515.4-1

METHOD 515.4 DETERMINATION OF CHLORINATED ACIDS IN DRINKING

WATER BY LIQUID-LIQUID MICROEXTRACTION,

DERIVATIZATION, AND FAST GAS CHROMATOGRAPHY WITH

ELECTRON CAPTURE DETECTION

Revision 1.0

April 2000

S.C. Wendelken, M.V. Bassett, T.A. Dattilio, and B.V. Pepich (IT Corporation) 

D.J. Munch (US EPA, Office of Ground Water and Drinking Water) - Method 515.4, Revision 1.0

(2000)

A. M. Pawlecki-Vonderheide (ICI) and D.J. Munch (US EPA, Office of Ground Water and Drinking

Water) - Method 515.3, Revision 1.0 (1996)

J.W. Hodgeson - Method 515.2, Revision 1.0 (1992)

R.L. Graves - Method 515.1, Revision 4.0 (1989)

T. Engels (Batelle Columbus Laboratory) and D.J. Munch (US EPA, Office of Ground Water and

Drinking Water) - National Pesticide Survey Method 3, Revision 3.0 (1987)

J.W. Hodgeson - Method 515, Revision 2.0 (1986)

TECHNICAL SUPPORT CENTER

OFFICE OF GROUND WATER AND DRINKING WATER

U. S. ENVIRONMENTAL PROTECTION AGENCY

CINCINNATI, OHIO 45268
515.4-2

METHOD 515.4 DETERMINATION OF CHLORINATED ACIDS IN DRINKING

WATER BY LIQUID-LIQUID MICROEXTRACTION,

DERIVATIZATION, AND FAST GAS CHROMATOGRAPHY WITH

ELECTRON CAPTURE DETECTION

1. SCOPE AND APPLICATION

1.1 This is a fast gas chromatography (GC) method for the determination of chlorinated

acids in drinking waters. Accuracy, precision, and Detection Limit data have been

generated for the following compounds in reagent water and finished ground and

surface waters.

Analyte Chemical Abstracts Service

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(b)

Dicamba 1918-00-9

3,5-Dichlorobenzoic acid 51-36-5

Dichlorprop 120-36-5

Dinoseb 88-85-7

Pentachlorophenol 87-86-5

Picloram 1918-02-1

2,4,5-T 93-76-5

2,4,5-TP (Silvex) 93-72-1

Quinclorac 84087-01-4

(a) The herbicide Lactofen will be quantitated as Acifluorfen as their

structures represent different esters of the same acid herbicide.

(b) Dacthal mono-acid and di-acid metabolites are included in method

scope; Dacthal di-acid was used for validation studies.
515.4-3

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. This method is

able to quantify the mono- and di-acid forms of Dacthal but not the parent compound. 

1.3 Detection Limits are compound, instrument, and matrix dependent. The Detection

Limit is defined as the statistically calculated minimum amount that can be measured

with 99% confidence that the reported value is greater than zero.(1) Experimentally

determined Detection Limits for the above listed analytes are provided in Section 17,

Table 4. The Detection Limit differs from, and is usually lower than (but never

above), the minimum reporting level (MRL) (Sect. 3.17). The concentration range

for target analytes in this method was evaluated between 0.5 ug/L and 20 ug/L for a

40-mL sample. Precision and accuracy data are presented in Section 17, Tables 5 -

10.

1.4 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 method was developed using fast GC but

“conventional” GC may be used as long as the laboratory meets the requirements of

the IDC (Sect. 9.2). 

2. SUMMARY OF METHOD

2.1 A 40-mL volume of sample is adjusted to pH $ 12 with 4 N sodium hydroxide and

allowed to sit for one hour at room temperature to hydrolyze derivatives. Following

hydrolysis, a wash step using a hexane:MtBE mixture is performed as a sample

cleanup and to remove Dacthal. The aqueous sample is then acidified with sulfuric

acid to a pH of less than 1 and extracted with 4-mL of methyl tert-butyl ether (MtBE). 

The chlorinated acids that have been partitioned into the MtBE are then converted to

methyl esters by derivatization with diazomethane. The target esters are separated

and identified by fast capillary column gas chromatography using an electron capture

detector (GC/ECD). Analytes are quantified using a procedural standard calibration

technique with an internal standard.

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. 

3. DEFINITIONS

3.1 EXTRACTION BATCH – A set of up to 20 field samples (not including QC
515.4-4

samples) extracted together by the same person(s) during a work day using the same

lots of solvents, surrogate solution, and fortifying solutions. Required QC samples

include Laboratory Reagent 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.

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 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
515.4-5

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.

3.9 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 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.

3.11 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 DETECTION LIMIT – 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
515.4-6

reported as a quantified 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.

3.18 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. 

3.19 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. A final rinse with solvents

may be needed. In place of a solvent rinse, non-volumetric glassware can be muffled

at 400 oC for 2 hours. Volumetric glassware should not be heated in an oven above

120 oC. Store inverted or capped with aluminum foil.

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 chromatograms. All items

such as these must be routinely demonstrated to be free from interferences (less than

1

/3

 the MRL for each target) under the conditions of the analysis by analyzing

laboratory reagent blanks as described in Section 9. Subtracting blank values from

sample results is not permitted. 

4.2.1 During method development it was found that sodium sulfate from several 

sources contained multiple interferences. After screening several brands, it

was found that a grade suitable for pesticide residue analysis provided the best

results. If the suitability of the available sodium sulfate is in question, extract

and analyze a laboratory reagent blank (section 3.5) to test for interferences

prior to sample processing.

4.3 Matrix interferences may be caused by contaminants that are extracted from the

sample. The extent of matrix interferences will vary considerably from source to

source, depending upon the water sampled. 
515.4-7

4.3.1 During method development a contaminant was found in local ground water

and surface water that interfered with the analysis of Dalapon on the primary

column. Due to this interferent, it was necessary to carefully optimize the

chromatographic conditions (see Table 1). A slow initial temperature program

was needed to ensure separation of the interferent from Dalapon. Analyte

identifications should be confirmed using the confirmation column specified

in Table 2 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 (ECD). 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)

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 a current awareness file of OSHA regulations regarding

the safe handling of the chemicals specified 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.(4-6)

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 breathe the

vapors or ingest the materials.

5.3 The toxicity of the extraction solvent, MTBE, has not been well defined. Susceptible

individuals may experience adverse affects upon skin contact or inhalation of vapors.

Therefore protective clothing and gloves should be used and MTBE should be used

only in a chemical fume hood or glove box. The same precaution applies to pure

standard materials.

5.4 Diazomethane is a toxic carcinogen which can explode under certain conditions. The

following precautions must be followed.

5.4.1 Use the diazomethane generator behind a safety shield in a well ventilated
515.4-8

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 (Sect 11.2) produces

micro molar amounts of diazomethane in solution. If the procedure is

followed carefully, no possibility for explosion exists.

5.5 Although hydrogen can be safely used 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.

6. APPARATUS AND EQUIPMENT

6.1 SAMPLE CONTAINERS – Amber glass bottles, approximately 40 mL, fitted with

PTFE (polytetrafluoroethylene) lined screw caps.

6.2 EXTRACTION VIALS – 60-mL clear glass vials with PTFE lined screw caps.

6.3 AUTOSAMPLER VIALS – 2.0-mL vials with screw or crimp cap and a PTFE faced

seal.

6.4 STANDARD SOLUTION STORAGE CONTAINERS – 10 to 20-mL amber glass

vials with PTFE lined screw caps.

6.5 CLEAR VIALS – 7-mL glass, disposable, with PTFE lined screw caps for extract

drying and derivatization. 

6.6 PASTEUR PIPETTES – Glass, disposable.

6.7 PIPETTES – Class A, 2.0-mL and 4.0-mL glass, or adjustable volume dispensers.

6.8 VOLUMETRIC FLASKS – Class A, suggested sizes include 5 mL, 10 mL, 100 mL.

6.9 MICRO SYRINGES – Various sizes.

6.10 BALANCE – Analytical, capable of weighing to the nearest 0.0001 g.

6.11 DIAZOMETHANE GENERATOR – See Figure 4 for a diagram of an all glass

system custom made for these validation studies. Micro molar generators are also

available from commercial sources (Aldrich Cat.#: Z10,889-8 or equivalent).

6.12 GAS CHROMATOGRAPH – Capillary GC (Hewlett Packard Model 6890 or
515.4-9

equivalent), if the fast GC option is used the modifications should include a high

pressure ( 50 psi) split/splitless injector, fast temperature ramp oven (50 oC/minute) 

and a low volume (150 μL ) micro ECD detector. Additionally, a data system capable

of fast sampling (20 points/peak) is required.

6.13 PRIMARY GC COLUMN – RTX-1701, 180 um i.d., fused silica capillary with

chemically bonded (14% cyanopropylphenyl-methylpolysiloxane), or equivalent

bonded, fused silica column.

6.14 CONFIRMATION GC COLUMN – DB-5, 180 um i.d., fused silica capillary with

chemically bonded (5% phenyl-methylpolysiloxane), or equivalent bonded, fused

silica column.

7. REAGENTS AND STANDARDS

7.1 REAGENTS AND SOLVENTS – Reagent grade or better chemicals should be used

in all analyses. 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 greater than 1/3 the

MRL for each compound of interest. 

7.1.2 METHYL tert-BUTYL ETHER (MtBE) – High purity, demonstrated to be

free from analytes and interferences (HPLC grade or better).

7.1.3 ACETONE – High purity, demonstrated to be free from analytes and

interferences (HPLC grade or better).

7.1.4 CARBITOL (DIETHYLENE GLYCOL MONOETHYL ETHER) – High

purity, demonstrated to be free from analytes and interferences (HPLC grade

or better).

7.1.5 HEXANE:MtBE (90:10, v/v) WASH SOLVENT – High purity, unpreserved,

demonstrated to be free from analytes and interferences (HPLC grade or

better).

7.1.6 HEXANE – High purity, demonstrated to be free from analytes and

interferences (HPLC grade or better).
515.4-10

7.1.7 SODIUM SULFATE, Na2SO4 – Pesticide grade, granular, anhydrous. 

Interferences have been observed when lower quality grades have been used. 

If interferences are observed, it may be necessary to heat the sodium sulfate in

a shallow tray at 400 oC 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.

7.1.8 ACIDIFIED SODIUM SULFATE – Acidify by slurrying 100 g of muffled

sodium sulfate with enough ethyl ether to just cover the solid. Add 0.5-mL

concentrated sulfuric acid dropwise while mixing thoroughly. Remove the

ether under vacuum. Mix 1 g 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

in a desiccator or at 100°C to keep the reagent dry.

7.1.9 COPPER II SULFATE PENTAHYDRATE, CUS04-5H20 – ACS reagent

grade or better.

7.1.10 4 N SODIUM HYDROXIDE SOLUTION – Dissolve 16 g sodium hydroxide

(NaOH) pellets (ACS grade or equivalent) in reagent water and dilute to 100

mL.

7.1.11 POTASSIUM HYDROXIDE SOLUTION (37%, w/v) – Dissolve 37 g of 

potassium hydroxide (KOH) pellets (ACS grade or equivalent) in reagent

water and dilute to 100 mL.

7.1.12 SODIUM SULFITE, Na2S03

 – ACS reagent grade, used as a dechlorinating

agent in this method.

7.1.13 DIAZALD SOLUTION – Prepare a solution containing 5 g diazald (ACS

reagent grade) in 50 mL of a 50:50 (v/v) mixture of MtBE and carbitol. This

solution is stable for one month or longer when stored at 4 °C in an amber

bottle with a PTFE lined screw cap.

7.1.14 SULFURIC ACID – Concentrated, ACS reagent grade.

7.1.15 SILICA GEL – ACS reagent grade, 35-60 mesh. 

7.1.16 HYDROGEN – 99.999% pure or better, GC carrier gas.

7.1.17 NITROGEN – 99.999% pure or better, ECD make-up gas.

7.2 STANDARD SOLUTIONS – When a compound purity is assayed to be 96% or
515.4-11

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. Laboratories

should use standard QC procedures to determine when Standard Solutions

described in this section need to be replaced.

7.2.1 INTERNAL STANDARD SOLUTIONS – 4,4'-Dibromooctafluorobiphenyl,

99+% is used as an internal standard for the method. 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.

7.2.1.1 INTERNAL STANDARD STOCK SOLUTION (2.0 mg/mL) –

Prepare an internal standard a stock solution by accurately weighing

approximately 0.0200 g of neat 4,4'- dibromooctafluorobiphenyl

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 PTFE-lined screw cap and store at # 0 °C. The resulting

concentration of the stock internal standard solution will be

approximately 2.0 mg/mL. 

7.2.1.2 INTERNAL STANDARD PRIMARY DILUTION STANDARD (2.5

ug/mL) – Prepare a internal standard fortification solution at

approximately 2.5 ug/mL by the addition of 12.5 uL of the stock

standard to 10 mL of MtBE. Transfer the primary dilution to an amber

glass vial with a PTFE lined screw cap and store at # 0 °C. The

solution should be replaced when ongoing QC indicates a problem.

7.2.1.3 MtBE EXTRACTION SOLVENT WITH INTERNAL STANDARD

(2.5 ug/mL) – The internal standard 4,4'-dibromooctafluorobiphenyl is

added to the extraction solvent prior to analyte extraction to

compensate for any volumetric differences encountered during sample

processing. This solution should be made fresh prior to extraction. 

The addition of 1 mL of the primary dilution standard (2.5 ug/mL) to

99-mL MtBE results in a final internal standard concentration of 25

ng/mL. This solution is used to extract the samples (Sect. 11.1).

7.2.2 SURROGATE (SUR) ANALYTE STANDARD SOLUTION –

2,4-Dichlorophenylacetic acid (99+%) is used as a surrogate compound in this

method to evaluate the extraction and derivatization procedures. This

compound has been shown to be an effective surrogate for the method

analytes, but other compounds may be used if the QC requirements in Section
515.4-12

9 are met. 

7.2.2.1 SURROGATE STOCK SOLUTION (1.0 mg/mL) – Prepare a

surrogate stock standard solution of 2,4-dichlorophenylacetic acid by

weighing 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 PTFE lined screw cap and store

at # 0 °C. The resulting concentration of the stock surrogate solution

will be 1.0 mg/mL. 

7.2.2.2 SURROGATE PRIMARY DILUTION STANDARD/SUR SAMPLE

FORTIFICATION SOLUTION (100 ug/mL) – Prepare a primary

dilution standard at approximately 100 ug/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 PTFE lined screw cap and store

at # 0 °C. Addition of 10 ul, of the primary dilution standard to the

40-mL aqueous sample results in a surrogate concentration of 25

ng/mL. The solution should be replaced when ongoing QC indicates a

problem.

7.2.3 ANALYTE STANDARD SOLUTIONS – Obtain the analytes listed in the

table in Section 1.1 as neat or solid free acid standards or as commercially

prepared ampulized solutions from a reputable standard manufacturer. The

use of pre-methylated standards is not allowed for the preparation of analyte

standards. Prepare the Analyte Stock and Primary Dilutions Standards as

described below. 

7.2.3.1 ANALYTE STOCK STANDARD SOLUTION – 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 suppliers. These stock standard solutions should be stored

at # 0 °C.

7.2.3.1.1 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-

mL volumetric flask. Dilute to volume with acetone.

7.2.3.1.2 For analytes which are liquid in their pure form at room

temperature, place about 9.8 mL of acetone into a 10-mL

volumetric flask. Allow the flask to stand, unstoppered, for

about 10 minutes to allow solvent film to evaporate from
515.4-13

the inner walls of the volumetric, and weigh to the nearest

0.1 mg. Use a 10-uL syringe and immediately add 10.0 uL

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. 

Calculate the concentration in milligrams per milliliter from

the net gain in weight. Dilute to volume, stopper, then mix

by inverting the flask several times.

7.2.3.2 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 # 0 °C. As a guideline to the analyst, the analyte

concentrations used in the primary dilution standard solution during

method development are given below.

Analyte Concentration

(ug/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

Dichlorprop 10

Dinoseb 10

Pentachlorophenol 1.0

Picloram 5.0

2,4,5-T 2.5

2,4,5-TP (Silvex) 2.5

Quinclorac 5.0
515.4-14

This primary dilution standard is used to fortify reagent water for

calibration standards. The lowest calibration standard concentration

must be at or below the MRL 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.

7.2.4 CALIBRATION STANDARDS (CAL) – A five-point calibration curve is to

be prepared by fortifying reagent water with the primary dilution standard. A

designated amount of each calibration standard in acetone is spiked into

separate 40-mL aliquots of reagent water to produce a calibration curve

ranging from below or at the MRL 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 described in Section 11. The

calibration standard solutions in acetone should be stored at # 0 °C. 

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(7) using amber glass containers with PTFE lined screw caps and

capacities of at least 40 mL. 

8.1.2 Add sodium sulfite crystals (approximately 2 mg/40 mL of sample) to the

sample bottle prior to collecting the sample. This reagent eliminates residual

chlorine in the sample.

8.2. SAMPLE COLLECTION

 8.2.1 Fill sample bottles but take care not to flush out the sodium sulfite. 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, 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 1 liter beaker with sample from a representative area, and carefully fill

sample vials from the container.
515.4-15

8.2.4 After collecting the sample, seal the bottle and agitate by hand for 15 seconds.

8.3 SAMPLE SHIPMENT AND STORAGE – All samples should be iced during

shipment and must not exceed 10 o C during the first 48 hours after collection. 

Samples should be confirmed to be at or below 10 oC when they are received at the

laboratory. Samples stored in the lab must be held at or below 6 oC and protected

from light until extraction. Samples should not be frozen.

8.4 SAMPLE AND EXTRACT HOLDING TIMES – Because of the several pH

adjustments made to the samples in the course of this method, the addition of organic

or inorganic biocides, including hydrochloric acid, have been omitted. The analyst

should be aware of the potential for the biological degradation of the analytes. 

Accordingly, samples should be extracted as soon as possible. Samples must be

extracted within 14 days. Extracts must be stored at # 0 oC or less and protected from

light in glass vials with PTFE lined caps. Holding time studies indicate that the

analytes are stable for up to 21 days in the extracts.

9. QUALITY CONTROL

9.1 Quality control (QC) requirements include the Initial Demonstration of Capability

(Sect. 17, Table 13), the determination of the Detection Limit, and subsequent

analysis in each analysis batch of a Laboratory Reagent Blank (LRB), Continuing

Calibration Check Standards (CCC), 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 13 and 14. These criteria are considered the minimum acceptable

QC criteria, and laboratories are encouraged to institute additional QC practices to

meet their specific needs.

9.1.1 Process all quality control samples through all steps of Section 11, including

hydrolysis and methylation. Sample preservatives as described in Section 8.1

must be added prior to extracting and analyzing the quality control samples.

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 13.

9.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND –

Before any samples are analyzed, it must be demonstrated that a laboratory

reagent blank (LRB) is reasonably free of contamination and that the criteria

in Section 9.4 are met. Process all quality control samples through all all steps

of the method (Sect. 9.1.1).
515.4-16

9.2.2 INITIAL DEMONSTRATION OF PRECISION – Prepare, extract, and

analyze 4-7 replicate LFBs fortified at 5 ug/L, or near the mid-range of the

initial calibration curve. Process all quality control samples through all all

steps of the method (Sect. 9.1.1). 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 DETECTION LIMIT – Prepare, extract and analyze at least seven replicate

LFBs at a concentration estimated to be near the Detection Limit, 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. Process

all quality control samples through all all steps of the method (Sect. 9.1.1). 

The fortification level may be estimated by selecting a concentration with a

signal of 2 to 5 times the noise level but must be at or below the laboratory’s

MRL (Sect. 9.3). The appropriate concentration will be dependent upon the

sensitivity of the GC/ECD system being used. Sample preservatives as

described in Section 8.1 must be added to these samples. Calculate the

Detection Limit using the equation

Detection Limit = St(n - 1, 1 - alpha = 0.99)

where

t

(n - 1, 1 - alpha = 0.99) = Students t value for the 99% confidence level with n1 degrees of freedom,

n = number of replicates, and 

S = standard deviation of replicate analyses.

 

NOTE: Calculated Detection Limits need only be less than 1/3 of the

laboratory’s MRL to be considered acceptable. Do not subtract blank values

when performing Detection Limit calculations. The Detection Limit is a

statistical determination of precision only.(1) If the Detection Limit replicates

are fortified at a low enough concentration, it is likely that they will not meet

precision and accuracy criteria, and may result in a calculated Detection Limit

that is higher than the fortified concentration 

9.2.5 METHOD MODIFICATIONS – The analyst is permitted to modify GC

columns, GC conditions, 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).
515.4-17

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 not be established at an analyte concentration that is less than either

three times the Detection Limit or a concentration which would yield a response less

than a signal-to-noise (S/N) ratio of five. Depending upon the study’s data quality

objectives it may be set at a higher concentration. Although the lowest calibration

standard must be at or 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. 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 standard prepared in the same

extraction batch as the samples of interest that contains all compounds of interest and

is extracted in the same manner as the procedural standards used to prepare the initial

calibration curve. Calibration checks, prepared with the samples being analyzed, are

required at the beginning of each day that samples are analyzed, after every ten

samples, and at the end of any group of sample analyses. See Section 10.3 for

concentration requirements, frequency requirements, and acceptance criteria. 

9.6 LABORATORY FORTIFIED BLANK (LFB) – Since this method utilizes procedural

calibration standards, which are fortified reagent waters, there is no difference

between the LFB and the continuing calibration check standard. Consequently, the

analysis of an LFB is not required ; however the acronym LFB is used for clarity in

the IDC.

9.7 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.
515.4-18

9.7.1 If the reinjected aliquot produces an acceptable internal standard response,

report results for that aliquot.

9.7.2 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 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.

9.8 SURROGATE RECOVERY – The surrogate standard is fortified into the aqueous

portion of all samples, LRBs, and LFMs and LFMDs prior to extraction. It is 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.

9.8.1 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.

9.8.2 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 injecting the last calibration standard that

passed. If the calibration standard fails the criteria of Section 9.8.1,

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 to

inform the data user that the results are suspect due to surrogate recovery.

9.9 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, 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
515.4-19

Duplicate (Sect. 9.10). 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. 

9.9.1 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. 7.2.3.2). Select a

spiking concentration that is at least twice the matrix background

concentration, if known. Use historical data and rotate through the designated

concentrations when selecting a fortifying concentration.

9.9.2 Calculate the percent recovery (R) for each analyte using the equation 

where

A = measured concentration in the fortified sample,

B = measured concentration in the unfortified sample, and

C = fortification concentration. 

9.9.3 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 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 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.

9.9.4 If an LFMD is analyzed instead of a Field Duplicate (Sect. 9.10), calculate 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
515.4-20

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.

9.10 FIELD DUPLICATES (FD1 AND FD2) – Within each extraction batch, a minimum

of one Field Duplicate (FD) or LFMD (Sect. 9.9) 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 will not require the analysis of a Field Duplicate.

9.10.1 Calculate the relative percent difference (RPD) for duplicate measurements

(FD1 and FD2) using the equation

9.10.2 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.

9.11 QUALITY CONTROL SAMPLES (QCS) – A QCS sample should be analyzed each

time that new Primary Dilution Standards (Sect. 7.2.3.2) are prepared. The source of

the QCS sample should ideally be a second vendor. If a second vendor is not

available then a different lot of the standard should be used. Although the use of premethylated standards is prohibited for preparing analyte standard solutions, premethylated standards may be used to prepare the QCS. 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 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.
515.4-21

10. CALIBRATION AND STANDARDIZATION

10.1 After initial calibration is successful, a Continuing Calibration Check (CCC) is

required at the beginning and end of each analysis batch, and after every tenth sample

(Sect. 10.3). Because this is a procedural standard method, the analyst will need to

make a decision to include an appropriate number of CCCs or an entire initial

calibration curve with each extraction batch. Initial calibration should be repeated

each time a major instrument modification or maintenance is performed.

10.2 INITIAL CALIBRATION

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 columns or conditions may be used if

equivalent or better performance can be demonstrated. 

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 CALIBRATION – Use the GC data system software to generate a linear

regression or quadratic calibration curve using the internal standard. The

analyst may choose whether or not to force zero to obtain a curve that best fits

the data. Examples of common GC system calibration curve options are: 

1) Ax /Ais vs Qx /Qis and 2) RRF vs Ax /Ais.

where:

Ax

 = integrated peak area of the analyte,

Ais = integrated peak area of the internal standard,

 Qx = quantity of analyte injected in ng or concentration units,

and 

 Qis = quantity of internal standard injected in ng or concentration

units.

10.2.4 As an alternative, concentrations may be calculated through the use of average

relative response factor (RRF). Calculate the RRFs using the equation 

 

Average RRF calibrations may only be used if the RRF values over the
515.4-22

calibration range are relatively constant (<30% RSD). Average RRF is

determined by calculating the mean RRF of a minimum of five calibration

concentrations.

10.2.5 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. 

10.3 CONTINUING CALIBRATION CHECK (CCC) – An appropriate number of CCCs

must be prepared with each extraction batch. The CCC verifies the initial calibration

at the beginning and end of each group of analyses, and after every 10th sample

during analyses. In this context, a “sample” is considered to be a field sample. LRBs, 

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 check standard

solution prepared with the extraction batch 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 level 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, 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.
515.4-23

11. PROCEDURE

11.1 SAMPLE EXTRACTION AND HYDROLYSIS

11.1.1 Remove the samples from storage (Sect. 8.3) 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

PTFE lined screw cap using a graduated cylinder.

11.1.3 Add 10 uL of surrogate standard (100 ug/mL, 2,4-dichlorophenylacetic acid in

acetone per Section 7.2.2) to the aqueous sample.

11.1.4 Add 1 mL, of the 4 N NaOH solution prepared in Section 7.1.10 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 

4 N 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. Failure to perform this step

may result in data that are biased low for some targets in field samples.(8)

11.1.5 Following hydrolysis, add 5 mL of (90:10, v:v) hexane:MtBE (Section 7.1.5)

and shake vigorously for three minutes. Allow the phases to separate for

approximately 5 minutes then remove and discard the top hexane/MtBE layer. 

This wash aids in sample cleanup and removes any Dacthal from the sample

which would interfere with the quantitation of the Dacthal metabolites. 

11.1.6 Adjust the pH to approximately 1 by adding concentrated sulfuric acid. Cap,

shake and then check the pH with a pH meter or narrow range pH paper. Add

additional sulfuric acid as needed to properly adjust the pH.

11.1.7 Quickly add approximately 2 g of copper II sulfate pentahydrate and shake

until dissolved. This colors the aqueous phase blue and allows the analyst to

better distinguish between the aqueous phase and the organic phase in this

micro extraction.

11.1.8 Quickly add approximately 16 g of muffled sodium sulfate (Sect. 7.1.7) and

shake until almost all is dissolved. Sodium sulfate is added to increase the

ionic strength of the aqueous phase and thus further drive the chlorophenoxy
515.4-24

acids into the organic phase. The addition of salt also decreases the solubility

of MtBE in the aqueous phase and allows greater volumetric recovery. The

addition of this salt and the copper II sulfate pentahydrate should be done

quickly so that the heat generated from the addition of the acid (Section

11.1.6) will help dissolve the salts.

11.1.9 Add exactly 4.0-mL MtBE and shake vigorously for three minutes. 

11.1.10 Allow the phases to separate for approximately 5 minutes.

11.2 SAMPLE METHYLATION WITH DIAZOMETHANE

11.2.1 GENERATION OF DIAZOMETHANE

11.2.1.1 Assemble the diazomethane generator (Figure 4) in a hood. The

collection vessel is a 10- or 15-mL, glass vial equipped with a

PTFE lined screw cap that is maintained at 0 - 5 °C.

11.2.1.2 Add a sufficient amount of MtBE (approximately 7 mL) to tube 1

to cover the first impinger. Add 10 mL of MtBE to the collection

vial. Set the nitrogen flow at 5-10 mL/min. Add 4-mL Diazald

solution (Sect. 7.1.13) and 3 mL of 37% KOH solution (Sect.

7.1.11) 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

72 hours.

11.2.1.3 Several commercial sources of glassware are available for

diazomethane generation. These include a mini Diazald apparatus

from Aldrich (Cat. #:Z10,889-8).

11.2.2 Using a Pasteur pipette, transfer the sample extract (upper MtBE layer) to a 7-

mL, screw cap vial. Add 0.6 g acidified sodium sulfate (Sect. 7.1.8) and

shake. This step is included to dry the MtBE extract.

11.2.3 Using a Pasteur pipette, transfer the extract to a second, 7-mL glass vial.

11.2.4 Add 250 uL of the diazomethane solution prepared in Section 11.2.1 to each

vial. The contents of the vial should remain slightly yellow in color indicating

an excess of diazomethane. Additional diazomethane may be added if

necessary. Let the esterification reaction proceed for 30 minutes.
515.4-25

11.2.5 Remove any unreacted diazomethane by adding 0.1 g of 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 Transfer the extract to an autosampler vial. A duplicate vial may be filled

using the excess extract.

11.2.7 Analyze the sample extracts as soon as possible. The sample extract may be

stored up to 21 days if kept at 0 °C or less. Keep the extracts away from light

in amber glass vials with PTFE lined caps.

11.3 GAS CHROMATOGRAPHY

11.3.1 If the fast GC option is used, several important changes from “conventional

GC” should be made to aid in the rapid analysis of the analytes. The

instrument should have a fast temperature ramp (50 °C/minute) oven and a

high pressure ( 50 psi) split/splitless injector. When using columns with

diameters less than 180 um the instrument should be equipped with a low

volume (150 μL ) micro ECD. Additionally, the column diameter and film

thickness should be decreased and the carrier gas should be changed to

hydrogen. 

11.3.2 Use of Hydrogen Safely – 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. 

11.3.3 Column Selection and Installation – During method development the RTX1701 columns provided baseline resolution for the 2,4-DB /Chloramben pair

(peaks 11 and 12 in Figure 1). Columns from other manufacturers were not as

effective in separating these analytes. 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. Additionally, a

loss of response over time was noted for Acifluorifen and Dinoseb. Trimming

approximately 1 meter from the head of the column restored the response for

these analytes. If conditions in the laboratory necessitate frequent column

trimming, a guard column is recommended.

11.4 ANALYSIS OF EXTRACTS

11.4.1 Establish operating conditions as described in Section 17, Table 1 (Table 2 if

performing confirmation). Confirm that retention times, compound separation

and resolution are similar to those summarized in Table 1 and Figure1. 
515.4-26

11.4.2 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 GC 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.

11.4.3 Check system calibration by analyzing a CCC (Sect. 10.3) and calibrate the

system by either the analysis of a calibration curve (Sect. 10. 2) or by

confirming the initial calibration is still valid by analyzing a continuing

calibration check as described in Section 10.3. Begin analyzing field and QC

samples at their appropriate frequency by injecting the same size aliquots

under the same conditions used to analyze the initial calibration. 

11.4.4 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 MtBE containing the internal standard (Sect. 7.2.1.3), and

the diluted extract injected. Acceptable surrogate performance (Sect. 9.8)

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 CALCULATION

12.1 Identify the method analytes in the sample chromatogram by comparing the retention

time of the suspect peak to retention time of an analyte peak in a calibration standard. 

Surrogate retention times should be confirmed to be within acceptance limits (Sect.

11.4.2) 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. 11.4.4). 

12.3 Analyte identifications should be confirmed using the confirmation column specified

in Table 2 or another column that is dissimilar to the primary column or by GC/MS if

the concentrations are sufficient.

12.4 Adjust the calculated concentrations of the detected analytes to reflect the initial
515.4-27

sample volume and any dilutions performed. 

12.5 Analyte concentrations are reported in ug/L as the total free acid. Calculations should

use all available digits of precision. 

13. METHOD PERFORMANCE

13.1 PRECISION, ACCURACY, AND Detection Limits – Tables for these data are

presented in Section 17. Single laboratory precision and accuracy data are presented

in Tables 5 -10. Detection Limits are presented in Table 4 and were calculated using

the formula presented in Section 9.2.4.

14. POLLUTION PREVENTION

14.1 This method utilizes liquid:liquid microextraction 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 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.
515.4-28

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. 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. "OSHA Safety and Health Standards, General Industry," (29CRF1910). Occupational

Safety and Health Administration, OSHA 2206, (Revised, Jan.1976).

5. “Carcinogens-Working with Carcinogens,” Publication No. 77-206, Department of

Health, Education, and Welfare, Public Health Service, Center for Disease Control,

National Institute of Occupational Safety and Health, Atlanta, Georgia, August 1977.

6. “Safety In Academic Chemistry Laboratories,” 3rd Edition, American Chemical

Society Publication, Committee on Chemical Safety, Washington, D.C., (1979).

7. 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.

8. Bassett, M.V.; Pepich, B.V. and Munch, D.M., “The Role Playedby Basic Hydrolysis

in the Validity of Acid herbicide Data - Recommendations for Future Preparation of

Herbicide Performance Evaluation Standards.” Environ. Sci. Technol., Vol.34, 1117,

(2000).
515.4-29

17. TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA

TABLE 1: CHROMATOGRAPHIC CONDITIONS AND AVERAGE RETENTION TIME

DATA FOR THE PRIMARY COLUMN (RTX-1701, 40m x 180 um i.d.)

Peak Number

(Figure 1) Compound

Average Tr

(min)a % RSD

1 Dalapon 7.06 0.017

2 3,5 Dichlorobenzoic acid 11.32 0.002

3 2,4-Dichlorophenylacetic acid (SUR) 11.80 0.002

4 Dicamba 11.91 0.002

5 Dichlorprop 12.21 0.002

6 4,4'-Dibromooctafluorobiphenyl (IS) 12.25 0.002

7 2,4-D 12.37 0.002

8 Pentachlorophenol 12.49 0.002

9 Silvex 12.70 0.002

10 2,4,5-T 12.90 0.002

11 2,4-DB 13.10 0.003

12 Chloramben 13.15 0.003

13 Dinoseb 13.44 0.003

14 Bentazon 13.60 0.003

15 Dacthal 13.68 0.002

16 Quinclorac 13.85 0.004

17 Picloram 14.02 0.003

18 Acifluorfen 15.20 0.004

a

 The average reten tion time represents the average of 7 runs.

Primary Column:

DB-1701, 40 m x 0.180 mm i.d., 0.20 μm film thickness, injector temp. 200 oC, liner 2 mm straight siltek®

deactivated, injection volume 1 μL of 20 0 ng/m L (high est level com ponen t) standard , splitless injection hold to

1 min then purge @ 50 mL/min, detector temp 360 oC, detecto r make up gas n itrogen at 2 0 mL/minute. 

Temperature program: 45 oC initial, program at 5 oC/minute to 80 oC, Then 50 oC/minute to 220 oC, Then 20

oC/minute to 280 oC and Hold for 3 min, Data collection via HP GC Chemstation at a rate of 20Hz.

Carrier gas: Hydrogen (UHP)

Detector makeup gas: Nitrogen (UHP)
515.4-30

TABLE 2: CHROMATOGRAPHIC CONDITIONS AND AVERAGE RETENTION TIME

DATA FOR THE CONFIRMATION COLUMN (DB-5, 40 m x 180 um i.d.)

Peak Number

(Figure 1) Compound

Average Tr

(min)a % RSD

1 Dalapon 5.04 0.057

2 3,5-Dichlorobenzoic acid 10.33 0.007

3 2,4-Dichlorophenylacetic acid (SUR) 11.31 0.004

4 Dicamba 11.46 0.006

5 Dichlorprop 12.19 0.006

6 4,4'-Dibromooctafluorobiphenyl (IS) 12.71 0.004

7 2,4-D 12.34 0.005

8 Pentachlorophenol 13.16 0.006

9 Silvex 13.29 0.004

10 2,4,5-T 13.48 0.005

11 2,4-DB 13.94 0.005

12 Chloramben 13.34 0.004

13 Dinoseb 14.01 0.004

14 Bentazon 14.28 0.006

15 Dacthal 15.00 0.004

16 Quinclorac 14.88 0.004

17 Picloram 14.59 0.004

18 Acifluorfen 16.38 0.003

a

 The average reten tion time represents the average of 7 runs.

Confirmation Column:

DB-5, 40 m x 0.180 mm i.d., 0.20 μm film thickness, injector temp. 200 oC, liner 2 mm straight siltek®

deactivated, injection volume 1 μL of 10 0 ng/m L (high est level com ponen t) standard , splitless injection hold to

1 min then purge @ 50 mL/min, detector temp 360 oC, detecto r make up gas n itrogen at 2 0 mL/minute. 

Temperature program: 45 oC initial, program at 4 oC/minute to 80 oC then program at 30 oC/minute to 280 and

Hold for 2 min, Data collection via HP GC Chemstation at a rate of 20Hz.

Carrier gas: Hydrogen (UHP)

Detector makeup gas: Nitrogen (UHP)
515.4-31

TABLE 3: CHROMATOGRAPHIC CONDITIONS AND AVERAGE RETENTION TIME

DATA FOR A CONVENTIONAL PRIMARY COLUMN (DB-5, 30 m x 250 um i.d.)

Peak Number

(Figure 3) Compound

Tr

(min)

1 Dalapon 14.28

2 3,5-Dichlorobenzoic acid 20.35

3 2,4-Dichlorophenylacetic acid (SUR) 20.98

4 Dicamba 21.10

5 Dichlorprop 21.46

6 4,4'-Dibromooctafluorobiphenyl (IS) 21.53

7 2,4-D 21.64

8 Pentachlorophenol 21.81

9 Silvex 22.03

10 2,4,5-T 22.27

11 2,4-DB 22.51

12 Chloramben 22.55

13 Dinoseb 22.94

14 Bentazon 23.17

15 Dacthal 23.30

16 Quinclorac 23.55

17 Picloram 23.82

18 Acifluorfen 25.90

Conventional Primary Column:

DB-1701, 30 m x 0.250 mm i.d., 0.25 μm film thickness, injector temp. 200 oC, liner 2 mm straight deactivated,

injection volume 1 μL of 100 ng/mL (highest level component) standard, splitless injection hold to .5 min then

purge @ 30 mL/min, detector temp 290 oC, detector make up gas P-5 at 60 mL/minute. Temperature program:

45 oC initial, hold 10 min then program at 5 oC/minute to 70 oC, Then 30 oC/minute to 260 oC, and Hold for 5

min, Data c ollection via T urbochro m 4.1

Carrier gas: Helium (UHP)

Detector makeup gas: 5% methane/argon (UHP)
515.4-32

TABLE 4: DETECTION LIMITS IN REAGENT WATER

Compound

Fortification

Level (ug/L)

Primary

Column

Detection

Limita

 (ug/L)

Secondary

Column

Detection

Limita

 (ug/L)

Dalapon 0.100 0.054 0.074

3,5-Dichlorobenzoic acid 0.050 0.212 0.049

Dicamba 0.050 0.032 0.042

Dichlorprop 0.100 0.433 0.12

2,4-D 0.100 0.055 0.066

Pentachlorophenol 0.010 0.014 0.084

Silvex 0.025 0.033 0.018

2,4,5-T 0.025 0.024 0.033

2,4-DB 0.100 0.246 0.182

Chloramben 0.050 0.057 0.083

Dinoseb 0.100 0.166 0.081

Bentazon 0.100 0.064 0.185

Dacthal 0.050 0.113 0.105

Quinclorac 0.050 0.080 0.113

Picloram 0.050 0.076 0.055

Acifluorfen 0.250 0.307 0.092

aDetection Limits were extracted and analyzed over 3 days for 9 replicates following

the procedure outlined in Section 9. 
515.4-33

TABLE 5: LOW LEVEL PRECISION AND ACCURACY IN REAGENT WATER

Compound Fortification

Level (ug/L)

 Mean %

Recovery 

% RSD

(n=7)

S/N

Ratioa

Dalapon 1.0 108 2.2 578

3,5-Dichlorobenzoic acid 0.50 117 16 9

Dicamba 0.50 97 3.7 35

Dichlorprop 1.0 95 5.7 19

2,4-D 1.0 98 5.2 23

Pentachlorophenol 0.10 76 7.4 21

Silvex 0.25 84 20 9

2,4,5-T 0.25 96 7.3 16

2,4-DB 1.0 97 8.4 6

Chloramben 0.50 90 9.0 113

Dinoseb 1.0 103 6.7 8

Bentazon 1.0 100 2.1 41

Dacthal 0.50 92 6.5 100

Quinclorac 0.10 106 9.3 20

Picloram 1.0 92 2.6 23

Acifluorfen 0.50 77 5.9 80

2,4-Dichlorophenylacetic acid (SUR) 25 104 8.0 -

aSignal-to-noise ratios were calculated for each target compound 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.
515.4-34

TABLE 6: MID LEVEL PRECISION AND ACCURACY IN REAGENT WATER

Compound Fortification

Level (ug/L)

Mean %

Recovery

% RSD

(n=7)

Dalapon 10 107 2.3

3,5-Dichlorobenzoic acid 5.0 96 2.3

Dicamba 5.0 102 1.5

Dichlorprop 10 107 0.6

2,4-D 10 106 1.4

Pentachlorophenol 1.0 103 1.4

Silvex 2.5 107 4.4

2,4,5-T 2.5 105 1.3

2,4-DB 10 93 1.6

Chloramben 5.0 105 2.9

Dinoseb 10 119 4.6

Bentazon 10 98 0.7

Dacthal 5.0 100 1.9

Quinclorac 1.0 101 2.9

Picloram 10 99 3.1

Acifluorfen 5.0 107 4.5

2,4-Dichlorophenylacetic acid (SUR) 25 112 1.3
515.4-35

TABLE 7: LOW LEVEL PRECISION AND ACCURACY IN FORTIFIED CHLORINATED

SURFACE WATER

Compound Fortification

Level (ug/L)

 Mean %

Recovery

% RSD

(n=7)

Dalapon 1.0 104 2.1

3,5-Dichlorobenzoic acid 0.50 110 3.0

Dicamba 0.50 93 4.4

Dichlorprop 1.0 93 11

2,4-D 1.0 97 5.2

Pentachlorophenol 0.10 86 3.2

Silvex 0.25 103 4.3

2,4,5-T 0.25 100 3.5

2,4-DB 1.0 104 5.9

Chloramben 0.50 93 2.0

Dinoseb 1.0 99 1.2

Bentazon 1.0 91 2.2

Dacthal 0.50 92 4.6

Quinclorac 0.10 96 15

Picloram 1.0 102 2.4

Acifluorfen 0.50 98 4.1

2,4-Dichlorophenylacetic acid (SUR) 25 107 1.5
515.4-36

TABLE 8: MID LEVEL PRECISION AND ACCURACY IN FORTIFIED CHLORINATED

SURFACE WATER

Compound Fortification

Level (ug/L)

Mean %

Recovery

% RSD

(n=7)

Dalapon 10 100 2.5

3,5-Dichlorobenzoic acid 5.0 104 2.6

Dicamba 5.0 102 2.9

Dichlorprop 10 101 1.2

2,4-D 10 101 1.2

Pentachlorophenol 1.0 100 1.0

Silvex 2.5 101 1.0

2,4,5-T 2.5 100 1.6

2,4-DB 10 101 2.3

Chloramben 5.0 102 2.6

Dinoseb 10 101 1.6

Bentazon 10 98 1.8

Dacthal 5.0 99 2.0

Quinclorac 1.0 105 2.5

Picloram 10 107 3.2

Acifluorfen 5.0 92 2.4

2,4-Dichlorophenylacetic acid (SUR) 25 106 1.4
515.4-37

TABLE 9: LOW LEVEL PRECISION AND ACCURACY IN FORTIFIED CHLORINATED

GROUND WATER

Compound Fortification

Level (ug/L)

 Mean %

Recovery 

% RSD

(n=7)

Dalapon 1.0 105 1.5

3,5-Dichlorobenzoic acid 0.50 104 6.0

Dicamba 0.50 100 2.2

Dichlorprop 1.0 109 3.5

2,4-D 1.0 108 10

Pentachlorophenol 0.10 103 17

Silvex 0.25 96 3.0

2,4,5-T 0.25 91 3.4

2,4-DB 1.0 94 3.1

Chloramben 0.50 88 2.7

Dinoseb 1.0 100 4.0

Bentazon 1.0 101 2.8

Dacthal 0.50 62a

9.9

Quinclorac 0.10 107 5.7

Picloram 1.0 97 2.8

Acifluorfen 0.50 92 4.2

2,4-Dichlorophenylacetic acid (SUR) 25 95 3.1

aDacthal was present in the chlorinated surface water at about 1/5th the fortification

level. This was taken into account in the determination of the Mean % Recovery.
515.4-38

TABLE 10: MID LEVEL PRECISION AND ACCURACY IN FORTIFIED CHLORINATED

GROUND WATER

Compound Fortification

Level (ug/L)

Mean %

Recovery

% RSD

(n=7)

Dalapon 10 97 5.5

3,5-Dichlorobenzoic acid 5.0 95 2.1

Dicamba 5.0 98 1.6

Dichlorprop 10 79 0.7

2,4-D 10 99 1.6

Pentachlorophenol 1.0 100 4.2

Silvex 2.5 97 1.6

2,4,5-T 2.5 97 1.5

2,4-DB 10 92 1.1

Chloramben 5.0 102 2.6

Dinoseb 10 105 2.6

Bentazon 10 101 1.8

Dacthal 5.0 99 1.2

Quinclorac 1.0 94 1.5

Picloram 10 103 2.5

Acifluorfen 5.0 84 2.5

2,4-Dichlorophenylacetic acid (SUR) 25 106 1.1
515.4-39

TABLE 11. SAMPLE HOLDING TIME DATA FOR SAMPLES FROM A

CHLORINATED SURFACE WATER FORTIFIED WITH METHOD

ANALYTESa

Compound

Day 0

% Rec

Day 2

% Rec

Day 7

% Rec

Day 14

% Rec

Dalapon 91 90 91 108

3,5-Dichlorobenzoic acid 98 97 85 91

Dicamba 92 92 96 99

Dichlorprop 89 88 91 95

2,4-D 88 85 87 92

Pentachlorophenol 92 85 86 93

Silvex 91 88 86 94

2,4,5-T 89 89 85 91

2,4-DB 87 84 86 93

Chloramben 86 86 88 100

Dinoseb 105 95 99 111

Bentazon 91 87 95 99

Dacthal 92 91 86 94

Quinclorac 90 80 84 91

Picloram 94 85 85 99

Acifluorfen 109 96 103 123

a

 All samples fortified at the same level used to collect the mid level precision and

accuracy data (see Table 10 for example). 
515.4-40

TABLE 12. EXTRACT HOLDING TIME DATA FOR SAMPLES FROM A

CHLORINATED SURFACE WATER FORTIFIED WITH METHOD

ANALYTESa,b

Compound

Initial a, b

Injection

%Rec

Day 21

Reinjection

%Rec

Dalapon 100 89

3,5-Dichlorobenzoic acid 104 113

Dicamba 102 104

Dichlorprop 100 101

2,4-D 101 102

Pentachlorophenol 100 102

Silvex 101 103

2,4,5-T 99 102

2,4-DB 101 102

Chloramben 102 105

Dinoseb 99 81

Bentazon 98 100

Dacthal 99 103

Quinclorac 105 127

Picloram 107 98

Acifluorfen 92 104

2,4-Dichlorophenylacetic acid

(SUR)

106 105

aSample storage stability is expressed as a percent recovery (%Rec) value calculated as

described in Section 9.9.2.

bAll samples fortified at the same level used to collect the mid level precision and

accuracy data (see Table 10 for example). 
515.4-41

TABLE 13: INITIAL DEMONSTRATION OF CAPABILITY (IDC) REQUIREMENTS

Method

Reference

Requirement Specification and

Frequency

Acceptance Criteria

Section

9.2.1

Initial

Demonstration of

Low Method

Background

Analyze LRB prior to any

other IDC steps

Demonstrate that all target

analytes are below 1

/3

 the

reporting limit or lowest CAL

standard, and that possible

interference from extraction

media do not prevent the

identification and

quantification of method

analytes.

Section

9.2.2

Initial

Demonstration of

Precision (IDP)

Analyze 4-7 replicate LFBs

fortified at midrange

concentration.

%RSD must be #20%

Section

9.2.3

Initial

Demonstration of

Accuracy

Calculate average recovery

for replicates used in IDP

Mean recovery must be within

± 20% of true value.

Section

9.2.4

 Detection Limit 

 Determination

Over a period of three days,

prepare a minimum of 7

replicate LFBs fortified at a

concentration estimated to

be near the Detection Limit. 

Analyze the replicates

through all steps of the

analysis. Calculate the

Detection Limit using the

equation in Section 9.2.4.

Must be # 1/3 of the

established MRL.

Note: Data from Detection

Limit replicates are not

required to meet method

precision and accuracy criteria. 

If the Detection Limit

replicates are fortified at a low

enough concentration, it is

likely that they will not meet

precision and accuracy criteria.
515.4-42

TABLE 14: QUALITY CONTROL REQUIREMENTS (SUMMARY)

Method

Referenc

e

Requirement Specification and

Frequency

Acceptance Criteria

Section

10.2

Initial Calibration Use internal standard

calibration technique to

generate a 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 1/

3

 the

method reporting limit or

lowest CAL standard, and that

possible interferences do not

prevent quantification of

method analytes.

If targets exceed 1/3

 the MRL,

results for all subject analytes in

extraction batch are invalid.

Sections

9.5 and

 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.
515.4-43

TABLE 14: QUALITY CONTROL REQUIREMENTS (SUMMARY)

Method

Reference

Requirement Specification and

Frequency

Acceptance Criteria

Section 9.7 Internal

Standard

(IS)

4,4'-dibromooctafluorobiphenyl is added to the

extraction solvent.

Peak area counts for the IS in LFBs,

LRBs, and sample extracts must be

within 50-150% of the average peak

area calculated during the initial

calibration. If the IS does not meet

criteria, corresponding target results

are invalid.

Section 9.8 Surrogate

Standard

(SUR)

2,4-dichlorophenylacetic

acid is added to all

calibration standards and

samples, including QC

samples. 

Surrogate recovery must be 70-130%

of the true value. If surrogate fails

this criterion, report all results for

sample as suspect/surrogate recovery.

Section 9.9 Laboratory

Fortified

Sample

Matrix (LFM)

and

Laboratory

Fortified

Matrix

Duplicate

(LFMD)

Analyze one LFM per

analysis batch (20

samples or less) fortified

with method analytes at a

concentration close to but

greater than the native

concentration.

LFMD should be used in

place of Field Duplicate if

frequency of detects for

targets is low.

Recoveries at mid and high levels not

within 70-130% or low-level

recoveries not within 50-150% of the

fortified amount may indicate a matrix

effect.

Target analyte RPDs for LFMD

should be within ±30% at mid and

high levels of fortification and within

±50% near MRL. 

Section

9.10

Field

Duplicates

(FD)

Extract and analyze at

least one FD with each

extraction batch (20

samples or less). A

LFMD may be substituted

for a FD when the 

frequency of detects for

target analytes is low.

Target analyte RPDs for FD should be

within ±30% at mid and high levels of

fortification and within ±50% near

MRL. 

Section

9.11

Quality

Control

Sample

(QCS)

Analyzed when new

Primary Dilution

Standards (PDS) are

prepared.

Results must be 70-130% of the

expected value.
Method

Reference

Requirement Specification and

Frequency

Acceptance Criteria

515.4-44

Section 8.4 Sample

Holding Time

14 days with appropriate

preservation and storage 

Sample results are valid only if

samples are extracted within sample

hold time.

Section 8.4 Extract

Holding Time

 21 days with appropriate

storage

Sample results are valid only if

extracts are analyzed within extract

hold time.
515.4-45

FIGURE 1

CHROMATOGRAM OF THE CHLORPHENOXY HERBICIDES ON A RTX-1701 COLUMN 

CHROMATOGRAPHIC CONDITIONS ARE GIVEN IN TABLE 1.
515.4-46

FIGURE 2

CHROMATOGRAM OF THE CHLORPHENOXY HERBICIDES ON A DB-5 COLUMN 

CHROMATOGRAPHIC CONDITIONS ARE GIVEN IN TABLE 2.
515.4-47

FIGURE 3

CHROMATOGRAM OF THE CHLORPHENOXY HERBICIDES ON A CONVENTIONAL PRIMARY COLUMN 

(30 m x 250 um) CHROMATOGRAPHIC CONDITIONS ARE GIVEN IN TABLE 3.
515.4-48

FIGURE 4

DIAZOMETHANE GENERATOR