SEPA
United States
Environmental Protection
Agency
www.epa.gov
Method 614: The Determination of
Organophosphorus Pesticides in
Municipal and Industrial
Waste water
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Method 614
The Determination of
Organophosphorus Pesticides in
Municipal and Industrial
Wastewater
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Method 614
The Determination of Organophosphorus Pesticides in Municipal and
Industrial Wastewater
1. SCOPE AND APPLICATION
1.1 This method covers the determination of certain organophosphorus pesticides. The
following parameters can be determined by this method:
Parameter STORET No. CAS No.
Azinphos methyl 39580 86-50-0
Demeton 39560 8065-48-3
Diazinon 39570 333-41-5
Disulfoton 39010 298-04-4
Ethion - 563-12-2
Malathion 39530 121-75-5
Parathion ethyl 39540 56-38-2
Parathion methyl 39600 298-00-0
1.2 This is a gas chromatographic (GC) method applicable to the determination of the
compounds listed above in industrial and municipal discharges as provided under
40 CFR 136.1. Any modification of this method beyond those expressly permitted shall
be considered a major modification subject to application and approval of alternative test
procedures under 40 CFR 136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 15) for several parameters are listed
in Table 1. The MDL for a specific wastewater may differ from those listed, depending
upon the nature of interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are essentially the same as
in Method 617. Thus, a single sample may be extracted to measure the parameters
included in the scope of both of these methods. When cleanup is required, the
concentration levels must be high enough to permit selecting aliquots, as necessary, in
order to apply appropriate cleanup procedures. Under gas chromatography, the analyst
is allowed the latitude to select chromatographic conditions appropriate for the
simultaneous measurement of combinations of these parameters (see Section 12).
1.5 This method is restricted to use by or under the supervision of analysts experienced in
the use of gas chromatography and in the interpretation of gas chromatograms. Each
analyst must demonstrate the ability to generate acceptable results with this method
using the procedure described in Section 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or all of the compounds
above, compound identifications should be supported by at least one additional
qualitative technique. This method describes analytical conditions for a second gas
chromatographic column that can be used to confirm measurements made with the
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Method 614
primary column. Section 14 provides gas chromatograph/mass spectrometer (GC/MS)
criteria appropriate for the qualitative confirmation of compound identifications.
2. SUMMARY OF METHOD
2.1 A measured volume of sample, approximately 1 L, is extracted with 15% methylene
chloride in hexane using a separatory funnel. The extract is dried and concentrated to
a volume of 10 mL or less. Gas chromatographic conditions are described which permit
the separation and measurement of the compounds in the extract by flame photometric
or thermionic bead gas chromatography.
2.2 Method 614 represents an editorial revision of a previously promulgated U.S. EPA
method for organophosphorus pesticides.1 While complete method validation data is not
presented herein, the method has been in widespread use since its promulgation, and
represents the state of the art for the analysis of such materials.
2.3 This method provides selected cleanup procedures to aid in the elimination of
interferences which may be encountered.
3. INTERFERENCES
3.1 Method interferences may be caused by contaminants in solvents, reagents, glassware,
and other sample-processing apparatus that lead to discrete artifacts or elevated baselines
in gas chromatograms. All reagents and apparatus must be routinely demonstrated to
be free from interferences under the conditions of the analysis by running laboratory
reagent blanks as described in Section 8.5.
3.1.1 Glassware must be scrupulously cleaned.2 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 and reagent
water. Drain dry, and heat in an oven or muffle furnace at 400°C for 15 to 30
minutes. Do not heat volumetric ware. Thermally stable materials, such as PCBs,
may not be eliminated by this treatment. Thorough rinsing with acetone and
pesticide-quality hexane may be substituted for the heating. After drying and
cooling, seal and store glassware in a clean environment to prevent any
accumulation of dust or other contaminants. Store inverted or capped with
aluminum foil.
3.1.2 The use of high-purity reagents and solvents helps to minimize interference
problems. Purification of solvents by distillation in all-glass systems may be
required.
3.2 Matrix interferences may be caused by contaminants that are coextracted from the
sample. The extent of matrix interferences will vary considerably from source to source,
depending upon the nature and diversity of the industrial complex or municipality
sampled. The cleanup procedure in Section 11 can be used to overcome many of these
interferences, but unique samples may require additional cleanup approaches to achieve
the MDL listed in Table 1.
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Method 614
4. SAFETY
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely
defined; however, each chemical compound must be treated as a potential health hazard.
From this viewpoint, exposure to these chemicals must be reduced to the lowest possible
level by whatever means available. The laboratory is responsible for maintaining a
current awareness file of OSHA regulations regarding the safe handling of the chemicals
specified in this method. A reference file of material data handling sheets should also
be made available to all personnel involved in the chemical analysis. Additional
references to laboratory safety are available and have been identified35 for the
information of the analyst.
5. APPARATUS AND MATERIALS
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab-sample bottle: Amber borosilicate or flint glass, 1-L or 1-quart volume,
fitted with screw-caps lined with TFE-fluorocarbon. Aluminum foil may be
substituted for TFE if the sample is not corrosive. If amber bottles are not
available, protect samples from light. The container and cap liner must be
washed, rinsed with acetone or methylene chloride, and dried before use to
minimize contamination.
5.1.2 Automatic sampler (optional): Must incorporate glass sample containers for the
collection of a minimum of 250 mL. Sample containers must be kept refrigerated
at 4°C and protected from light during compositing. If the sampler uses a
peristaltic pump, a minimum length of compressible silicone rubber tubing may
be used. Before use, however, the compressible tubing must be thoroughly rinsed
with methanol, followed by repeated rinsings with reagent water to minimize the
potential for contamination of the sample. An integrating flow meter is required
to collect flow-proportional composites.
5.2 Glassware. (All specifications are suggested. Catalog numbers are included for
illustration only.)
5.2.1 Separatory funnel: 125-mL, 1000-mL, and 2000-mL, with TFE-fluorocarbon
stopcock, ground-glass or TFE stopper.
5.2.2 Drying column: Chromatographic column 400 mm long by 19 mm ID with
coarse-fritted disc.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with coarse-fritted disc at
bottom and TFE-fluorocarbon stopcock (Kontes K-420540-0224 or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish: 10-mL, graduated (Kontes K-570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test.
Ground-glass stopper is used to prevent evaporation of extracts.
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Method 614
5.2.5 Evaporative flask, Kuderna-Danish: 500-mL (Kontes K-570001-0500 or
equivalent). Attach to concentrator tube with springs.
5.2.6 Snyder column, Kuderna-Danish: Three-ball macro (Kontes K-503000-0121 or
equivalent).
5.2.7 Snyder column, Kuderna-Danish: Two-ball micro (Kontes K-569001-0219 or
equivalent).
5.2.8 Pipette, disposable: 140 mm long by 5 mm ID.
5.2.9 Vials: Amber glass, 10- to 15-mL capacity with TFE-fluorocarbon-lined screw-cap.
5.3 Boiling chips: Approximately 10/40 mesh. Heat at 400°C for 30 minutes or perform a
Soxhlet extraction with methylene chloride.
5.4 Water bath: Heated, with concentric ring cover, capable of temperature control (±2°C).
The bath should be used in a hood.
5.5 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.
5.6 Gas chromatograph: Analytical system complete with gas chromatograph suitable for
on-column injection and all required accessories including syringes, analytical columns,
gases, detector, and strip-chart recorder. A data system is recommended for measuring
peak areas.
5.6.1 Column 1: 180 cm long by 4 mm ID glass, packed with 3% OV-1 on Gas Chrom
Q (100/120 mesh) or equivalent. This column was used to develop the method
performance statements in Section 15. Alternative columns may be used in
accordance with the provisions described in Section 12.1.
5.6.2 Column 2: 180 cm long by 4 mm ID glass, packed with 1.5% OV-17/1.95% QF-1
on Gas Chrom Q (100/120 mesh) or equivalent.
5.6.3 Detector: Phosphorus-specific; flame photometric detector (FPD, with 526 nm
filter) or thermionic bead detector in the nitrogen mode. These detectors have
proven effective in the analysis of wastewaters for the parameters listed in the
scope. The FPD was used to develop the method performance statements in
Section 15. Alternative detectors, including a mass spectrometer, may be used in
accordance with the provisions described in Section 12.1.
6. REAGENTS
6.1 Reagent water: Reagent water is defined as a water in which an interferent is not
observed at the method detection limit of each parameter of interest.
6.2 Acetone, hexane, isooctane, methylene chloride: Pesticide-quality or equivalent.
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Method 614
6.3 Ethyl ether: Nanograde, redistilled in glass if necessary. Must be free of peroxides as
indicated by EM Quant test strips (available from Scientific Products Co., Cat.
No. PI 126-8, and other suppliers). Procedures recommended for removal of peroxides
are provided with the test strips. After cleanup, 20 mL of ethyl alcohol preservative must
be added to each liter of ether.
6.4 Acetonitrile, hexane-saturated: Mix pesticide-quality acetonitrile with an excess of hexane
until equilibrium is established.
6.5 Sodium sulfate: ACS, granular, anhydrous. Condition by heating in a shallow tray at
400°C for a minimum of 4 hours to remove phthalates and other interfering organic
substances. Alternatively, heat 16 hours at 450-500°C in a shallow tray or perform a
Soxhlet extraction with methylene chloride for 48 hours.
6.6 Sodium chloride solution, saturated: Prepare saturated solution of NaCl in reagent water
and extract with hexane to remove impurities.
6.7 Alumina: Woelm, neutral; deactivate by pipetting 1 mL of distilled water into a 125-mL
ground-glass stoppered Erlenmeyer flask. Rotate flask to distribute water over surface
of glass. Immediately add 19.0 g fresh alumina through small powder funnel. Shake
flask containing mixture for 2 hours on a mechanical shaker.
6.8 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in dark in glass
container with ground-glass stopper or foil-lined screw-cap. Before use, activate each
batch at least 16 hours at 130°C in a foil-covered glass container.
6.9 Stock standard solutions (1.00 ug/uL): Stock standard solutions may be prepared from
pure standard materials or purchased as certified solutions.
6.9.1 Prepare stock standard solutions by accurately weighing approximately 0.0100 g
of pure material. Dissolve the material in pesticide-quality isooctane or acetone
and dilute to volume in a 10-mL volumetric flask. Larger volumes may be used
at the convenience of the analyst. If compound purity is certified at 96% or
greater, the weight may be used without correction to calculate the concentration
of the stock standard. Commercially prepared stock standards may be used at
any concentration if they are certified by the manufacturer or by an independent
source.
6.9.2 Transfer the stock standard solutions into TFE-fluorocarbon-sealed screw-cap
vials. Store at 4°C and protect from light. Frequently check stock standard
solutions for signs of degradation or evaporation, especially just prior to
preparing calibration standards from them.
6.9.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison
with check standards indicates a problem.
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Method 614
7. CALIBRATION
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in
Table 1. The gas chromatographic system may be calibrated using either the external
standard technique (Section 7.2) or the internal standard technique (Section 7.3).
7.2 External standard calibration procedure.
7.2.1 For each parameter of interest, prepare calibration standards at a minimum of
three concentration levels by adding accurately measured volumes of one or more
stock standards to a volumetric flask and diluting to volume with isooctane or
other suitable solvent. One of the external standards should be representative of
a concentration near, but above, the method detection limit. The other
concentrations should correspond to the range of concentrations expected in the
sample concentrates or should define the working range of the detector.
7.2.2 Using injections of 1 to 5 uL of each calibration standard, tabulate peak height or
area responses against the mass injected. The results can be used to prepare a
calibration curve for each parameter. Alternatively, the ratio of the response to
the mass injected, defined as the calibration factor (CF), may be calculated for
each parameter at each standard concentration. If the relative standard deviation
of the calibration factor is less than 10% over the working range, the average
calibration factor can be used in place of a calibration curve.
7.2.3 The working calibration curve or calibration factor must be verified on each
working shift by the measurement of one or more calibration standards. If the
response for any parameter varies from the predicted response by more than
±10%, the test must be repeated using a fresh calibration standard. Alternatively,
a new calibration curve or calibration factor must be prepared for that parameter.
7.3 Internal standard calibration procedure: To use this approach, the analyst must select
one or more internal standards similar in analytical behavior to the compounds of
interest. The analyst must further demonstrate that the measurement of the internal
standard is not affected by method or matrix interferences. Due to these limitations, no
internal standard applicable to all samples can be suggested.
7.3.1 Prepare calibration standards at a minimum of three concentration levels for each
parameter of interest by adding volumes of one or more stock standards to a
volumetric flask. To each calibration standard, add a known constant amount of
one or more internal standards, and dilute to volume with isooctane or other
suitable solvent. One of the standards should be representative of a concentration
near, but above, the method detection limit. The other concentrations should
correspond to the range of concentrations expected in the sample concentrates, or
should define the working range of the detector.
7.3.2 Using injections of 1 to 5 uL of each calibration standard, tabulate the peak height
or area responses against the concentration for each compound and internal
standard. Calculate response factors (RF) for each compound as follows:
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Method 614
Equation 1
RF = —
where
As = Response for the parameter to be measured
Als = Response for the internal standard
Cls = Concentration of the internal standard, in ug/L
Cs = Concentration of the parameter to be measured, in ug/L
If the RF value over the working range is constant, less than 10% relative
standard deviation, the RF can be assumed to be invariant and the average RF
may be used for calculations. Alternatively, the results may be used to plot a
calibration curve of response ratios, AS/A is against RF.
7.3.3 The working calibration curve or RF must be verified on each working shift by
the measurement of one or more calibration standards. If the response for any
parameter varies from the predicted response by more than ±10%, the test must
be repeated using a fresh calibration standard. Alternatively, a new calibration
curve must be prepared for that compound.
7.4 The cleanup procedure in Section 11 utilizes Florisil chromatography. Florisil from
different batches or sources may vary in adsorptive capacity. To standardize the amount
of Florisil which is used, the use of the lauric acid value is suggested. This procedure6
determines the adsorption from hexane solution of lauric acid, in milligrams, per gram
of Florisil. The amount of Florisil to be used for each column is calculated by dividing
this factor into 110 and multiplying by 20 g.
7.5 Before using any cleanup procedure, the analyst must process a series of calibration
standards through the procedure to validate elution patterns and the absence of
interference from the reagents.
8. QUALITY CONTROL
8.1 Each laboratory using this method is required to operate a formal quality control
program. The minimum requirements of this program consist of an initial demonstration
of laboratory capability and the analysis of spiked samples as a continuing check on
performance. The laboratory is required to maintain performance records to define the
quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must demonstrate the ability to
generate acceptable accuracy and precision with this method. This ability is
established as described in Section 8.2.
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Method 614
8.1.2 In recognition of the rapid advances occurring in chromatography, the analyst is
permitted certain options 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 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of all samples to
monitor continuing laboratory performance. This procedure is described in
Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and precision, the analyst must
perform the following operations.
8.2.1 Select a representative spike concentration for each compound to be measured.
Using stock standards, prepare a quality control check sample concentrate in
acetone, 1000 times more concentrated than the selected concentrations.
8.2.2 Using a pipette, add 1.00 mL of the check sample concentrate to each of a
minimum of four 1000-mL aliquots of reagent water. A representative
wastewater may be used in place of the reagent water, but one or more additional
aliquots must be analyzed to determine background levels, and the spike level
must exceed twice the background level for the test to be valid. Analyze the
aliquots according to the method beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the standard deviation of the
percent recovery (s), for the results. Wastewater background corrections must be
made before R and calculations are performed.
8.2.4 Table 2 provides single-operator recovery and precision for diazinon, parathion
methyl, and parathion ethyl. Similar results should be expected from reagent
water for all organophosphorus compounds listed in this method. Compare these
results to the values calculated in Section 8.2.3. If the data are not comparable,
review potential problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define the performance of
the laboratory for each spike concentration and parameter being measured.
8.3.1 Calculate upper and lower control limits for method performance as follows:
Upper Control Limit (UCL) = R + 3s
Lower Control Limit (LCL) = R - 3s
where R and S are calculated as in Section 8.2.3. The UCL and LCL can be used
to construct control charts7 that are useful in observing trends in performance.
8.3.2 The laboratory must develop and maintain separate accuracy statements of
laboratory performance for wastewater samples. An accuracy statement for the
method is defined as R ± s. The accuracy statement should be developed by the
analysis of four aliquots of wastewater as described in Section 8.2.2, followed by
the calculation of R and s. Alternatively, the analyst may use four wastewater
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Method 614
data points gathered through the requirement for continuing quality control in
Section 8.4. The accuracy statements should be updated regularly.7
8.4 The laboratory is required to collect in duplicate a portion of their samples to monitor
spike recoveries. The frequency of spiked sample analysis must be at least 10% of all
samples or one spiked sample per month, whichever is greater. One aliquot of the
sample must be spiked and analyzed as described in Section 8.2. If the recovery for a
particular parameter does not fall within the control limits for method performance, the
results reported for that parameter in all samples processed as part of the same set must
be qualified as described in Section 13.3. The laboratory should monitor the frequency
of data so qualified to ensure that it remains at or below 5%.
8.5 Before processing any samples, the analyst must demonstrate through the analysis of a
1-L aliquot of reagent water that all glassware and reagent interferences are under
control. Each time a set of samples is extracted or there is a change in reagents, a
laboratory reagent blank must be processed as a safeguard against laboratory
contamination.
8.6 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. Field duplicates may be analyzed
to monitor the precision of the sampling technique. When doubt exists over the
identification of a peak on the chromatogram, confirmatory techniques such as gas
chromatography with a dissimilar column, specific element detector, or mass
spectrometer must be used. Whenever possible, the laboratory should perform analysis
of quality control materials and participate in relevant performance evaluation studies.
9. SAMPLE COLLECTION, PRESERVATION, AND HANDLING
9.1 Grab samples must be collected in glass containers. Conventional sampling practices8
should be followed; however, the bottle must not be prerinsed with sample before
collection. Composite samples should be collected in refrigerated glass containers in
accordance with the requirements of the program. Automatic sampling equipment must
be as free as possible of plastic and other potential sources of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of collection until
extraction.
9.3 All samples must be extracted within 7 days and completely analyzed within 40 days of
extraction.
10. SAMPLE EXTRACTION
10.1 Mark the water meniscus on the side of the sample bottle for later determination of
sample volume. Pour the entire sample into a 2-L separatory funnel.
10.2 Add 60 mL of 15% (v/v) methylene chloride in hexane to the sample bottle, seal, and
shake 30 seconds to rinse the inner walls. Transfer the solvent to the separatory funnel
and extract the sample by shaking the funnel for 2 minutes with periodic venting to
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Method 614
release excess pressure. Allow the organic layer to separate from the water phase for a
minimum of 10 minutes. If the emulsion interface between layers is more than one third
the volume of the solvent layer, the analyst must employ mechanical techniques to
complete the phase separation. The optimum technique depends upon the sample, but
may include stirring, filtration of the emulsion through glass wool, centrifugation, or
other physical methods. Drain the aqueous phase into a 1000-mL Erlenmeyer flask and
collect the extract in a 250-mL Erlenmeyer flask. Return the aqueous phase to the
separatory funnel.
10.3 Add a second 60-mL volume of 15% methylene chloride in hexane to the sample bottle
and repeat the extraction procedure a second time, combining the extracts in the 250-mL
Erlenmeyer flask. Perform a third extraction in the same manner.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL concentrator tube
to a 500-mL evaporative flask. Other concentration devices or techniques may be used
in place of the K-D if the requirements of Section 8.2 are met.
10.5 Pour the combined extract through a drying column containing about 10 cm of
anhydrous sodium sulfate, and collect the extract in the K-D concentrator. Rinse the
Erlenmeyer flask and column with 20 to 30 mL of hexane to complete the quantitative
transfer.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball
Snyder column. Prewet the Snyder column by adding about 1 mL methylene chloride
to the top. Place the K-D apparatus on a hot water bath, 80 to 85°C, so that the
concentrator tube is partially immersed in the hot water, and the entire lower rounded
surface of the flask is bathed with hot vapor. Adjust the vertical position of the
apparatus and the water temperature as required to complete the concentration in 15 to
20 minutes. At the proper rate of distillation, the balls of the column will actively chatter
but the chambers will not flood with condensed solvent. When the apparent volume of
liquid reaches 1 mL, remove the K-D apparatus and allow it to drain and cool for at least
10 minutes.
10.7 Remove the Snyder column and rinse the flask and its lower joint into the concentrator
tube with 1 to 2 mL of hexane and adjust the volume to 10 mL. A 5-mL syringe is
recommended for this operation. Stopper the concentrator tube and store refrigerated
if further processing will not be performed immediately. If the extracts will be stored
longer than two days, they should be transferred to PTFE-sealed screw-cap bottles. If the
sample extract requires no further cleanup, proceed with gas chromatographic analysis.
If the sample requires cleanup, proceed to Section 11.
10.8 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the water to a 1000-mL graduated cylinder. Record the sample volume to
the nearest 5 mL.
11. CLEANUP AND SEPARATION
11.1 Cleanup procedures may not be necessary for a relatively clean sample matrix. The
cleanup procedure recommended in this method has been used for the analysis of
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Method 614
various industrial and municipal effluents. If particular circumstances demand the use
of an alternative cleanup procedure, the analyst must determine the elution profile and
demonstrate that the recovery of each compound of interest for the cleanup procedure
is no less than 85%.
11.2 Acetonitrile partition: The following acetonitrile partitioning procedure may be used to
isolate fats and oils from the sample extracts. The applicability of this procedure to
organophosphorus pesticides is indicated in Table 3.
11.2.1 Quantitatively transfer the previously concentrated extract to a 125-mL separatory
funnel with enough hexane to bring the final volume to 15 mL. Extract the
sample 4 times by shaking vigorously for 1 minute with 30-mL portions of
hexane-saturated acetonitrile.
11.2.2 Combine and transfer the acetonitrile phases to a 1-L separatory funnel and add
650 mL of reagent water and 40 mL of saturated sodium chloride solution. Mix
thoroughly for 30 to 45 seconds. Extract with two 100-mL portions of hexane by
vigorously shaking for 15 seconds.
11.2.3 Combine the hexane extracts in a 1 L separatory funnel and wash with two
100 mL portions of reagent water. Discard the water layer and pour the hexane
layer through a drying column containing 7-10 cm of anhydrous sodium sulfate
into a 500 mL K-D flask equipped with a 10-mL concentrator tube. Rinse the
separatory funnel and column with three 10-mL portions of hexane.
11.2.4 Concentrate the extracts to 6 tolO mL in the K-D as directed in Section 10.6.
Adjust the extract volume to 10 mL with hexane.
11.2.5 Analyze by gas chromatography unless a need for further cleanup is indicated.
11.3 Florisil column cleanup: The following Florisil column cleanup procedure has been
demonstrated to be applicable to the seven organophosphorus pesticides listed in Table 3.
It should also be applicable to the cleanup of extracts for ethion.
11.3.1 Add a weight of Florisil (nominally 20 g) predetermined by calibration
(Sections 7.4 and 7.5) to a chromatographic column. Settle the Florisil by tapping
the column. Add anhydrous sodium sulfate to the top of the Florisil to form a
layer 1 to 2 cm deep. Add 60 mL of hexane to wet and rinse the sodium sulfate
and Florisil. Just prior to exposure of the sodium sulfate to air, stop the elution
of the hexane by closing the stopcock on the chromatography column. Discard
the eluate.
11.3.2 Adjust the sample extract volume to 10 mL with hexane and transfer it from the
K-D concentrator tube to the Florisil column. Rinse the tube twice with 1 to 2 mL
hexane, adding each rinse to the column.
11.3.3 Place a 500-mL K-D flask and clean concentrator tube under the chromatography
column. Drain the column into the flask until the sodium sulfate layer is nearly
exposed. Elute the column with 200 mL of 6% (v/v) ethyl ether in hexane
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Method 614
(Fraction 1) using a drip rate of about 5 mL/min. Remove the K-D flask and set
aside for later concentration. Elute the column again, using 200 mL of 15% (v/v)
ethyl ether in hexane (Fraction 2) into a second K-D flask. Perform a third elution
using 200 mL of 50% (v/v) ethyl ether in hexane (Fraction 3) and a final elution
with 200 mL of 100% ethyl ether (Fraction 4) into separate K-D flasks. The
elution patterns for seven of the pesticides are shown in Table 3.
11.3.4 Concentrate the eluates by standard K-D techniques (Section 10.6), using the
water bath at about 85°C (75°C for Fraction 4). Adjust final volume to 10 mL
with hexane. Analyze by gas chromatography.
11.4 Removal of sulfur:9 Elemental sulfur will elute in Fraction 1 of the Florisil cleanup
procedure. If a large amount of sulfur is present in the extract, it may elute in all
fractions. If so, each fraction must be further treated to remove the sulfur.
11.4.1 Add one or two boiling chips to the 10-mL hexane solution contained in a
concentrator tube. Attach a micro-Snyder column and concentrate the extract to
about 0.2 mL in a hot water bath at 85°C. Remove the micro K-D from the bath,
cool, and adjust the volume to 0.5 mL with hexane.
11.4.2 Plug a disposable pipette with a small quantity of glass wool. Add enough
alumina to produce a 3-cm column after settling. Top the alumina with a 0.5-cm
layer of anhydrous sodium sulfate.
11.4.3 Quantitatively transfer the concentrated extract to the alumina microcolumn using
a 100- uL syringe. Rinse the ampule with 200 uL of hexane and add to the
microcolumn.
11.4.4 Elute the microcolumn with 3 mL of hexane and discard the eluate.
11.4.5 Elute the column with 5 mL of 10% hexane in methylene chloride, and collect the
eluate in a 10-mL concentrator tube. Adjust final volume to 10 mL with hexane.
Analyze by gas chromatography.
12. GAS CHROMATOGRAPHY
12.1 Table 1 summarizes the recommended operating conditions for the gas chromatograph.
Included in this table are estimated retention-times and method detection limits that can
be achieved by this method. Other packed columns, chromatographic conditions, or
detectors may be used if the requirements of Section 8.2 are met. Capillary
(open-tubular) columns may also be used if the relative standard deviations of responses
for replicate injections are demonstrated to be less than 6% and the requirements of
Section 8.2 are met.
12.2 Calibrate the system daily as described in Section 7.
12.3 If the internal standard approach is being used, add the internal standard to sample
extracts immediately before injection into the instrument. Mix thoroughly.
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Method 614
12A Inject 1 to 5 |_iL of the sample extract using the solvent-flush technique.10 Record the
volume injected to the nearest 0.05 uL, and the resulting peak size in area or peak height
units. An automated system that consistently injects a constant volume of extract may
also be used.
12.5 The width of the retention-time window used to make identifications should be based
upon measurements of actual retention-time variations of standards over the course of
a day. Three times the standard deviation of a retention time can be used to calculate
a suggested window size for a compound. However, the experience of the analyst
should weigh heavily in the interpretation of chromatograms.
12.6 If the response for the peak exceeds the working range of the system, dilute the extract
and reanalyze.
12.7 If the measurement of the peak response is prevented by the presence of interferences,
further cleanup is required.
13. CALCULATIONS
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used, calculate the amount of
material injected from the peak response using the calibration curve or calibration
factor in Section 7.2.2. The concentration in the sample can be calculated as
follows:
Equation 2
Concentration, \ig/L -
where
A = Amount of material injected, in ng
V, = Volume of extract injected, in uL
Vt = Volume of total extract, in uL
V, = Volume of water extracted, in ml
13.1.2 If the internal standard calibration procedure was used, calculate the
concentration in the sample using the response factor (RF) determined in Section
7.3.2 as follows:
Equation 3
(V) (V)
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Method 614
Concentration, \ig/L - s s
(Ais) (RF)
where
As = Response for parameter to be measured
Als = Response for the internal standard
Is = Amount of internal standard added to each extract, in ug
V0 = Volume of water extracted, in L
13.2 Report results in micrograms per liter without correction for recovery data. When
duplicate and spiked samples are analyzed, report all data obtained with the sample
results.
13.3 For samples processed as part of a set where the laboratory spiked sample recovery falls
outside of the control limits in Section 8.3, data for the affected parameters must be
labeled as suspect.
14. GC/MS CONFIRMATION
14.1 It is recommended that GC/MS techniques be judiciously employed to support
qualitative compound identifications made with this method. The mass spectrometer
should be capable of scanning the mass range from 35 amu to a mass 50 amu above the
molecular weight of the compound. The instrument must be capable of scanning the
mass range at a rate to produce at least 5 scans per peak but not to exceed 7 seconds per
scan utilizing a 70 V (nominal) electron energy in the electron impact ionization mode.
A GC-to-MS interface constructed of all glass or glass-lined materials is recommended.
A computer system should be interfaced to the mass spectrometer that allows the
continuous acquisition and storage on machine-readable media of all mass spectra
obtained throughout the duration of the chromatographic program.
14.2 Gas chromatographic columns and conditions should be selected for optimum separation
and performance. The conditions selected must be compatible with standard GC/MS
operating practices. Chromatographic tailing factors of less than 5.0 must be achieved.11
14.3 At the beginning of each day that confirmatory analyses are to be performed, the GC/MS
system must be checked to see that all decafluorotriphenyl phosphine (DFTPP)
performance criteria are achieved.12
14.4 To confirm an identification of a compound, the background-corrected mass spectrum
of the compound must be obtained from the sample extract and compared with a mass
spectrum from a stock or calibration standard analyzed under the same chromatographic
conditions. It is recommended that at least 25 ng of material be injected into the GC/MS.
The criteria below must be met for qualitative confirmation.
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Method 614
14.4.1 All ions that are present above 10% relative abundance in the mass spectrum of
the standard must be present in the mass spectrum of the sample with agreement
to ±10%. For example, if the relative abundance of an ion is 30% in the mass
spectrum of the standard, the allowable limits for the relative abundance of that
ion in the mass spectrum for the sample would be 20 to 40%.
14.4.2 The retention-time of the compound in the sample must be within 6 seconds of
the same compound in the standard solution.
14.4.3 Compounds that have very similar mass spectra can be explicitly identified by
GC/MS only on the basis of retention-time data.
14.5 Where available, chemical ionization mass spectra may be employed to aid in the
qualitative identification process.
14.6 Should these MS procedures fail to provide satisfactory results, additional steps may be
taken before reanalysis. These may include the use of alternate packed or capillary GC
columns or additional cleanup (Section 11).
15. METHOD PERFORMANCE
15.1 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
zero.13 The MDL concentrations listed in Table 1 were obtained using reagent watet.
15.2 In a single laboratory, Susquehanna University, using spiked tap water samples, the
average recoveries presented in Table 3 were obtained. The standard deviation of the
percent recovery is also included in Table 3.14
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Method 614
References
1. "Methods for Benzidine, Chlorinated Organic Compounds, Pentachlorophenol and
Pesticides in Water and Wastewater," U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory - Cincinnati, Ohio, September 1978.
2. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice for Preparation of
Sample Containers and for Preservation," American Society for Testing and Materials,
Philadelphia, PA, p. 679, 1980.
3. "Carcinogens - Working with Carcinogens," Department of Health, Education, and
Welfare, Public Health Service, Center for Disease Control, National Institute for
Occupational Safety and Health, Publication No. 77-206, August 1977.
4. "OSHA Safety and Health Standards, General Industry," (29 CFR 1910), Occupational
Safety and Health Administration, OSHA 2206, (Revised, January 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
6. ASTM Annual Book of Standards, Part 31, D3086, Appendix X3, "Standardization of
Florisil Column by Weight Adjustment Based on Adsorption of Laurie Acid," American
Society for Testing and Materials, Philadelphia, PA, p 765, 1980.
7. "Handbook for Analytical Quality Control in Water and Wastewater Laboratories,"
EPA-600/4-79-019, U. S. Environmental Protection Agency, Environmental Monitoring
and Support Laboratory - Cincinnati, Ohio, March 1979.
8. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling Water,"
American Society for Testing and Materials, Philadelphia, PA, p. 76, 1980.
9. Law, L. M. and D. F. Goerlitz. "Microcolumn Chromatographic Cleanup for the Analysis
of Pesticides in Water," Journal of the Association of Official Analytical Chemists, 53, 1276,
(1970).
10. Burke, J. A. "Gas Chromatography for Pesticide Residue Analysis; Some Practical
Aspects," Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).
11. McNair, H.M. and Bonelli, E. J. Basic Chromatography, Consolidated Printing, Berkeley,
California, p. 52, 1969.
12. Eichelberger, J.W., Harris, L.E., and Budde, W.L. "Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography-Mass Spectrometry," Analytical
Chemistry, 47, 995 (1975).
13. Glaser, J.A. et.al, "Trace Analysis for Wastewaters," Environmental Science & Technology,
15, 1426 (1981).
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Method 614
14. McGrath, T. F., "Recovery Studies of Pesticides From Surface and Drinking Waters," Final
Report for U.S. EPA Grant R804294, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio.
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Method 614
Table 1. Chromatographic Conditions and Method Detection Limits
Parameter
Diazinon
Disulfoton
Demeton
Parathion-
methyl
Malathion
Parathion-
ethyl
Ethion
Azinphos-
methyl
Retention Time
(min)
Column 1
1.8
1.9
2.3
2.5
2.9
3.1
6.8
14.5
Column 2
1.8
2.1
2.1
3.7
3.9
4.5
9.1
29.9
Method
Detection
Limit
0.012
ND
ND
0.012
ND
0.012
ND
ND
ND = Not determined
Column 1 conditions: Gas-Chrom Q (100/120 mesh) coated with 3% OV-1 packed in a glass
column 1.8 m long by 4 mm ID with nitrogen carrier gas at a flow rate of 60 mL/min. Column
temperature, isothermal at 200°C. A flame photometric detector was used with this column to
determine the MDL.
Column 2 conditions: Gas Chrom Q (100/120 mesh) coated with 1.5% OV-17/1.95% QF-1
packed in a glass column 1.8 m long by 4 mm ID with nitrogen carrier gas at a flow rate of
70 mL/min. Column temperature, isothermal at 212°C.
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Method 614
Table 2.
Single-Operator Accuracy and Precision
Parameter
Diazinon
Parathion
methyl
Parathion ethyl 102
Average
Percent
Recovery
94
95
Standard
Deviation
(%)
5.2
3.2
Spike
Range
(Vg/L)
0.04-40
0.06-60
Number of
Analyses
27
27
Matrix
Types
4
4
4.1
0.07-70
27
Table 3.
Florisil Fractionation Patterns and Acetonitrile Partition Applicability
Percent Recovery by Fraction
Parameter
Demeton
Disulfoton
Diazinon
Malathion
Parathion ethyl
Parathion methyl
Azinphos methyl
Ethion
ND = Not determined
No. 1
No. 2
No. 3
No. 4
100
100
ND
100
5
100
100
ND
95
20
ND
Florisil eluate composition by fraction:
Fraction 1 = 200 mL of 6% ethyl ether in hexane
Fraction 2 = 200 mL of 15% ethyl ether in hexane
Fraction 3 = 200 mL of 50% ethyl ether in hexane
Fraction 4 = 200 mL of ethyl ether
80
ND
Acetonitrile
Partition
Applicability
ND
ND
Yes
Yes
Yes
Yes
ND
Yes
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