United States
Environmental Protection
\r ^1 M^k. Agency
www.epa.gov August 1993
Method 633.1: The Determination
of Neutral Nitrogen-Containing
Pesticides in Municipal and
Industrial Wastewaters
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Method 633.1
The Determination of Neutral
Nitrogen-Containing Pesticides
in Municipal and Industrial
Wastewaters
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Method 633.1
The Determination of Neutral Nitrogen-Containing Pesticides in
Municipal and Industrial Wastewaters
1. Scope and Application
1.1 This method covers the determination of certain neutral nitrogen containing pesticides.
The following parameters can be determined by this method:
Parameter CAS No.
Fenarimol 60168-88-9
MGK 264-A 113-48-4
MGK 264-B 113-48-4
MGK 326 136-45-8
Pronamide 23950-58-5
1.2 This is a gas chromatographic (GC) method applicable to the determination of the
compounds listed above in municipal and industrial 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 each compound is listed in
Table 2. 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 similar to those in other
600-series methods. Thus, a single sample may be extracted to measure the compounds
included in the scope of the 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.
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
primary column. Section 14 provides gas chromatograph/mass spectrometer (GC/MS)
criteria appropriate for the qualitative confirmation of compound identifications.
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Method 633.1
2. Summary of Method
2.1 A measured volume of sample, approximately 1 L, is extracted with methylene chloride
using a separatory funnel. The methylene chloride extract is dried and concentrated to
1.0 mL. Gas chromatographic conditions are described which permit the separation and
measurement of the compounds in the extract by alkali flame detector gas
chromatography (GC/AFD).1
2.2 This method provides an optional Florisil column cleanup procedure 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 rinsing with the last solvent used in it. Follow by rinsing 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. Some 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 2.
4. Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method has not been precisely
defined; however, each chemical compound should be treated as a potential health
hazard. From this viewpoint, exposure to these chemicals must be reduced to the lowest
possible level by whatever means available. The laboratory is responsible for maintaining
a current awareness file of OSHA regulations regarding the safe handling of the
chemicals specified in this method. A reference file of materials data handling sheets
should also be made available to all personnel involved in the chemical analysis.
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Method 633.1
Additional references to laboratory safety are available and have been identified3 5 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 PTFE. Foil may be substituted for PTFE 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 should be thoroughly
rinsed with methanol, followed by repeated rinsings with distilled 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: 2000-mL, with PTFE stopcock.
5.2.2 Drying column: Chromatographic column 400 mm long by 10 mm ID with coarse
frit.
5.2.3 Chromatographic column: 400 mm long by 19 mm ID with 250-mL reservoir at
the top and PTFE stopcock (Kontes K-420290 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.
A ground-glass stopper is used to prevent evaporation of extracts.
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 Vials: Amber glass, 10- to 15-mL capacity with PTFE-lined screw-cap.
5.2.9 Erlenmeyer flask: 250-mL.
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Method 633.1
5.2.10 Graduated cylinder: 1000-mL.
5.2.11 Beaker: 250-mL.
5.3 Boiling chips: Approximately 10/40 mesh carborundum. Heat to 400°C for 4 hours or
perform a Soxhlet extraction with methylene chloride.
5.4 Water bath: Heated, capable of temperature control (±20°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 2 mm ID glass, packed with 3% SP-2250 on
Supelcoport (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 2 mm ID glass, packed with 3% SP-2100 on
Supelcoport (100/120 mesh) or equivalent.
5.6.3 Detector: Alkali flame detector (AFD), sometimes referred to as a nitrogen-
phosphorous detector (NPD) or a thermionic-specific detector (TSD). This
detector has proven effective in the analysis of wastewaters for the compounds
listed in the scope and was used to develop the method performance statements
in Section 15.
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 Methylene chloride, methanol, petroleum ether, ethyl ether, acetone: Distilled-in-glass
quality or equivalent. Ethyl ether must be free of peroxides as indicated by EM Quant
test strips (available from Scientific Products Co., Catalog No. PI 126-8 and other
suppliers). Procedures recommended for removal of peroxides are provided with the test
strips.
6.3 6N sodium hydroxide: Dissolve 24.0 g NaOH in 100 mL of reagent water.
6.4 6N sulfuric acid: Slowly add 16.7 mL of concentrated H2S04 (94%) to about 50 mL of
reagent water. Dilute to 100 mL with reagent water.
6.5 Sodium sulfate: (ACS), granular, anhydrous; heated in a muffle furnace at 400°C
overnight.
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Method 633.1
6.6 Florisil: PR grade (60/100 mesh). Purchase activated at 675°C and store in brown glass
bottle. To prepare for use, place 150 g in a wide-mouth jar and heat overnight at 160 to
170°C. Seal tightly with PTFE or aluminum-foil-lined screw-cap and cool to room
temperature.
6.7 Stock standard solutions (1.00 |ig/|_iL): Stock standard solutions can be prepared from
pure standard materials or purchased as certified solutions.
6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure
material. Dissolve the material in distilled-in-glass quality methanol and dilute
to volume in a 10-mL volumetric flask. Larger volumes can be used at the
convenience of the analyst. If compound purity is certified at 96% or greater, the
weight can be used without correction to calculate the concentration of the stock
standard. Commercially prepared stock standards can be used at any
concentration if they are certified by the manufacturer or by an independent
source.
6.7.2 Transfer the stock standard solutions into PTFE-sealed screw-cap bottles. 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.
6.7.3 Stock standard solutions must be replaced after 6 months, or sooner if comparison
with check standards indicates a problem.
7. Calibration
7.1 Establish gas chromatographic operating parameters equivalent to those indicated in
Table 2. The gas chromatographic system can be calibrated using 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 compound of interest, prepare calibration standards at a minimum of
three concentration levels by adding volumes of one or more stock standards to
a volumetric flask and diluting to volume with acetone. One of the external
standards should be at 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 |iL 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 compound. Alternatively, the ratio of the response to
the mass injected, defined as the calibration factor (CF), can be calculated for each
compound 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.
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Method 633.1
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 compound 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 compound.
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
compound 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 acetone. One of the
standards should be at 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 pL 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:
Equation 1
RF =
C4) (Q
VJ (CJ
where
As = Response for the parameter to be measured
Ats = Response for the internal standard
Cts = Concentration of the internal standard, in jig/L
Cs = Concentration of the parameter to be measured, in jig/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
can be used for calculations. Alternatively, the results can be used to plot a
calibration curve of response ratios, As/Ais 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
compound varies from the predicted response by more than ±10%, the test must
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Method 633.1
be repeated using a fresh calibration standard. Alternatively, a new calibration
curve must be prepared for that compound.
7.4 Before using any cleanup procedure, the analyst must process a series of calibration
standards through the procedure to validate elution patterns and the absence of
interferences from 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.
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
methanol, 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 s calculations are performed.
8.2.4 Using the appropriate data from Table 3, determine the recovery and single-
operator precision expected for the method, and compare these results to the
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Method 633.1
values measured in Section 8.2.3. If the data are not comparable, the analyst must
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 charts6 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
data points gathered through the requirement for continuing quality control in
Section 8.4. The accuracy statements should be updated regularly.6
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 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
compound does not fall within the control limits for method performance, the results
reported for that compound 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 should 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 should 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 standard reference materials and participate in relevant performance evaluation
studies.
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Method 633.1
9. Sample Collection, Preservation, and Handling
9.1 Grab samples must be collected in glass containers. Conventional sampling practices7
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 Adjust the pH of the sample to 6 to 8 with 6N sodium hydroxide or 6N sulfuric acid
immediately after sampling.
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. Check the pH of
the sample with wide range pH paper and adjust to 6 to 8 with 6N sodium hydroxide
or 6N sulfuric acid.
10.2 Add 60 mL of methylene chloride 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 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. Collect the methylene chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the
extraction procedure a second time, combining the extracts in the Erlenmeyer flask.
Perform a third extraction in the same manner.
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 methylene chloride to complete the
quantitative transfer. Once the flask rinse has passed through the drying column, rinse
the column with 30 to 40 mL of methylene chloride.
10.6 Add one or two clean boiling chips to the evaporative flask and attach a three-ball
Snyder column. Prewet the macro Snyder column by adding about 1 mL methylene
chloride to the top. Place the K-D apparatus on a hot water bath, 60 to 65°C, so that the
concentrator tube is partially immersed in the hot water, and the entire lower rounded
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Method 633.1
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 macro-Snyder column and rinse the flask and its lower joint into the
concentrator tube with 1 to 2 mL of methylene chloride. Add one or two clean boiling
chips and attach a two-ball micro-Snyder column to the concentrator tube. Prewet the
micro-Snyder column with methylene chloride and concentrate the solvent extract as
before. When an apparent volume of 0.5 mL is reached, or the solution stops boiling,
remove the K-D apparatus and allow it to drain and cool for 10 minutes.
10.8 Remove the micro-Snyder column and adjust the volume of the extract to 1.0 mL with
methylene chloride. Stopper the concentrator tube and store refrigerated if further
processing will not be performed immediately. If the extract is to be stored longer than
2 days, transfer the extract to a screw-capped vial with a PTFE-lined cap. If the sample
extract requires no further cleanup, proceed with solvent exchange to acetone as
described in Section 10.9. If the sample requires cleanup, proceed to Section 11.
10.9 Add one or two clean boiling chips to the concentrator tube along with 10 mL of acetone.
Attach the two-ball macro Snyder column and prewet the column with about 1 mL of
acetone. Adjust the temperature of the water bath to 85 to 95°C. Concentrate the solvent
extract as before to an apparent volume of 0.5 mL and allow it to drain and cool for 10
minutes. Add a second 10 mL of acetone to the concentrator tube and repeat the
concentration procedure a second time. Adjust the final volume of the extract to 1.0 mL
with acetone.
10.10 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
various clean waters and industrial 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 is no less than 85%.
11.2 The following Florisil cleanup procedure has been demonstrated to be applicable to the
four neutral nitrogen pesticides listed in Table 1.
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Method 633.1
11.2.1 Slurry 20 g of Florisil in 100 mL of ethyl ether and 400 |iL of reagent water.
Transfer the slurry to a chromatographic column (Florisil may be retained with
a plug of glass wool). Allow the solvent to elute from the column until the
Florisil is almost exposed to the air. Wash the column with 25 mL of petroleum
ether. Use a column flow rate of 2 to 2.5 mL/min throughout the wash and
elution profiles. Add an additional 50 mL of petroleum ether to the head of the
column.
11.2.2 Quantitatively transfer the sample from Section 10.8 to the petroleum ether
suspended over the column. Allow the solvent to elute from the column until the
Florisil is almost exposed to the air. Elute the column with 50 mL of 50% ethyl
ether in petroleum ether. Discard this fraction.
11.2.3 Elute the column with 50 mL of 100% ethyl ether (Fraction 1) and collect in a K-D
apparatus. Repeat procedure with 50 mL 6% acetone in ethyl ether (Fraction 2),
50 mL 15% acetone in ethyl ether (Fraction 3), 50 mL 50% acetone in ethyl ether
(Fraction 4), and 100 mL 100% acetone (Fraction 5), collecting each in a separate
K-D apparatus. The elution patterns for the neutral nitrogen compounds are
shown in Table 1. Concentrate each fraction to 1 mL as described in Sections 10.6
and 10.7. The fractions may be combined before concentration at the discretion
of the analyst. Solvent exchange Fraction 1 to acetone as described in Section 10.9
if the fractions are not combined.
11.2.4 Proceed with gas chromatographic analysis.
12. Gas Chromatography
12.1 Table 2 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. Examples of the separations achieved by Column 1 and
Column 2 are shown in Figures 1 and 2. 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 gas chromatographic system daily as described in Section 7.
12.3 If the internal standard approach is being used, the analyst must not add the internal
standard to the sample extracts until immediately before injection into the instrument.
Mix thoroughly.
12.4 Inject 1 to 5 |iL of the sample extract using the solvent flush technique.8 Record the
volume injected to the nearest 0.05 pL, and the resulting peak sizes in area or peak height
units. An automated system that consistently injects a constant volume of extract may
also be used.
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Method 633.1
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 for a compound can be
used to calculate a suggested window size; however, the experience of the analyst should
weigh heavily in the interpretation of chromatograms.
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
(A) (V)
Concentration, ug/L =
W (Fs)
where
A = Amount of material injected, in ng
Vi = Volume of extract injected, in pL
Vt = Volume of total extract, in {iL
Vs = 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:
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Method 633.1
Equation 3
(V (O
Concentration, ue/L =
(Ais) (RF) (Vo)
where
As = Response for parameter to be measured
Ats = Response for the internal standard
Is = Amount of internal standard added to each extract, in jig
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 compounds must be
labeled as suspect.
14. GC/MS Confirmation
14.1 It is recommended that GC/MS techniques be judiciously employed to support
qualitative 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.
When using a fused-silica capillary column, the column outlet should be threaded
through the interface to within a few mm of the entrance to the source ionization
chamber. 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.10
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 DFTPP performance criteria are achieved.9
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
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Method 633.1
conditions. It is recommended that at least 50 ng of material be injected into the GC/MS.
The criteria below must be met for qualitative confirmation.
14.4.1 The molecular ion and all other 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 alternative 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.11 The MDL concentrations listed in Table 2 were obtained using reagent water.
Similar results were achieved using representative wastewaters.
15.2 This method has been tested for linearity of recovery from spiked reagent water and has
been demonstrated to be applicable over the concentration range from 10 x MDL to 1000
x MDL.
15.3 In a single laboratory, Battelle's Columbus Laboratories, using spiked wastewater
samples, the average recoveries presented in Table 3 were obtained after Florisil cleanup.
Seven replicates of each of two different wastewaters were spiked and analyzed. The
standard deviation of the percent recovery is also included in Table 3.1
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Method 633.1
References
1. "Development of Methods for Pesticides in Wastewaters," EPA Contract Report
68-03-2956 (in preparation).
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, Pennsylvania, 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. "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.
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for Sampling
Water," American Society for Testing and Materials, Philadelphia, Pennsylvania, p. 76,
1980.
8. Burke, J.A. "Gas Chromatography for Pesticide Residue Analysis; Some Practical
Aspects," Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).
9. 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).
10. McNair, H.M. and Bonelli, E.J. Basic Chromatography, Consolidated Printing, Berkeley,
California, 52 (1969).
11. Glaser, J.A., et al. "Trace Analysis for Wastewaters," Environmental Science and Technology,
15, 1426 (1981).
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Method 633.1
Table 1. Elution Characteristics of the Neutral Nitrogen Compounds on 2% Deactivated
Florisil
Elution in Specified Fractiona
Parameter 1 1
Fl | F2 | F3
Fenarimol X X
MGK 264 X X
MGK326
Pronamide X
aElution solvents are 50 mL each of the following:
Fl = 100% ethyl ether
F2 = 6% acetone in ethyl ether
F3 = 15% acetone in ethyl ether
F4 = 50% acetone in ethyl ether
F5 = 100% acetone (100 mL)
F4
X
F5
X
Table 2. Chromatographic Conditions and Method Detection Limits
Retention Time (min) MDL
Parameter Column 1 | Column 2 (/ig/L)
Pronamide 19.9 22.0 4
MGK 326 21.9 23.8 6
MGK 264 23.0 and 25.5 and 2
23.5a 27.5a
Fenarimol 30.6 32.2 4
Column 1 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250 packed in a glass
column 1.8 m long by 2 mm ID with helium carrier gas at a flow rate of 30 mL/min. Column
temperature is programmed from 80 to 300°C at 8°C/min with a 4 minute hold at each extreme,
injector temperature is 250°C, and detector is 300°C. Alkali flame detector at bead voltage of 16
V.
Column 2 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2100 packed in a glass
column 1.8 m long by 2 mm ID with helium carrier gas at a flow rate of 30 mL/min. All other
conditions as for Column 1.
(a) Two isomers of MGK 264 are resolved from each other.
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Method 633.1
Table 3. Single-Laboratory Accuracy and Precision a
Spike
Mean
Standard
Number
Sample
Background
Level
Recovery
Deviation
of
Parameter
Typeb
(WW
(Vg/L)
(%)
(%)
Replicates
Fenarimol
1
1.8
20
98
4
7
2
ND
500
96
4
7
MGK 264
1
ND
20
96
23
7
2
ND
500
74
4
7
MGK 326
1
ND
20
108
7
7
2
ND
500
95
4
7
Pronamide
1
ND
20
102
5
7
2
210
500
86
3
7
(a) Column 1 conditions were used.
(b) 1 = Low-level relevant industrial effluent
2 = High-level relevant industrial effluent
(c) ND = Not detected
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Pronamide
MGK 326
MGK264
Fenarimol
—f T—~1 I I I I I I
19.0 21.0 23.0 25.0 27.0
17.0
29.0
31.0
33.0
35.0
Retention Time (minutes)
A52-002-32A
Figure 1. GC-AFD Chromatogram of 100 ng Each of the Neutral Nitrogen
Compounds (Column 1)
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Method 633.1
Pronamide
Fenarimol
MGK264
MGK 326
—i 1 1 1 1 r— i i i
21.0 24.0 27.0 30.0 33.0
Retention Time (minutes)
Figure 2. GC-AFD Chromatogram of 200 ng Each of the Neutral Nitrogen
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