY COLUMN - An HPLC column (4.6 x 150 mm) packed with
3. Sum dp C,g solid phase particles (Waters cat. # WAT200632). Any
532-8
-------
column that provides adequate resolution, peak shape, capacity, accuracy,
and precision (Sect. 9) may be used.
6.13.4 CONFIRMATION COLUMN - An HPLC column (4.6 x 150mm) packed
with 5 dp cyanopropyl stationary phase ( Supelco "Supelcosil LC-CN" cat.
# 58221-U). The secondary column must be chemically dissimilar to the
primary column and must yield a different elution order for some
compounds which will result in dissimilar retention times compared to the
primary column.
6.13.5 HPLC DATA SYSTEM - A computerized data system is recommended
for data acquisition and manipulation. The Waters Millennium software
system was used to generate all primary column data contained in the
Section 17 tables.
7. REAGENTS AND STANDARDS
7.1 REAGENTS AND SOLVENTS - Reagent grade or better chemicals should be
used in all tests. Unless otherwise indicated, it is intended that all reagents shall
conform to the specifications of the Committee on Analytical Reagents of the
American Chemical Society, where such specifications are available. Other
grades may be used, provided it is first determined that the reagent is of
sufficiently high purity to permit its use without lessening the quality of the
determination.
7.1.1 REAGENT WATER - Purified water which does not contain any
measurable quantities of any target analytes or interfering compounds at or
above 1/3 the MRL for each compound of interest.
7.1.2' ACETONITRILE - High purity, demonstrated to be free of analytes and
interferences (HPLC grade or better).
7.1.3 METHANOL - High purity, demonstrated to be free of analytes and
interferences (HPLC grade or better).
7.1.4 ACETONE - High purity, demonstrated to be free of analytes and
interferences (HPLC grade or better).
7.1.5 PHOSPHATE BUFFER SOLUTION, 25 mM - Used for HPLC mobile
phase. Add 100 mL 0.5 M potassium phosphate stock solution (Sect.
7.1.5.1) and 100 mL of 0.5 M phosphoric acid stock solution (Sect.
7.1.5.2) to reagent water and dilute to a final volume of 4 L . The pH
should be about 2.4 and should be confirmed with a pH meter. Filter
using a 0.45 urn nylon filter.
532-9
-------
7.2
7.1.5.1 POTASSIUM PHOSPHATE STOCK SOLUTION (0.5 M) -
Weigh 68 g KH2PO4 (Monobasic Potassium Phosphate) and dilute
to 1 L using reagent water.
7.1.5.2 PHOSPHORIC ACID STOCK SOLUTION (0.5M) - 34.0 mL of
phosphoric acid ( 85%, HPLC grade in reagent water) diluted to 1
L with reagent water.
7.1.6 SAMPLE PRESERVATION REAGENTS
7.1.6.1 CUPRIC SULFATE, CuSO4-5H2O ( ACS Grade or equivalent) -
Added as a biocide to guard against potential degradation of
method analytes by microorganisms (Sect. 8.1.2).
7.1.6.2 TRIZMA PRESET CRYSTALS, pH 7.0 (Sigma cat# T 3503 or
equivalent) — Reagent grade. A premixed blend of Tris
[Tris(hydroxymethyl)aminomethane] and Tris HCL
[Tris(hydroxymethyl)aminomethane hydrochloride]. Alternatively,
a mix of the two components with a weight ratio of 15.5/1; Tris
HCL/Tris may be used. These blends are targeted to produce a pH
of 7.0 at 25°C in reagent water. Tris functions as a buffer, binds
free chlorine in chlorinated finished waters, and prevents the
formation of a copper-based precipitate.
STANDARD SOLUTIONS - When a compound purity is assayed to be 96% or
greater, the weight can be used without correction to calculate the concentration of
the stock standard. Solution concentrations listed in this section were used to
develop this method and are included as an example. Standards for sample
fortification generally should be prepared in the smallest volume that can be
accurately measured to minimize the addition of organic solvent to aqueous
samples. Even though stability times for standard solutions are suggested in
the following sections, laboratories should used standard QC practices to
determine when their standards need to be replaced.
7.2.1 SURROGATE ANALYTES (SUR), MONURON (CAS #150-68-5) and
CARBAZOLE (CAS# 86-74-8) - Monuron and carbazole were chosen as
surrogates. Monuron is a phenylurea no longer in use in the U.S. that
elutes early in the chromatogram. Carbazole elutes late in the
chromatogram (Figure 1) and has been used as a surrogate in other EPA
drinking water methods. Alternate surrogates may be selected if there is a
problem with matrix interferences or chromatography. However, if an
alternate surrogate is used, it must have similar chemical properties
(structure, solubility, C18 retention, etc.) to the phenylureas, be
chromatographically resolved from all target analytes and matrix
interferences, and be highly unlikely to be found in any sample.
532-10
-------
7.2.1.1 SUR STOCK SOLUTION (5 to 7 mg/mL) - Accurately weigh
approximately 25 to 35 mg of the neat SUR to the nearest 0.1 mg
into a tared, 5 mL volumetric flask. Dilute to the mark with the
appropriate solvent: methanol should be used for monuron and
acetonitrile for carbazole. Prepare each compound individually, as
they will be combined in the SUR primary dilution standard. Stock
solutions have been shown to be stable for 6 months when stored at
-10°C or less. Laboratories should use standard QC practices to
determine when their standards need to be replaced.
7.2.1.2 SUR PRIMARY DILUTION STANDARD (500 ug/mL) - Prepare
the SUR Primary Dilution Standard (PDS) by dilution of the SUR
stock standards. Add enough of each of the SUR stock standards
to a volumetric flask partially filled with methanol to make a 500
ug/mL solution when filled to the mark with methanol. The PDS
has been shown to be stable for 3 months when stored at -10°C or
less.
7.2.2 ANALYTE STOCK STANDARD SOLUTION - Prepare analyte stock
standard solutions for all compounds in methanol except thidiazuron and
difiubenzuron. Thidiazuron and diflubenzuron should be prepared in
acetone due to their limited solubility in methanol. Acetone elutes early
in the chromatogram and should not interfere with compound quantitation
as long as its volume is minimized as specified in this method. Method
analytes may be obtained as neat materials or as ampulized solutions.
Stock solutions have been shown to be stable for 6 months when stored at
-10°Corless.
7.2.2.1 For analytes in their pure form that are soluble in methanol,
prepare stock solutions by accurately weighing 25 to 35 mg of pure
material to the nearest 0.1 mg in a 5 mL volumetric flask. Dilute to
volume with methanol.
7.2.2.2 Thidiazuron and diflubenzuron should be dissolved in acetone.
Accurately weigh neat material to the nearest 0.1 mg into
volumetric flasks, but using smaller amounts than those used for
other target analytes, approximately 10 to 12 mg. Thidiazuron is
especially difficult to dissolve, 10 mg of pure material should
dissolve in a 10 mL final volume of acetone. Sonnication may be
used to help dissolve these compounds.
7.2.3 ANALYTE PRIMARY DILUTION STANDARD (PDS, 200 ug/mL and
10 ug/mL) - Prepare the Analyte PDS by dilution of the stock standards
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(Sect. 7.2.2). Add enough of each stock standard to a volumetric flask
partially filled with methanol to make a 200 ug/mL solution when filled to
the mark with methanol. Once prepared, a dilution of the 200 ug/mL
solution may be used to prepare a 10 ug/mL solution used for low
concentration spiking. The PDSs can be used to prepare calibration and
fortification solutions. Analyte PDSs have been shown to be stable for 3
months when stored at -10°C or less.
7.2.4 CALIBRATION SOLUTIONS - At least 5 calibration concentrations will
be required to prepare the initial calibration curve (Sect. 10.2). Prepare at
least 5 Calibration Solutions over the concentration range of interest,
approximately 0.5-15 ug/mL, from dilutions of the analyte PDS in
methanol. The lowest concentration of calibration standard must be at or
below the MRL, which will depend on system sensitivity. In this method,
500 mL of an aqueous sample is concentrated to a 1 mL final extract
volume. The calibration standards for the development of this method
were prepared as specified below.
PREPARATION OF CALIBRATION (CAL) CURVE STANDARDS
CAL
Level
1
2
3
4
5
6
PDS Cone.
(ug/mL)
10
10
200
200
200
200
Volume PDS
Standard
(uL)
25
50
5.0 =
25
50
75
Final Volume of
CAL Standard
(mL)-'/-.,
1
i ;
i
i
i
i
'' '$ t ' '» - '
'Final Cone, of
CAlL Standard
(ug/mL),
0.25
0.50
1.00
5.00
10.0
15.0
Equivalent Cone,,,
in SOOonL sample
' (ug/L) , -/t.
0.50
1.00
2.00
10.0
20.0
30.0
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 SAMPLE BOTTLE PREPARATION
8.1.1 Grab samples must be collected in accordance with conventional sampling
practices(5) using 500 mL amber or clear glass bottles fitted with PTFE
lined screw caps.
8.1.2 Prior to shipment to the field, 0.25 g of cupric sulfate and 2.5 g of Trizma
crystals ( Sect. 7.1.6) must be added to each bottle for each 500 mL of
sample collected. Alternately, the Tris buffer may be prepared by adding
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2.35 g of Tris HC1 and 0.15 g Tris to the sample bottle in addition to the
0.25 g of cupric sulfate. Cupric sulfate acts as a biocide to inhibit
bacteriological decay of method analytes. Trizma functions as a buffering
reagent, binds the free chlorine, and helps to prevent the formation of a
precipitate. Add these materials as dry solids to the sample bottle. The
stability of these materials in concentrated aqueous solution has not been
verified.
8.2 SAMPLE COLLECTION
8.2.1 When sampling from a cold water tap, remove the aerator so that no air
bubbles will be trapped in the sample. Open the tap, and allow the system
to flush until the water temperature has stabilized (usually about 3-5
minutes). Collect samples from the flowing system.
8.2.2 When sampling from an open body of water, fill a 1 quart wide-mouth
bottle or 1L beaker with sample from a representative area, and carefully
fill sample bottles from the container. Sampling equipment, including
automatic samplers, must be free of plastic tubing, gaskets, and other parts
that may leach interfering analytes into the water sample.
8.2.3 Fill sample bottles, taking care not to flush out the sample preservation
reagents. Samples do not need to be collected headspace free.
8.2.4 After collecting the sample, cap carefully to avoid spillage, and agitate by
hand for 1 minute. Keep samples sealed from collection time until
extraction.
8.3 SAMPLE SHIPMENT AND STORAGE - All samples should be iced during
shipment and must not exceed 10° C during the first 48 hours after collection.
Samples should be confirmed to be at or below 10 °C when they are received at the
laboratory. Samples stored in the lab must be held at or below 6 °C until extraction,
but should not be frozen.
8.4 SAMPLE AND EXTRACT HOLDING TIMES - Results of the sample storage
stability study of all method analytes indicated that all compounds are stable for 14
days in water samples that are collected, dechlorinated, preserved, shipped and
stored as described in Sections 8.2 and 8.3. Samples must be extracted within 14
days. Sample extracts may be stored in methanol at 0°C or less for up to 21 days
after extraction. Samples that are exchanged into reagent water/acetonitrile (60/40)
for confirmational analysis may be stored 7 days at 0° or less; however, the
combined extract holding time may not exceed 21 days.
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9. QUALITY CONTROL
9.1 Quality control (QC) requirements include the Initial Demonstration of Capability,
the determination of the MDL, and subsequent analysis in each analysis batch of a
Laboratory Reagent Blank (LRB), Continuing Calibration Check Standards (CCC), a
Laboratory Fortified Blank (LFB), a Laboratory Fortified Sample Matrix (LFM), and
either a Laboratory Fortified Sample Matrix Duplicate (LFMD) or a Field Duplicate
Sample. This section details the specific requirements for each QC parameter. The
QC criteria discussed in the following sections are summarized in Section 17, Tables
10 and 11. These criteria are considered the minimum acceptable QC criteria, and
laboratories are encouraged to institute additional QC practices to meet their specific
needs.
9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) - Requirements for the
Initial Demonstration of Capability are described in the following sections and
summarized in Section 17, Table 10.
9.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND -
Any time a new lot of solid phase extraction (SPE) cartridges or disks is
used, it must be demonstrated that a laboratory reagent blank is reasonably
free of contamination and that the criteria in Section 9.4 are met.
9.2.2 INITIAL DEMONSTRATION OF PRECISION - Prepare, extract, and
analyze 4-7 replicate LFBs fortified at 5 to 10 ug/L, or near the mid-range
of the initial calibration curve, according to the procedure described in
Section 11. Sample preservatives as described in Section 8.1.2 must also
be added to these samples. The relative standard deviation (RSD) of the
results of the replicate analyses must be less than 20%.
9.2.3 INITIAL DEMONSTRATION OF ACCURACY - Using the same set of
replicate data generated for Section 9.2.2, calculate average recovery. The
average recovery of the replicate values must be within ± 20% of the true
value.
9.2.4 MDL DETERMINATION - Prepare, extract and analyze at least 7
replicate LFBs at a concentration estimated to be near the MDL over at
least 3 days (both extraction and analysis should be conducted over at least
3 days) using the procedure described in Section 11.. This fortification
level may be estimated by selecting a concentration with a signal of 2 to 5
times the noise level. The appropriate concentration will be dependent
upon the sensitivity of the HPLC system being used. Sample preservatives
as described in Section 8.1.2 must be added to these samples. Calculate
the MDL using the equation
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MDL-St(n.1;1.alpha =
where
Vu-aipha-o.99) = Student's t value for the 99% confidence level
with n-1 degrees of freedom
n = number of replicates, and
S = standard deviation of replicate analyses.
NOTE: Do not subtract blank values when performing MDL calculations.
This is a statistical determination based on precision only.(I) If
the MDL replicates are fortified at a low enough concentration, it
is likely that they will not meet method precision and accuracy
criteria.
9.2.5 METHOD MODIFICATIONS - The analyst is permitted to modify HPLC
columns, HPLC detector, HPLC conditions, evaporation techniques, and
surrogate standards, but each time such method modifications are made,
the analyst must repeat the procedures of the IDC (Sect. 9.2).
9.3 Minimum Reporting Level (MRL) - The MRL is the threshold concentration of an
analyte that a laboratory can expect to accurately quantitate in an unknown sample.
The MRL may be established at an analyte concentration either greater than three
times the MDL or at a concentration which would yield a response greater than a
signal to noise ratio of five. Although the lowest calibration standard for an
analyte may be below the MRL, the MRL must never be established at a
concentration lower than the lowest calibration standard.
9.4 LABORATORY REAGENT BLANK (LRB) - A LRB is required with each
extraction batch (Sect. 3.1) of samples to determine any background system
contamination. If within the retention time window of any analyte, the LRB
produces a peak that would prevent the determination of that analyte, determine the
source of contamination and eliminate the interference before processing samples.
Background contamination must be reduced to an acceptable level before
proceeding. Background from method analytes or contaminants that interfere with
the measurement of method analytes must be below V3 the MRL. If the target
analytes are detected in the LRB at concentrations equal to or greater than this level,
then all data for the problem analyte(s) must be considered invalid for all samples in
the extraction batch.
9.5 CONTINUING CALIBRATION CHECK (CCC) - A CCC is a standard prepared
with all compounds of interest which is analyzed during an analysis batch to ensure
the stability of the instrument initial calibration. See Section 10.3 for concentration
requirements, frequency requirements, and acceptance criteria.
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9.6 LABORATORY FORTIFIED BLANKS - A LFB is required with each extraction
batch. The fortified concentration of the LFB should be rotated between, low,
medium and high concentrations from day to day. The low concentration LFB must
be as near as practical to, but no more than two times the MRL. Similarly, the high
concentration should be near the high end of the calibration range established during
the initial calibration (Sect. 10.2). Results of the LFB for the low level fortification
must be 50-150% of the true value. The concentration determined for the medium
and high LFBs must be 70-130% of the true value. If LFB results do not meet these
criteria for target analytes, then all data for the problem analyte(s) must be
considered invalid for all samples in the extraction batch.
9.7 SURROGATE RECOVERY - The surrogate standards are fortified into the aqueous
portion of all samples, LRBs, and LFMs and LFMDs prior to extraction. They are
also added to the calibration curve and calibration check standards. The surrogate is
a means of assessing method performance from extraction to final chromatographic
measurement.
9.7.1 When surrogate recovery from a sample, blank, or CCC is <70% or
>130%, check (1) calculations to locate possible errors, (2) standard
solutions for degradation, (3) contamination, and (4) instrument
performance. If those steps do not reveal the cause of the problem,
reanalyze the extract.
9.7.2 If the extract reanalysis meets the surrogate recovery criterion, report only
data for the reanalyzed extract.
9.7.3 If the extract reanalysis fails the 70-130% recovery criterion, the analyst
should check the calibration by analyzing the most recently acceptable
calibration standard. If the calibration standard fails the criteria of Section
9.7.1, recalibration is in order per Section 10.2. If the calibration standard
is acceptable, extraction of the sample should be repeated provided the
sample is still within the holding time. If the sample re-extract also fails
the recovery criterion, report all data for that sample as suspect/surrogate
recovery.
9.8 LABORATORY FORTIFIED SAMPLE MATRIX AND DUPLICATE (LFM AND
LFMD) - Analysis of LFMs are required in each extraction batch and are used to
determine that the sample matrix does not adversely affect method accuracy. If the
occurrence of target analytes in the samples is infrequent, or if historical trends are
unavailable, a second LFM, or LMFD, must be prepared, extracted, and analyzed
from a duplicate of the field sample used to prepare the LFM to assess method
precision. Extraction batches that contain LFMDs will not require the analysis of a
Field Duplicate (Sect. 9.8). If a variety of different sample matrices are analyzed
regularly, for example, drinking water from groundwater and surface water sources,
532-16
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method performance should be established for each. Over time, LFM data should be
documented for all routine sample sources for the laboratory.
9.8.1 Within each extraction batch, a minimum of one field sample is fortified
• as a LFM for every 20 samples extracted. The LFM is prepared by spiking
: a sample with an appropriate amount of the appropriate Analyte PDS
(Sect. 7.2.3). Select a spiking concentration at least twice the matrix
background concentration, if known. Use historical data or rotate through
the designated concentrations to select a fortifying concentration.
Selecting a duplicate bottle of a sample that has already been analyzed aids
in the selection of appropriate spiking levels.
9.8.2 Calculate the percent recovery (R) for each analyte using the equation
c
• where
A = measured concentration in the fortified sample
B = measured concentration in the unfortified sample, and
C = fortification concentration.
9.8.3 Analyte recoveries may exhibit a matrix bias. For samples fortified at or
above their native concentration, recoveries should range between 70 -
130%, except for thidiazuron which should be recovered at 60 - 120%.
For LFM fortification at the MRLj 50 to 150% recoveries are acceptable.
If the accuracy of any analyte falls outside the designated range, and the
. laboratory-performance for that analyte is shown to be in control in the
LFB, the recovery, isjudged to be matrix biased. The result for that
• ' - analyte in the unfortified sample is labeled suspect/matrix to inform the
data user that the results are suspect due to matrix effects.
9.8.4 If a LFMD is analyzed instead of a Field Duplicate, calculate the relative
percent difference (RPD) for duplicate LFMs (LFM and LFMD) using the
••'.•' equation
RPD= -LFMD ^ Q
(LFM+LFMD)/2
RPDs for duplicate LFMs should fall in the range of + 30% for samples
fortified at or above their native concentration. Greater variability may be
observed when LFMs are spiked near the MRL. At the MRL, RPDs
532-17
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should fall in the range of + 50% for samples fortified at or above their
native concentration. If the RPD of any analyte falls outside the
designated range, and the laboratory performance for that analyte is shown
to be in control in the LFB, the recovery is judged to be matrix biased.
The result for that analyte in the unfortified sample is labeled
suspect/matrix to inform the data user that the results are suspect due to
matrix effects.
9.9 FIELD DUPLICATES (FD1 AND FD2) - Within each extraction batch, a minimum
of one field duplicate (FD) or LFMD (Sect. 9.8) must be analyzed. FDs serve as a
check the precision associated with sample collection, preservation, and storage, as
well as laboratory procedures. If target analytes are not routinely observed in field
samples, a LFMD should be analyzed to substitute for this requirement. Extraction
batches that contain LFMDs will not require the analysis of a Field Duplicate.
9.9.1 Calculate the relative percent difference (RPD) for duplicate
measurements (FD1 and FD2)using the equation
SPD- FD1-FD2 .(100)
FD2~)/2
RPDs for duplicates should fall hi the range of ± 30%. Greater variability
may be observed when analyte concentrations are near the MRL. At the
MRL, RPDs should fall in the range of + 50%. If the RPD of any analyte
falls outside the designated range, and the laboratory performance for that
analyte is shown to be in control in the LFB, the recovery is judged to be
matrix biased. The result for that analyte in the unfortified sample is
labeled suspect/matrix to inform the data user that the results are suspect
due to matrix effects.
9.10 QUALITY CONTROL SAMPLES (QCS) - Each time that new standards are
prepared or a new calibration curve is run, analyze a QCS from a source different
from the source of the calibration standards. The QCS may be injected as a
calibration standard, or fortified into reagent water and analyzed as a LFB. If the
QCS is analyzed as a continuing calibration check, then the acceptance criteria are
the same as for the CCC. If the QCS is analyzed as a LFB, then the acceptance
criteria are the same as for an LFB. If measured analyte concentrations are not of
acceptable accuracy, check the entire analytical procedure to locate and correct the
problem source.
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10. CALIBRATION AND STANDARDIZATION
10.1 After initial calibration is successful, a Continuing Calibration Check is required at
the beginning and end of each analysis batch, and after every tenth sample (Sect.
10.3). Initial calibration should be repeated each time a major instrument
modification or maintenance is performed.
10.2 INITIAL CALIBRATION
10.2.1 Establish HPLC operating parameters equivalent to the suggested
conditions in Section 17, Table 1. The system is calibrated using the
external standard technique. For this method, a PDA detector was used
and the analyte absorbance at 240 or 245 nm was used in order to
maximize target compound signal relative to the background interferences.
Other HPLC conditions may be used as long as all QC requirements in
Section 9 are met.
10.2.2 Prepare a set of at least 5 calibration standards as described in Section
7.2.4. The lowest concentration of calibration standard must be at or
below the MRL, which will depend on system sensitivity.
10.2.3 INJECTION VOLUME - Optimum injection volume for the primary
column may vary between HPLC instruments when a sample is dissolved
in an organic solvent such as methanol. Prior to establishing the initial
calibration, the injection volume must first be determined. Peak
asymmetry on the primary column was occasionally noted on one of the
two instruments used to develop this method. This asymmetry only
occurred with 20 uL injections. This phenomena is attributed to the
difference in elutropic strength between the initial mobile phase
composition (phosphate buffer/acetonitrile; 60/40) and the extract solvent
composition (100% methanol). In all cases, the asymmetry was eliminated
by reducing the injection volume to 10 uL. Prior to establishing the initial
calibration curve, acceptable chromatographic performance is determined
by calculating the Peak Gaussian Factor (PGF).
10.2.3.1 Section 17 (Table 3) lists MDLs on the primary column using two
injection sizes: 20 uL and 10 uL. Inject a 20 uL aliquot of the
medium level calibration standard on the primary column using
the suggested conditions listed in Section 17, Table 1. Determine
the PGF for the analyte fluometuron using the equation
1.83 x W05
PGF =
W0.,
532-19
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where,
W 0.5 is the peak width at half height, and
W 01 is the peak width at tenth height.
If fluometuron has not been included in the calibration standards, one of
the surrogate compounds may be substituted.
NOTE: Values for W „ 5 and W 0-1 can be attained via most data
acquisition software packages. If these values are manually
measured, the analyst should limit the retention time window to
enlarge the peak of interest allowing accurate determination of the
PGF. Inaccurate measurements may result when using a
chromatogram of the entire analysis run.
10.2.3.2 If the PGF is in the range of 0.90 to 1.10, the peak shape is
considered acceptable, and a 20 uL injection may be used. If not,
injection volume should be reduced, and the PGF redetermined as
described in Section 10.2.3.1.
10.2.4 Generate a calibration curve for each analyte by plotting the peak response
(area is recommended) against analyte concentration. Both instruments
used during method development yielded linear curves for the target
analytes over the concentration range of interest. However, data may be fit
with either a linear regression (response vs concentration) or quadratic fit
(response vs concentration). Alternately, if the ratio of the analyte peak
area to concentration (or response factor) is relatively constant (RSD <
30%) an average response factor may be used to calculate analyte
concentration. Siduron separates into two isomers (labeled A & B in Sect.
17, Figure 1). The responses of the two isomers should be added together
before plotting against the concentration.
10.2.5 Repeat steps 10.2.1 through 10.2.4 for the confirmation column.
Laboratories may choose to wait to establish an initial calibration curve for
the confirmation column until they have samples with positive results that
require confirmation; however, this step must be successfully completed
prior to confirming sample results.
10.3 CONTINUING CALIBRATION CHECK (CCC) - Minimum daily calibration
verification is as follows. Verify the initial calibration at the beginning and end of
each group of analyses, and after every tenth sample during analyses. (In this
context, a "sample" is considered to be a Field sample. LRBs, LFBs, LFMs, LFMDs
and CCCs are not counted as samples.) The beginning CCC each day should be at
or below the MRL in order to verify instrument sensitivity prior to any analyses.
532-20
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Subsequent CCCs should alternate between a medium and high concentration
standard.
10.3.1 Inject an aliquot of the appropriate concentration calibration solution and
analyze with the same conditions used during the initial calibration.
10.3.2 Calculate the concentration of each analyte and surrogate in the check
standard. The calculated amount for each analyte for medium and high
level CCCs must be ± 30% of the true value. The calculated amount for
the lowest calibration point for each analyte must be within ±50% of the
true value. If these conditions do not exist, then all data for the problem
analyte must be considered invalid, and remedial action should be taken
which may require recalibration. Any field sample extracts that have been
analyzed since the last acceptable calibration verification should be
reanalyzed after adequate calibration has been restored.
11. PROCEDURE
11.1 Important aspects of this analytical procedure include proper preparation of
laboratory glassware and sample containers (Sect. 4.1), and sample collection and
storage (Sect. 8). This section details the procedures for sample preparation, solid
phase extraction (SPE) using cartridges or disks, and extract analysis.
11.2 SAMPLE PREPARATION
11.2.1 Samples are preserved, collected and stored as presented in Section 8. All
field and QC samples must contain the preservatives listed in Section
8.1.2, including the LRB and LFB. Determine sample volume. The
sample volume may be measured directly in a graduated cylinder to the
nearest 10 mL. To minimize the need to use a different graduated cylinder
for each sample, an indirect measurement may be done in one of two
ways: by marking the level of the sample on the bottle or by weighing the
sample and bottle to the nearest 10 g. After extraction, proceed to Section
11.3.5 for final volume determination. The LRB and LFB may be
prepared by measuring 500 mL of reagent water into an erlenmeyer flask.
11.2.2 Add an aliquot of the SUR PDS (Sect. 7.2.1.2) to all samples and mix by
swirling the sample. Addition of 10 uL of a 500 ug/mL SUR PDS to a 500
mL sample will result in a concentration of 10 ug/L.
11.2.3 If the sample is a LFB or LFM, add the necessary amount of analyte PDS.
Swirl each sample to ensure all components are properly mixed.
11.2.4 Proceed with sample extraction. Refer to Section 11.3 if SPE cartridges
are being used. Refer to Section 11.4 if SPE disks are being used.
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11.3 CARTRIDGE SPE PROCEDURE - Proper conditioning of the solid phase can have
a marked effect on method precision and accuracy. This section describes the SPE
procedure using the equipment outlined in Section 6.9 in its simplest, least
expensive mode without the use of the alternate transfer system or robotics systems
(Sect. 6.9.2 and 6.9.6) This configuration was used to collect data presented in
Section 17.
11.3.1 CARTRIDGE CONDITIONING - Once the conditioning of the cartridge
is started, the cartridge must not be allowed to gos dry until the last portion
of the sample is filtered through it. If the cartridge goes dry during the
conditioning phase, the conditioning must be started over. However, if the
cartridge goes dry during sample extraction, the analyte and surrogate
recoveries may be affected. If this happens the analyst should make note
of this as this sample may require re-extraction due to low surrogate
recoveries.
11.3.1.1 CONDITIONING WITH METHANOL - Assemble the
extraction cartridges into the vacuum manifold. Rinse each
cartridge with two, 5 mL aliquots of methanol, allowing the
sorbent to soak in the methanol for about 30 seconds by turning
off the vacuum temporarily during the first rinsing. Do not allow
the methanol level to go below the top of the cartridge packing.
11.3.1.2 CONDITIONING WITH REAGENT WATER - Follow the
methanol rinse with two, 5 mL aliquots of reagent water being
careful not to allow the water level to go below the cartridge
packing. Turn off the vacuum. Add approximately 5 mL
additional reagent water to the cartridge, and attach a reservoir (or
transfer tube - Sect. 6.9.2) before adding sample to the cartridge.
11.3.2 CARTRIDGE EXTRACTION - Prepare samples, including QC samples,
as specified in Section 11.2. The samples may be added to the cartridge
using either a large reservoir attached to the cartridge or using a transfer
tube from the sample bottle to the cartridge.
11.3.2.1 SAMPLE ADDITION USING RESERVOIRS - Attach a
reservoir to the conditioned cartridge. Fill the reservoir (Sect.
6.9.2) with sample and then turn on the vacuum. Adjust the
vacuum so that the approximate flow rate is about 20 mL/min
(minus 9-10 in Hg.). Care must be taken to add additional aliquots
of sample to the reservoir to keep the cartridge packing from
going dry before all the sample has been extracted. Rinse the
sample container with reagent water and add to the reservoir after
532-22
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the last addition of sample, but before the cartridge goes dry.
After all of the sample has passed through the SPE cartridge,
detach the reservoir and draw air through the cartridge for 15
minutes at full vacuum (minus 10-15 in Hg). Turn off and release
the vacuum.
11.3.2.3 SAMPLE ADDITION USING TRANSFER TUBES - If the
sample transfer tubes are employed, make sure the transfer tube is
attached to the conditioned cartridge before turning on the
vacuum. Adjust the vacuum to a similar flow rate of
approximately 20 mL/min (minus 9-10 in Hg). Rinse down the
sample container with reagent water as it approaches dryness.
After all of the sample has passed through the SPE cartridge,
detach the transfer tube and draw air through the cartridge for 15
minutes at full vacuum (minus 10-15 in Hg). Turn off and release
the vacuum.
11.3.3 CARTRIDGE ELUTION - Lift the extraction manifold top and insert
collection tubes into the extraction tank to collect the extracts as they are
eluted from the cartridge. Add approximately 3 mL of methanol to the top
of each cartridge. Pull enough of the methanol into the cartridge at low
vacuum to soak the sorbent. Turn off the vacuum and vent the system.
Allow the sorbent to soak in methanol for approximately 30 seconds. Start
a low vacuum (minus 2-4 in Hg) and pull the methanol through in a
dropwise fashion into the collection tube. Repeat this elution a second
time with approximately 2 mL of methanol, and then a third time with
approximately 1 mL.
11.3.4 EXTRACT CONCENTRATION - Concentrate the extract to about 0.5
mL in a warm water bath (at about 40OC) under a gentle steam of nitrogen.
Transfer to a 1 mL volumetric flask, rinsing the collection tube with small
amounts of methanol. Adjust to volume with methanol. Filter the sample
using a 1 mL syringe and filter (Sects. 6.7 & 6.8) into an appropriate
autosampler vial.
11.3.5 SAMPLE VOLUME DETERMINATION - If the level of the sample was
marked on the sample bottle, use a graduated cylinder to measure the
volume of water required to fill the original sample bottle to the mark
made prior to extraction. Determine to the nearest 10 mL. If using weight
to determine volume, weigh the empty bottle to the nearest 10 g and
determine the sample weight by subtraction of the empty bottle from the
original weight (Sect. 11.2.1). In either case, the sample volume will be
used in the final calculations of analyte concentration (Sect. 12.4).
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11.4 DISK SPE PROCEDURE - Proper conditioning of the solid phase can have a
marked affect on method precision and accuracy. This section describes the SPE
procedure using the equipment outlined in Section 6.10 in its simplest, least
expensive mode without the use of robotics systems (Sect. 6.10.4). This
configuration was used to collect data presented in Section 17.
11.4.1 DISK CONDITIONING - Once the conditioning of the disk is started, the
disk must not be allowed to go dry until the last portion of the sample is
filtered through it. If the disk goes dry during the conditioning phase, the
conditioning must be started over. However, if the disk goes dry during
sample extraction, the analyte and surrogate recoveries may be affected. If
this happens the analyst should make note of this as this sample may
require re-extraction due to low surrogate recoveries.
11.4.1.1 CONDITIONING WITH METHANOL - Assemble the
extraction glassware in the vacuum manifold, placing the disks on
a support screen between the funnel and base. 'Rinse each disk
with two, 10 mL aliquots of methanol to the funnel, allowing the
sorbent to soak for about 30 seconds by pulling approximately
ImL through the disk and turning off the vacuum temporarily
during the first rinsing. Draw the methanol through the disk until
it is 3-5 mm above the disk surface, adding more methanol if
needed to keep the methanol from going below this level.
11.4.1.2 CONDITIONING WITH WATER - Follow the methanol rinse
with two, 10 mL aliquots of reagent water being careful to keep
the water level at 3-5 mm above the disk surface. Turn off the
vacuum.
11.4.2 DISK EXTRACTION - Prepare samples, including QC samples, as
specified in Section 11.2. Fill the extraction funnel containing the
conditioned disk with sample and turn on the vacuum. Care must be taken
to add additional aliquots of sample to the funnel to keep the disk from
going dry before all the sample has been extracted. Rinse the sample
container with reagent water and add to the funnel after the last addition of
sample, but before the disk goes dry. After all of the sample has passed
through the SPE disk, draw air through the disk for 15 minutes at full
vacuum (minus 10-15 in Hg). Turn off and release the vacuum.
11.4.3 DISK ELUTION - Detach the glassware base from the manifold without
disassembling the funnel from the base. Dry the underside of the base.
Insert collection tubes into the manifold to collect the extracts as they are
eluted from the disk. The collection tubes must fit around the drip tip of
the base to ensure the collection of all of the eluent. Reattach the base to
532-24
-------
the manifold. Add approximately 5 mL of methanol to the top of each
disk. Pull enough of the methanol into the disk to soak the sorbent. Turn
off the vacuum and vent the system. Allow the sorbent to soak in
methanol for approximately 30 seconds. Start the vacuum and pull the
methanol through. Attempt to pull the methanol through in a dropwise
fashion into the collection tube by slowly turning the valve (which controls
the flow through the funnel) until the methanol starts eluting into the
collection tube. Repeat this elution a second time with approximately 4
mL of methanol, and then a third time with approximately 3 mL.
11.4.4 EXTRACT CONCENTRATION - Proceed with extract concentration as
in Section 11.3.4.
11.4.5 SAMPLE VOLUME DETERMINATION - Proceed with sample volume
determination as in Section 11.3.5.
11.5 SOLVENT EXCHANGE FOR CONFIRMATION ANALYSIS - Samples that will
be confirmed must be exchanged into reagent water/acetonitrile (60/40). To
accomplish this, transfer the remaining 980 uL of the extract to a 1 mL volumetric
(or other appropriate collection tube). Mark the sample volume. Take the extract to
dryness in a warm water bath (at ~ 40°C) under a gentle steam of nitrogen.
Reconstitute the residue with a mixture of reagent water/acetonitrile (60/40) to the
. mark made before the extract was taken to dryness. Care must be taken to redissolve
the film as thoroughly as possible. Use of a vortex mixer is recommended. Transfer
to an appropriate autosampler vial.
Note: Recovery experiments have been conducted to investigate the effect
of drying time on compound recoveries by allowing the extract to sit an
additional 1,2, or 4 hours in the bath after being taken to dryness. These
studies indicate that: 1) the best recoveries are obtained when extracts are
reconstituted immediately after the methanol is removed; 2) recoveries
decrease with increasing time in the bath; and 3) carbazole recovery is the
most sensitive to the additional time. In our experiments, carbazole had
. acceptable recovery criteria (Sect. 12.3) at 2 additional hours of drying, but
had exceeded the acceptable range after 4 hours of additional tune.
11.6 ANALYSIS OF SAMPLE EXTRACTS
11.6.1 Establish operating conditions as summarized in Table 1 of Section 17 for
the HPLC system. Confirm that retention times, compound separation and
resolution on the primary and secondary columns are similar to those listed
in Tables 1 and 2 and Figures 1 and 2, respectively.
11.6.2 Determine the optimal injection volume. Establish a valid initial
calibration following the procedures outlined in Section 10.2 using the
532-25
-------
optimum injection size. Complete the IDC requirements described in
Section 9.2.
11.6.3 Establish an appropriate retention time window for each target and
surrogate to identify them in the QC and field samples. This should be
based on measurements of actual retention time variation for each
compound in standard solutions analyzed on the HPLC over the course of
time. Plus or minus three times the standard deviation of the retention
time for each compound while establishing the initial calibration and
completing the IDC can be used to calculate a suggested window size;
however, the experience of the analyst should weigh heavily on the
determination of the appropriate retention window size.
11.6.4 Check system calibration by analyzing a CCC (Sect. 10.3) and begin to
inject aliquots of field and QC samples using the same injection volume
and conditions used to analyze the initial calibration.
11.6.5 The analyst must not extrapolate beyond the established calibration range.
If an analyte peak area exceeds the range of the initial calibration curve,
the extract may be diluted with methanol. Acceptable surrogate
performance (Sect. 9.7) should be determined from the undiluted sample
extract. If confirmation analyses require dilution, this must be done using
reagent water/acetonitrile (Sect. 11.5) Incorporate the dilution factor into
final concentration calculations. Any dilutions will also affect analyte
MRL.
12. DATA ANALYSIS AND CALCULATION
12.1 Identify the method analytes hi the sample chromatogram by comparing the retention
time of the suspect peak to retention time of an analyte peak in a calibration standard
or the laboratory fortified blank. Surrogate retention times should be confirmed to
be within acceptance limits (Sect. 11.6.3) even if no target compounds are detected.
12.2 Calculate the analyte concentrations using the initial calibration curve generated as
described in Section 10.2. Quantitate only those values that fall between the MRL
and the highest calibration standard. Samples with target analyte responses that
exceed the highest standard require dilution and reanalysis (Sect. 11.6.5).
12.3 Positive results should be confirmed on the confirmation column (Sect. 17, Table 2)
that has been initially calibrated, and confirmed to still be in calibration by analyzing
appropriate CCCs (Sect. 10.3) prior to the confirmation analysis. Quantitated
values for the targets and surrogates on the confirmation column should be 50 -
150% of the primary column result. If so, report the more accurate primary column
result. If not, report the lower of the 2 values and mark the results as
532-26
-------
suspect/confirmation to inform the data user that the results are suspect due to lack
of confirmation. If values are taken from the confirmation column, both surrogates
must meet recovery acceptance criteria.
Note: The PDA spectra of these compounds do not have sufficient resolution to
definitively identify each target compound in this method.
12.4 Adjust the calculated concentrations of the detected analytes to reflect the initial
sample volume and any dilutions performed.
12.5 Prior to reporting the data, the chromatogram should be reviewed for any incorrect
peak identification or poor integration.
12.6 Analyte concentrations are reported in ug/L. Calculations should use all available
digits of precision, but final concentrations should be rounded to an appropriate
number of significant figures.
13. METHOD PERFORMANCE
13.1 PRECISION, ACCURACY, AND MDLs - Method detection limits (MDLs) are
presented in Table 3 and were calculated using the formula present in Section 9.2.4.
Tables for these data are presented in Section 17. Single laboratory precision and
accuracy data are presented for three water matrices: reagent water (Table 5);
chlorinated, "finished" surface water (Table 6); and chlorinated, "finished" ground
water (Table 7).
13.2 COMPOUND STABILITY - Thidiazuron and diflubenzuron are extremely sensitive
to free chlorine. LFM recoveries were found to drop to 0% for thidiazuron and
about 5% for diflubenzuron in the time required to extract samples in the presence of
free chlorine. Other analytes are sensitive to free chlorine over time, even within the
recommended sample holding time of 14 days. For example, LFM recoveries for
diuron drop to about 12% on day 14 in a finished surface water with a residual free
chlorine concentration of 0.8 mg/L. This illustrates the importance of proper sample
preservation (Sect. 8). During method development, it was experimentally
determined that the 2.5 g Trizma preset crystals (Sect. 7.1.6.2) was sufficient to
dechlorinate a 500 mL sample with up to approximately 10 mg/L free chlorine as
measured colorimetrically. This level is about 2.5 times the maximum allowable
free chlorine residual in municipal tap waters promulgated in Stage I of the
Disinfectant/Disinfection By-product Rule(6) and so should be sufficient to reduce
free chlorine in samples from public water systems.
13.3 ANALYTE STABILITY STUDIES
13.3.1 FIELD SAMPLES - Chlorinated finished surface water samples, fortified
with method analytes at 10 ug/L, were preserved and stored as required in
532-27
-------
Section 8. The average of triplicate analyses, conducted on days 0, 2, 7,
and 14, are presented in Section 17, Table 8. These data document the 14-
day sample holding time. . - .
13.3.2 EXTRACTS - Extracts from the day 0 extract holding time study
described above were stored below 0 °C and analyzed on.days 0, 8,14, and
21. The data presented in Section 17, Table 9, document the 21-day
extract holding time. .. ' ,
14. POLLUTION PREVENTION .. •,
14.1 This method utilizes solid phase extraction technology to extract analytes from
water. It requires the use of very small volumes of organic solvent and very small
quantities of pure analytes, thereby minimizing the potential hazards to both the
analyst and the environment as compared to the use of large volumes of organic
solvents in conventional liquid-liquid extractions.
14.2 For information about pollution prevention that may be applicable to laboratory .
operations, consult "Less is Better: Laboratory Chemical Management for Waste
Reduction" available from the American Chemical Society's Department of
Government Relations and Science Policy, 1155 16th Street NW, Washington, D.C.,
20036.
15. WASTE MANAGEMENT
15.1 The analytical procedures described in this method generate relatively small amounts
of waste since only small amounts of reagents and solvents are used. The matrices
of concern are finished drinking water or source water. However, the Agency
requires that laboratory waste management practices be conducted consistent with all
applicable rules and regulations, and that laboratories protect the air, water, and land
by minimizing and controlling all releases from fume hoods and bench operations.
Also, compliance is required with any sewage discharge permits and regulations,
particularly the hazardous waste identification rules and land disposal restrictions.
For further information on waste management, consult "The Waste Management
Manual for Laboratory Personnel" also available from the American Chemical
Society at the address in Section 14.2.
16. REFERENCES
1. Glaser, J.A., Foerst, D.L., McKee, G.D., Quave, S.A., and Budde, W.L., "Trace
Analyses for Wastewaters," Environ. Sci- Technol. 1981, 15, 1426-1435.
2. "OSHA Safety and Health Standards, General Industry," (29CRF1910).
Occupational Safety and Health Administration, OSHA 2206, (Revised, Jan. 1976).
532-28
-------
3. "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.
4. "Safety In Academic Chemistry Laboratories," 3rd Edition, American Chemical
Society Publication, Committee on Chemical Safety, Washington, D.C., 1979.
5. 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.
6. Federal Register, December 16, 1998, 63 (241) 69390-69476.
532-29
-------
17. TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. CHROMATOGRAPHIC CONDITIONS AND RETENTION TIME
DATA FOR THE PRIMARY COLUMN
Peak
Number
(Figure 1)
1
2
3
4
5
6
7
8
9
10
11
Analyte ,
Tebuthiuron
Thidiazuron
Monuron (SUR)
Fluometuron
Diuron
Propanil
Siduron A
Siduron B
Linuron
Carbazole (SUR)
Diflubenzuron
Retention ;
-'-Time (min.*)
x * \ "•
2.03
2.48
2.80
4.45
5.17
8.53
8.91
9.76
11.0
12.8
13.9
Primary Column: Symmetry 4.6 * 150 mm packed with 3.5 um C18 stationary phase.
Conditions:
Solvent A
Solvent B
40% B
linear gradient 40-50% B
linear gradient 50-60% B
linear gradient 60-40%B
Flow rate
Wavelength
25 mM phosphate buffer
acetonitrile
0-9.5 minutes
9.5-10.0 minutes
10.0-14.0 minutes
14.0-15.0 minutes
1.5 mL/min
245 nm
Equilibration time prior to next injection 15 minutes.
532-30
-------
TABLE 2. CHROMATOGRAPHIC CONDITIONS AND RETENTION TIME
DATA FOR THE CONFIRMATION COLUMN
4J;. ••>--:•<
1,,-^^aa^-^'-
^fiS^:?
1 .
2
3
4
5
6
7
8
9
10
11
- /".'""•^iX"* *••'••<-, • . i—'^j^j*
%4^T"'^< • -v • -^
f r\~/ f tfy ^J.rr'-i f ' • '* .j,_ ^' ^jv'jj, - <^
'&.;...'.* 'fbialyte^,;2%g>,^
:;^^*>w;; .' • •*•'',- ,, *•*
-:.:,„. ,^'%>-^!*rs,_ . . -^w
— , . ,. . yrs..'. «. w?***",' '',.,•* *«*&' •»?"
*--l-4;/ 'S*- 'sl^v ':
ws • <; ~>f?5, v;l->'
2.56
3.98
4.93
5.94
7.67
9.53
10.1
10.8
12.2
14.3
15.2
Confirmation Column: Supelcosil 4.6 x 150 mm packed with 5 um cyanopropyl
stationary phase.
Conditions:
Solvent A
Solvent B
linear gradient 20% B
linear gradient 20-40% B
40% B
linear gradient 40-20% B
Wavelength
25 mM phosphate buffer
acetonitrile
0-11.0 minutes, 1 .5 mL/min. flow rate
1 1 .0-12.0 minutes, 1 .5 mL/min flow rate
hold to 16 minutes at 1.5 mL/min. flow rate,
step to 2.0 mL/min flow rate at 16 min. and
hold to 20 min.
20.0-20.1 minutes, 2.0 mL/min. flow rate
240 nm
Equilibration time prior to next injection 15 minutes.
532-31
-------
TABLE 3. METHOD DETECTION LIMITS FOR 2 INJECTION VOLUMES IN
REAGENT WATER WITH CARTRIDGE AND DISK EXTRACTION
TECHNIQUES ON BOTH THE PRIMARY AND CONFIRMATION
COLUMNS
'** ,'A-S t ""^
Table 3 A. Cartridge Extraction, Primary Column Y/< -x^-r.
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&B
Linuron
Diflubenzuron
**<. '~ '•
Spiking Cone, (ug/L) * ,
20uLinj.
0.050
0.100
0.050
0.050
0.050
0.100
0.050
0.050
10 uL inj.
0.100
0.100
0.100
0.100
0.300
0.600
0.300
0.100
MDiyxug/L) ;; —
20uLinj.
0.032
0.035
0.013
0.010
0.023
0.024
0.062
0.014
10 uL inj.
0.071
0.047
0.027
0.026
0.084
0.091
0.067
0.033
Table 3B. Disk Extractions,, Primary Column, 20 uCrlnjectipii, / ,
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&B
Linuron
Diflubenzuron
Spiking Conel (ug/L)
0.050
0.100
0.050
0.050
0.100
0.100
0.100
0.050
;":— -MDLa'(ug/L) -,-
0.046
0.047
0.028
0.018
0.071
0.067
0.032
0.035
a Method detection limit samples extracted over 3 days for 7 replicates and analyzed using
conditions described in Table 1.
532-32
-------
Table 3C. Caijtriffge Extractio,n^Gonfirinatidii5Coluiiin^20 uL b^ctiqta^
-'Analyte /
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&B
Linuron
Diflubenzuron
. Spiking Cone* (wg/L)
0.300
0.300
0.300
0.300
0.300
0.300
0.300
0.300
^ MBLbVtng^X'>^ .
0.145
0.143
0.065°
0.056
0.066
0.136
0.085
0.126
b Method detection limit samples extracted over 3 days for 7 replicates and analyzed using
conditions described in Table 2.
0 MDLs for fluometuron were reinjected due to an interfering peak which tended to coelute
with fluometuron.
532-33
-------
TABLE 4. PRECISION, ACCURACY AND SIGNAL-TO-NOISE COMPARISON FOR
TWO HPLC SYSTEMS FOR LOW LEVEL SPIKES IN REAGENT WATER
EXTRACTED WITH CARTRIDGES AND ANALYZED USING THE
PRIMARY COLUMN
Table 4. Precision, Accuracy and S/N Comparison; 1 0;iiLIn|ecti6n/ Primary Column ,
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
SiduronA&Ba
Linuron
Diflubenzuron
Monuron (SUR)a
Carbazole (SUR)a
HPLC System #F
Concentration = 1.0 ug/L(n=7)
Mean
%Rec.c
100
103
99
102
96
100
108
100
105
97
RSD
(%)
1.8
2.4
2.2
1.8
3.6
3.0
1.6
2.0
2.8
2.7
S/N,
Ratio" >
84
33
43
44
28
16
12
22
NC
NC
,,..,_ HP^CiSyftem #2?>
Concentration = ^^ Lft/(ug/L(n=7)
" Mean '^
~;,%;Rec/
105
102
104
104
100
109
103
98
94
104
RSD
'• 1%)^
2.7
!-9
2.3
3.8
6.4
11
4.1
3.3
2.3'
2.7
,S/N "
:/llati6f '
17
36
35
34
16
40
21
11
NC
NC
NC: Not Calculated
"HPLC System 1 was equipped with a photodiode array detector that employed a 1 cm path
length.
bHPLC System 2 was equipped with a photodiode array detector that employed a 5 cm path
length.
cSeven replicates of the low level reagent water spikes were processed through the cartridge
extraction procedure and injected on both instruments.
dSignal-to-noise ratios were calculated for each peak by dividing the peak height for each
compound by the peak-to-peak noise for each peak, which was determined for each
component from the method blank over a period of time equal to the full peak width.
532-34
-------
TABLES. PRECISION AND ACCURACY DATA IN REAGENT WATER
'*\, /Table SAr Cartndge, Extract to >: ;
*^ '» "<^
•*' ~' •". "'
Analyte, ' „ "i"~ - "
% " r '^ i"^/
«,/, / ^ A
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&B
Linuron
Diflubenzuron
Monuron (SUR)a
Carbazole (SUR)a
e^oncentratiotf =1 «g/L (n=7> '
Mean
X;RecoveryX
'^ (%1
107
106
106
107
105
106
104
107
100
96.7
Relative ;, •
^ ,"Si|andard ""'"
'Deiiatipnt(%X(
2.4
1.1
0.9
1.0
.1.7
1.9
1.6
1.2
1.8
1.0
''Coneenlr^pn^^ ug^/(p=7)-,,
" --,' Mean •-' f
-&ec'owry:f%)::
>< ^ ^^
97.9
96.7
97.6
97.5
97.2
97.8
97.4
96.0
100.
96.1
Relative1, c
'^ta'ndalrd-,^.
Bevlatioii.Cyo^
1.4
1.6
1.5
0.0
1.4
1.6
1.5
1.4
2.0
1.5
*t Table-SB. BiskrExtractions, Prima^^dlunin, 2tt uE Ilnjectiott, V'^l, V"
y, %7 ''»„
" J" '' C-f " <
Analyte ;x "^-V^
if . '"- ""-~.- .
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&B
Linuron
Diflubenzuron
Monuron (SUR)a ,
Carbazole (SUR)a
;(EonceritratioS^X,ti_g/L (n=7),
: '-Me^an;. ,,,
^ecovery"l%f
104
99.8
100
104
101
110
99.2
102
102
96.9
'RelatiiaB"? j7
v Standard
:bey|a:tion'(%l
2.2
4.8
4.2
5.9
2.7
5.0
4.8
4.2
2.6
3.3
Concentration, ="-30 iig^(a=7)
/, . Mean ' .>^,
-" Recoveify
'i%r:
101
101
101
101
101
103
99.0
100
99.0
95.1
• JRelattye"!"
* Slandard ' -
Deviation (%)
2.3
2.4
2.3
3.2
2.3
2.3
2.5
2.5
2.8
3.0
"Surrogate concentration in all samples is 10 ug/L. Chromatographic conditions are described in
Table 1.
532-35
-------
Table 5C. Cartridge Extraction, Confirmation Column, 2d ul^Injection "/<*
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&Ba
Linuron
Diflubenzuron
Monuron(SUR)b
Carbazole (SUR)b
Concentration - 1 ug/L (a=7)
Mean%
Recovery
100
93.6
87.6
96.9
88.0
91.0
79.8
83.7
105
93.8
Relative
Standard
Deviation (*&) -
4.9
5.1
12.4
3.9
5.9
7.0
13.3
6.9
4.3
6.0
Concentration = 30 ug/L (ja~7)
Mean % ~ '<
Recovery
103
101
97.0
102
103
103
99.8
80.4
102
83.6
Relative
Stajidard/r ,
( Deviation ,(%)
4.8
4.8
5.7
2.7
4.9
5.0
5.6
18.6
4.5
7.6
"Total siduron concentration is twice the concentration of the other target analytes: 2.0 and
60.0 ug/L.
bSurrogate concentration in all samples is 10 ug/L. Conditions described in Table 2.
532-36
-------
TABLE 6. PRECISION AND ACCURACY8 OF LOW AND HIGH LEVEL FORTIFIED
CHLORINATED SURFACE WATER USING CARTRIDGES
Kf > O <• «%^
f ^ f ^ ^
"'- "-f" ,-""'?*„,
"•Analyte"* -. ' - ^
'~K " ' «>
„ %$,^
*'? \ *tf&4^ - ^ 3&&X*^r
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
SiduronA&B
Linuron
Diflubenzuron
Monuron (SUR)b
Carbazole (SUR)b
Conjcentration = 1 u^6.(BF*?)5^
* s-f^ ^<^ K "^-^
, '^Mean',%^ *
.^.Mecovery % '?.
'*^ S<-^J,'^_
108
81
109
110
104
102
102
109
103
98
?%" ^Relative, ^
Standard ;
"Dc^yiatiOBL|%)
1.6
2.9
1.0
1.7
1.8
1.8
1.3
1.5
2.3
2.7
' "*•* ~il x^> ' x ^
'ConceBttratidnt^ 30 ug^Ek, (n=7)>
**' ^ ***"» X ^ >"
_ Mean,% *\
'•-*<> J8Lecorejt^s
'/ "X -k
96
84
96
96
96
96
96
95
97
94
*:•' "Rllative"; ~v
;sS|andard •
Devi&tioii (%)
1.7
1.9
1.5
1.6
1.5
1.6
1.6
1.7
2.3
1.9
"All data collected using a 20 uL injection volume on the primary column and conditions
described in Table 1.
Surrogate concentration in all samples is 10 ug/L.
532-37
-------
TABLE 7. PRECISION AND ACCURACY" OF LOW AND HIGH LEVEL FORTIFIED
CHLORINATED GROUND WATER USING CARTRIDGES
Analyte
Tebuthiuron
Thidiazuron
Fluometuron
Diuron
Propanil
Siduron A&B
Linuron
Diflubenzuron
Monuron (SUR)b
Carbazole (SUR)b
Concentration = 1 ug/L (n=7)
Mean%
Recovery
109
95
103
106
106
106
101
104
100
95
Relative, i
Standard^ '
Deviation
1.5
2.9
1.9
2.1
1.9
4.2
2.7
2.6
1.3
1.3
Concentration = 3JKwg/L (n=7^-
Mean% /
>»> •
^. Recovery^ ,
99
94
97
99
99
99
98
98
99
89
V Relive, .
* X ' "^
, ^Standard r/
/"D',eviatiqn.;Xj,
1.2
1.2
1.9
1.1
1.1
1.2
1.9
1.0
1.0
4.7
"All data collected using a 20 uL injection volume on the primary column and conditions
described in Table 1.
bSurrogate concentration in all samples is 10 ug/L.
532-38
-------
TABLE 8. SAMPLE HOLDING TIME DATA3 FOR SAMPLES FROM A
CHLORINATED SURFACE WATER, FORTIFIED WITH METHOD
ANALYTES AT 10 ug/L, WITH CUPRIC SULFATE AND TRIZMA (Sect.
8.1.2)
' J^r /• S~^" , •:<.**
Airalyfe ' /"- ~ „ ;"<-<
'"If ' S'lx > 1^ "x
Tebuthiuron
Thidiazuron
Fluometuron
Dinron
Propanil
Siduron A&B
Linuron
Diflubenzuron
Monuron (SUR)b
Carbazole (SUR)b
5;-f;:Day,'0"S:if
S^lijit6^
93
72
93
94
93
92
93
93
96
88
$$/"**"'{ f. * * i?
;;;^rRe^vl^
95
76
95
97
97
96
97
95
101
96
_* 5 Day-7 . ' *
,^% Recovery, ,
98
84
98
99
98
98
98
98
103
92
^"^'tf- ^
~% Recovery t
96
77
97
97
98
98
99
97
102
95
aStorage stability is expressed as a percent recovery value. Each percent recovery value
represents the mean of 3 replicate analyses. Relative Standard Deviations ([Standard
Deviation/Recovery] x 100) for replicate analyses were all less than 7.0%.
Samples analyzed using a 20 uL injection volume and conditions described in Table 1.
bSurrogate concentration hi all samples is 10 ug/L.
532-39
-------
TABLE 9. EXTRACT HOLDING TIME DATA3 FOR SAMPLES FROM A
CHLORINATED SURFACE WATER, FORTIFIED WITH METHOD
ANALYTES AT 10 ug/L, WITH CUPRIC SULFATE AND TRIZMA (Sect.
8.1.2)
Analyte
Tebutbiuron
Thidiazuron
Fluometuron
Diuron
Propanil
SiduronA&B
Linuron
Diflubenzuron
Monuron (SUR)
Carbazole (SUR)
Initial
Injection11
% Recovery
93
72
93
94
93
92
93
93
96
88
;,j Days".,,,,
Reinfection : ,/•
% Recovery
95
73
95
96
96
94
97
95
98
90
^.-Pay'ltfr-
^einjectioir ,
%Rex>^eiyl
103
80
107
99
105
106
102
107
94
91
"Storage stability is expressed as a percent recovery value. Each percent recovery value
represents the mean of 3 replicate analyses. Relative Standard Deviations ([Standard
Deviation/Recovery] * 100) for replicate analyses were all less than 5.0%.
bSame as day 0 sample hold time analysis.
Extract stability study consisted of the analysis of day 0 extracts stored at < 0° C and reinjected.
Samples were analyzed using a 20 uL injection volume and conditions described in Table 1.
532-40
-------
TABLE 10. INITIAL DEMONSTRATION OF CAPABILITY (IDC) REQUIREMENTS
fetttod "'
Reference
\v^
Sect. 9.2.1
Sect. 9.2.2
Sect. 9.2.3
Sect. 9.2.4
Sect. 10.2.3
f •»•* " *"» J&S&g's
^equjfremejtyt, ,
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-------
TABLE 11. QUALITY CONTROL REQUIREMENTS (SUMMARY)
Method
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Sect. 8.4
Sample and
Extract Holding
Times
Properly preserved samples
must be shipped at or below
10°C and may be held in the
lab at or below 6°C for 14
days. Extracts in methanol
may be stored at or below 0°C
for up to 21 days after
extraction. Samples
exchanged into 60/40 reagent
water/acetonitrile may be held
at or below 0°C for 7 days,
with the combined extract hold
time not to exceed 21 days.
Do not report data for
samples or extracts that
have not been properly
preserved or stored, or
that have exceeded their
holding time.
Sect. 9.4
Laboratory
Reagent Blank
(LRB)
Include LRB with each
extraction batch (up to 20
samples). Analyze prior to
analyzing samples and
determine to be free from
interferences. Each analysis
batch (Sect. 3.2) must include
either a LRB or an instrument
blank after the initial low level
CCC injection.
Demonstrate that all
target analytes are below
1/3 the intended MRL or
lowest CAL standard,
and that possible
interference from
extraction media do not
prevent the identification
and quantitation of
method analytes.
Sect. 9.7
Surrogate
Standards
Surrogate standards are added
to all calibration standards and
samples, including QC
samples.
Surrogate recovery must
be 70-130% of the true
value.
532-42
-------
Method, "'
feeierenee <
•s
Requirement
Acceptance iSritejria
Sect 9.8
Laboratory
Fortified Sample
Matrix (LFM)
With each extraction batch
(Sect. 3.1), a minimum of one
LFM is extracted and
analyzed. A duplicate LFM,
or LFMD, should be extracted
when occurrence of target
analytes is low. Laboratory
duplicate analysis is not
required for extraction batches
containing a LFMD.
Recoveries not within
70-130% of the fortified
amount may indicate a
matrix effect, with the
exception of thidiazuron
which should have
recoveries of 60-120%;
If a LFMD is analyzed
instead of a Laboratory
Duplicate, target RPDs
should be + 30%.
Sect. 9.9
Field Duplicates
(LDlandLD2)
Extract and analyze at least
one duplicate with each
extraction batch (20 samples
or less). A Laboratory
Fortified Sample Matrix
Duplicate may be substituted
for a Field Duplicate when the
occurrence of target analytes is
low.
RPDs should be + 30%.
Sect. 9.10
Quality Control
Sample
Analyze either as a CCC or a
LFB. Analyze at least.
quarterly, with each initial
calibration, or when preparing
new standards.
A QCS analyzed as a
CCC will have the same
acceptance criteria as a
CCC, while a QCS
analyzed as a LFB will
have the same criteria as
a LFB.
532-43
-------
Method
Reference
Requirement
Specification and Frequency
Aceeptance;Crite5ria
f*-t "st'sitf**/
Sect. 10.2
Initial Calibration
Use external standard
calibration technique to
generate a calibration curve
with five standards that span
the approximate range of 1 to
30 ug/L sample concentration.
Calibration curve fit options
are discussed in Sect. 10.2.4.
Analyze a QCS.
QCS must be + 30% of
true value.
When each calibration
standard is calculated as
an unknown using the
calibration curve, the
results must be 70-130%
of the true value for all
but the lowest standard.
The lowest standard must
be 50-150% of the true
value. The lowest CAL
standard concentration
must be as low or lower
than the intended MRL.
Sect. 10.2.3
Peak Gaussian
Factor (PGF)
Calculated prior to
establishing the initial
calibration.
A PGF range of 0.90 to
1.10 is acceptable.
Sect. 10.3
Continuing
Calibration
Check (CCC)
Verify initial calibration by
analyzing a low level CCC
prior to analyzing samples.
CCCs are then injected after
every 10 samples and after the
last sample, rotating
concentrations to cover the
calibrated range of the
instrument.
Recovery for each
analyte must be 70-130%
of the true value for all
but the lowest level of
calibration. The lowest
calibration level CCC
must be 50-150% of the
true value.
Sect. 12.3
Confirmation
Column Results
Positive results should be
confirmed using a chemically
dissimilar column.
Quantitated target values
should be ±50% of the
primary result. Primary
column results should be
reported. If reporting
confirmation results,
both surrogates must
meet + 30% criteria.
532-44
-------
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532-46
-------
METHOD 549.2 DETERMINATION OF DIQUAT AND PARAQUAT IN DRINKING
WATER BY LIQUID-SOLID EXTRACTION AND HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY WITH
ULTRAVIOLET DETECTION
Revision 1.0
June 1997
J.W. Hodgeson (USEPA), W.J. Bashe (Technology Applications Inc.), and J.W.
Eichelberger (USEPA) - Method 549.1, Revision 1.0 (1992)
J.W. Munch, USEPA, Office of Research and Development and W.J. Bashe, DynCorp/TAI
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
549.2-1
-------
METHOD 549.2
DETERMINATION OF DIQUAT AND PARAQUAT IN DRINKING WATER
BY LIQUID-SOLID EXTRACTION AND HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY WITH ULTRAVIOLET DETECTION
1. SCOPE AND APPLICATION
1.1 This is a high performance liquid chromatography (HPLC) method for the
determination of diquat (l,r-ethylene-2,2'-bipyridilium dibromide salt) and paraquat
(l,r-dimethyl-4,4'- bipyridilium dichloride salt) in drinking water sources and finished
drinking water (1).
Chemistry Abstract Services
Analytes Registry Number
Diquat 85-00-7
Paraquat 1910-42-5
1.2 When this method is used to analyze unfamiliar samples, compound identification
should be supported by at least one additional qualitative technique. The use of a
photodiode array detector provides ultraviolet spectra that can be used for the
qualitative corifirmation.
1.3 The method detection limits (MDL, defined in Sect. 13) (2) for diquat and paraquat are
listed in Table 1.
1.4 This method is restricted to use by or under the supervision of analysts experienced in
the use of HPLC. Each analyst must demonstrate the ability to generate acceptable
results with this method using the procedure described in Sect. 9.3.
2. SUMMARY OF METHOD
2.1 A measured volume of liquid sample, approximately 250 mL, is extracted using a C8
solid sorbent cartridge or a C8 disk which has been specially prepared for the reversed-
phase, ion-pair mode. The disk or cartridge is eluted with 4.5 mL of an acidic aqueous
solvent. After the ion-pair reagent is added to the eluate, the final volume is adjusted
to 5.0 mL. Liquid chromatographic conditions are described which permit the
separation and measurement of diquat and paraquat in the extract by absorbance
detection at 308 nm and 257 nm, respectively. A photodiode array detector is utilized
to provide simultaneous detection and confirmation of the method analytes (1).
549.2-2
-------
2.2 Analysis of diquat and paraquat is complicated by their ionic nature. Glassware should
be deactivated to prevent loss of analytes. The substitution of polyvinylchloride (PVC)
for glass is recommended.
3. DEFINITIONS
3.1 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or
other blank matrix that is treated exactly as a sample including exposure to all
glassware, equipment, solvents, reagents, internal standards, and surrogates that are
used with other samples. The LRB is used to determine if method analytes or other
interferences are present in the laboratory environment, the reagents, or the apparatus.
3.2 FIELD REAGENT BLANK (FRB) -- An aliquot of reagent water or other blank matrix
that is placed in a sample container in the laboratory and treated as a sample in all
respects, including shipment to the sampling site, exposure to sampling site conditions,
storage, preservation and all analytical procedures. The purpose of the FRB is to
determine if method analytes or other interferences are present in the
field environment.
3.3 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water or other
blank matrix to which known quantities of the method analytes are added in the
laboratory. The LFB is analyzed exactly like a sample, and its purpose is to determine
whether the methodology is in control, and whether the laboratory is capable of making
accurate and precise measurements.
3.4 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) - An aliquot of an
environmental sample to which known quantities of the method analytes are added in
the laboratory. The LFM is analyzed exactly like a sample, and its purpose is to
determine whether the sample matrix contributes bias to the analytical results. The
background concentrations of the analytes in the sample matrix must be
determined in a separate aliquot and the measured values in the LFM corrected for
background concentrations.
3.5 STOCK STANDARD SOLUTION (SSS) - A concentrated solution containing one or
more method analytes prepared in the laboratory using assayed reference materials or
purchased from a reputable commercial source. •
3i6 PRIMARY DILUTION STANDARD SOLUTION (PDS) - A solution of several
analytes prepared in the laboratory from stock standard solutions and diluted as needed
to prepare calibration solutions and other needed analyte solutions.
3.7 CALIBRATION STANDARD (CAL) - A solution prepared from the primary dilution
standard solution and stock standard solutions and the internal standards and surrogate
549.2-3
-------
analytes. The CAL solutions are used to calibrate the instrument response with respect
to analyte concentration.
3.8 QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of known
concentration which is used to fortify an aliquot of LRB or sample matrix. The QCS
is obtained from a source external to the laboratory and different from the source of
calibration standards. It is used to check laboratory performance with externally
prepared test materials.
3.9 EXTERNAL STANDARD (ES) - A pure analyte(s) that is measured in an experiment
separate from the experiment used to measure the analyte(s) in the sample. The signal
observed for a known quantity of the pure external standard(s) is used to calibrate the
instrument response for the corresponding analyte(s). The instrument response is used
to calculate the concentrations of the analyte(s) in the sample.
4. INTERFERENCES
4.1 Method 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 chromatogram. All of these materials must be routinely demonstrated
to be free from interferences under the conditions of the analysis by analyzing
laboratory reagent blanks as described in Sect. 9.2.
4.1.1 Glassware must be scrupulously cleaned (3). Clean all glassware as soon as
possible after use by rinsing with the last solvent used in it. This should be
followed by detergent washing with hot water and rinses with tap water and
distilled water. It should then be drained dry and heated in a laboratory oven
at 130°C for several hours before use. Solvent rinses with methanol may be
substituted for the oven heating. After drying and cooling, glassware should
be stored in a clean environment to prevent any accumulation of dust or other
contaminants.
4.1.2 Before the initial use of all glassware, the procedure described in Sect. 4.1.1
should be followed. Silanization of all glassware which will come in contact
with the method analytes is necessary to prevent adsorption of the diquat and
paraquat cations onto glass surfaces (7.13).
4.1.3 Plasticware should be washed with detergent and rinsed in tap water and
distilled water. It should be drained dry before use.
4.1.4 The use of high purity reagents and solvents helps to minimize interference
problems. Purification of solvents by distillation in all-glass systems may be
required.
549.2-4
-------
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.
4.3 This method has been shown to be susceptible to interferences from Ca+2 and Mg+2 ions
which may be present in hard water samples. These divalent cations can cause low
recovery of method analytes, by interfering with the ion exchange process. Use of
LFM samples can assist the analyst in evaluating the affect of these interferences in
different matrices.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been
precisely defined. Each chemical compound should be treated as a potential health
hazard. From this viewpoint, exposure to these chemicals must be minimized. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this method. A reference file
of material data handling sheets should also be made available to all personnel
involved in the chemical analysis.
6. EQUIPMENT AND SUPPLIES
6.1 SAMPLING EQUIPMENT, discrete or composite sampling.
6.1.1 Grab sample bottle — Amber polyvinylchloride (PVC) high density, 1-L, fitted
with screw caps. If amber bottles are not available, protect samples from
light. The container must be washed, rinsed with deionized water, and dried
before use to minimize contamination.
6.2 GLASSWARE
6.2.1 Volumetric flask — 5 mL, silanized
6.2.2 Autosampler vials — 4 mL, silanized
6.3 BALANCE -- analytical, capable of accurately weighing 0.0001 g
6.4 pH METER — capable of measuring pH to 0.1 units
549.2-5
-------
6.5 HPLC APPARATUS
6.5.1 Isocratic pumping system, constant flow (Waters M6000A HPLC pump or
equivalent).
6.5.2 Manual injector or automatic injector, capable of delivering 200 (J-L.
6.5.3 Analytical column -- any column which produces results equal to or better
than those listed below may be used.
6.5.3.1 Phenomenex Spherisorb (3 u, 100mm X 4.6mm), Hamilton PRP-1,
(5 u, 150 mm x 4.1 mm), or MICRA NPS RP-C18 (1.5 u, 33 mm X
4.6mm) or equivalent.
6.5.3.2 Guard column, C8 packing
6.5.4 Column Oven (Fiatron, Model CH-30 and controller, Model TC-50, or
equivalent).
6.5.5 Photodiode array detector (LKB 2140 Rapid Spectral Detector or equivalent).
Any detector which has the capability to switch between 257 nm and 308 nm
may be used.
6.5.6 Data system -- Use of a data system to report retention times and peak areas is
recommended but not required.
6.6 EXTRACTION APPARATUS
6.6.1 Liquid solid extraction cartridges, C8, 500 mg or equivalent.
Note: EPA has observed significant variability between brands of C8 LSE
media, and also between lots of the same brand of C8 LSE media. Verification
of analyte recovery should be performed any time a new brand or lot of LSE
media is used.
6.6.2 Liquid solid extraction system (Baker - 10 SPE, or equivalent).
6.6.3 Liquid solid extraction disks (C-8 Empore, 47 mm, or equivalent).
Note: EPA has observed significant variability between brands of C8 LSE
media, and also between lots of the same brand of C8 LSE media. Verification
of analyte recovery should be performed any time a new brand or lot of LSE
media is used.
549.2-6
-------
6.6.4 Liquid solid extraction system, Empore, 47 mm, 6 position manifold (Varian
Associates or equivalent).
6.6.5 Vacuum pump, 100 VAC, or other source of vacuum, capable of maintaining
a vacuum of 8-10 mm of Hg.
6.6.6 Membrane Filters, 0.45 [im pore-size, 47 mm diameter, Nylon.
7. REAGENTS AND STANDARDS
7.1 DEIONIZED WATER -- Water which has been processed through a series of
commercially available filters including a particulate filter, carbon bed, ion exchange
resin and finally a bacterial filter to produce deionized, reagent grade water. Any other
source of reagent water may be used provided the requirements of Sect. 9 are met.
7.2 METHANOL - HPLC grade or higher purity
7.3 ORTHOPHOSPHORIC ACID, 85% (w/v) - Reagent grade
7.4 DffiTHYLAMTNE - 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 TRDVIETHYL 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.
549.2-7
-------
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 Disk or cartridge eluting solution — Add 13.5 mL of orthophosphoric acid and
10.3 mL of diethylamine to 500 mL of deionized water and dilute to 1000 mL
in a volumetric flask.
7.14.6 Ion-pair concentrate — Dissolve 3.75 g of 1-hexanesulfonic acid in 15 mL of
the disk or cartridge eluting solution and dilute to 25 mL in a volumetric flask
with the disk eluting solution.
7.15 STOCK STANDARD SOLUTIONS
7.15.1 Diquat dibromide and 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 hr. Cool in a desiccator. Repeat process to a
constant weight. Weigh 0.1968 g of dried diquat salt and 0.1770 g of dried
paraquat salt and place into a silanized glass or polypropylene 100-mL
volumetric flask. Dissolve with approximately 50 mL of deionized water.
Dilute to the mark with deionized water.
7.15.3 The salts used in preparing the stock standards (Sect. 7.15.2) were taken to be
diquat dibromide monohydrate and paraquat dichloride tetrahydrate (4). The
drying procedure described in Sect. 7.15.2 will provide these hydration levels.
7.16 MOBILE PHASE — Make mobile phase by adding the following to 500 mL of
deionized water: 13.5 mL of orthophosphoric acid; 10.3 mL of diethylamine; 3.0 g of
1-hexanesulfonic acid, sodium salt. Mix and dilute with deionized water to a final
volume of 1 L.
8. 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 (5).
Automatic sampling equipment must be free as possible of adsorption sites which
might be extracted into the sample.
549.2-8
-------
8.2 The samples must be iced or refrigerated at approximately 4°C from the time of
collection until extraction. The analytes are light-sensitive, particularly diquat.
8.3 Samples which are known or suspected to contain residual chlorine must be preserved
with sodium thiosulfate (100 mg/L). Samples which are biologically active must be
preserved by adding sulfuric acid to pH 2 to prevent adsorption of method analytes by
the humectant material.
8.4 Analyte stability over time may depend on the matrix tested. All samples must be.
extracted within 7 days of collection. Extracts must be analyzed within 21 days of
extraction (6).
9. QUALITY CONTROL
9.1 Minimum quality control (QC) requirements are initial demonstration of laboratory
capability, analysis of laboratory reagent blanks, laboratory fortified matrix samples,
and laboratory fortified blanks. The MDL (2) must also be determined for each
analyte. The analyst should institute quality control practices to ensure that the brand
and lot of LSE media being used show reliable recoveries of method analytes. The
laboratory must maintain records to document the quality of the data generated.
Additional quality control practices are recommended.
9.2 LABORATORY REAGENT BLANKS (LRB) - Before processing any samples, the
analyst must analyze a LRB to demonstrate that all deactivated glassware or
plasticware, and reagent interferences are reasonably free of contamination. In
addition, each time a set of samples is extracted or reagents are changed, a LRB must
be analyzed. If within the retention time window (Sect. 11.4.2) of the analyte of
interest, the LRB produces a peak that would prevent the determination of that analyte,
determine the source of contamination and eliminate the interference before
processing samples.
9.3 INITIAL DEMONSTRATION OF CAPABILITY
9.3.1 Prepare laboratory fortified blanks (LFBs) at analyte concentrations of 100
jig/L. With a syringe, add 25 uL of the stock standard (Sect. 7.15.2) to at least
four 250 mL aliquots of reagent water and analyze each aliquot according to
procedures beginning in Sect. 11.2.
9.3.2 Calculate the recoveries and relative standard deviation (RSD). The recovery
(R) value for each sample, should be within ± 30% of the fortified amount.
The RSD of the mean recovery should be less than 30%. For analytes that
fail this critera, initial demonstration procedures should be repeated.
9.3.3 For each analyte, determine the MDL. Prepare a minimum of 4-7 LFBs (7 is
recommended) at a low concentration. Fortification concentrations in Table 1
549.2-9
-------
9.4
maybe used as a guide, or use calibration data obtained in Section 10 to
estimate a concentration for each analyte that will produce a peak with a 3-5
times signal to noise response. Extract and analyze each according to Sections
11 and 12. It is recommended that these LFBs be prepared and analyzed over
a period of several days, so that day to day variations are reflected in precision
measurements. Calculate mean recovery and standard deviation for each
analyte. Use the standard deviation and the equation in Section 13 to calculate
the MDL.
9.3.4 The initial demonstration of capability is used primarily to preclude a
laboratory from analyzing unknown samples via a new, unfamiliar method
prior to obtaining some experience with it. As laboratory personnel gain
experience with this method the quality of the data should improve beyond the
requirements stated in Sect. 9.3.2.
The analyst is permitted to use other HPLC columns, HPLC conditions, or HPLC
detectors to improve separations or lower analytical costs. Each time such method
modifications are made, the analyst must repeat the procedures in Sect. 9.3.
9.5 LABORATORY FORTIFIED BLANKS
9.5.1 The laboratory must analyze at least one laboratory fortified blank (LFB)
sample per sample set (all samples extracted within a 24-hr period). The
fortified concentration of each analyte in the LFB should be 10 times the
MDL. If the recovery of either analyte falls outside the control limits (Sect.
9.5.2), that analyte is judged out of control, and the source of the problem
must be identified and resolved before continuing analyses.
9.5.2 Until sufficient data become available, usually a minimum of results from 20
to 30 analyses, the laboratory should assess laboratory performance against the
control limits in Sect. 9.3.2. When sufficient internal performance data
become available, develop control limits from the mean percent recovery (R)
and standard deviation (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 = 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. These calculated control
limits should not exceed the limits established in Sect. 9.3.2.
549.2-10
-------
9.6 LABORATORY FORTIFIED SAMPLE MATRIX
9.6.1 The laboratory must add a known fortified concentration to a minimum of
10% of the samples or one fortified sample per set, whichever is greater. The
fortified concentration should not be less than the background concentration of
the original sample. Ideally, the fortified concentration should be the same as
that used for the laboratory fortified blank (Sect. 9.5). Over time, samples
from all routine samples sources should be fortified.
9.6.2 Calculate the accuracy as percent recovery (R) for each analyte, corrected for
background concentrations measured in the original sample, and compare
these values to the control limits established in Sect. 9.5.2 from the analyses of
LFBs, :
9.6.3 If the recovery of any such analyte falls outside the designated range, and the
laboratory performance for that analyte is shown to be in control .(Sect. 9.5),
the recovery problem encountered with the dosed sample is judged to be
matrix related, not system related. The result for that analyte in the original
sample is labeled suspect/matrix to inform the data user that the results are
suspect due to matrix effects.
9.7 QUALITY CONTROL SAMPLES (QCS) - Each quarter the laboratory should
analyze one or more QCS. If criteria provided with the QCS are not met, corrective
action should be taken and documented.
9.8 The laboratory may adopt additional quality control practices for use with this method.
The specific, practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. For example, field or laboratory duplicates may be
analyzed to assess the precision of the environmental measurements or field reagent
blanks may be used to assess contamination of samples under site conditions,
transportation and storage.
10. CALIBRATION AND STANDARDIZATION
10.1 Establish HPLC operating conditions indicated in Table 1. The chromatographic
system can be calibrated using the external standard technique.
10.2 In order to closely match calibration standards to samples, process standards by the
following method: Using C8 disks or Cg cartridges conditioned according to Sect.
11.2.1, pass 250 mL of reagent water through the disk or cartridge and discard the
water. Dry the disk or cartridge by passing 5 mL of methanol through it. Discard the
methanol. Pass 4.0 mL of the eluting solution through the disk or cartridge and catch
in a 5 mL silanized volumetric flask. Fortify the eluted solution with 100 [iL of the
ion-pair concentrate and with 500 uL of the stock standard and dilute to the mark with
549.2-11
-------
eluting solution. This provides a 10:1 dilution of the stock. Use serial dilution of the
calibration standard by the same method to achieve lower concentration standards.
10.3 Analyze a minimum of three calibration standards prepared by the procedure described
in Sect. 10.2 utilizing the HPLC conditions given in Table 1. From full spectral data
obtained, extract the 308 nm chromatographic trace for diquat and the 257 nm trace
for paraquat. Integrate and record the analyte peak areas. Any mathematical
manipulations performed to aid in data reduction must be recorded and performed on
all sample chromatograms. Tabulate the peak area against quantity injected. The
results may be used to prepare calibration curves for diquat and paraquat.
10.4 The working calibration curve must be verified on each working day by measurement
of a calibration check standard, at the beginning of the analysis day. These check
standards should be at two different concentration levels to verify the calibration curve.
For extended periods of analysis (greater than 8 hr), it is strongly recommended that
check standards be interspersed with samples at regular intervals. If the response for
any analyte varies from the predicted response by more than ±20%, the test must be
repeated using a fresh calibration standard. If the results still do not agree, generate a
new calibration curve.
11. PROCEDURE
11.1 SAMPLE CLEANUP ~ Cleanup procedures 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 \im Nylon membrane' filter into a
silanized glass or plastic container.
11.1.2 Store all samples at 4°C unless extraction is to be performed immediately.
11.2 CARTRIDGE EXTRACTION
11.2.1 Before sample extraction, the C8 extraction cartridges must be conditioned by
the following procedure.
11.2.1.1 Place a C8 cartridge on the cartridge extraction system manifold.
11.2.1.2 Elute the following solutions through the cartridge in the stated order.
Take special care not to let the column go dry. The flow rate through
the cartridge should be approximately 10 mL/min.
549.2-12
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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, 20 mL
11.2.1.2.2 Retain conditioning solution B in the Cg cartridge to keep it
activated.
11.2.2 The Cg cartridges should not be prepared more than 48 hr prior to use. After
conditioning, the cartridge should be capped and stored at 4PC.
11.2.3 Measure a 250-mL aliquot of the sample processed through Sect.l 1.1 in a
silanized, volumetric flask.
11.2.4 Measure the pH of the sample. If the pH is between 7.0 and 9.0, the sample
can be analyzed without adjustment. If the pH is not in this range, or if the
sample has been acidified for preservation purposes, adjust the pH of sample
to between 7.0 and 9.0 with 10% w/v NaOH (aq) or 10% v/v HC1 (aq) before
extracting.
11.2.5 Place a conditioned C8 cartridge on the solid phase extraction vacuum
manifold. Attach a 60-mL reservoir to the C8 cartridge with the appropriate
adapter. Put a 250- mL beaker inside the extraction manifold to catch waste
solutions and sample. Transfer the measured volume in aliquots to the
reservoir. Turn on the vacuum pump or house vacuum and adjust the flow
rate to 3 to 6 mL/min. Filter the sample through the Cg cartridge, and wash the
column with 5 mL of HPLC grade methanol. Continue to draw the vacuum
through the cartridge for one additional minute to dry the cartridge. Release
the vacuum and discard the sample waste and methanol.
11.2.6 Place a silanized 5-mL volumetric flask beneath the collection stem in the
vacuum manifold. Add 4.5 mL of the eluting solution to the sample cartridge.
Turn on the vacuum and adjust the flow rate to 1 to 2 mL/min.
11.2.7 Remove the 5-mL volumetric flask with the extract. Fortify the extract with
100 [iL of the ion-pair concentrate. Adjust the volume to the mark with
cartridge eluting solution, mix thoroughly, and seal tightly until analyzed.
549.2-13
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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 Sect. 10.3.
11.3 DISK EXTRACTION » The top surface of the disk matrix must remain covered with
liquid at all times. If the disk is exposed to air at any step in the disk cleanup
procedure, the elution procedure should be restarted. Eluants applied to the disk
should be allowed to soak into the disk before drawing them through. Vacuum should
then be applied to draw most of the eluant through the disk, leaving a thin layer of
solution on the top of the disk. Flow rate through the disk is not critical.
11.3.1 Assemble the 47 mm disk in the disk holder or a filter apparatus. Be sure that
the surfaces of the holder are either silanized glass or Teflon coated to avoid
adsorption or decomposition of the analytes.
11.3.2 Measure the pH of the sample. If the pH is between 7.0 and 9.0, the sample
can be analyzed without adjustment. If the pH is not in this range, or if the
sample has been acidified for preservation purposes, adjust the pH of sample
to between 7.0 and 9.0 with 10% w/v NaOH (aq) or 10% v/v HC1 (aq) before
extracting. ,
11.3.3 Apply 10 mL of methanol to the disk. Apply vacuum to begin elution, then
immediately vent the vacuum when drops of liquid appear at the drip tip.
Allow the methanol to soak into the disk for a minimum of 1 min, then
reapply the vacuum to bring the methanol to, just above the top surface of the
disk. , .
11.3.4 Draw 2 10-mL aliquots of reagent water through to just above the top surface
of the disk to remove the methanol.
11.3.5 Apply 10 mL of Conditioning Solution A to the disk. As with the methanol,
draw a few drops through, then allow the disk to soak for at least 1 min. Draw
the Conditioning Solution A through the disk to just above its top surface.
11.3.6 Draw 2 10-mL aliquots of reagent water through to just above the top surface
of the disk.
11.3.7 Apply 20 mL of Conditioning Solution B to the disk. Draw a few drops
through using vacuum and allow the disk to soak for at least 1 min. Draw the
remaining Conditioning Solution B through to just above the top surface of
the disk.
11.3.8 Measure 250 mL of the sample using a polypropylene graduated cylinder.
Pour the sample aliquot into the filtration apparatus reservoir and apply
549.2-14
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vacuum to draw the sample through the disk. Pass the entire sample through
the disk, leaving no liquid on the top of the disk, then vent the vacuum.
11.3.9 Assemble a graduated collection tube under the drip tip with the tip
descending into the tube slightly to prevent losses of eluants. Be sure the tube
will hold at least'10 mL of eluate.
11.3.10 With the vacuum vented, drip enough methanol onto the disk to cover it
completely (0.5-1.0 mL). Allow the methanol to soak into the disk for 1 min.
Add more methanol as needed to keep the disk covered as it soaks.
11.3.11 Pipet 4 mL of Disk Eluting Solvent onto the disk. Apply vacuum until drops
appear at the drip tip. Vent the vacuum and allow the disk to soak for 1 min.
11.3.12 Draw the Disk Eluting Solution through to just above the top surface of the
disk. Add 4 mL of Disk Eluting Solution and draw it completely through the
disk. Tap the disk holder assembly gently to loosen adhering drops into the
collection tube.
11.3.13 Vent the vacuum, disassemble the disk extraction device, and remove the
collection tube. Fortify the extract with 200 p,L of the ion-pair concentrate.
Add Disk Eluting Solution to the tube to a final volume of 10 mL.
11.3.14 Analyze samples by HPLC. Some suggested conditions, which were used in
developing this method, are listed in Table 1. This table includes the retention
times andMDLs that were obtained using the suggested conditions.
11.4 IDENTIFICATION OF ANALYTES
11.4.1 Identify a sample component by comparison of its retention time to the
retention time of a reference chromatogram. If the retention time of an
unknown compound corresponds, within limits (Sect. 11.4.2), to the retention
time of a standard compound, then identification is considered positive.
11.4.2 The width of the retention time window used to make identification should be
based upon measurements of actual retention time variations of standards over
the course of a day. Three times the standard deviation of a retention time
can be used to calculate a suggested window size for a compound. However,
the experience of the analyst should weigh heavily in the interpretation of
chromatograms.
11.4.3 Identification requires expert judgment when sample components are not
resolved chromatographically. When peaks obviously represent more than
one sample component (i.e., broadened peak with shoulder(s) or valley
549.2-15
-------
between two or more maxima), or any time doubt exists over the identification
of a peak in a chromatogram, a confirmatory technique must be employed.
Through the use of the photodiode array detector, full spectra of the analyte
peaks are obtained (Figure 2). When a peak of an unknown sample falls
within the retention time windows of method analytes, confirm the peak
identification by spectral comparison with analyte standards.
If additional confirmation is required, replace the 1-hexanesulfonic acid salt
with 1-heptanesulfonic acid, sodium salt in the mobile phase and reanalyze the
samples. Comparison of the ratio of retention times in the samples by the two
mobile phases with that of the standards will provide additional confirmation.
11.4.4 If the peak area exceeds the linear range of the calibration curve, a smaller
sample volume should be used. Alternatively, the final solution may be
diluted with mobile phase and reanalyzed.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Determine the concentration of the analytes in the sample.
12.1.1 Calculate the concentration of each analyte injected from the peak area using
the calibration curves in Sect. 10.3 and the following equation.
Concentration, [ig/L = (A') x (VF)
(VS)
where: A = Peak area of analyte in sample extract
VF = Final volume of sample extract, in mL
VS = Sample volume, hi 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 (2). The MDL data listed in
Table 1 were obtained using disks with reagent water as the matrix.
MDL = S t
549.2-16
-------
where:
t(n-u-aipha=o.99)= Student's t value for the 99% confidence level with n-1 degrees
of freedom
n = number of replicates
S = standard deviation of replicate analyses.
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 approximately 4 x MDL to
lOOxMDL.
13.3 Single-laboratory precision and accuracy results at several concentration levels in
drinking water matrices using disks are presented in Table 2.
13.4 This methodology has been shown to be sensitive to brand differences and even lot
differences in C8 LSE media. This is presumed to be due to variations in
manufacturing processes. If the method does not demonstrate performance data similar
to those demonstrated in Sect. 17, C8 LSE media should be obtained from a different
source.
14. POLLUTION PREVENTION
14.1 Only an extremely small volume of an organic solvent is used in this method. A
maximum of 15 mL of methanol is used per sample to condition each cartridge or disk.
Methanol is not considered to be a toxic or hazardous solvent. All other chemicals
used in this method are can be handled in a non-hazardous way when used in the
prescribed manner and amounts.
14.2 For information about pollution prevention that may be applicable to laboratory
operations, consult "Less is Better: Laboratory Chemical Management for Waste
Reduction" available from the American Chemical Society's Department of
Government Relations and Science Policy, 1155 16th Street N.W., Washington, D.C.
20036.
15. WASTE MANAGEMENT
15.1 There are generally no waste management problems involved with discarding spent or
left over samples in this method since most often the sample matrix is drinking water.
If a sample is analyzed which appears to be highly contaminated with chemicals,
analyses should be carried out to assess the type and degree of contamination so that
the samples may be discarded properly. The Agency requires that laboratory waste
management practices be conducted consistent with all applicable rules and
regulations, and that laboratories protect the air, water, and land by minimizing and
controlling all releases from fume hoods and bench operations. Also, compliance is
549.2-17
-------
required with any sewage discharge restrictions. For further information on waste
management, consult "The Waste Management Manual for Laboratory Personnel" also
available from the American Chemical Society at the address in Sect. 14.2.
16. REFERENCES
1. 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.
2. Glaser, J. A., D. L. Foerst, G. M. McKee, S. A. Quave, and W. L. Budde, "Trace
Analyses for Wastewaters", Environ. Sci. Technol.. 15., 1426,1981.
3. 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.
4. Worobey, B. L., "Analytical Method for the Simultaneous Determination of Diquat and
Paraquat Residues in Potatoes by High Pressure Liquid Chromatography", Pestic. Sci
18(41 245,1987.
5. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling
Water", American Society for Testing and Materials, Philadelphia, PA, p. 76,1980.
6. Hodgeson, J.W., Bashe, W.J. and J.W. Eichelberger, "Method 549.1 - Determination of
Diquat and Paraquat in Drinking Water by Liquid-Solid Extraction and High
Performance Liquid Chromatography with Ultraviolet Detection", Methods for the
Determination of Organic Compounds in Drinking Water. Supplement E. EPA/600/R-
92/129, U.S. Environmental Protection Agency, Envirinmental Monitoring Systems
Laboratory, Cincinnati, Ohio, 45268,1992.
549.2-18
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17. TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
CONDITIONS AND METHOD DETECTION LIMITS
Method Detection Limits3
Analyte G-ig/L)
(disks)
Diquat 0.72
Paraquat 0.68
HPLC Conditions:
Column: Phenomenex Spherisorb, 3|i, 4.6 mm x 100 mm
Column Temperature 3 5.0° C
Flow Rate: 2.0 mL/min., Ion-Pair Mobile Phase
(Sect. 7.16)
Injection Volume: 200 |iL
Photodiode Array Detector Settings:
Wavelength Range: 210 - 370 nm
Sample Rate: 1 scan/sec.
Wavelength Step: 1 nm
Integration Time: 1 sec.
Run Time: 5.0 min.
Quantitation Wavelengths: Diquat - 308 nm
Paraquat - 257 nm
aMDL data were obtained from five samples fortified at 2.5 |xg/L diquat and 2.5 \ig/L paraquat.
549.2-19
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TABLE 2. SINGLE OPERATOR ACCURACY AND PRECISION
USING DISK (N = 5 FOR EACH TYPE OF WATER)
Type of Fortified 2.5 (j-g/L
Water Mean%Rec. %RSD
DIQUAT
Fortified 10.5 \igfL
Mean%Rec. %RSD
PARAQUAT
Type of Fortified 2.5 \igfL Fortified 10 jig/L
Water Mean%Rec. %RSD Mean%Rec. %RSD
RW = Reagent Water
T\y = Tap Water (Dechlorinated with sodium thiosulfate)
GW = Ground Water
All samples adjusted to pH 7.
Fortified 52.5 |ig/L
Mean%Rec. %RSD
RW
TW
GW
90.9
91.7
91.4
8.4
6.5
6.4
94.1
93.6
93.7
5.2
3.1
3.0
92.1
93.0
90.2
2.9
5.3
3.3
Fortified 50
Mean%Rec. %RSD
RW
DW
GW
94.7
93.5
92.0
7.7
6.6
8.1
92.3
89.7
89.9
5.5
3.6
2.5
88.8
91.4
90.4
4.2
6.5
2.5
549.2-20
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METHOD 556. DETERMINATION OF CARBONYL COMPOUNDS IN DRINKING
WATER BY PENTAFLUOROBENZYLHYDROXYLAMINE
DERIVATIZATION AND CAPILLARY GAS
CHROMATOGRAPHY WITH ELECTRON CAPTURE
DETECTION
Revision 1.0
June 1998
J.W. Munch, USEPA Office of Research and Development and
D. J. Munch, USEPA, Office of Ground Water and Drinking Water and
S.D. Winslow, S.C. Wendelken, B.V. Pepich, ICF Kaiser Engineers, Inc.
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
556-1
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METHOD 556
DETERMINATION OF CARBONYL COMPOUNDS IN DRINKING WATER BY
PENTAFLUORBENZYLHYROXYLAMINE DERIVATIZATION AND CAPILLARY
GAS CHROMATOGRAPHY AND ELECTRON CAPTURE DETECTION
1. SCOPE AND APPLICATION
1.1 This is a gas chromatographic method optimized for the determination of selected
carbonyl compounds in finished drinking water and raw source water. The
analytes applicable to this method are derivatized to their corresponding
pentafluorobenzyl oximes. The oxime derivatives are then extracted from the
water with hexane. The hexane extracts are analyzed by capillary gas
chromatography with electron capture detection (GC-ECD) and quantitated using
procedural standard calibration. Accuracy, precision, and method detection limit
(MDL) data have been generated for the following compounds:
Chemical Abstract Services
Analvte Registry Number
Formaldehyde 50-00-0
Acetaldehyde 75-07-0
Propanal 123-38-6
Butanal 123-72-8
Pentanal 110-62-3
Hexanal 66-25-1
Heptanal 111-71-7
Octanal 124-13-0
Nonanal 124-19-6
Decanal 112-31-2
Cyclohexanone 108-94-1
Crotonaldehyde 123-73-9
Benzaldehyde 100-52-7
Glyoxal (ethanedial) 107-22-2
Methyl glyoxal (2-oxopropanal or pyruvicaldehyde) 78-98-8
1.2 This method applies to the determination of target analytes over the concentration
ranges typically found in drinking water. Analyte retention times are in Section
17, Table 1. Other method performance data are presented in Sectionl7, Tables
2-6. Experimentally determined method detection limits (MDLs) for the above
listed analytes are provided in Section 17, Table 3. The MDL is defined as the
556-2
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statistically calculated minimum amount that can be measured with 99%
confidence that the reported value is greater than zero.{1) However, it should be
noted that background levels of some method analytes (usually formaldehyde and
acetaldehyde) are problematic. The minimum reporting level (MRL) for method
analytes, for each analyst/laboratory that uses this method, will depend on their
ability to control background levels (Sect. 4).
1.3 This method is restricted to use by or under the supervision of analysts skilled in
liquid-liquid extractions, derivatization procedures and the use of GC and
interpretation of gas chromatograms. Each analyst must demonstrate the ability to
generate acceptable results with this method, using the procedures described in
Section 9.
2.0 SUMMARY OF METHOD
2.1 A 20 mL volume of water sample is adjusted to pH 4 with potassium hydrogen
phthalate (KHP) and the analytes are derivatized at 35 °C for 2 hr with 15 mg of
O-(2,3,4,5,6-Pentafluorobenzyl)-hydroxylamine (PFBHA) reagent. The oxime
derivatives are extracted from the water with 4 mL hexane. The extract is
processed through an acidic wash step, and then analyzed by GC-ECD. The target
analytes are identified and quantitated by comparison to a procedural standard
(Sect. 3.9). Two chromatographic peaks will be observed for many of the target
analytes. Both (E) and (Z) isomers are formed for carbonyl compounds that are
asymmetrical, and that are not sterically hindered. However, the (E) and (Z)
isomers may not be chromatographically resolved in a few cases. Compounds
where two carbonyl groups are derivatized, such as glyoxal and methyl glyoxal,
have even more possible isomers. See SectionlT, Table 1 and Figure 1 for the
chromatographic peaks used for analyte identification.
NOTE: The absolute identity of the (E) and (Z) isomers was not
determined during method development. Other researchers(2>3>4)
have reported the first eluting peak as (E), and the second peak as
(Z). For convenience, this method will follow this convention.
Because more than 2 isomers are formed for glyoxal and methyl
glyoxal, the peaks used for identification are referred to as "peak 1"
and "peak 2."
2.2 All results should be confirmed on a second, dissimilar capillary GC column.
3. DEFINITIONS
3.1 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or
other blank matrix that is treated exactly as a sample including exposure to all
556-3
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glassware, equipment, solvents and reagents, sample preservatives, internal
standards, and surrogates that are used with other samples. The LRB is used to
determine if method analytes or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.2 FIELD REAGENT BLANK (FRB) - An aliquot of reagent water or other blank
matrix that is placed in a sample container in the laboratory and treated as a
sample in all respects, including shipment to the sampling site, storage,
preservation, and all analytical procedures. The purpose of the FRB is to
determine if method analytes or other interferences are introduced during sample
shipping or storage. For this analysis the FRB should not be opened at the
sampling site.
3.3 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water or
other blank matrix to which known quantities of the method analytes are added in
the laboratory. The LFB is analyzed exactly like a sample, and its purpose is to
determine whether the methodology is in control, and whether the laboratory is
capable of making accurate and precise measurements.
3.4 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) - An aliquot of an
environmental sample to which known quantities of the method analytes are
added in the laboratory. The LFM is analyzed exactly like a sample, and its
purpose is to determine whether the sample matrix contributes bias to the
analytical results. The background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the measured values in the
LFM corrected for background concentrations.
3.5 STOCK STANDARD SOLUTION (SSS) -- A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed reference
materials or purchased from a reputable commercial source.
3.6 PRIMARY DILUTION STANDARD SOLUTION (PDS) - A solution of several
analytes prepared in the laboratory from stock standard solutions and diluted as
needed to prepare calibration solutions and other needed analyte solutions.
3.7 CALIBRATION STANDARD (CAL) - A solution prepared from the primary
dilution standard solution and stock standard solutions and the internal standards
and surrogate analytes. The CAL solutions are used to calibrate the instrument
response with respect to analyte concentration.
3.8 QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of
known concentrations which is used to fortify an aliquot of LRB or sample
matrix. The QCS is obtained from a source external to the laboratory and
556-4
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different from the source of calibration standards. It is used to check laboratory
performance with externally prepared test materials.
3.9 PROCEDURAL STANDARD CALIBRATION - A calibration method where
aqueous calibration standards are prepared and processed (e.g. purged, extracted,
and/or derivatized) in exactly the same manner as a sample. All steps in the
process from addition of sampling preservatives through instrumental analyses are
included in the calibration. Using procedural standard calibration compensates
for any inefficiencies in the processing procedure.
3.10 INTERNAL STANDARD (IS) - A pure analyte added to a sample, extract, or
standard solution in known amount(s) and used to measure the relative responses
of other method analytes and surrogates that are components of the same sample
or solution. The internal standard must be an analyte that is not a sample
component.
3.11 SURROGATE ANALYTE (SUR) - A pure analyte, which is extremely unlikely
to be found in any sample, and which is added to a sample aliquot in known
amount(s) before extraction or other processing and is measured with the same
procedures used to measure other sample components. The purpose of the SA is
to monitor method performance with each sample.
3.12 METHOD DETECTION LIMIT (MDL) - The minimum concentration of an
analyte that can be identified, measured and reported with 99% confidence that
the analyte concentration is greater than zero.
3.13 MATERIAL SAFETY DATA (MSDS) - Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical properties, fire,
and reactivity data including storage, spill, and handling precautions.
3.14 CONTINUING CALIBRATION CHECK (CCC) - A calibration standard
containing one or more method analytes, which is analyzed periodically to verify
the accuracy of the existing calibration for those analytes.
3.15 MINIMUM REPORTING LEVEL (MRL) - The minimum concentration of an
analyte that should be reported. This concentration is determined by the
background level of the analyte in the LRBs and the sensitivity of the method to
the analyte. Ideally, the MRL will be at or near the concentration of the lowest
calibration standard.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in laboratory air, solvents,
reagents (including reagent water), glassware, sample bottles and caps, and other
556-5
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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 (less than 1/2 the MRL) under the
conditions of the analysis by analyzing laboratory reagent blanks as described in
Section 9.3. Subtracting blank values from sample results is not permitted.
4.1.1 Before attempting analyses by this method, the analyst must obtain a
source of reagent water free from carbonyl compounds and other
interferences. The most likely interferences are the presence of
formaldehyde and acetaldehyde in the reagent water. The most successful
techniques for generating aldehyde free water are (1) exposure to UV light,
or (2) distillation from permanganate.
4.1.2 Commercially available systems for generating reagent grade water have
proved adequate, if a step involving exposure to UV light is included. For
the data presented hi this method, a Millipore Elix 3 reverse osmosis
system followed by a Milli-Q TOC Plus polishing unit provided reagent
water with background levels of 1 ug/L or less for each method analyte.
Other researchers have reported typical blank values of 1-3 ug/L.(3>4)
4.1.3 Distillation of reagent water from acidified potassium permanganate has
been reported as an effective method of eliminating background levels of
aldehydes.(2) Distill 500 mL of reagent water to which 64 mg potassium
permanganate and 1 mL cone, sulfuric acid have been added, hi our
laboratory, this procedure reduced formaldehyde levels to approximately 3
ug/L.
4.1.4 It may be necessary to purchase reagent grade water. If acceptably clean
reagent grade water is purchased, care must also be taken to protect it from
contamination caused by contact with laboratory air.
4.2 Formaldehyde is typically present in laboratory air and smaller amounts of other
aldehydes may also be found. Care should be taken to minimize exposure of
reagents and sample water with laboratory air. Because latex is a potential
aldehyde contaminant source, protective gloves should not contain latex. Nitrile
gloves, such as N-Dex Plus, are acceptable. Bottle caps should be made of
polypropylene. Commonly used phenolic resin caps must be avoided because
they can introduce formaldehyde contamination into samples.
4.3 Reagents must also be free from contamination. Many brands of solvents may
contain trace amounts of carbonyl compounds.
4.4 Glassware must be scrupulously cleaned by detergent washing with hot water, and
rinses with tap water and distilled water. Glassware should then be drained, dried,
556-6
-------
and heated in a laboratory oven at 130 °C for several hours before use. Solvent
rinses with methanol or acetonitrile, followed by air drying, may be substituted
for the oven heating. After cleaning, glassware should be stored in a clean
environment to prevent any accumulation of dust or other contaminants.
4.5 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 matrix being sampled.
4.6 An interferant that elutes just prior to the acetaldehyde (E) isomer peak on the
primary column is typically observed in chlorinated or chloraminated waters. If
this peak interferes with the integration of the acetaldehyde (E) isomer peak, then
acetaldehyde should be quantitated using only the acetaldehyde (Z) isomer, or
from the confirmation column data.
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 safety data sheets should also be made available to all
personnel involved in the chemical analysis. Additional references to laboratory
safety are available.(5"8)
5.2 Formaldehyde and acetaldehyde have been tentatively classified as known or
suspected human or mammalian carcinogens. Glyoxal and methyl glyoxal have
been shown to be mutagenic in in-vitro tests.(2)
6. EQUIPMENT AND SUPPLIES (All specifications are suggested. Brand names and/or
catalog numbers are included for illustration only.)
6.1 SAMPLE CONTAINERS - Grab Sample Bottle (aqueous samples) - 30 mL
amber glass, screw cap bottles and caps equipped with Teflon-faced silicone
septa. Screw caps should be polypropylene. Typical phenolic resin caps should
be avoided due to the possibility of sample contamination from
formaldehyde. Prior to use, wash bottles and septa according to Section 4.4.
6.2 VIALS - 8 mL or 12 mL vials for the acid wash step (Sect. 11.1.10), and GC
autosampler vials, both types must be glass with Teflon-lined polypropylene caps.
6.3 VOLUMETRIC FLASKS - various sizes used for preparation of standards.
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6.4 BALANCE ~ Analytical, capable of accurately weighing to the nearest 0.0001 g.
6.5 WATER BATH or HEATING BLOCK - Capable of maintaining 35 ± 2 °C
6.6 GAS CHROMATOGRAPH -- Capillary Gas Chromatograph equipped with a
split/splitless injector, or other injector suitable for trace analysis, and an electron
capture detector.
6.6.1 Primary Column - 30 m x 0.25mm J&W DB-5ms, 0.25 um film thickness
(or equivalent). Note: The J&W DB-5 was not found to be equivalent for
this application. The surrogate analyte is not resolved from octanal with
the DB-5 column.
6.6.2 Confirmation Column - 30 m x 0.25 mm Restek Rtx-1701, 0.25 um film
thickness (or equivalent)
7. REAGENTS AND STANDARDS
7.1 Reagent grade or better chemicals should be used in all tests. Unless otherwise
indicated, it is intended that all reagents shall conform to the specifications of the
Committee on Analytical Reagents of the American Chemical Society, where
such specifications are available. Other grades may be used, provided it is first
ascertained that the reagent is of sufficiently high purity to permit its use without
lessening the accuracy of the determination.
7.2 REAGENT WATER --. Reagent water as free as possible from interferences
and contamination is critical to the success of this method. See Section 4.1.
7.3 ACETONTTRILE - High purity, demonstrated to be free of analytes and
interferences.
7.4 HEXANE — High purity, demonstrated to be free of analytes and interferences:
B&J Brand, GC2 grade or equivalent.
7.5 POTASSIUM HYDROGEN PHTHALATE (KHP) - ACS Grade or better.
7.6 0-(2,3,5,6-PENTAFLUOROBENZYL)-HYDROXYLAMINE
HYDROCHLORIDE (PFBHA) - 98+%, Aldrich cat# 19,448-4. (Store in a
desiccator - Do not refrigerate).
7.7 SULFURIC ACID ~ ACS Grade, or better.
7.8 COPPER SULFATE PENTAHYDRATE ~ ACS Grade or better.
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7.9 AMMONIUM CHLORIDE, NH4C1 or AMMONIUM SULFATE, (NH4)2SO4.
7.10 SOLUTIONS
7.10.1 PFBHA REAGENT - Prepare a fresh 15 mg/mL solution in reagent water
daily. Prepare an amount appropriate to the number of samples to be
derivatized. OneniLof solution is added per sample. For example, if 14
sample vials are being extracted, prepare 15 mL of solution. For a 15 mL
volume of solution, weigh 0.225 grams of PFBHA into a dry 40 mL vial,
add 15 mL water and shake to dissolve.
7.10.2 0.2 N SULFURIC ACID -- Add 5 mL of concentrated sulfuric acid to 900
mL of reagent water.
7.11 STOCK STANDARD SOLUTIONS - When a compound purity is assayed to be
96% or greater, the weight can be used without correction to calculate the
concentration of the stock standard.
7.11.1 INTERNAL STANDARD (IS) - 1,2-DIBROMOPROPANE, 98+%
purity. An alternate compound may be used as the IS at the discretion of
the analyst. If an alternate is selected, an appropriate concentration will
need to be determined.
7.11.1.1 INTERNAL STANDARD STOCK SOLUTION, (10,000
ug/mL) — Accurately weigh approximately 0.1 gram to the
nearest 0.000 Ig, into a tared 10 mL volumetric flask containing
hexane up to the neck. After determining weight difference,
fill to mark with hexane. Stock solutions can be used for up to
6 months when stored at-10 °C.
7.11.1.2 INTERNAL STANDARD FORTIFIED EXTRACTION
SOLVENT, 400 ug/L in hexane - This is the solvent used to
extract the derivatized samples. The internal standard is added
to the solvent prior to performing the extraction. The volume
of this solvent to be prepared should be determined by the
sample workload. The following example illustrates
preparation of 1 L of fortified solvent. If fewer samples are to
be analyzed each month, prepare smaller batches of working
solvent. Add 40 uL of internal standard stock solution directly
to 1 L of hexane in a volumetric flask. Cap flask and invert
three times to ensure thorough mixing. Transfer to 1L storage
bottle with Teflon lined cap. This solution can be used up to 4
weeks. As a check, runasample of this working solvent on the
GC before the first extraction of aqueous samples. Have
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enough working solvent available to extract all calibration and
aqueous samples in each extraction set. Never use two
different batches of working solvent for one set of extractions.
7.11.2 SURROGATE (SUR) - 2',4',5' -TRIFLUOROACETOPHENONE
This compound was found to be an appropriate surrogate analyte for these
analyses. However, the chromatograms for this analysis are very crowded,
and all possible matrix interferences cannot be anticipated. An alternate
carbonyl compound may be selected as the surrogate analyte if matrix
interferences or chromatographic problems are encountered. Any
surrogate analyte selected must form an oxime derivative, because its
purpose is to monitor the derivatization process. If an alternate surrogate
is selected, its concentration may also be adjusted to meet the needs of the
laboratory.
7.11.2.1 SURROGATE STOCK SOLUTION, 10,000 ug/mL -
Accurately weigh approximately 0.1 gram SUR to the nearest
O.OOOlg, into a 10 mL tared volumetric flask containing
acetonitrile up to the neck. After determining weight
difference, fill to mark with acetronitrile. Stock solutions can
be used for up to 6 months when stored at -10 °C or less.
7.11.2.2 SURROGATE ADDITIVE SOLUTION, 20 ug/mL - Dilute
the surrogate stock solution to 20 ug/mL in acetonitrile. This
solution can be used up to 3 months when stored at 4°C or less.
7.11.3 STOCK STANDARD SOLUTION (SSS)
Prepare stock standard solutions for each analyte of interest at a
concentration of 1 to 10 mg/mL in acetonitrile, or purchase SSSs or
primary dilution standards (PDSs) from a reputable supplier. Method
analytes may be obtained as neat materials or as ampulized solutions from
commercial suppliers. The stock standard solutions should be stored at
-10 °C or less and protected from light. Standards prepared in this manner
were stable for at least 60 days. Standards may be used for longer periods
of time if adequate records of stability are kept. Laboratories should use
standard QC practices to determine when their standards need to be
replaced.
7.11.3.1 For analytes which are solids in their pure form, prepare stock
standard solutions by accurately weighing approximately 0.1
gram of pure material to the nearest O.OOOlg in a 10 mL
volumetric flask. Dilute to volume with acetonitrile.
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7.11.3.2 Stock standard solutions for analytes which are liquid in their
pure form at room temperature can be accurately prepared in
the following manner.
7.11.3.2.1 Place about 9.8 mL of aeetonitrile into a 10- mL
volumetric flask. Allow the flask to stand, unstoppered,
for about 10 min. to allow solvent film to evaporate
from the inner walls of the volumetric, and weigh to the
nearest 0.0001 gram.
7.11.3.2.2 Usea 100-uL syringe and immediately add lOOuLof
standard material to the flask by keeping the syringe
needle just above the surface of the aeetonitrile. Be
sure the standard material falls dropwise directly into
the aeetonitrile without contacting the inner wall of the
volumetric.
7.11.3.2.3 Reweigh, dilute to volume, stopper, then mix by
inverting several times. Calculate the concentration in
milligrams per milliliter from the net gain in weight.
7.11.4 PRIMARY DILUTION STANDARD (PDS) - The PDS for this method
should include all method analytes of interest to the analyst. The PDS is
prepared by combining and diluting stock standard solutions with
aeetonitrile to a concentration of 100 ug/mL. Store at -10 °C or less and
protect from light. Standards prepared in this manner were stable for at
least 60 days. Standards may be used for longer periods of time if
adequate records of stability are kept. Laboratories should use standard
QC practices to determine when their standards need to be replaced. This
primary dilution standard is used to prepare calibration spiking solutions,
which are prepared at 5 concentration levels for each analyte, and are used
to spike reagent water to prepare the aqueous calibration standards.
7.11.5 CALIBRATION SPIKING SOLUTIONS - Five calibration spiking
solutions are prepared, each at a different concentration, and are used to
spike reagent water to prepare the calibration standards. The calibration
spiking solutions are prepared from the PDS. Store the calibration spiking
solutions at -10 °C or less and protect from light. Solutions prepared in
this manner were stable for at least 60 days. Solutions may be used for
longer periods of time if adequate records of stability are kept.
Laboratories should use standard QC practices to determine when
solutions need to be replaced. An example of how the calibration spiking
solutions are prepared is given in the following table. Modifications of
this preparation scheme maybe made to meet the needs of the laboratory.
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PREPARATION OF CALIBRATION SPIKING SOLUTIONS
Cal.
Level
1
2
3
4
5
PDS Cone.,
ug/mL
100
100
100
100
100
Vol. PDS
Std., uL
250
500
1000
1500
2000
Final Vol.,
Cal Spike
Sol'n, mLs
5
5
5
5
5
Final Cone.,
Cal Spike
Sol'n, ug/mL
5
10
20
30
40
7.11.6 PROCEDURAL CALIBRATION STANDARDS -- A designated amount
of each calibration spiking solution is spiked into five separate 20 mL
aliquots of reagent water in a 30 mL sample container, to produce
aqueous calibration standards. The reagent water used to make the
calibration standards should contain the preservation reagents described in
Section 8.1.2 (ammonium chloride or ammonium sulfate at 500 mg/L and
copper sulfate pentahydrate at 500 mg/L). Aqueous calibration standards
are processed and analyzed according to the procedures in Section 11.
Resulting data are used to generate a calibration curve. An example of the
preparation of aqueous calibration standards is given below. The lowest
concentration calibration standard should be at or near (within 25% of)
the MRL. Modifications of this preparation scheme may be made to meet
the needs of the laboratory. Preparing aqueous calibration standards using
varying volumes of one calibration spiking solution is an acceptable
alternative to the example below.
PREPARATION OF PROCEDURAL (AQUEOUS)
CALIBRATION STANDARDS
Cal.
Level
1
2
3
4
5
Cal. Spike
Sol'n Cone.,
ug/mL
5
10
20
30
40
Vol. Cal.
Spike
Sol'n., uL
20
20
20
20
20
Final Vol.,
Cal Std
mL
20
20
20
20
20
Final Cone.,
Cal Std
ug/L
5
10
20
30
40
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8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 SAMPLE VIAL PREPARATION
8.1.1 Grab samples must be collected in accordance with conventional sampling
practices (6) using amber glass 30 mL containers with PTFE-lined screw-
caps, or caps with PTFE-faced silicon septa.
8.1.2 Prior to shipment to the field, 15 mg of copper sulfate pentahydrate must
be added to each bottle. This material acts as a biocide to inhibit
bacteriological decay of method analytes. If samples to be collected
contain free chlorine, then 15mg of ammonium chloride or ammonium
sulfate must also be added to the bottle prior to sample collection. The
ammonium compound will react with the free chlorine to form
monochloramine, and retard the formation of additional carbonyl
compounds. Add these materials as dry solids to the sample bottle. The
stability of these materials in concentrated aqueous solution has not been
verified.
NOTE: Aldehydes have been demonstrated to be extremely susceptible
to microbiological decay. The use of other chlorine reducing
agents such as sodium thiosulfate or ascorbic acid, has also
been shown to produce invalid data. Proper sample collection
and preser-vation is important to obtaining valid data. The data
in Section! 7, Table 6 illustrates the importance of proper
sample preservation.
8.2 SAMPLE COLLECTION
8.2.1 Fill sample bottles to just overflowing but take care not to flush out the
sample preservation reagents. The capped sample should be head-space
free.
8.2.2 When sampling from a water tap, remove the aerator so that no air bubbles
will be trapped in the sample. Open the tap, and allow the system to flush
until the water temperature has stabilized (usually about 3-5 min). Collect
samples from the flowing system.
8.2.3 When sampling from an open body of water, fill a 1 quart wide-mouth
bottle or 1L beaker with sample from a representative area, and carefully
fill sample bottles from the container.
8.2.4 After collecting the sample, cap carefully to avoid spillage, and agitate by
hand for 1 min.
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8.3 SAMPLE STORAGE/HOLDING TIMES
8.3.1 Samples must be iced or refrigerated at 4°C and maintained at these
conditions away from light until extraction. Samples must be extracted
within 7 days of sampling. However, since aldehydes are subject to decay
in stored samples, all samples should be derivatized and extracted as soon
as possible.
NOTE: A white or blue precipitate is likely to occur. This is normal
and does not indicate any problem with sample collection or
storage.
8.3.2 Extracts (Sect. 11.1.11) must be stored at 4°C or less away from light in
glass vials with Teflon-lined caps. Extracts must be analyzed within 14
days of extraction.
8.4 FIELD REAGENT BLANKS - Processing of a field reagent blank (FRB) is
required along with each sample set. A sample set is composed of the samples
collected from the same general sampling site at approximately the same time.
Field reagent blanks are prepared at the laboratory before sample vials are sent to
the field. At the laboratory, fill a sample container with reagent water (Sect. 7.2),
add sample preservatives as described in Section 8.1.2, seal and ship to the
sampling site along with the empty sample containers. FRBs should be confirmed
to be free (less than 1/2 the MRL) of all method analytes prior to shipping them to
the field. Return the FRB to the laboratory with filled sample bottles. DO NOT
OPEN THE FRB AT THE SAMPLING SITE. If any of the analytes are
detected at concentrations equal to or greater than 1/2 the MRL, then all data for
the problem analyte(s) should be considered invalid for all samples in the shipping
batch.
9. QUALITY CONTROL
9.1 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 (which includes calculation of the MDL), analysis of
laboratory reagent blanks, laboratory fortified blanks, field reagent blanks,
laboratory fortified sample matrices, and QC samples. Additional QC practices
are encouraged.
9.2 INITIAL DEMONSTRATION OF CAP ABILITY (IDC) - Requirements for the
initial demonstration of laboratory capability are described in the following
sections and summarized in Section 17, Table 7.
9.2.1 Initial demonstration of low system background. (See Sect. 9.3)
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9.2.2 Initial demonstration of precision. Prepare, derivatize, extract, and analyze
4-7 replicate LFBs fortified at 20 ug/L, or other mid-range concentration,
over a period of at least 2 days. Generating the data over a longer period
of time, e.g. 4 or 5 days may produce a more realistic indication of day to
day laboratory performance.' The relative standard deviation (RSD) of the
results of the replicate analyses must be less than 20%.
9.2.3 Initial demonstration of accuracy. Using the same set of replicate data
generated for Section 9.2.2, calculate average recovery. The average
recovery of the replicate values must be within ± 20% of the true value.
9.2.4 MDL(1) determination. Replicate analyses for this procedure should be
done over at least 3 days (both the sample derivatization/extraction and the
GC analyses should be done over at least 3 days). Prepare at least 7
replicate LFBs at a concentration estimated to be near the MDL. This
concentration may be estimated by selecting a concentration at 2-5X the
noise level. Analyze the seven replicates through all steps of Section 11.
Calculate the MDL '
MDL=St(n.1; il alpha = 0.99)
where:
t(n-u-aipha=o.99) ~ Student's t value for me 99% confidence level with
n-1 degrees of freedom
n = number of replicates
S = standard deviation of replicate analyses.
NOTE: Do not subtract blank values when performing MDL
calculations.
9.2.5 Minimum Reporting Level (MRL) — Although an MDL can be calculated
for analytes. that commonly occur as background contaminants, the
calculated MDLs should not be used as the MRL for each analyte.
Method analytes that are seen in the background (typically formaldehyde,
' acetaldehyde) should be reported as present in field samples, only after
careful evaluation of the background levels. It is recommended that a
MRL be established at the mean LRB concentration + 3a, or three times
the mean LRB concentration, whichever is greater. This value should be
calculated over a period of time, to reflect variability in the blank
measurements. It is recommended that this value be used as a minimum
reporting level in order to avoid reporting false positive results.
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9.3 LABORATORY REAGENTS BLANKS (LRB) - Each time a set of samples is
extracted or reagents are changed, a LRB must be analyzed. If within the reten-
tion time window of any analyte, the LRB produces a peak that would prevent the
determination of that analyte, determine the source of contamination and eliminate
the interference before processing samples. Because background contamination is
a significant problem for several method analytes, it is highly recommended that
the analyst maintain a historical record of LRB data. If target analytes are
detected hi the LRB at concentrations equal to or greater than 1/2 the MRL (Sect.
9.2.5), then all data for the problem analyte(s) should be considered invalid for all
samples in the extraction batch.
9.4 CONTINUING CALIBRATION CHECK/LABORATORY FORTIFIED BLANK -
Since this methodology is based on procedural standard calibration, a LFB and the
calibration check sample (CCC) are prepared and analyzed in the same manner.
Laboratory fortified blank QC requirements are therefore omitted. Calibration
procedure options and the QC acceptance criteria associated with them are fully
described hi Sect 10.3. Please refer to that section for these criteria.
9.5 INTERNAL STANDARD~The analyst must monitor the IS response peak area of
all injections during each analysis day. A mean IS response is determined from the
five point calibration curve. The IS response for any chromatographic run should
not deviate from this mean IS response by more than 30%. If a deviation greater
than 30% occurs with an individual extract inject a second aliquot of that extract.
9.5.1 If the reinjected aliquot produces an acceptable internal standard response,
report results for that aliquot.
9.5.2 If a deviation of greater than 30% is obtained for the reinj ected extract, the
analyst should check the calibration by analyzing the most recently
acceptable calibration standard. If the calibration standard fails the criteria
of Section 9.5, recalibration is in order per Section 10. If the calibration
standard is acceptable, extraction of the sample should be repeated
provided the sample is still within the holding time. Otherwise, report
results obtained from the reinjected extract, but annotate as suspect.
9.6 SURROGATE RECOVERY~The surrogate standard is fortified into the aqueous
portion of all calibration standards, samples, FRBs and LRBs. The surrogate is a
means of assessing method performance from derivatization to final
chromatographic measurement.
9.6.1 When surrogate recovery from a sample, blank, or CCC is <70% or
>130%, check (1) calculations to locate possible errors, (2) standard
solutions for degradation, (3) contamination, and (4) instrument
556-16
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performance. If those steps do not reveal the cause of the problem,
reanalyze the extract.
9.6.2 If the extract reanalysis meets the surrogate recovery criterion, report only
data for the reanalyzed extract.
9.6.3 If the extract reanalysis fails the 70-130% recovery criterion, the analyst
should check the calibration by analyzing the most recently acceptable
calibration standard. If the calibration standard fails the criteria of Section
9.6.1, recalibration is in order per Section 10. If the calibration standard is
acceptable, it may be necessary to extract another aliquot of sample if
sample holding time has not been exceeded. If the sample reextract also
fails the recovery criterion, report all data for that sample as suspect.
9.7 LABORATORY FORTIFIED SAMPLE MATRIX (LFM)
9.7.1 Within each analysis set, a minimum of one field sample is fortified as a
LFM for every 20 samples analyzed. The LFM is prepared by spiking a
sample with an appropriate amount of the calibration standard. The
concentrations 5, 10, and 20 ug/L are suggested spiking concentrations.
Select the spiking concentration that is closest to, and at least twice the
matrix background concentration. Use historical data or rotate through the
designated concentrations to select a fortifying concentration. Selecting a
duplicate vial of a sample that has already been analyzed, aids in the
selection of appropriate spiking levels.
9.7.2 Calculate the percent recovery (R) for each analyte, after correcting the
measured concentration, A, from the fortified sample for the background
concentration, B, measured in the unfortified sample, i.e.,
where C is the fortified concentration. Compare these values to
control limits appropriate for reagent water data collected in the
same fashion.
9.7.3 Recoveries may exhibit a matrix dependence. For samples fortified at or
above their native concentration, recoveries should range between 70 -
130%. If the accuracy of any analyte falls outside the designated range,
and the laboratory performance for that analyte is shown to be in control,
the accuracy problem encountered with the fortified sample is judged to be
556-17
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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. Repeated failure to meet the
suggested recovery criteria indicates potential problems with the extraction
procedure and should be investigated.
9.8 FIELD DUPLICATES - Within each analysis batch, a minimum of one field
sample should be analyzed in duplicate. Duplicate sample analyses serve as a
check on sampling and laboratory precision.
9.8.1 Calculate the relative percent difference (RPD) for duplicate
measurements (FD1 and FD2) as shown below.
FD1-FD2 ^
(FD1 + FD2)I2
9.8.2 Relative percent differences for laboratory duplicates should fall in the
range of ± 30 %. Greater variability may be observed for target analytes
with concentrations near their MRL.
9.9 QUALITY CONTROL SAMPLE (QCS) - At least quarterly, analyze a QCS
from an external source. If measured analyte concentrations are not of acceptable
accuracy (60-140% of the expected value), check the entire analytical procedure to
locate and correct the problem source.
9.10 ASSESSDSTGfZ/E) RATIOS -- In addition to monitoring analyte response from
CCC/LFB, the ratio of the peak areas of each isomef pah- should be monitored.
When samples and standards are processed and analyzed by exactly the same
procedure, the ratio of the (Z/E) isomers produced by each method analyte will be
reproducible. This information can be used as a QC check to avoid biased results
caused by an interferant with one isomer of the pair. Calculate and record the ratio
of the peak area of the first eluting isomer (designated (E)) to the second eluting
isomer (designated (Z)). This ratio will be used in data evaluation Section 12.4.
10. CALIBRATION AND STANDARDIZATION
10.1 Demonstration and documentation of acceptable initial calibration is required
before any samples are analyzed, and is required intermittently throughout sample
analysis. After initial calibration is successful, the analyst may choose one of two
options for maintaining on-going calibration. The first option is to verify the
initial calibration daily using a minimum of 2 calibration standards. The other
option is daily calibration of the method with all 5 calibration standards. These
options are further described in Section 10.3.
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10.2 INITIAL CALIBRATION CURVE
10.2.1 Establish GC operating parameters equivalent to the suggested
specifications in Section 17, Table 1. The GC system must be calibrated
using the internal standard (IS) technique. Other GC columns or GC
conditions may be used if equivalent or better performance can be
demonstrated.
10.2.2 Five calibration standards are recommended to calibrate over the range of
approximately 2-40 ug/L. The lowest level standard will depend upon the
level of blank contamination for each analyte (Sect. 7.11.6).
10.2.3 Prepare each calibration standard by the procedural standard calibration
method. Method analytes are fortified into reagent water and carried
through the entire extraction and derivatization procedure described in
Section 11.
10.2.4 Inject 1 uL of each calibration standard extract into the GC and tabulate
peak area response and concentration for each analyte and the internal
standard. NOTE: The formaldehyde peak will be much larger (for the
same concentration) than the other analyte peaks. The formaldehyde peak
may need to be attenuated on some instruments/data systems to avoid
signal saturation.
10.2.5 (Z/E) ISOMERS - Two isomers, referred to as (E) and (Z), are formed for
most asymmetrical carbonyl compounds derivatized with PFBHA.
Chromatographic resolution is usually obtained with the columns
suggested in Section 6.6 for acetaldehyde, propanal, butanal, pentanal,
hexanal, heptanal, octanal, and crotonaldehyde (see chromatograms in Fig.
1 and Fig 2). With dicarbonyl species such as glyoxal and methyl glyoxal,
(E) and (Z) isomerism occurs from oxime formation with both carbonyl
groups, increasing the number of isomers. The demonstration data
included in this method has used two distinct isomer peaks each for
glyoxal and methyl glyoxal. Use one of the following methods for both
calibration and quantitation of each method analyte.
(a) Use the sum of the isomer peak areas for each constituent for both
calibration and quantitation.
(b) Use the peak area of each individual isomer to independently
calculate a concentration for each isomer. Then average the
amount of the two isomers to report one value for the analyte.
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10.2.6 Generate a calibration curve for each analyte by plotting the area ratios
(Aa/Ajs) against the concentration ratios (Ca/Cis) of the five calibration
standards where:
Aj is the peak area of the analyte (or analyte isomer pair),
A,-s is the peak area of the internal standard,
Ca is the concentration of the analyte, and
Cis is the concentration of the internal standard.
10.2.7 This curve must always be forced through zero and can be defined as
either first or second order. Forcing zero allows for a better estimate of the
background level of method analytes.
10.2.8 A data system is recommended to collect the chromatographic data, to
calculate relative response factors, and calculate either linear or second
order calibration curves.
10.2.9 VERIFICATION OF CALIBRATION STANDARD MATERIALS -
Analyze a LFB prepared from standard materials from a source other than
those used to prepare the initial calibration curve (Sect. 3.8, QCS).
Calculate the concentration of this QCS from the calibration curve. The
calculated concentration of the QCS must agree within 60-140% of its true
value. This step verifies the validity of calibration standard materials and
the calibration curve prior to sample analyses.
10.3 OPTIONS FOR ON-GOING CALIBRATION
The time, temperature, pH, and PFBHA concentration will all affect the rate,
efficiency and reproducibility of the derivatization reaction. It is critical that those
parameters be controlled. Calibration frequency will depend upon the
laboratory's ability to control these parameters so that continuing calibration
check standard criteria can be met. Some laboratories may find it more
productive to prepare and analyze a calibration curve with each batch of samples.
A batch of samples for this methodology should not exceed 20 samples, including
field samples, FRBs, laboratory duplicates, and fortified sample matrices.
10.3.1 CONTINUING CALIBRATION CHECK (CCC) OPTION-The analyst
must periodically verify calibration during the analysis of samples in order
to ensure accuracy of analytical results. Prepare a minimum of one low-
level (suggested concentration 2-5 ug/L) and one mid-level (suggested
concentration 10-30 ug/L) calibration standard with each batch of samples.
Verify calibration using these two standards, prior to analyzing any of the
sample extracts from the batch. In addition, reanalyze one of these two
standard extracts after every tenth sample extract, and after the last sample
in an analysis batch to ensure instrument stability throughout the analysis
556-20
-------
batch. Recovery must be within 70-130% of the true value for the mid-
level standard, and within 50-150% of the true value for the low-level
standard.
10.3.2 DAILY CALIBRATION OPTION - The analyst may choose to create a
new calibration curve for each batch of samples by preparing and
analyzing a standard at all five calibration concentrations, with each batch
of samples. If this option is selected, the calibration standard extracts
should be analyzed prior to the analysis of sample extracts. To ensure that
sensitivity and performance of the method has not changed significantly
between sample batches , or changed since the IDC, the following
performance check is required. The response (peak area) of the internal
standard, surrogate and each method analyte in the mid-level standard
(suggested concentration 10-30 ug/L), must be within 50-150% of the
mean peak area for that analyte in the initial demonstration of precision
replicates (Sect. 9.2.2). One of the calibration standard extracts must be
reanalyzed after every tenth sample extract, and after the last sample in an
analysis batch to ensure instrument stability throughout the analysis batch.
Recovery must be within 70 to 130% of the true value for mid- and high-
level calibration standards, and within 50-150% of the true value for the
low-level standard (suggested concentration 2-5 ug/L).
11. PROCEDURE
11.1 SAMPLE EXTRACTION - Once samples have been opened, process the
samples straight through to step 11.1.11. There is no known "safe" stopping point
once sample processing has begun. Samples are derivatized and extracted in the
sample bottle in which they were collected. Transferring the sample to another
container for derivatization and extraction has been shown to cause a loss of
method analytes.
11.1.1 Remove the samples from storage and allow them to equilibrate to room
temperature.
11.1.2 Remove 10 mL of sample and discard. Mark the level of the remaining
sample volume on the outside of the bottle, for later sample volume
determination.
11.1.3 Add 200 mg KHP to the sample for pH adjustment.
11.1.4 Add 20 uL surrogate solution (Sect 7.11.2.2).
11.1.5 Add 1 mL of freshly prepared PFBHA Reagent as per Section 7.10.1.
Cap and swirl gently to mix.
556-21
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11.1.6 Place all samples in a constant-temperature water bath set at 3 5 ± 2 °C
for 2 hrs. Remove vials and cool to room temperature for 10 min.
11.1.7 To each vial add 0.05 mL (approximate 2 to 4 drops) of concentrated
sulfuric acid. This prevents the extraction of excess reagent, which will
cause chromatographic interferences.
11.1.8 Add 4 mL of hexane that contains the internal standard (as per Sect.
7.11.1.2).
11.1.9 Shake manually for 3 min. Let stand for approximately 5 min to permit
phases to separate.
11.1.10 Draw off hexane layer (top layer) using a clean disposable Pasteur pipet
for each sample into a smaller 8 mL vial containing 3 mL 0.2 N sulfuric
acid. Shake for 30 sec and let stand for 5 min for phase separation.
NOTE: This acid wash step further reduces the reagent and other
interferants from the final extract. Do not skip this step.
11.1.11 Draw off top hexane layer using another clean disposable pipet for each
sample and place in two 1.8 mL autosampler vials per sample. Store
extra autosampler vials as a backup extract. Extracts may be stored for
up to 14 days at 4 °C.
11.1.12 Sample Volume Determination ~ Discard remaining water sample and
hexane hi each sample bottle. Fill with water to the level indicated by
the mark made hi Section 11.1.2. Pour the water into a 25 mL graduated
cylinder and measure the volume to the nearest mL. Record the sample
volume for each sample.
Alternately, if a laboratory has control over the brand and style of the
sample bottles being used, the exact volume of a number of bottles from
the same manufacturer and lot may be measured, and the average bottle
volume minus 10 mL may be used as the sample volume for all samples
using the same lot of sample bottles. A minimum of 10 % of the sample
bottles obtained from the same manufacturer, from the same lot should
be measured.
11.2 GAS CHROMATOGRAPHY
11.2.1 Analyze the extracts by GC/ECD. Table 1 (Sect. 17) summarizes
recommended GC operating conditions and retention times observed
using this method. Figure 1 illustrates the performance of the
recommended primary column with the method analytes. Figure 2
556-22
-------
illustrates the performance of the recommended confirmation column
•\ with the method analytes. Other GC columns or chromatographic
conditions may be used if the requirements of Section 9 are met.
11.2.2 The width of the retention time window used to make identifications
should be based on measurements of actual retention time variations of
standards over the course of time. Plus or minus three times the standard
deviation of the 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.
11.2.3 If an analyte peak area exceeds the range of the calibration curve, the
extract may be diluted with the hexane extraction solvent (that contains
the internal standard) and reanalyzed. Incorporate the dilution factor into
final concentration calculations. The analyst must not extrapolate beyond
the calibration range established.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Identify the method analytes in the sample chromatogram by comparing the
retention time of the suspect peak to the retention time of an analyte peak (or
isomer peaks) in a calibration standard or the laboratory fortified blank.
12.2 Calculate the analyte concentrations using the first or second order calibration
curves generated as described in Section 10.
12.3 For any analytes that are found, adjust the calculated concentration to reflect the
true sample volume determined in Section 11.1.12.
12.4 Prior to reporting the data, the chromatograrn should be reviewed for any incorrect
peak identification or poor integration. If a confirmation column has been used,
all identifications should be verified using the retention time data from that
, .analysis. In addition, the (Z/E) isomer ratio should be within 50% of the ratio
observed in standards. If the (Z/E) ratio does not meet these criteria, it is likely
that an interferant occurred at the retention time of one of the isomer peaks. In
this case, the amount indicated by the lower of the 2 isomer peaks should be
reported. (This may require that the analyst recalculate the analyte amount using
individual isomer peaks for quantitation.) If one peak of the isomeric pair is
missing, the identification is not confirmed and should not be reported.
12.5 Analyte concentrations are reported in ug/L.
556-23
-------
13. METHOD PERFORMANCE
13.1 Precision and accuracy data are presented in Section 17. Data are presented for
three water matrices: reagent water (Table 2), chlorinated "finished" surface water
(Table 4) , and untreated "raw" surface water (Table 5). These data, as well as the
MDL data in Table 3, were generated in two laboratories. Data in Table 2 and
column A of Table 3, were generated in one laboratory, while data in column B
of Table 3 and in Tables 4 and 5 were generated in a second laboratory. Method
performance in both laboratories was similar.
13.2 DERTVATIZATION PARAMETERS - This method is a procedural standard
method that will generate accurate and precise results when used as written. The
time, temperature, pH, and PFBHA concentration will all affect the rate,
efficiency and reproducibility of the derivatization reaction. It is critical that those
parameters be controlled. Calibration frequency will depend upon the
laboratory's ability to control these parameters. Some laboratories may need to
prepare and analyze a calibration curve with each batch of samples. Of all the
method analytes, glyoxal, methyl glyoxal, benzaldehyde, crotonaldehyde and
cyclohexanone are the most difficult to derivatize. Poor sensitivity for any of
these compounds indicates that there may be a problem with the reaction
conditions. Measurements of nonanal, decanal, glyoxal and methyl glyoxal
appear to be less precise than the measurement of other analytes.
13.3 The importance of low background levels of formaldehyde and acetaldehyde
cannot be overemphasized. Some laboratories or reagent waters may also contain
background amounts of other method analytes. Care must be taken to avoid
reporting false positive results that result from background contamination.
13.4 The importance of proper sample collection and preservation also cannot be
overemphasized. Holding time studies in various matrices showed better than
70% recovery of all method analytes when samples were collected, preserved, and
stored according to Section 8, and analyzed within 7 days. There were variations
in the recovery of analytes from fortified samples from different matrices.
Therefore, it is strongly recommended that samples be analyzed as soon as
possible after collection. The data in Section 17, Table 6 illustrates the dramatic
difference between a preserved and a non-preserved sample.
13.5 Data for crotonaldehyde is not listed in Section 17, Tables 4-6, because it was not
included in the standard mixtures being used at the time that those data were
collected. However, crotonaldehyde was included in many other studies not
presented here, and its performance was similar to other method analytes.
556-24
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14. POLLUTION PREVENTION
14.1 This method uses a micro-extraction procedure which requires very small
quantities of organic solvents. ,
14.2 For information about pollution prevention that may be applicable to laboratory
operations, consult "Less is Better: Laboratory Chemical Management for Waste
Reduction" available from the American Chemical Society's Department of
Government Relations and Science Policy, 1155 16th Street N.W., Washington,
D.C. 20036.
15. WASTE MANAGEMENT . ...
15.1 Due to the nature of this method tHere is little need for waste management. Only
small volumes of solvents are used. The matrices of concern are finished drinking
water or source water. However, the Agency requires that laboratory waste
management practices be conducted consistent with all applicable rules and
regulations, and that laboratories protect the air, water, and land by minimizing
and controlling all releases from fume hoods and bench operations. Also,
compliance is required with any sewage discharge permits and regulations,
particularly the hazardous waste identification rules and land disposal restrictions.
For further information on waste management, consult "The Waste Management
Manual for Laboratory Personnel" also available from the American Chemical
Society at the address in Section 14.2.
16. REFERENCES
1. Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde, "Trace
Analyses for Wastewaters," Environ. Sci. Technol. 1981, 15, 1426-1435.
2. Standard Method Number 6252B, "PFBHA Liquid-Liquid Extraction Gas
Chromatographic Method", Standard Methods for the Examination of Water and
Wastewater. pp. 6-77 to 6-83, American Public Health Assoc., Washington, D.C.,
1995.
3. Sclimenti, M.J., S.W. Krasner, W.H. Glaze, and H.S. Weinberg,"Ozone
Disinfection By-Products: Optimization of the PFBHA Derivatization Method for
the Analysis of Aldehydes", In Advances in Water Analysis and Treatment, Proc.
AWWA Water Quality Technology Conf., 1990,pp 477-501.
4. Glaze, W.H. and H.S. Weinberg, Identification and Occurrence of Ozonation By-
Products in Drinking Water. American Water Works Assoc. Research Foundation,
Denver, CO., 1993, pp!9-22.
556-25
-------
5. "OSHA Safety and Health Standards, General Industry," (29CRF1910).
Occupational Safety and Health Administration, OSHA 2206, (Revised, Jan.1976).
6. ASTM Annual Book of Standards, Part n? Volume 11.01, D3370-82, "Standard
Practice for Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, 1986.
7. "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. . ..,'....
8. "Safety In Academic Chemistry Laboratories", 3rd Edition, American Chemical
Society Publication, Committee on Chemical Safety, Washington, D.C., 1979.
556-26
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17. TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. CHROMATOGRAPHIC CONDITIONS AND RETENTION DATA FOR
ANALYTE DERIVATIVES
ANALYTE
Formaldehyde
Acetaldehyde (E)
Acetaldehyde (Z)
Propanal (E)
Propanal (Z)
Butanal (E)
Butanal (Z)
Crotonaldehyde (E)
Crotonaldehyde (Z)
Pentanal (E)
Pentanal (Z)
Hexanal (E)
Hexanal (Z)
Cyclohexanone
Heptanal (E)
Heptanal (Z)
Octanal (E)
Octanal (Z)
Benzaldehyde
NTonanal
Decanal
Column A
RT(min)
10.69
14.23
14.49
17.36
17.62
20.58
20.81
22.67
23.00
23.82
24.01
26.99
27.16
29.42
30.05
30.16
33.01
33.09
33.88
35.89
38.63
Peak #, Fig 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
19
20
21
22
23
Column B
RT (min)
13.35
16.71
17.00
19.59
19.86
22.62
22.88
24.79
25.31
25.54
25.70
28.74
28.93
31.40
31.64
31.78
34.45
34.57
36.74
37.18
39.80
Peak#,Fig.2
2
3
4
5
6
7
8
not shown
not shown
9
10
11
12
13
14
15
16
18
19
20
21
556-27
-------
ANALYTE
Glyoxal (peakl)
Glyoxal (peak 2)
Methyl glyoxal
(peakl)
Methyl glyoxal
(peak 2)
1.2 dibromopropane
(Internal standard)
2',4',5I trifluoro-
acetophenone
(Surrogate)
Column A
RT (min)
40.87
41.09
41.22
41.88
6.14
32.84
Peak #, Fig 1
24
25
26
27
1
18
Column B
RT (min)
43.74
43.86
43.86
44.38
7.84
34.45
Peak #, Fig.2
22
23
24
25
1
17
Column A- DB-5ms, 30 m x 0.25mm i.d., 0.25 urn film thickness, injector temp. 220°C, head
pressure 15 psi, detector temp. 300 °C, splitless injection, 1 min split delay.
Temperature program: 50 °C for 1 min, program at 4°C/min to 220 °C, program at
20 °C/min to 250 °C and hold at 250 °C for 10 min. (Figure 1)
Column B- Rtx-1701, 30 m x 0.25 mm i.d., 0.25 urn film thickness, injector temp. 180 °C,
head pressure 15 psi, detector temp. 300°C,splitless injection, 1 min split delay.
Temperature program: 50 °C for 1 min, program at 4°C/min to 220 °C, program at
20 °C/min to 250 °C and hold at 250 °C for 10 min. (Figure 2)
Carrier gas- Helium
Detector gas- P5 Argon/Methane
556-28
-------
TABLE 2. PRECISION AND ACCURACY IN REAGENT WATER (n=8)
ANALYTE
Formaldehyde
Acetaldehyde
Propanal
Butanal
Crotonaldehyde
Pentanal
Hexanal
Cyclohexanone
Heptanal
Octanal
Benzaldehyde
Nonanal
Decanal
Glyoxal
Methyl glyoxal
Fortified
Cone. (ug/L)
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
Mean Cone.
Measured
(ug/L)
19.5
19.2
19.5
19.8
19.8
20.0
19.7
19.2
19.2
19.1
19.1
18.8
18.7
18.4
17.9
Standard
Deviation
0.792
0.784
0.873
1.12
1.11
1.10
1.25
0.729
1.37
1.32
0.362
1.09
1.20
0.571
1.36
Relative
Std Dev (%)
4.0
4.1
4.5
5.6
5.6
5.5
6.4
3.8
7.1
6.9
1.9
5.8
6.4
3.1
7.6
Mean
Accuracy
(%)
98
96
97
99
99
100
98
96
98
95
95
94
93
92
90
a- Analyzed over 2 days on the primary chromatographic column (DB-5ms).
556-29
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TABLE 3. METHOD DETECTION LIMITS IN REAGENT WATER (n=8)
ANALYTE
Formaldehyde
Acetaldehyde
Propanal
Butanal
Crotonaldehyde
Pentanal
Hexanal
Cyclohexanone
Heptanal
Octanal
Benzaldehyde
Nonanal
Decanal
Glyoxal
Methyl glyoxal
Fortified
Cone. (ug/L)
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
MDL(ug/L)
Column A
0.36
0.21
0.41
0.35
0.28
0.47
0.42
0.29
0.43
0.60
0.31
0.74
1.0
0.59
0.81
MDL(ug/L)
ColumnB
0.11
0.14
0.06
0.12
0.09
0.17
0.35
0.10
0.71
0.12
0.06
0.40
0.82
0.21
0.22
Column A= DB-5ms
Column B=Rtx-1701
556-30
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TABLE 4. PRECISION AND ACCURACY IN CHLORINATED TAP WATER(n=4)
ANALYTE
Formaldehyde
Acetaldehyde*
Propanal
Butanal
Pentanal
Hexanal
Cyclohexanone
Heptanal
Octanal
Benzaldehyde
Nonanal
Decanal
Glyoxal
Methyl glyoxal
Fortified
Cone. (ug/L)
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
Mean Cone.
Measured
(ug/L)
21.6
19.6
19.5
20.2
20.3
20.2
20.7
20.5
20.3
21.2
20.0
19.8
22.3
21.0
Standard
Deviation
0.197
0.263
0.178
0.254
0.318
0.382
0.247
3.073
0.979
0.432
0.828
1.203
0.783
0.775
Relative
Std Dev (%)
0.9
1.3
0.9
1.3
1.6
1.9
1.2
2.5
4.8
2.0
4.2
6.1
3.5
3.7
Mean
Accuracy
(%)
108
98
98
101
101
101
103
103
102
106
100
99
112
105
a- Analyzed on the primary chromatographic column (DB-5ms).
*- Values taken from the confirmation column.
556-31
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TABLE 5. PRECISION AND ACCURACY IN UNTREATED SURFACE WATER (n=4)
ANALYTE
Formaldehyde
Acetaldehyde*
Propanal
Butanal
Pentanal
Hexanal
Cyclohexanone
Heptanal
Octanal
Benzaldehyde
Nonanal
Decanal
Glyoxal
Methyl glyoxal
Fortified
Cone. (ug/L)
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
Mean Cone.
Measured
(ug/L)
19.4
20.0
19.5
18.9
18.7
18.2
17.3
18.0
18.3
17.2
18.4
18.1
20.5
23.3
Standard
Deviation
0.941
0.891
0.966
0.965
1.00
1.080
0.943
0.995
1.017
1.118
0.881
0.908
1.10
0.799
Relative
StdDev(%)
4.8
4.4
5.0
5.1
5.4
6.0
5.4
5.5
5.6
6.5
4.8
5.0
5.4
3.4
Mean
Accuracy
(%)
97
100
97
94
93
91
87
90
92
86
92
90
102
116
a- Analyzed on the primary chromatographic column (DB-5ms).
*- Values taken from the confirmation column.
556-32
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TABLE 6. HOLDING TIME DATA FOR SAMPLES FROM AN UNTREATED
SURFACE WATER SOURCE, FORTIFIED WITH METHOD ANALYTES
AT 20 ug/L, WITH AND WITHOUT COPPER SULFATE BIOCIDE
Analyte
Formaldehyde
Acetaldehyde
Propanal
Butanal
Pentanal
Hexanal
Cyclohexanone
Heptanal
Octanal
Benzaldehyde
Nonanal
Decanal
Glyoxal
Methyl glyoxal
% Recovery without Copper
S u If ate
DayO
104
96
94
92
87
83
94
83
82
94
72
50
103
108
Day 6
144
23
21
20
19
21
99
20
18
83
15
<10
98
68
Day 14
569
21
19
18
16
17
84
16
<10
." 74
<10
<10
37
<10
Day 21
619
24
22
21
21
22
78
17
11
79
<10
<10
<10
<10
% Recovery with Copper Sulfate
DayO
105
98
98
99
96
93
96
96
99
98
104
107
106
111
Day 6
105
99
98
98
98
97
101
94
96
100
98
101
108
105
Day 14
109
103
103
102
100
100
98
97
96
104
92
93
106
94
Day 21
106
98
96
91
94
92
94
91
93
92
84
82
90
73
- All samples were stored headspace free at 4 °C.
- Values at all time points are the mean of 5 replicate analyses. RSDs for replicate analyses of
samples containing copper sulfate were <10%. RSDs for unpreserved samples were higher due
to the degradation process.
556-33
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TABLE 7. INITIAL DEMONSTRATION OF CAPABILITY REQUIREMENTS
Reference
Sect. 9.3
Sect 9.2.2
Sect. 9.2.3
Sect. 9.2.4
Sect. 9.2.5
Requirement
Initial
Demonstration of
Low System
Background
Initial
Demonstration of
Precision (TOP)
Initial
Demonstration of
Accuracy
Method Detection
Limit (MDL)
Determination
Minimum
Reporting Levels
(MRLs)
Specification and Frequency
Analyze method blank and
determine that all target
analytes are below 1/2 the MRL
prior to performing IDC
Analyze 4-7 replicate LRBs
fortified at 20.0 ug/L (or mid
cal.) on at least 2 different days
Calculate average recovery of
IDP replicates
a) select a fortifying level at 2 -
5 x the noise level .
b) analyze 7 replicates in
reagent water taken thru all
steps
c) calculate MDL via equation -
do not subtract blank
d) replicate extractions and
analyses must be conducted
over at least 3 days
MRLs should be established for
all analytes during IDC, and be
updated as additional LRB data
is available.
Acceptance Criteria
The LRB concentration
must be < 1/2 of the
intended MRL
RSD must be <: 20 %
Mean recovery ± 20% of
true value
Establish the MRL for
each analyte, as the LRB
concentration + 3 a or 3
times the mean LRB
concentration, whichever
is greater.
556-34
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TABLE 8. QUALITY CONTROL REQUIREMENTS (SUMMARY)
Reference
Sect. 10.2
Sect. 9.3
Sect. 10.3.1
Sect. 10.3.2
Sect. 8.4
Sect. 9.5
Requirement
Initial
Calibration
Laboratory
Reagent Blank
(LRB)
Continuing
Calibration
Check (CCC)
Option
Daily
Calibration
Option
Field Reagent
Blanks (FRB)
Internal
Standard (IS)
Specification and Frequency
Use internal standard technique
to generate curve with five
standards that span the
approximate range of 2-40 ug/L.
First or second order curves must
be forced through zero. Either
sum E/Z isomer areas or average
the amount of each isomer.
Calculate E/Z ratios for analytes.
Run QCS.
Include LRB with each extraction
batch (up to 20 samples).
Analyze prior to analyzing
samples and determine to be free
of interferences.
Verify initial calibration by
running CCCs prior to analyzing
samples, after 10 samples, and
after the last sample.
Calibrate daily, but verify that
sensitivity and performance have
not changed significantly since
IDC.
1 per shipping batch
1,2-Dibromopropane is added to
all samples, blanks and standards
Acceptance Criteria
QCS must agree within
60 -140 %.'
Lowest concentration
should be near MRL.
All analytes < 1/2 MRL
Recovery for mid-level
CCC must be 70-130%
of the true value,
recovery for low level
must be 50-150% of the
true value.
Peak areas for IS, SUR,
and method analytes for
mid-level C AL std must
be +/- 50% of the peak
areas obtained for that
CAL std during IDC.
All analytes < 1/2 MRL
IS area counts must be
70 - 130% of the average
initial calibration area
counts
556-35
-------
Reference
Sect. 9.6
Sect. 9.7
Sect. 9.8
Sect. 9.9
Sect.9.10,
Sect 10.2.5
and
Sect 12.4
Sect 8.3.1
Sect 8.3.2
Requirement
Surrogate
Standard
(SUR)
Laboratory
Fortified
Sample Matrix
(LFM)
Field
Duplicates
Quality
Control
Sample (QCS)
E/Z Isomer
Ratio
Agreement
Sample
Holding Time
Extract
Holding Time
Specification and Frequency
2',4',5' -Trifluoroacetophenone is
added samples, blanks and
standards
Fortify at least one sample per
analysis batch (20 samples or
less) at a concentration close to
that in the native sample.
Extract and analyze at least one
duplicate sample with every
analysis batch (20 samples or
less)
Analyze a QCS at least quarterly
from an external/second source.
Calculate the E/Z isomer ratio for
target analytes and compare to
E/Z ratio in initial calibration
Properly preserved samples may
be stored in the dark at 4 ° C for 7
days.
Extracts may be stored in the dark
at 4 °C for 14 days.
Acceptance Criteria
Surrogate recovery must
be 70 - 130 % of the true
value.
Recoveries not within
70-130% may indicate
matrix effect
Suggested RPD <; 30 %
QCS must agree within
60 -140 %.
E/Z ratio in standards,
blanks, and samples must
be within ± 50% of E/Z
ratio in initial calibration.
Do not report value if
one isomer is missing.
Do not report data for
samples that have
exceeded their holding
time, or that have not
been properly preserved
or stored.
Do not report data for
extracts that have
exceeded their holding
time.
556-36
-------
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556-38
-------
METHOD 556.1 DETERMINATION OF CARBONYL COMPOUNDS IN DRINKING
WATER BY FAST GAS CHROMATOGRAPHY
Revision 1.0
September 1999
J.W. Munch, USEPA Office of Research and Development, and
D.J. Munch, USEPA, Office of Ground Water and Drinking Water and
S.D. Winslow, S.C. Wendelken, B.V. Pepich, ICF Kaiser Engineers, Inc. - Method 556,
Revision 1.0 (1998)
S.C. Wendelken and B.V. Pepich (IT Corporation) and D.J. Munch, USEPA, Office of
Ground Water and Drinking Water
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
556.1-1
-------
METHOD 556.1
DETERMINATION OF CARBONYL COMPOUNDS IN DRINKING WATER BY FAST
GAS CHROMATOGRAPHY
1.
SCOPE AND APPLICATION
1.1
1.2
This is a fast gas chromatographic method optimized for the determination of
selected carbonyl compounds in finished drinking water and raw source water.
The analytes applicable to this method are derivatized to their corresponding
pentafluorobenzyl oximes. The oxime derivatives are then extracted from the
water with hexane. The hexane extracts are analyzed by fast gas chrornatography
with electron capture detection (FGC-ECD). Fast GC uses a small diameter
capillary column (< 100 jam i.d.), hydrogen carrier gas and a fast oven ramp rate
to dramatically decrease analysis tune. Accuracy, precision, and method detection
limit (MDL) data have been generated for the following compounds:
Analyte
Formaldehyde
Acetaldehyde
Propanal
Butanal
Pentanal
Hexanal
Heptanal
Octanal
Nonanal
Decanal
Cyclohexanone
Benzaldehyde
Glyoxal (ethanedial)
Chemical Abstract Services
Registry Number
50-00-0
75-07-0
123-38-6
123-72-8
110-62-3
66-25-1
111-71-7
.124-13-0
124-19-6
112-31-2
108-94-1
100-52-7
107-22-2
Methyl glyoxal (2-oxopropanal or pyruvic aldehyde) 78-98-8
This method applies to the determination of target analytes over the concentration
ranges typically found in drinking water. Analyte retention times are in Section
17, Tables 1 and 2. Other method performance data are presented in Section 17,
Tables 3-7. Experimentally determined method detection limits (MDLs) for the
above listed analytes are provided in Section 17, Table. 4. The MDL is defined as
the statistically calculated minimum amount that can be measured with 99%
556.1-2
-------
confidence that the reported value is greater than zero.(1>2) However, it should be
noted that background levels of some method analytes (usually formaldehyde and
acetaldehyde) are problematic. The minimum reporting level (MRL) for method
analytes, for each analyst/laboratory that uses this method, will depend on their
ability to control background levels (Section 4).
1.3 This method is restricted to use by or under the supervision of analysts skilled in
liquid-liquid extractions, derivatization procedures and the use of GC and
interpretation of gas chromatograms. The analyst should be thoroughly familiar
with the practice of fast GC before significantly modifying the conditions in Table
1. Each analyst must demonstrate the ability to generate acceptable results with
this method, using the procedures described in Section 9.
2. SUMMARY OF METHOD
2.1 A 20 mL volume of water sample is adjusted to pH 4 with potassium hydrogen
phthalate (KHP) and the analytes are derivatized at 35 °C for 2 hr with 15 mg of
O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine (PFBHA) reagent. The oxime
derivatives are extracted from the water with 4 mL hexane. The extract is
processed through an acidic wash step, and then analyzed by FGC-ECD. The
target analytes are identified and quantified by comparison to a procedural
standard (Section 3.9). Two chromatographic peaks will be observed for many of
the target analytes. Both (E) and (Z) isomers are formed for carbonyl compounds
that are asymmetrical, and that are not sterically hindered. The (E) and (Z)
isomers may not be chromatographically resolved in a few cases. Compounds
with two carbonyl groups, such as glyoxal and methyl glyoxal, can produce even
more isomers. See Section 17, Table 1 and Figure 1 for the chromatographic
peaks used for analyte identification.
NOTE: The absolute identity of the (E) and (Z) isomers was not determined
during method development. Other researchers (3>4>5) have reported the first eluting
peak as (E),and the second peak as (Z). For convenience, this method will follow
this convention. Because more than 2 isomers are formed for glyoxal and methyl
glyoxal, the peaks used for identification are referred to as "peak 1" and "peak 2."
2.2 All results should be confirmed on a second, dissimilar capillary GC column.
3. DEFINITIONS
3.1 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or
other blank matrix that is treated exactly as a sample including exposure to all
glassware, equipment, solvents and reagents, sample preservatives, internal
standards, and surrogates that are used with other samples. The LRB is used to
556.1-3
-------
determine if method analytes or other interferences are present in the laboratory
environment, the reagents, or the apparatus.
3.2 FIELD REAGENT BLANK (FRB) - An aliquot of reagent water or other blank
matrix that is placed in a sample container in the laboratory and treated as a
sample in all respects, including shipment to the sampling site, storage,
preservation, and all analytical procedures. The purpose of the FRB is to
determine if method analytes or other interferences are introduced during sample
shipping or storage. For this analysis the FRB should not be opened at the
sampling site.
3.3 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water or
other blank matrix to which known quantities of the method analytes are added in
the laboratory. The LFB is analyzed exactly like a sample, and its purpose is to
determine whether the methodology is in control, and whether the laboratory is
capable of making accurate and precise measurements.
3.4 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) -- An aliquot of an
environmental sample to which known quantities of the method analytes are
added in the laboratory. The LFM is analyzed exactly like a sample, and its
purpose is to determine whether the sample matrix contributes bias to the
analytical results. The background concentrations of the analytes in the sample
matrix must be determined in a separate aliquot and the measured values in the
LFM corrected for background concentrations.
3.5 STOCK STANDARD SOLUTION (SSS) - A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed reference
materials or purchased from a reputable commercial source.
3.6 PRIMARY DILUTION STANDARD SOLUTION (PDS) -- A solution of several
analytes prepared in the laboratory from stock standard solutions and diluted as
needed to prepare calibration solutions and other needed analyte solutions.
3.7 CALIBRATION STANDARD (CAL) — A solution prepared from the primary
dilution standard solution and stock standard solutions and the internal standards
and surrogate analytes. The CAL solutions are used to calibrate the instrument
response with respect to analyte concentration.
3.8 QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of
known concentrations which is used to fortify an aliquot of LRB or sample
matrix. The QCS is obtained from a source external to the laboratory and
different from the source of calibration standards. It is used to check laboratory
performance with externally prepared test materials.
556.1-4
-------
3.9 PROCEDURAL STANDARD CALIBRATION - A calibration method where
aqueous calibration standards are prepared and processed (e.g. purged, extracted,
and/or derivatized) in exactly the same manner as a sample. All steps in the
process from addition of sampling preservatives through instrumental analyses are
included in the calibration. Using procedural standard calibration compensates
for any inefficiencies in the processing procedure.
3.10 INTERNAL STANDARD (IS) - A pure analyte added to a sample, extract, or
standard solution in known amount(s) and used to measure the relative responses
of other method analytes and surrogates that are components of the same sample
or solution. The internal standard must be an analyte that is not a sample
component.
3.11 SURROGATE ANALYTE (SUR) -- A pure analyte, which is extremely unlikely
to be found in any sample, and which is added to a sample aliquot in known
amount(s) before extraction or other processing and is measured with the same
procedures used to measure other sample components. The purpose of the SUR is
to monitor method performance with each sample.
3.12 METHOD DETECTION LIMIT (MDL) - The minimum concentration of an
analyte that can be identified, measured and reported with 99% confidence that
the analyte concentration is greater than zero.
3.13 MATERIAL SAFETY DATA (MSDS) - Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical properties, fire,
and reactivity data including storage, spill, and handling precautions.
3.14 CONTINUING CALIBRATION CHECK (CCC) - A calibration standard
containing one or more method analytes, which is analyzed periodically to verify
the accuracy of the existing calibration for those analytes.
3.15 MINIMUM REPORTING LEVEL (MRL) - The minimum concentration of an
analyte that should be reported. This concentration is determined by the
background level of the analyte in the LRBs and the sensitivity of the method to
the analyte. The MRL will be at or near the concentration of the lowest calibration
standard.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in laboratory air, solvents,
reagents (including reagent water), glassware, sample bottles and caps, and other
sample processing hardware that lead to discrete artifacts and/or elevated
baselines in the chromatograms. All items such as these must be routinely
demonstrated to be free from interferences (less than 1/2 the MRL) under the
556.1-5
-------
conditions of the analysis by analyzing laboratory reagent blanks as described in
Section 9.3. Subtracting blank values from sample results is not permitted.
4.1.1 Before attempting analyses by this method, the analyst must obtain a
source of sufficiently pure reagent water. This water must not contain
target carbonyl compounds or interferences at a concentration greater than
1/2 of the MRL. The most likely interferents are formaldehyde and
acetaldehyde in the reagent water. The most successful techniques for
generating aldehyde free water are (1) exposure to UV light, or (2)
distillation from permanganate.
4.1.2 Commercially available systems for generating reagent grade water have
proved adequate, if a step involving exposure to UV light is included. For
the data presented in this method, a Millipore Elix 3 reverse osmosis
system followed by a Milli-Q TOC Plus polishing unit provided reagent
water with background levels of 1 \igfL or less for each method analyte.
Other researchers have reported typical blank values of 1-3 |j,g/L.(4'5)
4.1.3 Distillation of reagent water from acidified potassium permanganate has
been reported as an effective method of eliminating background levels of
aldehydes.® Distill 500 mL of reagent water to which 64 nig potassium
permanganate and 1 mL concentrated sulfuric acid have been added. In
our laboratory, this procedure reduced formaldehyde levels to
approximately 3 [ig/L.
4.1.4 It may be necessary to purchase reagent grade water. If acceptably clean
reagent grade water is purchased, care must also be taken to protect it from
contamination caused by contact with laboratory air.
4.2 Formaldehyde is typically present in laboratory air and smaller amounts of other
aldehydes may also be found. Care should be taken to minimize exposure of
reagents and sample water with laboratory air. Because latex is a potential
aldehyde contaminant source, protective gloves should not contain latex. Nitrile
gloves, such as N-Dex Plus, are acceptable. Bottle caps should be made of
polypropylene. Commonly used phenolic resin caps must be avoided because
they can introduce formaldehyde contamination into samples.
4.3 Reagents must also be free from contamination. Many brands of solvents may
contain trace amounts of carbonyl compounds.
4.4 Glassware must be scrupulously cleaned by detergent washing with hot water, and
rinses with tap water and distilled water. Glassware should then be drained, dried,
and heated in a laboratory oven at 130 °C for several hours before use. Solvent
rinses with methanol or acetonitrile, followed by air drying, may be substituted
556.1-6
-------
for the oven heating. After cleaning, glassware should be stored in a clean
environment to prevent any accumulation of dust or other contaminants.
4.5 Matrix interferences may be caused by contaminants that are co-extracted from
the sample. The extent of matrix interferences will vary considerably from source
to source, depending upon the nature and diversity of the matrix being sampled.
4.6 An interferant that elutes just prior to the acetaldehyde (E) isomer peak on the
primary column is typically observed in chlorinated or chloraminated waters. If
this peak interferes with the integration of the acetaldehyde (E) isomer peak, then
acetaldehyde should be quantified using only the acetaldehyde (Z) isomer, or
from the confirmation column data.
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 safety data sheets should also be made available to all
personnel involved in the chemical analysis. Additional references to laboratory
safety are available.(6"9)
5.2 Formaldehyde and acetaldehyde have been tentatively classified as known or
suspected human or mammalian carcinogens. Glyoxal and methyl glyoxal have
been shown to be mutagenic in in-vitro tests.(3)
5.3 Although hydrogen can be used as a carrier gas safely, the potential for fire or
explosion does exist if the gas system is mishandled. If you are unsure of the
safety guidelines for using hydrogen as a carrier gas, seek advice from your
instrument manufacturer regarding its use.
6. EQUIPMENT AND SUPPLIES (All specifications are suggested. Brand names and/or
catalog numbers are included for illustration only.)
6.1 SAMPLE CONTAINERS - Grab Sample Bottle (aqueous samples) -- 30 mL
amber glass, screw cap bottles and caps equipped with Teflon-faced silicone
septa. Screw caps should be polypropylene. Typical phenolic resin caps should
be avoided due to the possibility of sample contamination from
formaldehyde. Prior to use, wash bottles and septa according to Section 4.4.
556.1-7
-------
6.2 VIALS - 8 mL or 12 mL vials for the acid wash step (Section 11.1.10), and GC
autosampler vials, both types must be glass with Teflon-lined polypropylene caps.
6.3 VOLUMETRIC FLASKS - various sizes used for preparation of standards.
6.4 BALANCE - Analytical, capable of accurately weighing to the nearest 0.0001 g.
6.5 WATER BATH or HEATING BLOCK - Capable of maintaining 35 ± 2 °C
6.6 GAS CHROMATOGRAPH - Capillary Gas Chromatograph (Hewlett Packard
6890 or equivalent), equipped for fast gas chromatography. Modifications will
include a high pressure (>50 psi) split/splitless injector, fast temperature ramp (30
"C/minute) oven and a low volume (150 \\L ) micro BCD detector. Additionally, a
data system capable of fast sampling (20 points/peak) is required.
6.6.1 Primary Column -lOmxO.10 mm J&W DB-5, 0.10 urn film thickness
(or equivalent).
6.6.2 Confirmation Column - 10 m x 0.10 mm Alltech AT-1701, 0.10 urn film
thickness (or equivalent)
6.6.3 Straight injection port liners (< 2 mm i.d.) packed with a central 2 cm plug
of silanized glass wool. Hewlett Packard PN 5181-3317 or equivalent.
7. REAGENTS AND STANDARDS
7.1 Reagent grade or better chemicals should be used in all tests. Unless otherwise
indicated, it is intended that all reagents shall conform to the specifications of the
Committee on Analytical Reagents of the American Chemical Society, where
such specifications are available. Other grades may be used, provided it is first
ascertained that the reagent is of sufficiently high purity to permit its use without
lessening the accuracy of the determination.
7.2 REAGENT WATER — Reagent water as free as possible from interferences and
contamination is critical to the success of this method. See Section 4.1.
7.3 ACETONTTRILE ~ High purity, demonstrated to be free of analytes and
interferences.
7.4 HEXANE -- High purity, demonstrated to be free of analytes and interferences:
B&J Brand, GC2 grade or equivalent.
7.5 POTASSIUM HYDROGEN PHTHALATE (KHP) ~ ACS Grade or better.
556.1-8
-------
7.6 0-(2,3,5,6-PENTAFLUOROBENZYL)-EYDROXYLAMINE
HYDROCHLORIDE (PFBHA) - 98+%, Aldrich cat. #19,448-4. (Store in a
desiccator - Do not refrigerate).
7.7 SULFURIC ACID - ACS Grade or better.
7.8 COPPER SULFATE PENTAHYDRATE - ACS Grade or better.
7.9 AMMONIUM CHLORIDE, NH4C1 or AMMONIUM SULFATE, (NH4)2SO4-
ACS grade or better.
7.10 SOLUTIONS
7.10.1 PFBHA REAGENT - Prepare a fresh 15 mg/mL solution in reagent water
daily. Prepare an amount appropriate to the number of samples to be
derivatized. One mL of solution is added per sample. For example, if 14
sample vials are being extracted, prepare 15 mL of solution. For a 15 mL
volume of solution, weigh 0.225 grams of PFBHA into a dry 40 mL vial,
add 15 mL water and shake to dissolve.
7.10.2 0.2 N SULFURIC ACID - Add 5 mL of concentrated sulfuric acid to 900
mL of reagent water.
7.11 STOCK STANDARD SOLUTIONS - When a compound purity is assayed to be
96% or greater, the weight can be used without correction to calculate the
concentration of the stock standard.
7.11.1 INTERNAL STANDARD (IS) - 1,2-DffiROMOPROPANE, 98+%
purity. An alternate compound may be used as the IS at the discretion of
the analyst. If an alternate is selected, an appropriate concentration will
need to be determined.
7.11.1.1 INTERNAL STANDARD STOCK SOLUTION, (10,000
Hg/mL) -- Accurately weigh approximately 0.1 gram to the
nearest 0.000 Ig, into a tared 10 mL volumetric flask containing
hexane up to the neck. After determining weight difference,
fill to mark with hexane. Stock solutions can be used for up to
6 months when stored at-10 °C.
7.11.1.2 INTERNAL STANDARD FORTIFIED EXTRACTION
SOLVENT, 400 |ig/L in hexane - This is the solvent used to
extract the derivatized samples. The internal standard is added
to the solvent prior to performing the extraction. The volume
of this solvent to be prepared should be determined by the
sample workload. The following example illustrates
556.1-9
-------
preparation of 1 L of fortified solvent. If fewer samples are to
be analyzed each month, prepare smaller batches of working
solvent. Add 40 |iL of internal standard stock solution directly
to 1 L of hexane in a volumetric flask. Cap flask and invert
three times to ensure thorough mixing. Transfer to 1 L storage
bottle with Teflon lined cap. This solution can be used up to 4
weeks. As a check, run a sample of this working solvent on the
FGC before the first extraction of aqueous samples. Have
enough working solvent available to extract all calibration and
aqueous samples in each extraction set. Never use two
different batches of working solvent for one set of extractions.
7.11.2 SURROGATE (SUR) - 2',4',5' -TRIFLUOROACETOPHENONE
This compound was found to be an appropriate surrogate analyte for these
analyses. However, the chromatograms for this analysis are very crowded,
and all possible matrix interferences cannot be anticipated. An alternate
carbonyl compound maybe selected as the surrogate analyte if matrix
interferences or chromatographic problems are encountered. Any
surrogate analyte selected must form an oxime derivative, because one of
its purposes is to monitor the derivatization process. If an alternate
surrogate is selected, its concentration may also be adjusted to meet the
needs of the laboratory.
7.11.2.1 SURROGATE STOCK SOLUTION, 10,000 ng/mL -
Accurately weigh approximately 0.1 gram SUR to the nearest
0.000 Ig, into a 10 mL tared volumetric flask containing room
temperature (25 °C) acetonitrile up to the neck. After
determining weight difference, fill to mark with acetonitrile.
Stock solutions can be used for up to 6 months when stored at -
10 "Cor less.
7.11.2.2 SURROGATE ADDITIVE SOLUTION, 20 ^g/mL - Dilute
the surrogate stock solution to 20 [ig/mL in acetonitrile. This
solution can be used up to 3 months when stored at 4 °C or less.
7.11.3 STOCK STANDARD SOLUTION (SSS)
Prepare stock standard solutions for each analyte of interest at a
concentration of 1 to 10 mg/mL. Acetonitrile should be used as the
solvent for all analytes except glyoxal. Glyoxal standards should be
prepared using a volumetric 90:10 acetonitrile:water mixture due to the
limited solubility of glyoxal in pure acetonitrile. Method analytes may be
obtained as neat materials or as ampulized solutions from commercial
suppliers. The stock standard solutions should be stored at -10 °C or less
and protected from light. Standards prepared in this manner were stable
556.1-10
-------
for at least 60 days. Laboratories should use standard QC practices to
determine when their standards need to be replaced.
7.11.3.1 For analytes which are solids in their pure form, prepare stock
standard solutions by accurately weighing approximately 0.1
gram of pure material to the nearest O.OOOlg in a 10 mL
volumetric flask. Dilute to volume with solvent.
7.11.3.2 Stock standard solutions for analytes which are liquid in their
pure form at room temperature can be accurately prepared in
the following manner.
7.11.3.2.1 Place about 9.8 mL of solvent into a 10- mL
volumetric flask. Allow the flask to stand,
unstoppered, for about 10 min to allow solvent film
to evaporate from the inner walls of the volumetric,
and weigh to the nearest 0.0001 gram.
7.11.3.2.2 Use a 100-uJL syringe and immediately add 100 |iL
of standard material to the flask by keeping the
syringe needle just above the surface of the solvent.
Be sure the standard material falls dropwise directly
into the solvent without contacting the inner wall of
the volumetric.
7.11.3.2.3 Reweigh, dilute to volume, stopper, then mix by
inverting several times. Calculate the concentration
in milligrams per milliliter from the net gain in
weight.
7.11.4 PRIMARY DILUTION STANDARD (PDS) - The PDS for this method
should include all method analytes of interest. The PDS is prepared by
combining and diluting stock-standard solutions with acetonitrile to a
concentration of 100 |4,g/mL. Store at -10 "C or less and protect from light.
Standards prepared in this manner were stable for at least 60 days.
Laboratories should use standard QC practices to determine when their
standards need to be replaced. This primary dilution standard is used to
prepare "calibration spiking solutions, which are prepared at 5
concentration levels for each analyte, and are used to spike reagent water
to prepare the aqueous calibration standards.
7.11.5 CALIBRATION SPIKING SOLUTIONS - Five calibration spiking
solutions are prepared, each at a different concentration, and are used to
spike reagent water to prepare the calibration standards. The calibration
spiking solutions are prepared from the PDS. Store the calibration spiking
556.1-11
-------
solutions at -10 °C or less and protect from light. Solutions prepared in
this manner were stable for at least 60 days. Laboratories should use
standard QC practices to determine when solutions need to be replaced.
An example of how the calibration spiking solutions are prepared is given
hi the following table. Modifications of this preparation scheme may be
made to meet the needs of the laboratory.
PREPARATION OF CALIBRATION SPIKING SOLUTIONS
Calibration
Level
1
2
3
4
5
PDS
Concentration,
Og/mL)
100
100
100
100
100
Volume PDS
Standard,
OiL)
250
500
1000
1500
2000
Final Volume
Calibration
Spike
Solution, (mL)
5
5
5
5
5
Final
Concentration
Calibration
Spike Solution,
(jig/mL)
5
10
20
30
40
7.11.6 PROCEDURAL CALIBRATION STANDARDS - A designated amount
of each calibration spiking solution is spiked into five separate 20 mL
aliquots of reagent water in a 30 mL sample container, to produce
aqueous calibration standards. The reagent water used to make the
calibration standards should contain the preservation reagents described in
Section 8.1.2 (ammonium chloride or ammonium sulfate at 500 mg/L and
copper sulfate pentahydrate at 500 mg/L). Aqueous calibration standards
are processed and analyzed according to the procedures in Section 11.
Resulting data are used to generate a calibration curve. An example of the
preparation of aqueous calibration standards is given below. The lowest
concentration calibration standard must be at or below the MRL.
Modifications of this preparation scheme may be made to meet the needs
of the laboratory. Preparing aqueous calibration standards using varying
volumes of one calibration spiking solution is an acceptable alternative to
the example below.
556.1-12
-------
PREPARATION OF PROCEDURAL (AQUEOUS) CALIBRATION
STANDARDS
Calibration
Level
1
2
3
4
5
Calibration Spike
Solution
Concentration,
Og/mL)
5
10
20
30
40
Volume
Calibration
Spike
Solution,
(uL)
20
20
20
20
20
Final Volume
Calibration
Standard
(mL)
20
20
20
20
20
Final
Concentration
Calibration
Standard
(Hg/L)
5
10
20
30
40
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 SAMPLE VIAL PREPARATION
8.1.1 Grab samples must be collected in accordance with conventional sampling
practices(7) using amber glass 30 mL containers with PTFE-lined screw-
caps, or caps with PTFE-faced silicon septa.
8.1.2 Prior to shipment to the field, 15 mg of copper sulfate pentahydrate must
be added to each bottle. This material acts as a biocide to inhibit
bacteriological decay of method analytes. If samples to be collected
contain free chlorine, then 15 mg of ammonium chloride or ammonium
sulfate must also be added to the bottle prior to sample collection. The
ammonium compound will react with the free chlorine to form
monochloramine, and retard the formation of additional carbonyl
compounds. Add these materials as dry solids to the sample bottle. The
stability of these materials in concentrated aqueous solution has not been
verified.
NOTE: Aldehydes have been demonstrated to be extremely susceptible to
microbiological decay. The use of other chlorine reducing agents such as
sodium thiosulfate or ascorbic acid, has also been shown to produce
invalid data. Proper sample collection and preservation is important to
obtaining valid data. The data in Section 17, Table 7 illustrates the
importance of proper sample preservation.
556.1-13
-------
8.2 SAMPLE COLLECTION
8.2.1 Fill sample bottles to just overflowing but take care not to flush out the
sample preservation reagents. The capped sample should be head-space
free.
8.2.2 When sampling from a water tap, remove the aerator so that no air bubbles
will be trapped in the sample. Open the tap, and allow the system to flush
until the water temperature has stabilized (usually about 3-5 minutes).
Collect samples from the flowing system.
8.2.3 When sampling from an open body of water, fill a 1 quart wide-mouth
bottle or 1L beaker with sample from a representative area, and carefully
fill sample bottles from the container.
8.2.4 After collecting the sample, cap carefully to avoid spillage, and agitate by
hand for 1 minute.
8.3 SAMPLE STORAGE/HOLDING TIMES
8.3.1 Samples must be iced or refrigerated at 4 + 2 °C and maintained at these
conditions away from light until extraction. Samples must be extracted
within 7 days of sampling. However, since aldehydes are subject to decay
in stored samples, all samples should be derivatized and extracted as soon
as possible.
NOTE: A white or blue precipitate is likely to occur. This is normal and
does not indicate any problem with sample collection or storage.
8.3.2 Extracts (Section 11.1.11) must be stored at 4 ± 2 °C away from light in
glass vials with Teflon-lined caps. Extracts must be analyzed within 14
days of extraction.
8.4 FIELD REAGENT BLANKS ~ Processing of a field reagent blank (FRB) is
required along with each sample set. A sample set is composed of the samples
collected from the same general sampling site at approximately the same time.
Field reagent blanks are prepared at the laboratory before sample vials are sent to
the field. At the laboratory, fill a sample container with reagent water (Section
7.2), add sample preservatives as described in Section 8.1.2, seal and ship to the
sampling site along with the empty sample containers. FRBs should be confirmed
to be free (less than 1/2 the MRL) of all method analytes prior to shipping them to
the field. Return the FRB to the laboratory with filled sample bottles. DO NOT
OPEN THE FRB AT THE SAMPLING SITE. If any of the analytes are
detected at concentrations equal to or greater than 1/2 the MRL, then all data for
556.1-14
-------
the problem analyte(s) should be considered invalid for all samples in the shipping
batch.
9. QUALITY CONTROL
9.1 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 (which includes calculation of the MDL), analysis of
laboratory reagent blanks, laboratory fortified blanks, field reagent blanks,
laboratory fortified sample matrices, and QC samples. Additional QC practices
are encouraged.
9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) - Requirements for the
initial demonstration of laboratory capability are described in the following
sections and summarized in Section 17, Table 8.
9.2.1 Initial demonstration of low system background. (Section 9. 3)
9.2.2 Initial demonstration of precision. Prepare, derivatize, extract, and analyze
4-7 replicate LFBs fortified at 20 Mg/L, or other mid-range concentration,
over a period of at least 2 days. Generating the data over a longer period
of time, e.g., 4 or 5 days may produce a more realistic indication of day to
day laboratory performance. The relative standard deviation (RSD) of the
results of the replicate analyses must be less than 20%.
9.2.3 Initial demonstration of accuracy. Using the same set of replicate data
generated for Section 9.2.2, calculate average recovery. The average
recovery of the replicate values must be within ± 20% of the true value.
9.2.4 MDL determination(1>2). Replicate analyses for this procedure should be
done over at least 3 days (both the sample derivatization/extraction and the
GC analyses should be done over at least 3 days). Prepare at least 7
replicate LFBs at a concentration estimated to be near the MDL. This
concentration may be estimated by selecting a concentration at 2-5X the
noise level. Analyze the seven replicates through all steps of Section 11.
Calculate the MDL
MDL = St(n. ^ j. alpha = 099)
where:
t(n-u-aipha=o.99)= Student's t value for the 99% confidence level with n-1
degrees of freedom
n = number of replicates
S = standard deviation of replicate analyses.
556.1-15
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NOTE: Do not subtract blank values when performing MDL calculations.
9.2.5 Minimum Reporting Level (MRL) -- Although an MDL can be calculated
for analytes that commonly occur as background contaminants, the
calculated MDLs should not be used as the MRL for each analyte. Method
analytes that are seen in the background (typically formaldehyde,
acetaldehyde) should be reported as present in field samples, only after
careful evaluation of the background levels. It is recommended that a
MRL be established at the mean LRB concentration + 3 a, or three times
the mean LRB concentration, whichever is greater. This value should be
calculated over a period of time, to reflect variability in the blank
measurements. It is recommended that this value be used as a minimum
reporting level in order to avoid reporting false positive results.
9.3 LABORATORY REAGENTS BLANKS (LRB) - Each time a set of samples is
extracted or reagents are changed, a LRB must be analyzed. If within the reten-
tion tune window of any analyte, the LRB produces a peak that would prevent the
determination of that analyte, determine the source of contamination and eliminate
the interference before processing samples. Because background contamination is
a significant problem for several method analytes, it is highly recommended that
the analyst maintain a historical record of LRB data. If target analytes are
detected hi the LRB at concentrations equal to or greater than 1/2 the MRL
(Section 9.2.5), then all data for the problem analyte(s) should be considered
invalid for all samples in the extraction batch.
9.4 CONTINUING CALIBRATION CHECK/LABORATORY FORTIFIED BLANK -
Since this methodology is based on procedural standard calibration, a LFB and the
calibration check sample (CCC) are prepared and analyzed in the same manner.
Laboratory fortified blank QC requirements are therefore omitted. Calibration
procedure options and the QC acceptance criteria associated with them are fully
described in Section 10.3. Please refer to that section for these criteria.
9.5 INTERNAL STANDARD~The analyst must monitor the IS response peak area of
all injections during each analysis day. A mean IS response is determined from the
five point calibration curve. The IS response for any chromatographic run should
not deviate from this mean IS response by more than 30%. If a deviation greater
than 30% occurs with an individual extract, inject a second aliquot of that extract.
9.5.1 If the reinjected aliquot produces an acceptable internal standard response,
report results for that aliquot.
9.5.2 If a deviation of greater than 30% is obtained for the reinjected extract, the
analyst should check the calibration by analyzing the most recently
acceptable calibration standard. If the calibration standard fails the criteria
556.1-16
-------
of Section 10.3, recalibration is in order per Section 10. If the calibration
standard is acceptable, extraction of the sample should be repeated
provided the sample is still within the holding time. Otherwise, report
results obtained from the reinjected extract, but annotate as suspect.
9.6 SURROGATE RECOVERY-The surrogate standard is fortified into the aqueous
portion of all calibration standards, samples, FRBs and LRBs. The surrogate is a
means of assessing method performance from derivatization to final
chromatographic measurement.
9.6.1 When surrogate recovery from a sample, blank, or CCC is <70% or
>130%, check (1) calculations to locate possible errors, (2) standard
solutions for degradation, (3) contamination, and (4) instrument
performance. If those steps do not reveal the cause of the problem,
reanalyze the extract.
9.6.2 If the extract reanalysis meets the surrogate recovery criterion, report only
data for the reanalyzed extract.
9.6.3 If the extract reanalysis fails the 70-130% recovery criterion, the analyst
should check the calibration by analyzing the most recently acceptable
calibration standard. If the calibration standard fails the criteria of Section
9.6.1, recalibration is in order per Section 10. If the calibration standard is
acceptable, it may be necessary to extract another aliquot of sample if
sample holding time has not been'exceeded. If the sample re-extract also
fails the recovery criterion, report all data for that sample as suspect.
9.7 LABORATORY FORTIFIED SAMPLE MATRIX (LFM)
9.7.1 Within each analysis set, a minimum of one field sample is fortified as a
LFM for every 20 samples analyzed. The LFM is prepared by spiking a
sample with an appropriate amount of the calibration standard. The
concentrations 5, 10, and 20 [ig/L are suggested spiking concentrations.
Select the spiking concentration that is closest to, but greater than the
concentration in the unfortified sample. Use historical data or rotate
through the designated concentrations to select a fortifying concentration.
Selecting a duplicate vial of a sample that has already been analyzed, aids
in the selection of appropriate spiking levels.
9.7.2 Calculate the percent recovery (R) for each analyte, after correcting the
measured concentration, A, from the fortified sample for the background
concentration, B, measured in the unfortified sample, i.e.,
556.1-17
-------
c
where C is the fortified concentration. Compare these values to control limits
appropriate for reagent water data collected in the same fashion.
9.7.3 Recoveries may exhibit a matrix dependence. For samples fortified at or
above their native concentration, recoveries should range between 70 -
130%. If the accuracy of any analyte falls outside the designated range,
and the laboratory performance for that analyte is shown to be hi control,
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. Repeated failure to meet the
suggested recovery criteria indicates potential problems with the extraction
procedure and should be investigated.
9.8 FIELD DUPLICATES ~ Within each analysis batch, a minimum of one field
sample should be analyzed in duplicate. Duplicate sample analyses serve as a
check on sampling and laboratory precision.
9.8.1 Calculate the relative percent difference (RPD) for duplicate
measurements (Ldl and Ld2) as shown below.
*10Q
9.8.2 Relative percent differences for laboratory duplicates should fall in the
range of ± 30 %. Greater variability may be observed for target analytes
with concentrations near their MRL.
9.9 QUALITY CONTROL SAMPLE (QCS) - At least quarterly, analyze a QCS
from an external source. If measured analyte concentrations are not of acceptable
accuracy (70-130% of the expected value), check the entire analytical procedure to
locate and correct the problem source.
9.10 ASSESSING (Z/E) RATIOS -- In addition to monitoring analyte response from
CCC/LFB, the ratio of the peak areas of each isomer pair should be monitored.
When samples and standards are processed and analyzed by exactly the same
procedure, the ratio of the (Z/E) isomers produced by each method analyte will be
reproducible. This information can be used as a QC check to avoid biased results
caused by an interferant with one isomer of the pair. Calculate and record the ratio
of the peak area of the first eluting isomer (designated (E)) to the second eluting
isomer (designated (Z)). This ratio will be used in data evaluation Section 12.4.
556.1-18
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10. CALIBRATION AND STANDARDIZATION
10.1 Demonstration and documentation of acceptable initial calibration is required
before any samples are analyzed, and is required intermittently throughout sample
analysis. After initial calibration is successful, the analyst may choose one of two
options for maintaining on-going calibration. The first option is to verify the
initial calibration daily using a minimum of 2 calibration standards. The other
option is daily calibration of the method with all 5 calibration standards. These
options are further described in Section 10.3.
10.2 INITIAL CALIBRATION CURVE
• 10.2.1 Establish FGC operating parameters equivalent to the suggested
specifications in Section 17, Table 1. The GC system must be calibrated
using the internal standard (IS) technique. Other GC columns or GC
conditions maybe used if equivalent or better performance can be
demonstrated (Section 1.3).
10.2.2 Five calibration standards are recommended to calibrate over the range of
approximately 5-40 ng/L. The lowest level standard will depend upon the
level of blank contamination for each analyte (Section 7.1 1.6).
10.2.3 Prepare each calibration standard by the procedural standard calibration
method. Method analytes are fortified into reagent water and carried
through the entire extraction and derivatization procedure described in
Section 1 1 .
10.2.4 Inject 1 (xL of each calibration standard extract into the FGC and tabulate
peak area response and concentration for each analyte and the internal
standard. NOTE: The formaldehyde peak will be much larger (for the
same concentration) than the other analyte peaks. The formaldehyde peak
may need to be attenuated on some instruments/data systems to avoid
signal saturation.
10.2.5 (Z/E) ISOMERS - Two isomers, referred to "as (E) and (Z), are formed for
most asymmetrical carbonyl compounds derivatized with PFBHA.
Chromatographic resolution is usually obtained with the columns
suggested in Section 6.6 for acetaldehyde, propanal, butanal, pentanal,
hexanal, heptanal, and octanal, (see chromatograms in Section 17, Figure 1
and Figure 2). With dicarbonyl species such as glyoxal and methyl
glyoxal, (E) and (Z) isomerism occurs from oxime formation with both
carbonyl groups, increasing the number of isomers. The demonstration
data included in this method use two distinct isomer peaks each for
556.1-19
-------
glyoxal and methyl glyoxal. Use one of the following methods for both
calibration and quantitation of each method analyte.
(a) Use the sum of the isomer peak areas for each constituent for both
calibration and quantitation.
(b) Use the peak area of each individual isomer to independently
calculate a concentration for each isomer. Then average the
amount of the two isomers to report one value for the analyte.
10.2.6 Generate a calibration curve for each analyte by plotting the area ratios
(A-/A;,.) against the concentration ratios (Ca/Cis) of the five calibration
standards where:
Aa is the peak area of the analyte (or analyte isomer pair),
AJS is the peak area of the internal standard,
Ca is the concentration of the analyte, and
Cis is the concentration of the internal standard.
10.2.7 This curve must always be forced through zero and can be defined as
either first or second order. Forcing zero allows for a better estimate of the
background level of method analytes.
10.2.8 A data system is required to collect the chromatographic data, to calculate
relative response factors, and calculate either linear or second order
calibration curves.
10.2.9 VERIFICATION OF CALIBRATION STANDARD MATERIALS --
Analyze a LFB prepared from standard materials from a source other than
those used to prepare the initial calibration curve (Sections 3.8, and 9.9).
Calculate the concentration of this QCS from the calibration curve. The
calculated concentration of the QCS must agree within 70-130% of its true
value. This step verifies the validity of calibration standard materials and
the calibration curve prior to sample analyses.
10.3 OPTIONS FOR ON-GOING CALIBRATION~The time, temperature, pH, and
PFBHA concentration will all affect the rate, efficiency and reproducibility of the
derivatization reaction. It is critical that those parameters be controlled.
Calibration frequency will depend upon the laboratory's ability to control these
parameters so that continuing calibration check standard criteria can be met.
Some laboratories may find it more productive to prepare and analyze a
calibration curve with each batch of samples. A batch of samples for this
methodology should not exceed 20 samples, including field samples, FRBs,
laboratory duplicates, and fortified sample matrices.
556.1-20
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10.3.1 CONTINUING CALIBRATION CHECK (CCC) OPTION-The analyst
must periodically verify calibration during the analysis of samples in order
to ensure accuracy of analytical results. Prepare a minimum of one low-
level (suggested concentration 2-5 |Jg/L) and one mid-level (suggested
concentration 10-30u.g/L) calibration standard with each batch of samples.
Verify calibration using these two standards, prior to analyzing any of the
sample extracts from the batch. In addition, reanalyze one of these two
standard extracts after every tenth sample extract, and after the last sample
in an analysis batch to ensure instrument stability throughout the analysis
batch. Recovery must be within 70-130% of the true value for the mid-
level standard, and within 50-150% of the true value for the low-level
standard.
10.3.2 DAILY CALIBRATION OPTION - The analyst may choose to create a
new calibration curve for each batch of samples by preparing and
analyzing a standard at all five calibration concentrations, with each batch
of samples. If this option is selected, the calibration standard extracts
should be analyzed prior to the analysis of sample extracts. To ensure that
sensitivity and performance of the method has not changed significantly
between sample batches, or changed since the IDC, the following
performance check is required. The response (peak area) of the internal
: standard, surrogate and each method analyte in the mid-level standard
(suggested concentration 10-30u.g/L), must be within 50-150% of the
mean peak area for that analyte in the initial demonstration of precision
replicates (Section 9.2.2). One of the calibration standard extracts must be
reanalyzed after every, tenth sample extract, and after the last sample in an
analysis batch to ensure instrument stability throughout the analysis batch.
Recovery must be within 70 to 130% of the true value for mid- and high-
level calibration standards, and within 50-150% of the true value for the
low-level standard (suggested concentration 2-5p,g/L).
11. PROCEDURE
11.1 SAMPLE EXTRACTION - Once samples have been opened, process the
samples straight through to step 11.1.11. There is no known "safe" stopping point
once sample processing has begun. Samples are derivatized and extracted in the
sample bottle in which they were collected. Transferring the sample to another
container for derivatization and extraction has been shown to cause a loss of
method analytes.
11.1.1 Remove the samples from storage and allow them to equilibrate to
room temperature.
556.1-21
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11.1.2 Remove 10 mL of sample and discard. Mark the level of the remaining
sample volume on the outside of the bottle, for later sample volume
determination.
11.1.3 Add 200 mg KHP to adjust the sample pH to approximately 4.
11.1.4 Add 20 |aL surrogate solution (Sect 7.11.2.2).
11.1.5 Add 1 mL of freshly prepared PFBHA Reagent as per Section 7.10.1.
Cap and swirl gently to mix.
11.1.6 Place all samples in a constant-temperature water bath set at 35 ± 2 G
for 2 hours. Remove vials and cool to room temperature for 10
minutes.
11.1.7 To each vial add approximately 0.05 mL (2 to 4 drops) of concentrated
sulfuric acid. This prevents the extraction of excess reagent, which will
cause chromatographic interferences.
11.1.8 Add 4 mL of hexane that contains the internal standard (Section
7.11.1.2).
11.1.9. Shake manually for 3 minutes. Let stand for approximately 5 minutes
to permit phases to separate.
11.1.10 Draw off hexane layer (top layer) using a clean disposable Pasteur
pipette for each sample into a smaller 8 mL vial containing 3 mL 0.2 N
sulfuric acid. Shake for 30 seconds and let stand for 5 minutes for
phase separation. NOTE: This acid wash step further reduces the
reagent and other interferants from the final extract.
11.1.11 Draw off top hexane layer using another clean, disposable pipette for
each sample and place in two 1.8 mL autosampler vials per sample.
Store extra autosampler vials as a backup extract. Extracts may be
stored for up to 14 days at 4 "C.
11.1.12 Sample Volume Determination -- Discard remaining water sample and
hexane in each sample bottle. Fill with water to the level indicated by
the mark made in Section 11.1.2. Pour the water into a 25 mL
graduated cylinder and measure the volume to the nearest mL. Record
the sample volume for each sample.
Alternately, if a laboratory has control over the brand and style of the 30
mL sample bottles being used, the exact volume of a number of bottles
556.1-22
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from the same manufacturer and lot may be measured, and the average
bottle volume minus 10 mL may be used as the sample volume for all
samples using the same lot of sample bottles. A minimum of 10 % of
the sample bottles obtained from the same manufacturer, from the same
lot should be measured.
11.2 FAST GAS CHROMATOGRAPHY- This method uses fast gas chromatography
(FGC) for the analysis of the hexane extracts. Several important changes from
"conventional GC" must be made to allow for the rapid analysis of the analytes.
First the instrument must be capable of providing a fast temperature ramp (30
°C/minute) oven, a high pressure (>50 psi) split/splitless injector, and a low
volume (150 uL) micro BCD. Second, the column diameter, length and film
thickness must all be decreased. Third, the carrier gas must be changed to a
highly diffusive or "fast" gas such as hydrogen. Although hydrogen can be used
safely as a carrier gas, the potential for fire or explosion does exist if the gas
system is mishandled. If you are unsure of the safety guidelines for using
hydrogen as a carrier gas, seek advice from your instrument manufacturer
regarding its use. Finally, strict attention must be paid to established column
installation guidelines with regard to the proper cutting and placement of the
capillary columns within the instrument.
In addition to decreasing the column dimensions and changing the carrier gas,
successful FGC depends on the minimization of extracolumn variance.
Extracolumn variance refers to any source of bandbroadening other than those
which occur within the chromatographic column itself. The major source of
extracolumn variance in a properly designed FGC chromatographic system is the
injector/injection process. The best chromatographic results for the oxime
derivatives have been achieved using glass wool packed small i.d. liners (Section
6.6.3) combined with low split ratios. Although excellent precision and accuracy
have been demonstrated using the listed chromatographic conditions (Section 17,
Table 1.), it possible that the optimum conditions for a specific instrument will
need to be empirically determined by the user.
11.2.1 Analyze the extracts by FGC/ECD. Tables 1 and 2 (Section 17)
summarize recommended FGC operating conditions and retention times
observed using this method. Figure 1 illustrates the performance of the
recommended primary column with the method analytes. Figure 2
illustrates the performance of the recommended confirmation column with
the method analytes. Other GC columns or chromatographic conditions
may be used if the requirements of Section 9 are met.
11.2.2 The width of the retention time window used to make identifications
should be based on measurements of actual retention time variations of
standards over the course of time. Plus or minus three times the standard
556.1-23
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deviation of the 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.
11.2.3 If an analyte peak area exceeds the range of the calibration curve, the
extract may be diluted with the hexane extraction solvent (that contains the
internal standard) and reanalyzed. Incorporate the dilution factor into final
concentration calculations. The analyst must not extrapolate beyond the
calibration range established.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Identify the method analytes in the sample chromatogram by comparing the
retention time of the suspect peak to the retention time of an analyte peak (or
isomer peaks) in a calibration standard or the laboratory fortified blank.
12.2 Calculate the analyte concentrations using the first or second order calibration
curves generated as described in Section 10.
12.3 For any analytes that are found, adjust the calculated concentration to reflect the
true sample volume determined in Section 11.1.12.
12.4 Prior to reporting the data, the chromatogram should be reviewed for any incorrect
peak identification or poor integration. If a confirmation column has been used,
all identifications should be verified using the retention time data from that
analysis. In addition, the (Z/E) isomer ratio should be within 50% of the ratio
observed in standards. If the (Z/E) ratio does not meet these criteria, it is likely
that an interferent occurred at the retention time of one of the isomer peaks. In
this case, the amount indicated by the lower of the 2 isomer peaks should be
reported. (This may require that the analyst recalculate the analyte amount using
individual isomer peaks for quantitation.) If one peak of the isomeric pair is
missing, the identification is not confirmed and should not be reported.
12.5 Analyte concentrations are reported in \igfL.
13. METHOD PERFORMANCE
13.1 Precision and accuracy data are presented in Section 17. Data are presented for
three water matrices: reagent water (Table 3), chlorinated "finished" surface water
(Table 5) , chlorinated "finished" ground water (Table 6).
13.2 DERIVATIZATION PARAMETERS - This method is a procedural standard
method that will generate accurate and precise results when used as written. The
time, temperature, pH, and PFBHA concentration will all affect the rate,
556.1-24
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efficiency and reproducibility of the derivatization reaction. It is critical that those
parameters be controlled. Calibration frequency will depend upon the
laboratory's ability to control these parameters. Some laboratories may need to
prepare and analyze a calibration curve with each batch of samples. Of all the
method analytes, glyoxal, methyl glyoxal, benzaldehyde, and cyclohexanone are
the most difficult to derivatize. Poor sensitivity for any of these compounds
indicates that there may be a problem with the reaction conditions. Measurements
of nonanal, decanal, glyoxal and methyl glyoxal appear to be less precise than the
measurement of other analytes.
13.3 The importance of low background levels of formaldehyde and acetaldehyde
cannot be overemphasized. Some laboratories or reagent waters may also contain
background amounts of other method analytes. Care must be taken to avoid
reporting false positive results that result from background contamination.
13.4 The importance of proper sample collection and preservation also cannot be
overemphasized. Holding time studies in various matrices showed better than
70% recovery of all method analytes when samples were collected, preserved, and
stored according to Section 8, and analyzed within 7 days. There were variations
in the recovery of analytes from fortified samples from different matrices.
Therefore, it is strongly recommended that samples be analyzed as soon as
possible after collection. The data in Section 17, Table 7 illustrate the dramatic
difference between a preserved and a non-preserved sample. Although this data
was presented as Table 6 of Method 556, revision 1.0, it is included to illustrate
the importance of proper sample preservation.
14. POLLUTION PREVENTION
14.1 This method uses a micro-extraction procedure which requires very small
quantities of organic solvents.
14.2 For information about pollution prevention that may be applicable to laboratory
operations, consult "Less is Better: Laboratory Chemical Management for Waste
Reduction" available from the American Chemical Society's Department of
Government Relations and Science Policy, 1155 16th Street N.W., Washington,
B.C., 20036.
15. WASTE MANAGEMENT
15.1 The analytical procedures described in this method generate relatively small
amounts of waste since only small amounts of reagents and solvents are used.
The matrices of concern are finished drinking water or source water. However,
the Agency requires that laboratory waste management practices be conducted
consistent with all applicable rules and regulations, and that laboratories protect
556.1-25
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the air, water, and land by minimizing and controlling all releases from fume
hoods and bench operations. Also, compliance is required with any sewage
discharge permits and regulations, particularly the hazardous waste identification
rules and land disposal restrictions. For further information on waste
management, consult "The Waste Management Manual for Laboratory Personnel"
also available from the American Chemical Society at the address in Section 14.2.
16. REFERENCES
1. Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde, "Trace Analyses
for Wastewaters," Environ. ScL Technol. 1981, 15,1426-1435.
2. Definition and procedure for the determination of the method detection limit. 40 CFR
Partl36, Appendix B.
3. Standard Method Number 6252B, "PFBHA Liquid-Liquid Extraction Gas
Chromatographic Method," Standard Methods for the Examination of Water and
Wastewater. pp. 6-77 to 6-83, American Public Health Assoc., Washington, D.C., 1995.
4. Sclimenti, M.J., S.W. Krasner, W.H. Glaze, and H.S. Weinberg,"Ozone Disinfection By-
Products: Optimization of the PFBHA Derivatization Method for the Analysis of
Aldehydes," m Advances in Water Analysis and Treatment, Proc. AWWA Water Quality
Technology Conf.. 1990, pp 477-501.
5. Glaze, W.H. and H.S. Weinberg, Identification and Occurrence of Ozonation Bv-Products
in Drinking Water, American Water Works Assoc. Research Foundation, Denver, CO.,
1993,ppl9-22.
6. "OSHA Safety and Health Standards, General Industry," (29CRF1910). Occupational
Safety and Health Administration, OSHA 2206, (Revised, Jan. 1976).
7. ASTM Annual Book of Standards, PartE, Volume 11.01, D3370-82, "Standard Practice
for Sampling Water," American Society for Testing and Materials, Philadelphia, PA,
1986.
8. "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.
9. "Safety In Academic Chemistry Laboratories," 3rd Edition, American Chemical Society
Publication, Committee on Chemical Safety, Washington, D.C., 1979.
556.1-26
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17. TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. CHRQMATOGRAPHIC CONDITIONS AND RETENTION
TIME DATA FOR THE PRIMARY COLUMN (N=8)
Peak Number
(Figure 1.)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Analyte
1,2 dibromopropane (IS)
Formaldehyde
E-Acetaldehyde
Z-Acetaldehyde
E-Propanal
Z-Propanal
E-Butanal
Z-Butanal
E-Pentanal
Z-Pentanal
E-Hexanal
Z-Hexanal
Cyclohexanone
E-Heptanal
Z-Heptanal
2,4,5, trifluoroacetophenone (S)
E-Octanal
Z-Octanal
Benzaldehyde
Nonanal
Decanal
Glyoxal (peak 1)
Glyoxal (peak 2)
Methyl Glyoxal (peak 1)
Methyl Glyoxal (peak 2)
Average
Retention
Time (min)
0.673
1.08
1.46
1.50
1.85
1.88
2.28
2.31
2.73
2.76
3.19
3.21
3.54
3.64
3.65
3.97
4.07
4.08
4.19
4.50
4.91
5.23
5.27
5.29
5.41
Standard
Deviation
3.26E-03
1.67E-03
l.OOE-04
7.08E-04
4.51E-04
4.63E-04
5.07E-04
4.86E-04
3.36E-04
3.27E-04
3.18E-04
1.85E-04
4.33E-04
3.47E-04
1.59E-04
3.18E-04
3.25E-04
4.88E-04
2.13E-04
2.97E-04
2.76E-04
5.33E-04
4.91E-04
2.90E-04
2.85E-04
Relative
Standard
Deviation
0.49%
0.15%
0.01%
0.05%
0.02%
0.02%
0.02%
0.02%
0.01%
0.01%
0.01%
0.01%
0.01%
0.01%
0.00%
0.01%
0.01%
0.01%
0.01%
0.01%
0.01%
0.01%
0.01%
0.01%
0.01%
Primary Column: DB-5,10 m x 0.10 mm i.d., 0.10 jam film thickness, injector temp. 200 °C, liner
2 mm with a central 2 cm silanized glass wool plug, injection volume 1 |j,L, split ratio 30:1, constant
head pressure @ 32 psi, detector temp. 300 °C, detector make up flow 20 mL/minute.
Temperature program: 70 °C initial, program at 27 °C/minute to 220 °C, ballistic heating to 280 °C
for burnout and hold at 280 °C for 0.4 minutes. Data collection via HP GC Chemstation at a rate of
50 Hz.
Carrier gas: Hydrogen (UHP)
Detector gas: 95:5 Argon:Methane
556.1-27
-------
TABLE 2. CHROMATOGRAPHIC CONDITIONS AND RETENTION
TIME DATA FOR THE SECONDARY COLUMN (N=8)
Peak Number
(Figure 2.)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Analyte
1,2 dibromopropane (IS)
Formaldehyde
E-Acetaldehyde
Z-Acetaldehyde
E-Propanal
Z-Propanal
E-Butanal
Z-Butanal
E-Pentanal
Z-Pentanal
E-Hexanal
Z-Hexanal
Cyclohexanone
E-Heptanal
Z-Heptanal
E-Octanal
Z-Octanal
2,4,5, trifluoroacetophenone (S)
Benzaldehyde
E-Nonanal
Z-Nonanal
Decanal
Glyoxal
Methyl Glyoxal
Average
Retention
Time (min)
0.808
1.29
1.69
1.73
2.07
2.10
2.48
2.52
2.92
2.96
3.37
3.40
3.76
3.80
3.82
4.22
4.23
4.36
4.53
4.62
4.63
5.01
5.57
5.68
Standard
Deviation
1.48E-03
1.75E-03
1.87E-03
1.79E-03
1.79E-03
1.87E-03
1.76E-03
1.85E-03
1.56E-03
1.58E-03
1.59E-03
1.53E-03
1.43E-03
1.46E-03
1.48E-03
1.14E-03
7.25E-04
1.09E-03
1.29E-03
1.28E-03
1.31E-03
1.07E-03
9.91E-04
7.85E-04
Relative
Standard
Deviation
0.18%
0.14%
0.11%
0.10%
0.09%
0.09%
0.07%
0.07%
0.05%
0.05%
0.05%
0.05%
0.04%
0.04%
0.04%
0.03%
0.02%
0.03%
0.03%
0.03%
0.03%
0.02%
0.02%
0.01%
Secondary Column: AT-1701, lOmxO.lOmmi.d., 0.10 jjm film thickness, injector temp. 200 °C,
liner 2 mm with a central 2 cm silanized glass wool plug, injection volume 1 uL, split ratio 30:1,
constant head pressure @ 32 psi, detector temp. 300 "C, detector make up flow 20 mL/minute.
Temperature program: 70 °C initial, program at 27 "C/minute to 220 °C, ballistic heating to 280 °C
for burnout and hold at 280 °C for 0.4 minutes. Data collection via HP GC Chemstation at a rate of
50 Hz.
Carrier gas: Hydrogen (UHP)
Detector gas: 95:5 Argon:Methane
556.1-28
-------
TABLE 3. PRECISION AND ACCURACY IN REAGENT WATER (N=8)
5 ug/L Fortification
Analyte
Formaldehyde
Acetaldehyde
Propanal
Butanal
Pentanal
Hexanal
Cyclohexanone
Heptanal
Octanal
Benzaldehyde
Nonanal
Decanal
Glyoxal
Methyl Glyoxal
Fortified
Concentration
(Ug/L)
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
Average
Concentration
(Ug/L)
6.01
4.46
6.61
5.54
5.54
5.55
6.19
6.77
5.22
4.50
5.42
5.47
4.60
4.52
Unfortified
Sample
GigflL)
0.303
ND
1.46
ND
ND
ND
ND
1.68
ND
ND
0.403
ND
ND
ND
Relative
Standard
Deviation
1.9%
2.5%
2.0%
2.4%
2.9%
4.0%
3.2%
5.0%
5.4%
4.1%
4.6%
5.1%
6.3%
5.7%
Average
Percent
Recovery3
114%
89%
103%
111%
111%
111%
124%
102%
104%
90%
100%
109%
92%
90%
Analyte
Formaldehyde
Acetaldehyde
Propanal
Butanal
Pentanal
Hexanal
Cyclohexanone
Heptanal
Octanal
Benzaldehyde
Nonanal
Decanal
Glyoxal
Methyl Glyoxal
20 ug/L Fortification
Fortified
Concentration
(Ug/L)
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
Average
Concentration
(Ug/L)
20.8
20.7
20.6
20.0
20.2
20.3
20.7
20.8
20.2
19.6
20.3
20.3
19.5
19.3
Unfortified
Sample
(Ug/L)
0.303
ND
1.46
ND
ND
ND
ND
1.68
ND
ND
0.403
ND
ND
ND
Relative
Standard
Deviation
2.3%
6.3%
2.4%
2.6%
3.0%
3.2%
2.0%
3.1%
2.2%
2.7%
2.6%
3.2%
4.1%
4.0%
Average
Percent
Recovery3
102%
104%
96%
100%
101%
101%
104%
95%
101%
98%
100%
101%
98%
97%
a - These recovery values were calculated using the equation in Section 9.7.2.
556.1-29
-------
TABLE 4. METHOD DETECTION LIMITS IN REAGENT WATER (n = 7)
Analyte
Formaldehyde
Acetaldehyde
Propanal
Butanal
Pentanal
Hexanal
Cyclohexanone
Heptanal
Octanal
Benzaldehyde
Nonanal
Decanal
Glyoxal
Methyl Glyoxal
Fortified
Concentration
(ue/L)
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Primary
Column
MDL (ng/L)
0.09
0.18
0.11
0.09
0.09
0.10
0.19
0.40
0.22
0.19
0.62
0.46
0.39
0.26
Secondary
Column
MDL (ng/L)
0.08
0.12
0.06
0.06
0.06
0.04
0.09
0.24
0.84
0.04
0.64
0.35
0.13
0.12
556.1-30
-------
TABLE 5. PRECISION AND ACCURACY IN CHLORINATED SURFACE
WATER (N=7)
5 ng/L Fortification
Analyte
Formaldehyde
Acetaldehyde*
Propanal
Butanal
Pentanal
Hexanal
Cyclohexanone
Heptanal
Octanal
Benzaldehyde
Nonanal
Decanal
Glyoxal
Methyl Glyoxal
Fortified
Concentration
(Hg/L)
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
Average
Concentration
(Hg/L)
8.45
6.53
5.68
5.73
5.43
5.48
6.02
5.64
4.84
4.92
5.25
5.78
7.92
6.42
Unfortified
Sample
(Hg/L)
3.40
1.76
0.620
0.390
ND
ND
0.650
0.840
ND
ND
0.250
ND
1.40
0.380
Relative
Standard
Deviation
3.3%
2.7%
2.2%
2.4%
2.6%
2.8%
4.2%
4.1%
6.4%
3.1%
8.5%
8.9%
9.2%
9.2%
Average
Percent
Recovery"
101%
96%
101%
107%
109%
110%
107%
96%
97%
98%
100%
116%
130%
121%
20 jig/L Fortification
Analyte
Formaldehyde
Acetaldehyde*
Propanal
Butanal
Pentanal
Hexanal
Cyclohexanone
Heptanal
Octanal
Benzaldehyde
Nonanal
Decanal
Glyoxal
Methyl Glyoxal
Fortified
Concentration
(Hg/L)
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
Average
Concentration
(Hg/L)
22.6
20.1
20.4
20.1
20.5
20.7
20.5
19.1
18.8
20.4
19.9
20.8
25.9
23.0
Unfortified
Sample
(Hg/L)
3.40
1.76
0.620
0.390
ND
ND
0.650
0.840
ND
ND
0.250
ND
1.40
0.380
Relative
Standard
Deviation
1.1%
1.8%
1.3%
1.8%
1.9%
2.2%
2.1%
4.1%
7.7%
2.2%
11.2%
10.5%
6.5%
10.3%
Average
Percent
Recovery3
96%
91%
99%
99%
103%
103%
99%
91%
94%
102%
98%
104%
122%
113%
* = Data for acetaldehyde were taken from the secondary column due to an interference with E-
acetaldehyde.
a = These recovery values were calculated using the equation in Section 9.7.2.
556.1-31
-------
TABLE 6 . PRECISION AND ACCURACY IN CHLORINATED GROUND
WATER (N=7)
5 us/L Fortification
Analyte
Formaldehyde
Acetaldehyde*
Propanal
Butanal
Pentanal
Hexanal
Cyclohexanone
Heptanal
Octanal
Benzaldehyde
Nonanal
Decanal
Glyoxal
Methyl Glyoxal
Fortified
Concentration
(Hg/L)
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
Mean
Concentration
(Hg/L)
7.10
6.13
5.56
5.58
5.36
5.15
6.02
5.40
4.90
4.60
5.02
5.29
5.82
4.94
Unfortified
Sample
fag/L)
2.21
1.13
0.657
0.437
ND
0.120
0.534
0.883
ND
ND
ND
ND
0.471
0.202
Relative
Standard
Deviation
1.3%
3.8%
3.1%
3.5%
3.5%
3.7%
4.7%
7.0%
7.9%
5.3%
8.7%
10.4%
10.9%
10.7%
Average
Percent
Recovery"
97.8%
101%
98.0%
103%
107%
101%
110%
90.3%
98.0%
92.1%
100%
106%
107%
94.7%
20 ng/L Fortification
Analyte
Formaldehyde
Acetaldehyde*
Propanal
Butanal
Pentanal
Hexanal
Cyclohexanone
Heptanal
Octanal
Benzaldehyde
Nonanal
Decanal
Glyoxal
Methyl Glyoxal
Fortified
Concentration
(Hg/L)
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
Mean
Concentration
(Hg/L)
21.3
20.2
19.9
20.3
20.2
19.7
20.3
19.7
20.5
19.6
20.5
20.7
22.3
19.9
Unfortified
Sample
Oig/L)
2.21
1.13 .
0.657
0.437
ND
0.120
0.534
0.883
ND
ND
ND
ND
0.471
0.202
Relative
Standard
Deviation
2.8%
, 4.0%
3.3%
3.0%
3.7%
5.8%
4.8%
5.5%
6.2%
4.7%
6.6%
8.9%
10.3%
8.9%
Average
Percent
Recovery3
95%
96%
96%
99%
101%
98%
99%
94%
102%
98%
103%
104%
109%
98%
* = Data for acetaldehyde were taken from the secondary column due to an interference with E-
acetaldehyde.
a = These recovery values were calculated using the equation in Section 9.7.2.
556.1-32
-------
TABLE 7. HOLDING TIME DATA FOR SAMPLES FROM AN
UNTREATED SURFACE WATER SOURCE, FORTIFIED WITH
METHOD ANALYTES AT 20 jig/L, WITH AND WITHOUT COPPER
SULFATE BIOCIDE*
ANALYTE
Formaldehyde
Acetaldehyde
Propanal
Butanal
Pentanal
Hexanal
Cyclohexanone
Heptanal
Octanal
Benzaldehyde
Nonanal
Decanal
Glyoxal
Methyl glyoxal
% Recovery without Copper
Sulfate
DayO
104
96
94
92
87
83
94
83
82
94
72
50
103
108
Day 6
144
23
21
20
19
21
99
20
18
83
15
•
-------
TABLE 8. INITIAL DEMONSTRATION OF CAPABILITY
REQUIREMENTS
Reference
Section
9.3
Section
9.2.2
Section
9.2.3
Section
9.2.4
Section
9.2.5
Requirement
Initial
Demonstration of
Low System
Background
Initial
Demonstration of
Precision (TOP)
Initial
Demonstration of
Accuracy
Method Detection
Limit (MDL)
Determination
Minimum
Reporting Levels
(MRLs)
Specification and Frequency
Analyze method blank and
determine that all target analytes
are below 1/2 the MRL
prior to performing IDC
Analyze 4-7 replicate LRBs
fortified at 20.0 Mg/L (or mid
cal.) on at least 2 different days
Calculate average recovery of
IDP replicates
a) select a fortifying level at 2 -
5 x the noise level
b) analyze 7 replicates in
reagent water taken thru all
steps
c) calculate MDL via equation -
do not subtract blank
d) replicate extractions and
analyses must be conducted
over at least 3 days
MRLs should be established for
all analytes during IDC, and be
updated as additional LRB data
is available.
Acceptance Criteria
The LRB concentration
must be < 1/2 of the
intended MRL
RSD must be <; 20 %
Mean recovery ± 20% of
true value
Establish the MRL for
each analyte, as the LRB
concentration + 3 a or 3
times the mean LRB
concentration, whichever
is greater.
556.1-34
-------
TABLE 9. QUALITY CONTROL REQUIREMENTS (SUMMARY)
Reference
Section
10.2
Section
9.3
Section
10.3.1
Section
10.3.2
Section
8.4
Section
9.5
Section
9.6
Requirement
Initial
Calibration
Laboratory
Reagent Blank
(LRB)
Continuing
Calibration
Check (CCC)
Option
Daily
Calibration
Option
Field Reagent
Blanks (FRB)
Internal
Standard (IS)
Surrogate
Standard
(SUR)
Specification and Frequency
Use internal standard technique to
generate curve with five standards
that span the approximate range
of 5-40 ug/L. First or second
order curves must be forced
through zero. Either sum E/Z
isomer areas or average the
amount of each isomer.
Calculate E/Z ratios for analytes.
Run QCS.
Include LRB with each extraction
batch (up to 20 samples).
Analyze prior to analyzing
samples and determine to be free
of interferences .
Verify initial calibration by
running CCCs prior to analyzing
samples, after 10 samples, and
after the last sample.
Calibrate daily, but verify that
sensitivity and performance have
not changed significantly since
IDC.
1 per shipping batch
1,2-Dibromopropane is added to
all samples, blanks and standards
-
2',4',5' -Trifluoroacetophenone is
added samples, blanks and
standards
Acceptance Criteria
QCS must agree within
70-130 %.
Lowest concentration
should be near MRL.
All analytes < 1/2 MRL
Recovery for mid-level
CCC must be 70-130%
of the true value,
recovery for low level
must be 50-150% of the
true value.
Peak areas for IS, SUR,
and method analytes for
mid-level CAL std must
be +/- 50% of the peak
areas obtained for that
CAL std during IDC.
All analytes < 1/2 MRL
IS area counts must be 70
- 130% of the average
initial calibration area
counts
Surrogate recovery must
be 70-130 % of the true
value.
556.1-35
-------
Section
9.7
Section
9.8
Section
9.9
Section
9.10,
Section
10.2.5
and
Section
12.4
Section
8.3.1
Section
8.3.2
Laboratory
Fortified
Sample Matrix
(LFM)
Field
Duplicates
Quality
Control
Sample (QCS)
E/Z Isomer
Ratio
Agreement
Sample
Holding Time
Extract
Holding Time
Fortify at least one sample per
analysis batch (20 samples or
less) at a concentration close to
that hi the native sample.
Extract and analyze at least one
duplicate sample with every
analysis batch (20 samples or
less)
Analyze a QCS at least quarterly
from an external/second source.
Calculate the E/Z isomer ratio for
target analytes and compare to
E/Z ratio hi initial calibration
Properly preserved samples may
be stored hi the dark at 4 °C for 7
days.
Extracts may be stored in the dark
at 4 °C for 14 days.
Recoveries not, within 70-
130% may indicate
matrix effect
Suggested RPD < 30 %
QCS must agree within
70-130 %.
E/Z ratio in standards,
blanks, and samples must
be within ± 50% of E/Z
ratio in initial calibration.
Do not report value if one
isomer is missing.
Do not report data for
samples that have
exceeded their holding
time, or that have not
been properly preserved
or stored.
Do not report data for
extracts that have
exceeded their holding
time.
556.1-36
-------
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556.1-38
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