EPA 609.1

METHODS FOR ORGANIC CHEMICAL ANALYSIS OF MUNICIPAL AND

INDUSTRIAL WASTEWATER

METHOD 609—NITROAROMATICS AND ISOPHORONE

1.1.1

Scope and Application

This method covers the determination of certain nitroaromatics and isophorone. Thefollowing parameters may be determined by this method:

Parameter

2,4-Dinitrotoluene2,6-DinitrotolueneIsophorone.....Nitrobenzene....

1.2

................................................................................................................

STORET No.

34611346263440834447

CAS No.

121-14-2606-20-278-59-198-95-3

This is a gas chromatographic (GC) method applicable to the determination of thecompounds listed above in municipal and industrial discharges as provided under40 CFR Part 136.1. When this method is used to analyze unfamiliar samples for anyor all of the compounds above, compound identifications should be supported by atleast one additional qualitative technique. This method describes analytical conditionsfor a second gas chromatographic column that can be used to confirm measurementsmade with the primary column. Method 625 provides gas chromatograph/massspectrometer (GC/MS) conditions appropriate for the qualitative and quantitativeconfirmation of results for all of the parameters listed above, using the extractproduced by this method.

The method detection limit (MDL, defined in Section 14.1)1 for each parameter islisted in Table 1. The MDL for a specific wastewater may differ from those listed,depending upon the nature of interferences in the sample matrix.

The sample extraction and concentration steps in this method are essentially the sameas in Methods 606, 608, 611, and 612. Thus, a single sample may be extracted tomeasure the parameters included in the scope of each of these methods. When

cleanup is required, the concentration levels must be high enough to permit selectingaliquots, as necessary, to apply appropriate cleanup procedures. The analyst isallowed the latitude, under Section 12, to select chromatographic conditions

appropriate for the simultaneous measurement of combinations of these parameters.Any modification of this method, beyond those expressly permitted, shall be

considered as a major modification subject to application and approval of alternatetest procedures under 40 CFR Parts 136.4 and 136.5.

1.3

1.4

1.5

1.6

This method is restricted to use by or under the supervision of analysts experiencedin the use of a gas chromatograph and in the interpretation of gas chromatograms.Each analyst must demonstrate the ability to generate acceptable results with thismethod using the procedure described in Section 8.2.Summary of Method

A measured volume of sample, approximately 1 L, is extracted with methylenechloride using a separatory funnel. The methylene chloride extract is dried and

exchanged to hexane during concentration to a volume of 10 mL or less. Isophoroneand nitrobenzene are measured by flame ionization detector gas chromatography(FIDGC). The dinitrotoluenes are measured by electron capture detector gaschromatography (ECDGC).2

The method provides a Florisil column cleanup procedure to aid in the elimination ofinterferences that may be encountered.Interferences

Method interferences may be caused by contaminants in solvents, reagents, glassware,and other sample processing hardware that lead to discrete artifacts and/or elevatedbaseliles in gas chromatograms. All of these materials must be routinely

demonstrated to be free from interferences under the conditions of the analysis byrunning laboratory reagent blanks as described in Section 8.1.3.3.1.1

Glassware must be scrupulously cleaned.3 Clean all glassware as soon aspossible after use by rinsing with the last solvent used in it. Solvent rinsingshould be followed by detergent washing with hot water, and rinses with tapwater and distilled water. The glassware should then be drained dry, andheated in a muffle furnace at 400°C for 15-30 minutes. Some thermally stablematerials, such as PCBs, may not be eliminated by this treatment. Solventrinses with acetone and pesticide quality hexane may be substituted for themuffle furnace heating. Thorough rinsing with such solvents usually

eliminates PCB interference. Volumetric ware should not be heated in a mufflefurnace. After drying and cooling, glassware should be sealed and stored in aclean environment to prevent any accumulation of dust or other contaminants.Store inverted or capped with aluminum foil.

The use of high purity reagents and solvents helps to minimize interferenceproblems. Purification of solvents by distillation in all-glass systems may berequired.

2.2.1

2.23.3.1

3.1.2

3.2

Matrix interferences may be caused by contaminants that are co-extracted from thesample. The extent of matrix interferences will vary considerably from source tosource, depending upon the nature and diversity of the industrial complex ormunicipality being sampled. The cleanup procedure in Section 11 can be used toovercome many of these interferences, but unique samples may require additionalcleanup approaches to achieve the MDL listed in Table 1.

4.4.1

Safety

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 potentialhealth hazard. From this viewpoint, exposure to these chemicals must be reduced tothe lowest possible level by whatever means available. The laboratory is responsiblefor maintaining a current awareness file of OSHA regulations regarding the safehandling of the chemicals specified in this method. A reference file of material datahandling sheets should also be made available to all personnel involved in the

chemical analysis. Additional references to laboratory safety are available and havebeen identified4-6 for the information of the analyst.Apparatus and Materials

Sampling equipment, for discrete or composite sampling.5.1.1

Grab sample bottle—1 L or 1 qt, amber glass, fitted with a screw cap linedwith Teflon. Foil may be substituted for Teflon if the sample is not corrosive.If amber bottles are not available, protect samples from light. The bottle andcap liner must be washed, rinsed with acetone or methylene chloride, anddried before use to minimize contamination.

Automatic sampler (optional)—The sampler must incorporate glass samplecontainers for the collection of a minimum of 250 mL of sample. Samplecontainers must be kept refrigerated at 4°C and protected from light duringcompositing. If the sampler uses a peristaltic pump, a minimum length ofcompressible silicone rubber tubing may be used. Before use, however, thecompressible tubing should be thoroughly rinsed with methanol, followed byrepeated rinsings with distilled water to minimize the potential for

contamination of the sample. An integrating flow meter is required to collectflow proportional composites.

5.5.1

5.1.2

5.2

Glassware (All specifications are suggested. Catalog numbers are included forillustration only.)5.2.15.2.25.2.35.2.4

Separatory funnel—2 L, with Teflon stopcock.

Drying column—Chromatographic column, approximately 400 mm long x19 mm ID, with coarse frit filter disc.

Chromatographic column—100 mm long x 10 mm ID, with Teflon stopcock.Concentrator tube, Kuderna-Danish—10 mL, graduated (Kontes K-570050-1025or equivalent). Calibration must be checked at the volumes employed in thetest. Ground glass stopper is used to prevent evaporation of extracts.Evaporative flask, Kuderna-Danish—500 mL (Kontes K-570001-0500 orequivalent). Attach to concentrator tube with springs.

5.2.5

5.2.65.2.75.2.8

5.35.45.55.6

Snyder column, Kuderna-Danish—Three-ball macro (Kontes K-503000-0121 orequivalent).

Snyder column, Kuderna-Danish—Two-ball micro (Kontes K-569001-0219 orequivalent).

Vials—10-15 mL, amber glass, with Teflon-lined screw cap.

Boiling chips—Approximately 10/40 mesh. Heat to 400°C for 30 minutes or Soxhletextract with methylene chloride.

Water bath—Heated, with concentric ring cover, capable of temperature control(±2°C). The bath should be used in a hood.

Balance—Analytical, capable of accurately weighing 0.0001 g.

Gas chromatograph—An analytical system complete with gas chromatograph suitablefor on-column injection and all required accessories including syringes, analytical

columns, gases, detector, and strip-chart recorder. A data system is recommended formeasuring peak areas.5.6.1

Column 1—1.2 m long x 2 or 4 mm ID glass, packed with 1.95% QF-1/1.5%OV-17 on Gas-Chrom Q (80/100 mesh) or equivalent. This column was usedto develop the method performance statements given in Section 14. Guidelinesfor the use of alternate column packings are provided in Section 12.1.Column 2—3.0 m long x 2 or 4 mm ID glass, packed with 3% OV-101 onGas-Chrom Q (80/100 mesh) or equivalent.

Detectors—Flame ionization and electron capture detectors. The flameionization detector (FID) is used when determining isophorone and

nitrobenzene. The electron capture detector (ECD) is used when determiningthe dinitrotoluenes. Both detectors have proven effective in the analysis ofwastewaters and were used in develop the method performance statements inSection 14. Guidelines for the use to alternate detectors are provided inSection 12.1.

5.6.25.6.3

6.6.16.26.36.4

Reagents

Reagent water—Reagent water is defined as a water in which an interferent is notobserved at the MDL of the parameters of interest.

Sodium hydroxide solution (10 N)—Dissolve 40 g of NaOH (ACS) in reagent waterand dilute to 100 mL.

Sulfuric acid (1+1)—Slowly, add 50 mL of H2SO4 (ACS, sp. gr. 1.84) to 50 mL ofreagent water.

Acetone, hexane, methanol, methylene chloride—Pesticide quality or equivalent.

6.56.6

Sodium sulfate—(ACS) Granular, anhydrous. Purify by heating at 400°C for fourhours in a shallow tray.

Florisil—PR grade (60/100 mesh). Purchase activated at 1250°F and store in dark inglass containers with ground glass stoppers or foil-lined screw caps. Before use,activate each batch at least 16 hours at 200°C in a foil-covered glass container andallow to cool.

Stock standard solutions (1.00 µg/µL)—Stock standard solutions can be prepared frompure standard materials or purchased as certified solutions.6.7.1

Prepare stock standard solutions by accurately weighing about 0.0100 g ofpure material. Dissolve the material in hexane and dilute to volume in a

10 mL volumetric flask. Larger volumes can be used at the convenience of theanalyst. When compound purity is assayed to be 96% or greater, the weightcan be used without correction to calculate the concentration of the stockstandard. Commercially prepared stock standards can be used at any

concentration if they are certified by the manufacturer or by an independentsource.

Transfer the stock standard solutions into Teflon-sealed screw-cap bottles.Store at 4°C and protect from light. Stock standard solutions should bechecked frequently for signs of degradation or evaporation, especially justprior to preparing calibration standards from them.

Stock standard solutions must be replaced after six months, or sooner ifcomparison with check standards indicates a problem.

6.7

6.7.2

6.7.3

6.87.7.1

Quality control check sample concentrate—See Section 8.2.1.Calibration

Establish gas chromatographic operating conditions equivalent to those given inTable 1. The gas chromatographic system can be calibrated using the externalstandard technique (Section 7.2) or the internal standard technique (Section 7.3).External standard calibration procedure7.2.1

Prepare calibration standards at a minimum of three concentration levels foreach parameter of interest by adding volumes of one or more stock standardsto a volumetric flask and diluting to volume with hexane. One of the externalstandards should be at a concentration near, but above, the MDL (Table 1) andthe other concentrations should correspond to the expected range of

concentrations found in real samples or should define the working range of thedetector.

Using injections of 2-5 µL, analyze each calibration standard according toSection 12 and tabulate peak height or area responses against the massinjected. The results can be used to prepare a calibration curve for eachcompound. Alternatively, if the ratio of response to amount injected

7.2

7.2.2

(calibration factor) is a constant over the working range (<10% relative

standard deviation, RSD) linearity through the origin can be assumed and theaverage ratio or calibration factor can be used in place of a calibration curve.

7.3

Internal standard calibration procedure—To use this approach, the analyst must selectone or more internal standards that are similar in analytical behavior to the

compounds of interest. The analyst must further demonstrate that the measurementof the internal standard is not affected by method or matrix interferences. Because ofthese limitations, no internal standard can be suggested that is applicable to allsamples.7.3.1

Prepare calibration standards at a minimum of three concentration levels foreach parameter of interest by adding volumes of one or more stock standardsto a volumetric flask. To each calibration standard, add a known constantamount of one or more internal standards, and dilute to volume with hexane.One of the standards should be at a concentration near, but above, the MDLand the other concentrations should correspond to the expected range of

concentrations found in real samples or should define the working range of thedetector.

Using injections of 2-5 µL, analyze each calibration standard according to

Section 12 and tabulate peak height or area responses against concentration foreach compound and internal standard. Calculate response factors (RF) foreach compound using Equation 1.

Equation 1

7.3.2

where:

As = Response for the parameter to be measured.Ais = Response for the internal standard.

Cis = Concentration of the internal standard (µg/L).

Cs = Concentration of the parameter to be measured (µg/L).

If the RF value over the working range is a constant (<10% RSD), the RF canbe 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, vs. concentration ratios Cs/Cis.

7.4

The working calibration curve, calibration factor, or RF must be verified on each

working day by the measurement of one or more calibration standards. If the

response for any parameter varies from the predicted response by more than ±15%, anew calibration curve must be prepared for that compound.

This equation corrects an error made in the original method publication (49 FR 43234,

October 26, 1984). This correction will be formalized through a rulemaking in FY97.

7.5

Before using any cleanup procedure, the analyst must process a series of calibrationstandards through the procedure to validate elution patterns and the absence ofinterferences from the reagents.Quality Control

Each laboratory that uses this method is required to operate a formal quality controlprogram. The minimum requirements of this program consist of an initial

demonstration of laboratory capability and an ongoing analysis of spiked samples toevaluate and document data quality. The laboratory must maintain records todocument the quality of data that is generated. Ongoing data quality checks are

compared with established performance criteria to determine if the results of analysesmeet the performance characteristics of the method. When results of sample spikesindicate atypical method performance, a quality control check standard must be

analyzed to confirm that the measurements were performed in an in-control mode ofoperation.8.1.1

The analyst must make an initial, one-time, demonstration of the ability togenerate acceptable accuracy and precision with this method. This ability isestablished as described in Section 8.2.

In recognition of advances that are occurring in chromatography, the analyst ispermitted certain options (detailed in Sections 10.4, 11.1, and 12.1) to improvethe separations or lower the cost of measurements. Each time such amodification is made to the method, the analyst is required to repeat theprocedure in Section 8.2.

Before processing any samples, the analyst must analyze a reagent water blankto demonstrate that interferences from the analytical system and glassware areunder control. Each time a set of samples is extracted or reagents are changed,a reagent water blank must be processed as a safeguard against laboratorycontamination.

The laboratory must, on an ongoing basis, spike and analyze a minimum of10% of all samples to monitor and evaluate laboratory data quality. Thisprocedure is described in Section 8.3.

The laboratory must, on an ongoing basis, demonstrate through the analyses ofquality control check standards that the operation of the measurement systemis in control. This procedure is described in Section 8.4. The frequency of thecheck standard analyses is equivalent to 10% of all samples analyzed but maybe reduced if spike recoveries from samples (Section 8.3) meet all specifiedquality control criteria.

The laboratory must maintain performance records to document the quality ofdata that is generated. This procedure is described in Section 8.5.

8.8.1

8.1.2

8.1.3

8.1.4

8.1.5

8.1.6

8.2

To establish the ability to generate acceptable accuracy and precision, the analyst mustperform the following operations.8.2.1

A quality control (QC) check sample concentrate is required containing eachparameter of interest in acetone at a concentration of 20 µg/mL for eachdinitrotoluene and 100 µg/mL for isophorone and nitrobenzene. The QCcheck sample concentrate must be obtained from the U.S. EnvironmentalProtection Agency, Environmental Monitoring and Support Laboratory inCincinnati, Ohio, if available. If not available from that source, the QC checksample concentrate must be obtained from another external source. If notavailable from either source above, the QC check sample concentrate must beprepared by the laboratory using stock standards prepared independently fromthose used for calibration.

Using a pipet, prepare QC check samples at the test concentrations shown inTable 2 by adding 1.00 mL of QC check sample concentrate to each of four 1 Laliquots of reagent water.

Analyze the well-mixed QC check samples according to the method beginningin Section 10.

Calculate the average recovery () in µg/L, and the standard deviation of therecovery (s) in µg/L, for each parameter using the four results.

For each parameter compare s and with the corresponding acceptancecriteria for precision and accuracy, respectively, found in Table 2. If s and for all parameters of interest meet the acceptance criteria, the system

performance is acceptable and analysis of actual samples can begin. If anyindividual s exceeds the precision limit or any individual falls outside therange for accuracy, the system performance is unacceptable for that parameter.Locate and correct the source of the problem and repeat the test for allparameters of interest beginning with Section 8.2.2.

8.2.2

8.2.38.2.48.2.5

8.3

The laboratory must, on an ongoing basis, spike at least 10% of the samples from eachsample site being monitored to assess accuracy. For laboratories analyzing one to 10samples per month, at least one spiked sample per month is required.8.3.1

The concentration of the spike in the sample should be determined as follows:8.3.1.1If, as in compliance monitoring, the concentration of a specific

parameter in the sample is being checked against a regulatory

concentration limit, the spike should be at that limit or one to five timeshigher than the background concentration determined in Section 8.3.2,whichever concentration would be larger.8.3.1.2If the concentration of a specific parameter in the sample is not being

checked against a limit specific to that parameter, the spike should beat the test concentration in Section 8.2.2 or one to five times higher thanthe background concentration determined in Section 8.3.2, whicheverconcentration would be larger.

8.3.1.3If it is impractical to determine background levels before spiking (e.g.,

maximum holding times will be exceeded), the spike concentrationshould be (1) the regulatory concentration limit, if any; or, if none

(2) the larger of either five times higher than the expected backgroundconcentration or the test concentration in Section 8.2.2.

8.3.2

Analyze one sample aliquot to determine the background concentration (B) ofeach parameter. If necessary, prepare a new QC check sample concentrate(Section 8.2.1) appropriate for the background concentrations in the sample.Spike a second sample aliquot with 1.0 mL of the QC check sample concentrateand analyze it to determine the concentration after spiking (A) of each

parameter. Calculate each percent recovery (P) as 100 (A-B)%/T, where T isthe known true value of the spike.

Compare the percent recovery (P) for each parameter with the correspondingQC acceptance criteria found in Table 2. These acceptance criteria werecalculated to include an allowance for error in measurement of both the

background and spike concentrations, assuming a spike to background ratio of5:1. This error will be accounted for to the extent that the analyst's spike tobackground ratio approaches 5:1.7 If spiking was performed at a concentrationlower than the test concentration in Section 8.2.2, the analyst must use eitherthe QC acceptance criteria in Table 2, or optional QC acceptance criteria

calculated for the specific spike concentration. To calculate optional acceptancecriteria for the recovery of a parameter: (1) Calculate accuracy (X') using theequation in Table 3, substituting the spike concentration (T) for C; (2) calculateoverall precision (S') using the equation in Table 3, substituting X' for ;(3) calculate the range for recovery at the spike concentration as (100 X'/T)±2.44(100 S'/T)%.7

If any individual P falls outside the designated range for recovery, that

parameter has failed the acceptance criteria. A check standard containing eachparameter that failed the criteria must be analyzed as described in Section 8.4.

8.3.3

8.3.4

8.4.

If any parameter fails the acceptance criteria for recovery in Section 8.3, a QC checkstandard containing each parameter that failed must be prepared and analyzed.NOTE:

The frequency for the required analysis of a QC check standard willdepend upon the number of parameters being simultaneously tested,the complexity of the sample matrix, and the performance of thelaboratory.

Prepare the QC check standard by adding 1.0 mL of QC check sampleconcentrate (Section 8.2.1 or 8.3.2) to 1 L of reagent water. The QC check

standard needs only to contain the parameters that failed criteria in the test inSection 8.3.

Analyze the QC check standard to determine the concentration measured (A)of each parameter. Calculate each percent recovery (Ps) as 100 (A/T)%, whereT is the true value of the standard concentration.

8.4.1

8.4.2

8.4.3

Compare the percent recovery (Ps) for each parameter with the correspondingQC acceptance criteria found in Table 2. Only parameters that failed the testin Section 8.3 need to be compared with these criteria. If the recovery of anysuch parameter falls outside the designated range, the laboratory performancefor that parameter is judged to be out of control, and the problem must beimmediately identified and corrected. The analytical result for that parameterin the unspiked sample is suspect and may not be reported for regulatorycompliance purposes.

8.5

As part of QC program for the laboratory, method accuracy for wastewater samplesmust be assessed and records must be maintained. After the analysis of five spikedwastewater samples as in Section 8.3, calculate the average percent recovery () andthe standard deviation of the percent recovery (sp). Express the accuracy assessmentas a percent recovery interval from -2sp to +2sp. If =90% and sp = 10%, forexample, the accuracy interval is expressed as 70-110%. Update the accuracy

assessment for each parameter on a regular basis (e.g., after each 5-10 new accuracymeasurements).

It is recommended that the laboratory adopt additional quality assurance practices foruse with this method. The specific practices that are most productive depend uponthe needs of the laboratory and the nature of the samples. Field duplicates may beanalyzed to assess the precision of the environmental measurements. When doubtexists over the identification of a peak on the chromatogram, confirmatory techniquessuch as gas chromatography with a dissimilar column, specific element detector, ormass spectrometer must be used. Whenever possible, the laboratory should analyzestandard reference materials and participate in relevant performance evaluationstudies.

Sample Collection, Preservation, and Handling

Grab samples must be collected in glass containers. Conventional sampling practices8should be followed, except that the bottle must not be prerinsed with sample beforecollection. Composite samples should be collected in refrigerated glass containers inaccordance with the requirements of the program. Automatic sampling equipmentmust be as free as possible of Tygon tubing and other potential sources ofcontamination.

All samples must be iced or refrigerated at 4°C from the time of collection untilextraction.

All samples must be extracted within seven days of collection and completelyanalyzed within 40 days of extraction.2Sample Extraction

Mark the water meniscus on the side of the sample bottle for later determination ofsample volume. Pour the entire sample into a 2 L separatory funnel. Check the pHof the sample with wide-range pH paper and adjust to within the range of 5-9 withsodium hydroxide solution or sulfuric acid.

8.6

9.9.1

9.29.310.10.1

10.2

Add 60 mL of methylene chloride to the sample bottle, seal, and shake 30 seconds torinse the inner surface. Transfer the solvent to the separatory funnel and extract thesample by shaking the funnel for two minutes with periodic venting to release excesspressure. Allow the organic layer to separate from the water phase for a minimum of10 minutes. If the emulsion interface between layers is more than one-third thevolume of the solvent layer, the analyst must employ mechanical techniques tocomplete 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 mLErlenmeyer flask.

Add a second 60 mL volume of methylene chloride to the sample bottle and repeatthe extraction procedure a second time, combining the extracts in the Erlenmeyerflask. Perform a third extraction in the same manner.

Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10 mL concentratortube to a 500 mL evaporative flask. Other concentration devices or techniques may beused in place of the K-D concentrator if the requirements of Section 8.2 are met.Pour the combined extract through a solvent-rinsed drying column containing about10 cm of anhydrous sodium sulfate, and collect the extract in the K-D concentrator.Rinse the Erlenmeyer flask and column with 20-30 mL of methylene chloride tocomplete the quantitative transfer.

Sections 10.7 and 10.8 describe a procedure for exchanging the methylene chloridesolvent to hexane while concentrating the extract volume to 1.0 mL. When it is notnecessary to achieve the MDL in Table 2, the solvent exchange may be made by theaddition of 50 mL of hexane and concentration to 10 mL as described in Method 606,Sections 10.7 and 10.8.

Add one or two clean boiling chips to the evaporative flask and attach a three-ballSnyder column. Prewet the Snyder column by adding about 1 mL of methylene

chloride to the top. Place the K-D apparatus on a hot water bath (60-65°C) so that theconcentrator 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 ofthe apparatus and the water temperature as required to complete the concentration in15-20 minutes. At the proper rate of distillation the balls of the column will activelychatter but the chambers will not flood with condensed solvent. When the apparentvolume of liquid reaches 1 mL, remove the K-D apparatus and allow it to drain andcool for at least 10 minutes.

Remove the Snyder column and rinse the flask and its lower joint into theconcentrator tube with 1-2 mL of methylene chloride. A 5 mL syringe is

recommended for this operation. Add 1-2 mL of hexane and a clean boiling chip tothe concentrator tube and attach a two-ball micro-Snyder column. Prewet the columnby adding about 0.5 mL of hexane to the top. Place the micro-K-D apparatus on a hotwater bath (60-65°C) so that the concentrator tube is partially immersed in the hotwater. Adjust the vertical position of the apparatus and the water temperature asrequired to complete the concentration in 5-10 minutes. At the proper rate ofdistillation the balls of the column will actively chatter but the chambers will not

10.3

10.4

10.5

10.6

10.7

10.8

flood. When the apparent volume of liquid reaches 0.5 mL, remove the K-Dapparatus and allow it to drain and cool for at least 10 minutes.

10.9

Remove the micro-Snyder column and rinse its lower joint into the concentrator tubewith a minimum amount of hexane. Adjust the extract volume to 1.0 mL. Stopper theconcentrator tube and store refrigerated if further processing will not be performedimmediately. If the extract will be stored longer than two days, it should be

transferred to a Teflon-sealed screw-cap vial. If the sample extract requires no furthercleanup, proceed with gas chromatographic analysis (Section 12). If the samplerequires further cleanup, proceed to Section 11.

Determine the original sample volume by refilling the sample bottle to the mark andtransferring the liquid to a 1000 mL graduated cylinder. Record the sample volume tothe nearest 5 mL.Cleanup and Separation

Cleanup procedures may not be necessary for a relatively clean sample matrix. Ifparticular circumstances demand the use of a cleanup procedure, the analyst may usethe procedure below or any other appropriate procedure. However, the analyst firstmust demonstrate that the requirements of Section 8.2 can be met using the method asrevised to incorporate the cleanup procedure.Florisil column cleanup

11.2.1Prepare a slurry of 10 g of activated Florisil in methylene chloride/hexane

(1+9) (V/V) and place the Florisil into a chromatographic column. Tap thecolumn to settle the Florisil and add 1 cm of anhydrous sodium sulfate to thetop. Adjust the elution rate to about 2 mL/min.11.2.2Just prior to exposure of the sodium sulfate layer to the air, quantitatively

transfer the sample extract onto the column using an additional 2 mL of

hexane to complete the transfer. Just prior to exposure of the sodium sulfatelayer to the air, add 30 mL of methylene chloride/hexane (1+9) (V/V) andcontinue the elution of the column. Discard the eluate.11.2.3Next, elute the column with 30 mL of acetone/methylene chloride (1+9) (V/V)

into a 500 mL K-D flask equipped with a 10 mL concentrator tube. Concentratethe collected fraction as in Sections 10.6, 10.7, 10.8, and 10.9 including thesolvent exchange to 1 mL of hexane. This fraction should contain the

nitroaromatics and isophorone. Analyze by gas chromatography (Section 12).

12.12.1

Gas Chromatography

Isophorone and nitrobenzene are analyzed by injection of a portion of the extract intoan FIDGC. The dinitrotoluenes are analyzed by a separate injection into an ECDGC.Table 1 summarizes the recommended operating conditions for the gas

chromatograph. Included in this table are retention times and MDL that can be

achieved under these conditions. Examples of the separations achieved by Column 1are shown in Figures 1 and 2. Other packed or capillary (open-tubular) columns,

10.10

11.11.1

11.2

chromatographic conditions, or detectors may be used if the requirements ofSection 8.2 are met.

12.212.3

Calibrate the system daily as described in Section 7.

If the internal standard calibration procedure is being used, the internal standard

must be added to the same extract and mixed thoroughly immediately before injectioninto the gas chromatograph.

Inject 2-5 µL of the sample extract or standard into the gas chromatograph using thesolvent-flush technique.9 Smaller (1.0 µL) volumes may be injected if automaticdevices are employed. Record the volume injected to the nearest 0.05 µL, the totalextract volume, and the resulting peak size in area or peak height units.

Identify the parameters in the sample by comparing the retention times of the peaksin the sample chromatogram with those of the peaks in standard chromatograms. The width of the retention time window used to make identifications should be basedupon measurements of actual retention time variations of standards over the course ofa day. Three times the standard deviation of a retention time for a compound can beused to calculate a suggested window size; however, the experience of the analystshould weigh heavily in the interpretation of chromatograms.

If the response for a peak exceeds the working range of the system, dilute the extractand reanalyze.

If the measurement of the peak response is prevented by the presence of interferences,further cleanup is required.Calculations

Determine the concentration of individual compounds in the sample.

13.1.1If the external standard calibration procedure is used, calculate the amount of

material injected from the peak response using the calibration curve or

calibration factor determined in Section 7.2.2. The concentration in the samplecan be calculated from Equation 2.

Equation 2

12.4

12.5

12.612.713.13.1

where:

A = Amount of material injected (ng).Vi = Volume of extract injected (µL).Vt = Volume of total extract (µL).Vs = Volume of water extracted (mL).

13.1.2If the internal standard calibration procedure is used, calculate the

concentration in the sample using the response factor (RF) determined inSection 7.3.2 and Equation 3.

Equation 3

where:

As = Response for the parameter to be measured.Ais = Response for the internal standard.

Is = Amount of internal standard added to each extract (µg).Vo = Volume of water extracted (L).

13.214.14.1

Report results in µg/L without correction for recovery data. All QC data obtainedshould be reported with the sample results.Method Performance

The method detection limit (MDL) is defined as the minimum concentration of asubstance that can be measured and reported with 99% confidence that the value isabove zero.1 The MDL concentrations listed in Table 1 were obtained using reagentwater.10 Similar results were achieved using representative wastewaters. The MDLactually achieved in a given analysis will vary depending on instrument sensitivityand matrix effects.

This method has been tested for linearity of spike recovery from reagent water andhas been demonstrated to be applicable over the concentration range from 7 x MDL to1000 x MDL.10

This method was tested by 18 laboratories using reagent water, drinking water,

surface water, and three industrial wastewaters spiked at six concentrations over therange 1.0-515 µg/L.11 Single operator precision, overall precision, and method

accuracy were found to be directly related to the concentration of the parameter andessentially independent of the sample matrix. Linear equations to describe theserelationships are presented in Table 3.

14.2

14.3

References1.2.

40 CFR Part 136, Appendix B.

“Determination of Nitroaromatic Compounds and Isophorone in Industrial and

Municipal Wastewaters,” EPA 600/4-82-024, National Technical Information Service,PB82-208398, Springfield, Virginia 22161, May 1982.

ASTM Annual Book of Standards, Part 31, D3694-78. “Standard Practices forPreparation of Sample Containers and for Preservation of Organic Constituents,”American Society for Testing and Materials, Philadelphia.

3.

4.

“Carcinogens-Working With Carcinogens,” Department of Health, Education, andWelfare, Public Health Service, Center for Disease Control, National Institute forOccupational Safety and Health, Publication No. 77-206, August 1977.

“OSHA Safety and Health Standards, General Industry,” (29 CFR part 1910),

Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).“Safety in Academic Chemistry Laboratories,” American Chemical Society Publication,Committee on Chemical Safety, 3rd Edition, 1979.

Provost, L.P. and Elder, R.S. “Interpretation of Percent Recovery Data,” AmericanLaboratory, 15, 58-63 (1983). (The value 2.44 used in the equation in Section 8.3.3 istwo times the value 1.22 derived in this report.)

ASTM Annual Book of Standards, Part 31, D3370-76. “Standard Practices forSampling Water,” American Society for Testing and Materials, Philadelphia.Burke, J.A. “Gas Chromatography for Pesticide Residue Analysis; Some PracticalAspects,” Journal of the Association of Official Analytical Chemists, 48, 1037 (1965).“Determination of Method Detection Limit and Analytical Curve for EPAMethod 609-Nitroaromatics and Isophorone,” Special letter report for EPAContract 68-03-2624, U.S. Environmental Protection Agency, EnvironmentalMonitoring and Support Laboratory, Cincinnati, Ohio 45268, June 1980.

“EPA Method Study 19, Method 609 (Nitroaromatics and Isophorone),”

EPA 600/4-84-018, National Technical Information Service, PB84-176908, Springfield,Virginia 22161, March 1984.

5.6.7.

8.9.10.

11.

Table 1—Chromatographic Conditions and Method Detection Limits

Parameter

Nitrobenzene....2, 6-DinitrotolueneIsophorone.....2,4-Dinitrotoluene

.......................................................................................

Retention time (min)

Method detectionlimit (µg/L)

FIDGC3.6---5.7---13.7 0.0115.7 0.02

Column 1Column 2ECDGC3.313.524.495.35

4.314.755.726.

Column 1 conditions: Gas-Chrom Q (80/100 mesh) coated with 1.95% QF-1/1.5% OV-17packed in a 1.2 m long x 2 mm or 4 mm ID glass column. A 2 mm ID column and nitrogencarrier gas at 44 mL/min. flow rate were used when determining isophorone and

nitrobenzene by FIDGC. The column temperature was held isothermal at 85°C. A 4 mm IDcolumn and 10% methane/90% argon carrier gas at 44 mL/min. flow rate were used whendetermining the dinitrotoluenes by ECDGC. The column temperature was held isothermalat 145°C.

Column 2 conditions: Gas-Chrom Q (80/100 mesh) coated with 3% OV-101 packed in a3.0 m long x 2 mm or 4 mm ID glass column. A 2 mm ID column and nitrogen carrier gasat 44 mL/min. flow rate were used when determining isophorone and nitrobenzene byFIDGC. The column temperature was held isothermal at 100°C. A 4 mm ID column and10% methane/90% argon carrier gas at 44 mL/min. flow rate were used when determiningthe dinitrotoluenes by ECDGC. The column temperature was held isothermal at 150°C.

Table 2—QC Acceptance Criteria—Method 609

Parameter2,4-Dinitrotoluene2, 6-DinitrotolueneIsophorone.....Nitrobenzene...................................................................Test Conc.Limit for sRange for (µg/L)(µg/L)(µg/L)20201001005.14.832.333.33.6-22.83.8-23.08.0-100.025.7-100.0Range forP, Ps (%)6-1258-126D-1176-118s = Standard deviation of four recovery measurements, in µg/L (Section 8.2.4). = Average recovery for four recovery measurements, in µg/L (Section 8.2.4).P, Ps = Percent recovery measured (Section 8.3.2, Section 8.4.2).D = Detected; result must be greater than zero.NOTE:

These criteria are based directly upon the method performance data in Table 3.Where necessary, the limits for recovery have been broadened to assureapplicability of the limits to concentrations below those used to developTable 3.

Table 3—Method Accuracy and Precision as Functions of Concentration—Method 609

Parameter2,4-Dinitrotoluene2, 6-DinitrotolueneIsophorone.....Nitrobenzene...............................................................................Accuracy, asrecovery, X'(µg/L)0.65C+0.220.66C+0.200.49C+2.930.60C+2.00Single analystprecision, sr'(µg/L)0.200.190.280.25+0.08+0.06+2.77+2.53Overallprecision, S'(µg/L)0.370.360.460.37-0.07-0.00+0.31-0.78X' = Expected recovery for one or more measurements of a sample containing aconcentration of C, in µg/L.

sr' = Expected single analyst standard deviation of measurements at an averageconcentration found of , in µg/L.S' = Expected interlaboratory standard deviation of measurements at an averageconcentration found of , in µg/L.C = True value for the concentration, in µg/L. = Average recovery found for measurements of samples containing a concentration of C,in µg/L.