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1.3 The detection limit of this method is usually dependent on the level of interferences rather than instrumental limitations. The limits listed in Tables 3 and 4 represent the minimum quantity that can be detected with no interferences present.

1.4 The GC/MS portions of this method are for use only by analysts experienced with GC/MS or under the close supervision of such qualified persons. Laboratories unfamiliar with analyses of environmental samples by GC/MS should run the performance tests in reference 1 before beginning.

2. Summary of Method

2.1 Stable isotopically labeled analogs of the compounds of interest are added to a one liter wastewater sample. The sample is extracted at pH 12–13, then at pH <2 with methylene chloride using continuous extraction techniques. The extract is dried over sodium sulfate and concentrated to a volume of one mL. An internal standard is added to the extract, and the extract is injected into the gas chromatograph (GC). The compounds are separated by GC and detected by a mass spectrometer (MS). The labeled compounds serve to correct the variability of the analytical technique.

2.2 Identification of a compound (qualitative analysis) is performed by comparing the GC retention time and background corrected characteristic spectral masses with those of authentic standards.

2.3 Quantitative analysis is performed by GC/MS using extracted ion current profile (EICP) areas. Isotope dilution is used when labeled compounds are available; otherwise, an internal standard method is used.

2.4 Quality is assured through reproducible calibration and testing of the extraction and GC/MS systems.

3. Contamination and Interferences

3.1 Solvents, reagents, glassware, and other sample processing hardware may yield artifacts and/or elevated baselines causing misinterpretation of chromatograms and spectra. All materials shall be demonstrated to be free from interferences under the conditions of analysis by running method blanks initially and with each sample lot (samples started through the extraction process on a given 8 hr shift, to a maximum of 20). Specific selection of reagents and purification of solvents by distillation in all-glass systems may be required. Glassware and, where possible, reagents are cleaned by solvent rinse and baking at 450 °C for one hour minimum.

3.2 Interferences coextracted from samples will vary considerably from source to source, depending on the diversity of the industrial complex or municipality being samples.

4. Safety

4.1 The toxicity or carcinogenicity of each compound or reagent used in this method has not been precisely determined; however, each chemical compound should be treated as a potential health hazard. Exposure to these compounds should be reduced to the lowest possible level. 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 data handling sheets should also be made available to all personnel involved in these analyses. Additional information on laboratory safety can be found in references 2–4.

4.2 The following compounds covered by this method have been tentatively classified as known or suspected human or mammalian carcinogens: benzidine benzo(a)anthracene, 3,3'-dichlorobenzidine, benzo(a)pyrene, dibenzo(a,h)anthracene, N-nitrosodimethylamine, and ß-naphtylamine. Primary standards of these compounds shall be prepared in a hood, and a NIOSH/MESA approved toxic gas respirator should be worn when high concentrations are handled.

5. Apparatus and Materials

5.1 Sampling equipment for discrete or composite sampling.

5.1.1 Sample bottle, amber glass, 1.1 liters minimum. If amber bottles are not available, samples shall be protected from light. Bottles are detergent water washed, then solvent rinsed or baked at 450 °C for one hour minimum before use.

5.1.2 Bottle caps—threaded to fit sample bottles. Caps are lined with Teflon. Aluminum foil may be substituted if the sample is not corrosive. Liners are detergent water washed, then reagent water (Section 6.5) and solvent rinsed, and baked at approximately 200 °C for one hour minimum before use.

5.1.3 Compositing equipment—automatic or manual compositing system incorporating glass containers for collection of a minimum 1.1 liters. Sample containers are kept at 0 to 4 °C during sampling. Glass or Teflon tubing only shall be used. If the sampler uses a peristaltic pump, a minimum length of compressible silicone rubber tubing may be used in the pump only. Before use, the tubing is thoroughly rinsed with methanol, followed by repeated rinsings with reagent water (Section 6.5) to minimize sample contamination. An integrating flow meter is used to collect proportional composite samples.

5.2 Continuous liquid-liquid extractor—Teflon or glass conncecting joints and stopcocks without lubrication (Hershberg-Wolf Extractor) one liter capacity, Ace Glass 6841–10, or equivalent.

5.3 Drying column—15 to 20 mm i.d. Pyrex chromatographic column equipped with coarse glass frit or glass wool plug.

5.4 Kuderna-Danish (K-D) apparatus

5.4.1 Concentrator tube—10mL, graduated (Kontes K–570050–1025, or equivalent) with calibration verified. Ground glass stopper (size 19/22 joint) is used to prevent evaporation of extracts.

5.4.2 Evaporation flask—500 mL (Kontes K–570001–0500, or equivalent), attached to concentrator tube with springs (Kontes K–662750–0012).

5.4.3 Snyder column—three ball macro (Kontes K–503000–0232, or equivalent).

5.4.4 Snyder column—two ball micro (Kontes K–469002–0219, or equivalent).

5.4.5 Boiling chips—approx 10/40 mesh, extracted with methylene chloride and baked at 450 °C for one hr minimum.

5.5 Water bath—heated, with concentric ring cover, capable of temperature control ±2 °C, installed in a fume hood.

5.6 Sample vials—amber glass, 2–5 mL with Teflon-lined screw cap.

5.7 Analytical balance—capable of weighing 0.1 mg.

5.8 Gas chromatograph—shall have splitless or on-column injection port for capillary column, temperature program with 30 °C hold, and shall meet all of the performance specifications in Section 12.

5.8.1 Column—30 ±5 m×0.25 ±0.02 mm i.d. 5% phenyl, 94% methyl, 1% vinyl silicone bonded phase fused silica capillary column (J & W DB–5, or equivalent).

5.9 Mass spectrometer—70 eV electron impact ionization, shall repetitively scan from 35 to 450 amu in 0.95 to 1.00 second, and shall produce a unit resolution (valleys between m/z 441–442 less than 10 percent of the height of the 441 peak), backgound corrected mass spectrum from 50 ng decafluorotriphenylphosphine (DFTPP) introduced through the GC inlet. The spectrum shall meet the mass-intensity criteria in Table 5 (reference 5). The mass spectrometer shall be interfaced to the GC such that the end of the capillary column terminates within one centimeter of the ion source but does not intercept the electron or ion beams. All portions of the column which connect the GC to the ion source shall remain at or above the column temperature during analysis to preclude condensation of less volatile compounds.

5.10 Data system—shall collect and record MS data, store mass-intensity data in spectral libraries, process GC/MS data, generate reports, and shall compute and record response factors.

5.10.1 Data acquisition—mass spectra shall be collected continuously throughout the analysis and stored on a mass storage device.

5.10.2 Mass spectral libraries—user created libraries containing mass spectra obtained from analysis of authentic standards shall be employed to reverse search GC/MS runs for the compounds of interest (Section 7.2).

5.10.3 Data processing—the data system shall be used to search, locate, identify, and quantify the compounds of interest in each GC/MS analysis. Software routines shall be employed to compute retention times and peak areas. Displays of spectra, mass chromatograms, and library comparisons are required to verify results.

5.10.4 Response factors and multipoint calibrations—the data system shall be used to record and maintain lists of response factors (response ratios for isotope dilution) and multipoint calibration curves (Section 7). Computations of relative standard deviation (coefficient of variation) are useful for testing calibration linearity. Statistics on initial (Section 8.2) and on-going (Section 12.7) performance shall be computed and maintained.

6. Reagents and Standards

6.1 Sodium hydroxide—reagent grade, 6N in reagent water.

6.2 Sulfuric acid—reagent grade, 6N in reagent water.

6.3 Sodium sulfate—reagent grade, granular anhydrous, rinsed with methylene chloride (20 mL/g) and conditioned at 450 °C for one hour minimum.

6.4 Methylene chloride—distilled in glass (Burdick and Jackson, or equivalent).

6.5 Reagent water—water in which the compounds of interest and interfering compounds are not detected by this method.

6.6 Standard solutions—purchased as solutions or mixtures with certification to their purity, concentration, and authenticity, or prepared from materials of known purity and composition. If compound purity is 96 percent or greater, the weight may be used without correction to compute the concentration of the standard. When not being used, standards are stored in the dark at -20 to -10 °C in screw-capped vials with Teflon-lined lids. A mark is placed on the vial at the level of the solution so that solvent evaporation loss can be detected. The vials are brought to room temperature prior to use. Any precipitate is redissolved and solvent is added if solvent loss has occurred.

6.7 Preparation of stock solutions—prepare in methylene chloride, benzene, p-dioxane, or a mixture of these solvents per the steps below. Observe the safety precautions in Section 4. The large number of labeled and unlabeled acid, base/neutral, and Appendix C compounds used for combined calibration (Section 7) and calibration verification (12.5) require high concentratimns (approx 40 mg/mL) when individual stock solutions are prepared, so that dilutions of mixtures will permit calibration with all compounds in a single set of solutions. The working range for most compounds is 10–200 µg/mL. Compounds with a reduced MS response may be prepared at higher concentrations.

6.7.1 Dissolve an appropriate amount of assayed reference material in a suitable solvent. For example, weigh 400 mg naphthalene in a 10 mL ground glass stoppered volumetric flask and fill to the mark with benzene. After the naphthalene is completely dissolved, transfer the solution to a 15 mL vial with Teflon-lined cap.

6.7.2 Stock standard solutions should be checked for signs of degradation prior to the preparation of calibration or performance test standards. Quality control check samples that can be used to determine the accuracy of calibration standards are available from the US Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268.

6.7.3 Stock standard solutions shall be replaced after six months, or sooner if comparison with quality control check samples indicates a change in concentration.

6.8 Labeled compound spiking solution—from stock standard solutions prepared as above, or from mixtures, prepare the spiking solution at a concentration of 200 µg/mL, or at a concentration appropriate to the MS response of each compound.

6.9 Secondary standard—using stock solutions (Section 6.7), prepare a secondary standard containing all of the compounds in Tables 1 and 2 at a concentration of 400 µg/mL, or higher concentration appropriate to the MS response of the compound.

6.10 Internal standard solution—prepare 2,2'-difluorobiphenyl (DFB) at a concentration of 10 mg/mL in benzene.

6.11 DFTPP solution—prepare at 50 µg/mL in acetone.

6.12 Solutions for obtaining authentic mass spectra (Section 7.2)—prepare mixtures of compounds at concentrations which will assure authentic spectra are obtained for storage in libraries.

6.13 Calibration solutions—combine 0.5 mL of the solution in Section 6.8 with 25, 50, 125, 250, and 500 uL of the solution in section 6.9 and bring to 1.00 mL total volume each. This will produce calibration solutions of nominal 10, 20, 50, 100, and 200 µg/mL of the pollutants and a constant nominal 100 µg/mL of the labeled compounds. Spike each solution with 10 µL of the internal standard solution (Section 6.10). These solutions permit the relative response (labeled to unlabeled) to be measured as a function of concentration (Section 7.4).

6.14 Precision and recovery standard—used for determination of initial (Section 8.2) and on-going (Section 12.7) precision and recovery. This solution shall contain the pollutants and labeled compounds at a nominal concentration of 100 µg/mL.

6.15 Stability of solutions—all standard solutions (Sections 6.8–6.14) shall be analyzed within 48 hours of preparation and on a monthly basis thereafter for signs of degradation. Standards will remain acceptable if the peak area at the quantitation mass relative to the DFB internal standard remains within ±15 percent of the area obtained in the initial analysis of the standard.

7. Calibration

7.1 Assemble the GC/MS and establish the operating conditions in Table 3. Analyze standards per the procedure in Section 11 to demonstrate that the analytical system meets the detection limits in Tables 3 and 4, and the mass-intensity criteria in Table 5 for 50 ng DFTPP.

7.2 Mass spectral libraries—detection and identification of compounds of interest are dependent upon spectra stored in user created libraries.

7.2.1 Obtain a mass spectrum of each pollutant, labeled compound, and the internal standard by analyzing an authentic standard either singly or as part of a mixture in which there is no interference between closely eluted components. That only a single compound is present is determined by examination of the spectrum. Fragments not attributable to the compound under study indicate the presence of an interfering compound.

7.2.2 Adjust the analytical conditions and scan rate (for this test only) to produce an undistorted spectrum at the GC peak maximum. An undistorted spectrum will usually be obtained if five complete spectra are collected across the upper half of the GC peak. Software algorithms designed to “enhance” the spectrum may eliminate distortion, but may also eliminate authentic masses or introduce other distortion.

7.2.3 The authentic reference spectrum is obtained under DFTPP tuning conditions (Section 7.1 and Table 5) to normalize it to spectra from other instruments.

7.2.4 The spectrum is edited by saving the 5 most intense mass spectral peaks and all other mass spectral peaks greater than 10 percent of the base peak. This edited spectrum is stored for reverse search and for compound confirmation.

7.3 Analytical range—demonstrate that 20 ng anthracene or phenanthrene produces an area at m/z 178 approx one-tenth that required to exceed the linear range of the system. The exact value must be determined by experience for each instrument. It is used to match the calibration range of the instrument to the analytical range and detection limits required, and to diagnose instrument sensitivity problems (Section 15.4). The 20 ug/mL calibration standard (Section 6.13) can be used to demonstrate this performance.

7.3.1 Polar compound detection—demonstrate that unlabeled pentachlorophenol and benzidine are detectable at the 50 µg/mL level (per all criteria in Section 13). The 50 µg/mL calibration standard (Section 6.13) can be used to demonstrate this performance.

7.4 Calibration with isotope dilution—isotope dilution is used when (1) labeled compounds are available, (2) interferences do not preclude its use, and (3) the quantitation mass extracted ion current profile (EICP) area for the compound is in the calibration range. If any of these conditions preclude isotope dilution, internal standard methods (Section 7.5 or 7.6) are used.

7.4.1 A calibration curve encompassing the concentration range is prepared for each compound to be determined. The relative response (pollutant to labeled) vs concentration in standard solutions is plotted or computed using a linear regression. The example in Figure 1 shows a calibration curve for phenol using phenol-d5 as the isotopic diluent. Also shown are the ±10 percent error limits (dotted lines). Relative Reponse (RR) is determined according to the procedures described below. A minimum of five data points are employed for calibration.

7.4.2 The relative response of a pollutant to its labeled analog is determined from isotope ratio values computed from acquired data. Three isotope ratios are used in this process:

RX = the isotope ratio measured for the pure pollutant.

Ry = the isotope ratio measured for the labeled compound.

Rm = the isotope ratio of an analytical mixture of pollutant and labeled compounds.

The m/z's are selected such that RX> Ry. If Rm is not between 2Ry and 0.5RX, the method does not apply and the sample is analyzed by internal or external standard methods.

7.4.3 Capillary columns usually separate the pollutant-labeled pair, with the labeled compound eluted first (Figure 2). For this case, RX = [area m1/z]/1, at the retention time of the pollutant (RT2). Ry = 1/[area m2/z, at the retention time of the labeled compound RT1). Rm = [area at m1/z (at RT2)]/[area at RT1)], as measured in the mixture of the pollutant and labeled compounds (Figure 2), and RR = Rm.

7.4.4 Special precautions are taken when the pollutant-labeled pair is not separated, or when another labeled compound with interfering spectral masses overlaps the pollutant (a case which can occur with isomeric compounds). In this case, it is necessary to determine the respective contributions of the pollutant and labeled compounds to the respective EICP areas. If the peaks are separated well enough to permit the data system or operator to remove the contributions of the compounds to each other, the equations in Section 7.4.3 apply. This usually occurs when the height of the valley between the two GC peaks at the same m/z is less than 10 percent of the height of the shorter of the two peaks. If significant GC and spectral overlap occur, RR is computed using the following equation:

RR = (Ry - Rm) (RX + 1)/(Rm - RX) (Ry + 1), where RX is measured as shown in Figure 3A, Ry is measured as shown in Figure 3B, and Rm is measured as shown in Figure 3C. For example, RX = 46100/4780 = 9.644, Ry = 2650/43600 = 0.0608, Rm = 49200/48300 = 1.019. amd RR = 1.114.

7.4.5 To calibrate the analytical system by isotope dilution, analyze a 1.0 µL aliquot of each of the calibration standards (Section 6.13) using the procedure in Section 11. Compute the RR at each concentration.

7.4.6 Linearity—if the ratio of relative response to concentration for any compound is constant (less than 20 percent coefficient of variation) over the 5 point calibration range, and averaged relative response/concentration ratio may be used for that compound; otherwise, the complete calibration curve for that compound shall be used over the 5 point calibration range.

7.5 Calibration by internal standard—used when criteria for istope dilution (Section 7.4) cannot be met. The internal standard to be used for both acid and base/neutral analyses is 2,2'–difluorobiphenyl. The internal standard method is also applied to determination of compounds having no labeled analog, and to measurement of labeled compounds for intra-laboratory statistics (Sections 8.4 and 12.7.4).

7.5.1 Response factors—calibration requires the determination of response factors (RF) which are defined by the following equation:

RF = (As × Cis)/(Ais × Cs), where

As is the area of the characteristic mass for the compmund in the daily standard

Ais is the area of the characteristic mass for the internal standard

Cis is the concentration of the internal standard (µg/mL)

Cs is the concentration of the compound in the daily standard (µg/mL)

7.5.1.1 The response factor is determined for at least five concentrations appropriate to the response of each compound (Section 6.13); nominally, 10, 20, 50, 100, and 200 µg/mL. The amount of internal standard added to each extract is the same (100 µg/mL) so that Cis remains constant. The RF is plotted vs concentration for each compound in the standard (Cs) to produce a calibration curve.

7.5.1.2 Linearity—if the response factor (RF) for any compound is constant (less than 35 percent coefficient of variation) over the 5 point calibration range, an averaged response factor may be used for that compound; otherwise, the complete calibration curve for that compound shall be used over the 5 point range.

7.6 Combined calibration—by using calibration solutions (Section 6.13) containing the pollutants, labeled compounds, and the internal standard, a single set of analyses can be used to produce calibration curves for the isotope dilution and internal standard methods. These curves are verified each shift (Section 12.5) by analyzing the 100 µg/mL calibration standard (Section 6.13). Recalibration is required only if calibration verification (Section 12.5) criteria cannot be met.

8. Quality Assurance/Quality Control

8.1 Each laboratory that uses this method is required to operate a formal quality assurance program. The minimum requirements of this program consist of an initial demonstration of laboratory capability, analysis of samples spiked with labeled compounds to evaluate and document data quality, and analysis of standards and blanks as tests of continued performance. Laboratory performance is compared to established performance criteria to determine if the results of analyses meet the performance characteristics of the method.

8.1.1 The analyst shall make an initial demonstration of the ability to generate acceptable accuracy and precision with this method. This ability is established as described in Section 8.2.

8.1.2 The analyst is permitted to modify this method to improve separations or lower the costs of measurements, provided all performance specifications are met. Each time a modification is made to the method, the analyst is required to repeat the procedure in Section 8.2 to demonstrate method performance.

8.1.3 Analyses of blanks are required to demonstrate freedom from contamination. The procedures and criteria for analysis of a blank are described in Section 8.5.

8.1.4 The laboratory shall spike all samples with labeled compounds to monitor method performance. This test is described in Section 8.3. When results of these spikes indicate atypical method performance for samples, the samples are diluted to bring method performance within acceptable limits (Section 15).

8.1.5 The laboratory shall, on an on-going basis, demonstrate through calibration verification and the analysis of the precision and recovery standard (Section 6.14) that the analysis system is in control. These procedures are described in Sections 12.1, 12.5, and 12.7.

8.1.6 The laboratory shall maintain records to define the quality of data that is generated. Development of accuracy statements is described in Section 8.4.

8.2 Initial precision and accuracy—to establish the ability to generate acceptable precision and accuracy, the analyst shall perform the following operations:

8.2.1 Extract, concentrate, and analyze two sets of four one-liter aliquots (8 aliquots total) of the precision and recovery standard (Section 6.14) according to the procedure in Section 10.

8.2.2 Using results of the first set of four analyses, compute the average recovery (X ) in µg/mL and the standard deviation of the recovery (s) in Tg/µL for each compound, by isotope dilution for pollutants with a labeled analog, and by internal standard for labeled compounds and pollutants with no labeled analog.

8.2.3 For each compound, compare s and X with the corresponding limits for initial precision and accuracy in Table 8. If s and X for all compounds meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may begin. If, however, any individual s exceeds the precision limit or any individual X falls outside the range for accuracy, system performance is unacceptable for that compound.

Note: The large number of compounds in Table 8 present a substantial probability that one or more will fail the acceptance criteria when all compounds are analyzed. To determine if the analytical system is out of control, or if the failure can be attributed to probability, proceed as follows:

8.2.4 Using the results of the second set of four analyses, compute s and X for only those compounds which failed the test of the first set of four analyses (Section 8.2.3). If these compounds now pass, system performance is acceptable for all compounds and analysis of blanks and samples may begin. If, however, any of the same compoulds fail again, the analysis system is not performing properly for these compounds. In this event, correct the problem and repeat the entire test (Section 8.2.1).

8.3 The laboratory shall spike all samples with labeled compounds to assess method performance on the sample matrix.

8.3.1 Analyze each sample according to the method in Section 10.

8.3.2 Compute the percent recovery (P) of the labeled compounds using the internal standard methmd (Section 7.5).

8.3.3 Compare the labeled compound recovery for each compound with the corresponding limits in Table 8. If the recovery of any compounds falls outside its warning limit, method performance is unacceptable for that compound in that sample, Therefore, the sample is complex and is to be diluted and reanalyzed per Section 15.4.

8.4 As part of the QA program for the laboratory, method accuracy for wastewater samples shall be assessed and records shall be maintained. After the analysis of five wastewater samples for which the labeled compounds pass the tests in Section 8.3, compute the average percent recovery (P) and the standard deviation of the percent recovery (sp) for the labeled compounds only. Express the accuracy assessment as a percent recovery interval from P—2 sp to P+2sp. For example, if P=90% and sp=10%, the accuracy interval is expressed as 70–100%. Update the accuracy assessment for each compound on a regular basis (e.g. after each 5–10 new accuracy measurements).

8.5 Blanks—reagent water blanks are analyzed to demonstrate freedom from contamination.

8.5.1 Extract and concentrate a blank with each sample lot (samples started through the extraction process on the same 8 hr shift, to a maximum of 20 samples). Analyze the blank immediately after analysis of the precision and recovery standard (Section 6.14) to demonstrate freedom from contamination.

8.5.2 If any of the compounds of interest (Tables 1 and 2) or any potentially interfering compound is found in a blank at greater than 10 µg/L (assuming a response factor of 1 relative to the internal standard for compounds not listed in Tables 1 and 2), analysis of samples is halted until the source of contamination is eliminated and a blank shows no evidence of contamination at this level.

8.6 The specifications contained in this method can be met if the apparatus used is calibrated properly, then maintained in a calibrated state. The standards used for calibration (Section 7), calibration verification (Section 12.5), and for initial (Section 8.2) and on-going (Section 12.7) precision and recovery should be identical, so that the most precise results will be obtained. The GC/MS instrument in particular will provide the most reproducible results if dedicated to the settings and conditions required for the analysis of semi-volatiles by this method.

8.7 Depending on specific program requirements, field replicates may be collected to determine the precision of the sampling technique, and spiked samples may be required to determine the accuracy of the analysis when internal or external standard methods are used.

9. Sample Collection, Preservation, and Handling

9.1 Collect samples in glass containers following conventional sampling practices (Reference 7). Composite samples are collected in refrigerated glass containers (Section 5.1.3) in accordance with the requirements of the sampling program.

9.2 Maintain samples at 0–4 °C from the time collectimn until extraction. If residual chlorine is present, add 80 mg sodium thiosulfate per liter of water. EPA Methods 330.4 and 330.5 may be used to measure residual chlorine (Reference 8).

9.3 Begin sample extraction within seven days of collection, and analyze all extracts within 40 days of extraction.

10. Sample Extraction and Concentration (See Figure 4)

10.1 Labeled compound spiking—measure 1.00 ±0.01 liter of sample into a glass container. For untreated effluents, and samples which are expected to be difficult to extract and/or concentrate, measure an additional 10.0 ±0.1 mL and dilute to a final volume of 1.00 ±0.01 liter with reagent water in a glass container.

10.1.1 For each sample or sample lot (to a maximum of 20) to be extracted at the same time, place three 1.00 ±0.10 liter aliquots of reagent water in glass containers.

10.1.2 Spike 0.5 mL of the labeled compound spiking solution (Section 6.8) into all samples and one reagant water aliquot.

10.1.3 Spike 1.0 mL of the precision and recovery standard (Section 6.14) into the two remaining reagent water aliquots.

10.1.4 Stir and equilibrate all solutions for 1–2 hr.

10.2 Base/neutral extraction—place 100–150 mL methylene chloride in each continuous extractor and 200–300 in each distilling flask.

10.2.1 Pour the sample(s), blank, and standard aliquots into the extractors. Rinse the glass containers with 50–100 mL methylene chloride and add to the respective extractor.

10.2.2 Adjust the pH of the waters in the extractors to 12–13 with 6N NaOH while monitoring with a pH meter. Begin the extraction by heating the flask until the methylene chloride is boiling. When properly adjusted, 1–2 drops of methylene chloride per second will fall from the condensor tip into the water. After 1–2 hours of extraction, test the pH and readjust to 12–13 if required. Extract for 18–24 hours.

10.2.3 Remove the distilling flask, estimate and record the volume of extract (to the nearest 100 mL), and pour the contents through a drying column containing 7 to 10 cm anhydrous sodium sulfate. Rinse the distilling flask with 30–50 mL of methylene chloride and pour through the drying column. Collect the solution in a 500 mL K-D evaporator flask equipped with a 10 mL concentrator tube. Seal, label as the base/neutral fraction, and concentrate per Sections 10.4 to 10.5.

10.3 Acid extraction—adjust the pH of the waters in the extractors to 2 or less using 6N sulfuric acid. Charge clean distilling flasks with 300–400 mL of methylene chloride. Test and adjust the pH of the waters after the first 1–2 hr of extraction. Extract for 18–24 hours.

10.3.1 Repeat Section 10.2.3, except label as the acid fraction.

10.4 Concentration—concentrate the extracts in separate 500 mL K-D flasks equipped with 10 mL concentrator tubes.

10.4.1 Add 1 to 2 clean boiling chips to the flask and attach a three-ball macro Snyder column. Prewet the column by adding approximately one mL of methylene chloride through the top. Place the K-D apparatus in a hot water bath so that the entire lower rounded surface of the flask is bathed with steam. 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. When the liquid has reached an apparent volume of 1 mL, remove the K-D apparatus from the bath and allow the solvent to drain and cool for at least 10 minutes. Remove the Snyder column and rinse the flask and its lower joint into the concentrator tube with 1–2 mL of methylene chloride. A 5-mL syringe is recommended for this operation.

10.4.2 For performance standards (Sections 8.2 and 12.7) and for blanks (Section 8.5), combine the acid and base/neutral extracts for each at this point. Do not combine the acid and base/neutral extracts for samples.

10.5 Add a clean boiling chip and attach a two ball micro Snyder column to the concentrator tube. Prewet the column by adding approx 0.5 mL methylene chloride through the top. Place the apparatus in the hot water bath. Adjust the vertical position and the water temperature as required to complete the concentration in 5–10 minutes. At the proper rate of distillation, the balls of the column will actively chatter but the chambers will not flood. When the liquid reaches an apparent volume of approx 0.5 mL, remove the apparatus from the water bath and allow to drain and cool for at least 10 minutes. Remove the micro Snyder column and rinse its lower joint into the concentrator tube with approx 0.2 mL of methylene chloride. Adjust the final volume to 1.0 mL.

10.6 Transfer the concentrated extract to a clean screw-cap vial. Seal the vial with a Teflon-lined lid, and mark the level on the vial. Label with the sample number and fraction, and store in the dark at -20 to -10 °C until ready for analysis.

11. GC/MS Analysis

11.1 Establish the operating conditions given in Table 3 or 4 for analysis of the base/neutral or acid extracts, respectively. For analysis of combined extracts (Section 10.4.2), use the operating conditions in Table 3.

11.2 Bring the concentrated extract (Section 10.6) or standard (Sections 6.13 through 6.14) to room temperature and verify that any precipitate has redissolved. Verify the level on the extract (Sections 6.6 and 10.6) and bring to the mark with solvent if required.

11.3 Add the internal standard solution (Section 6.10) to the extract (use 1.0 uL of solution per 0.1 mL of extract) immediately prior to injection to minimize the possibility of loss by evaporation, adsorption, or reaction. Mix thoroughly.

11.4 Inject a volume of the standard solution or extract such that 100 ng of the internal standard will be injected, using on-column or splitless injection. For 1 mL extracts, this volume will be 1.0 uL. Start the GC column initial isothermal hold upon injection. Start MS data collection after the solvent peak elutes. Stop data collection after the benzo (ghi) perylene or pentachlorophenol peak elutes for the base/neutral or acid fraction, respectively. Return the column to the initial temperature for analysis of the next sample.

12. System and Laboratory Performance

12.1 At the beginning of each 8 hr shift during which analyses are performed, GC/MS system performance and calibration are verified for all pollutants and labeled compounds. For these tests, analysis of the 100 µg/mL calibration standard (Section 6.13) shall be used to verify all performance criteria. Adjustment and/or recalibration (per Section 7) shall be performed until all performance criteria are met. Only after all performance criteria are met may samples, blanks, and precision and recovery standards be analyzed.

12.2 DFTPP spectrum validity—inject 1 µL of the DFTPP solution (Section 6.11) either separately or within a few seconds of injection of the standard (Section 12.1) analyzed at the beginning of each shift. The criteria in Table 5 shall be met.

12.3 Retention times—the absolute retention time of 2,2'-difluorobiphenyl shall be within the range of 1078 to 1248 seconds and the relative retention times of all pollutants and labeled compounds shall fall within the limits given in Tables 3 and 4.

12.4 GC resolution—the valley height between anthracene and phenanthrene at m/z 178 (or the analogs at m/z 188) shall not exceed 10 percent of the taller of the two peaks.

12.5 Calibration verification—compute the concentration of each pollutant (Tables 1 and 2) by isotope dilution (Section 7.4) for those compounds which have labeled analogs. Compute the concentration of each pollutant which has no labeled analog by the internal standard method (Section 7.5). Compute the concentration of the labeled compounds by the internal standard method. These concentrations are computed based on the calibration data determined in Section 7.

12.5.1 For each pollutant and labeled compound being tested, compare the concentration with the calibration verification limit in Table 8. If all compounds meet the acceptance criteria, calibration has been verified and analysis of blanks, samples, and precision and recovery standards may proceed. If, however, any compound fails, the measurement system is not performing properly for that compound. In this event, prepare a fresh calibration standard or correct the problem causing the failure and repeat the test (Section 12.1), or recalibrate (Section 7).

12.6 Multiple peaks—each compound injected shall give a single, distinct GC peak.

12.7 On-going precision and accuracy.

12.7.1 Analyze the extract of one of the pair of precision and recovery standards (Section 10.1.3) prior to analysis of samples from the same lot.

12.7.2 Compute the concentration of each pollutant (Tables 1 and 2) by isotope dilution (Section 7.4) for those compounds which have labeled analogs. Compute the concentration of each pollutant which has no labeled analog by the internal standard method (Section 7.5). Compute the concentration of the labeled compounds by the internal standard method.

12.7.3 For each pollutant and labeled compound, compare the concentration with the limits for on-going accuracy in Table 8. If all compounds meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may proceed. If, however, any individual concentration falls outside of the range given, system performance is unacceptable for that compound.

Note: The large number of compounds in Table 8 present a substantial probability that one or more will fail when all compounds are analyzed. To determine if the extraction/concentration system is out of control or if the failure is caused by probability, proceed as follows:

12.7.3.1 Analyze the second aliquot of the pair of precision and recovery standard (Section 10.1.3).

12.7.3.2 Compute the concentration of only those pollutants or labeled compounds that failed the previous test (Section 12.7.3). If these compounds now pass, the extraction/concentration processes are in control and analysis of blanks and samples may proceed. If, however, any of the same compounds fail again, the extraction/concentration processes are not being performed properly for these compounds. In this event, correct the problem, re-extract the sample lot (Section 10) and repeat the on-going precision and recovery test (Section 12.7).

12.7.4 Add results which pass the specifications in Section 12.7.2 to initial and previous on-going data. Update QC charts to perform a graphic representation of continued laboratory performance (Figure 5). Develop a statement of laboratory accuracy for each pollutant and labeled compound by calculating the average percent recovery (R) and the standard deviation of percent recovery (sr). Express the accuracy as a recovery interval from R-2sr to R+2sr. For example, if R=95% and sr=5%, the accuracy is 85-105%.

13. Qualitative Determination

13.1 Qualititative determination is accomplished by comparison of data from analysis of a sample or blank with data from analysis of the shift standard (Section 12.1) and with data stored in the spectral libraries (Section 7.2.4). Identification is confirmed when spectra and retention times agree per the criteria below.

13.2 Labeled compounds and pollutants having no labeled analog:

13.2.1 The signals for all characteristic masses stored in the spectral library (Section 7.2.4) shall be present and shall maximize within the same two consecutive scans.

13.2.2 Either (1) the background corrected EICP areas, or (2) the corrected relative intensities of the mass spectral peaks at the GC peak maximum shall agree within a factor of two (0.5 to 2 times) for all masses stored in the library.

13.2.3 The retention time relative to the nearest eluted internal standard shall be within ±15 scans or ±15 seconds, whichever is greater of this difference in the shift standard (Section 12.1).

13.3 Pollutants having a labled analog:

13.3.1 The signals for all characteristic masses stored in the spectral library (Section 7.2.4) shall be present and shall maximize within the same two consecutive scans.

13.3.2. Either (1) the background corrected EICP areas, or (2) the corrected relative intensities of the mass spectral peaks at the GC peak maximum shall agree within a factor of two for all masses stored in the spectral library.

13.3.3. The retention time difference between the pollutant and its labeled analog shall agree within ±6 scans or ±6 seconds (whichever is greater) of this difference in the shift standard (Section 12.1).

13.4 Masses present in the experimental mass spectrum that are not present in the reference mass spectrum shall be accounted for by contaminant or background ions. If the experimental mass spectrum is contaminated, an experienced spectrometrist (Section 1.4) is to determine the presence or absence of the cmmpound.

14. Quantitative Determination

14.1 Isotope dilution—by adding a known amount of a labeled compound to every sample prior to extraction, correction for recovery of the pollutant can be made because the pollutant and its labeled analog exhibit the same effects upon extraction, concentration, and gas chromatography. Relative response (RR) values for mixtures are used in conjunction with calibration curves described in Section 7.4 to determine concentrations directly, so long as labeled compound spiking levels are constant. For the phenml example given in Figure 1 (Section 7.4.1), RR would be equal to 1.114. For this RR value, the phenol calibration curve given in Figure 1 indicates a concentration of 27 µg/mL in the sample extract (Cex).

14.2 Internal standard—compute the concentration in the extract using the response factor determined from calibration data (Section 7.5) and the following equation: Cex(µg/mL)=(As × Cis/(Ais × RF) where Cex is the concentration of the compound in the extract, and the other terms are as defined in Section 7.5.1.

14.3 The concentration of the pollutant in water is computed using the volumes of the original water sample (Section 10.1) and the final extract volume (Section 10.5), as follows: Concentration in water (µg/L)=(Cex × Vex)/Vs where Vex is the extract volume in mL, and Vs is the sample volume in liters.

14.4 If the EICP area at the quantitiation mass for any compound exceeds the calibration range of the system, the extract of the dilute aliquot (Section 10.1) is analyzed by isotope dilution; otherwise, the extract is diluted by a factor of 10, 9 µL of internal standard solution (Section 6.10) are added to a 1.0 mL aliquot, and this diluted extract is analyzed by the internal standard method (Section 14.2). Quantify each compound at the highest concentration level within the calibration range.

14.5 Report results for all pollutants and labeled compounds (Tables 1 and 2) found in all standards, blanks, and samples in µg/L, to three significant figures. Results for samples which have been diluted are reported at the least dilute level at which the area at the quantitation mass is within the calibration range (Section 14.4) and the labeled compound recovery is within the normal range for the method (Section 15.4).

15. Analysis of Complex Samples

15.1 Untreated effluents and other samples frequently contain high levels (>1000 µg/L) of the compounds of interest, interfering compounds, and/or polymeric materials. Some samples will not concentrate to one mL (Section 10.5); others will overload the GC column and/or mass spectrometer.

15.2 Analyze the dilute aliquot (Section 10.1) when the sample will not concentrate to 1.0 mL. If a dilute aliquot was not extracted, and the sample holding time (Section 9.3) has not been exceeded, dilute an aliquot of the sample with reagent water and re-extract (Section 10.1); otherwise, dilute the extract (Section 14.4) and analyze by the internal standard method (Section 14.2).

15.3 Recovery of internal standard—the EICP area of the internal standard should be within a factor of two of the area in the shift standard (Section 12.1). If the absolute areas of the labeled compounds are within a factor of two of the respective areas in the shift standard, and the internal standard area is less than one-half of its respective area, then internal standard loss in the extract has occurred. In this case, use one of the labeled compounds (perferably a polynuclear aromatic hydrocarbon) to compute the concentration of a pollutant with no labeled analog.

15.4 Recovery of labeled compounds—in most samples, labeled compound recoveries will be similar to those from reagent water (Section 12.7). If the labeled compound recovery is outside the limits given in Table 8, the dilute extract (Section 10.1) is analyzed as in Section 14.4. If the recoveries of all labeled compounds and the internal staldard are low (per the criteria above), then a loss in instrument sensitivity is the most likely cause. In this case, the 100 µg/mL calibration standard (Section 12.1) shall be analyzed and calibration verified (Section 12.5). If a loss in sensitivity has occurred, the instrument shall be repaired, the performance specifications in Section 12 shall be met, and the extract reanalyzed. If a loss in instrument sensitivity has not occurred, the method does not work on the sample being analyzed and the result may not be reported for regulatory compliance purposes.

16. Method Performance

16.1 Interlaboratory performance for this method is detailed in references 9 and 10.

16.2 A chromatogram of the 100 µg/mL acid/base/neutral calibration standard (Section 6.13) is shown in Figure 6.

References

1. “Performance Tests for the Evaluation of Computerized Gas Chromatography/Mass Spectrometry Equipment and Laboratories” USEPA, EMSL/Cincinnati, OH 45268, EPA–600/4–80–025 (April 1980).

2. “Working with Carcinogens,” DHEW, PHS, CDC, NIOSH, Publication 77–206, (August 1977).

3. “OSHA Safety and Health Standards, General Industry” OSHA 2206, 29 CFR part 1910 (January 1976).

4. “Safety in Academic Chemistry Laboratories, ” ACS Committee on Chemical Safety (1979).

5. “Reference Compound to Calibrate Ion Abundance Measurement in Gas Chromatography-Mass Spectrometry Systems,” J.W. Eichelberger, L.E. Harris, and W.L. Budde, Anal. Chem., 47, 955 (1975).

6. “Handbook of Analytical Quality Control in Water and Wastewater Laboratories,” USEPA, EMSL/Cincinnati, OH 45268, EPA–600/4–79–019 (March 1979).

7. “Standard Practice for Sampling Water,” ASTM Annual Book of Standards, ASTM, Philadelphia, PA, 76 (1980).

8. “Methods 330.4 and 330.5 for Total Residual Chlorine,” USEPA, EMSL/ Cincinnati, OH 45268, EPA 600/4–70–020 (March 1979).

9. Colby, B.N., Beimer, R.G., Rushneck, D.R., and Telliard, W.A., “Isotope Dilution Gas Chromatography-Mass Spectrometry for the determination of Priority Pollutants in Industrial Effluents.” USEPA, Effluent Guidelines Division, Washington, DC 20460 (1980).

10. “Inter-laboratory Validation of US Environmental Protection Agency Method 1625,” USEPA, Effluent Guidelines Division, Washington, DC 20460 (June 15, 1984).


Table 1_Base/Neutral Extractable Compounds
------------------------------------------------------------------------
CAS
Compound STORET registry EPA-EGD NPDES
------------------------------------------------------------------------
Acenaphthene................. 34205 83-32-9 001 B 001 B
Acenaphthylene............... 34200 208-96-8 077 B 002 B
Anthracene................... 34220 120-12-7 078 B 003 B
Benzidine.................... 39120 92-87-5 005 B 004 B
Benzo(a)anthracene........... 34526 56-55-3 072 B 005 B
Benzo(b)fluoranthene......... 34230 205-99-2 074 B 007 B
Benzo(k)fluoranthene......... 34242 207-08-9 075 B 009 B
Benzo(a)pyrene............... 34247 50-32-8 073 B 006 B
Benzo(ghi)perylene........... 34521 191-24-2 079 B 008 B
Biphenyl (Appendix C)........ 81513 92-52-4 512 B ........
Bis(2-chloroethyl) ether..... 34273 111-44-4 018 B 011 B
Bis(2-chloroethyoxy)methane.. 34278 111-91-1 043 B 010 B
Bis(2-chloroisopropyl) ether. 34283 108-60-1 042 B 012 B
Bis(2-ethylhexyl) phthalate.. 39100 117-81-7 066 B 013 B
4-bromophenyl phenyl ether... 34636 101-55-3 041 B 014 B
Butyl benzyl phthalate....... 34292 85-68-7 067 B 015 B
n-C10 (Appendix C)........... 77427 124-18-5 517 B ........
n-C12 (Appendix C)........... 77588 112-40-2 506 B ........
n-C14 (Appendix C)........... 77691 629-59-4 518 B ........
n-C16 (Appendix C)........... 77757 544-76-3 519 B ........
n-C18 (Appendix C)........... 77804 593-45-3 520 B ........
n-C20 (Appendix C)........... 77830 112-95-8 521 B ........
n-C22 (Appendix C)........... 77859 629-97-0 522 B ........
n-C24 (Appendix C)........... 77886 646-31-1 523 B ........ (continued)