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9.6.1.1 Retention times: The absolute retention time of 2,2'-difluorobiphenyl shall be within the range of 765 to 885 seconds, and the relative retention times of all pollutants and labeled compounds shall fall within the limits given in Table 2.

9.6.1.2 GC resolution: The valley height between 4,6-dichloroguaiacol and 3,4-dichloroguaiacol at m/z 192 shall not exceed 10% of the height of the taller of the two peaks.

9.6.1.3 Multiple peaks: Each compound injected shall give a single, distinct GC peak.

9.6.2 Compute the percent recovery of each pollutant (Table 1) by isotope dilution (Section 10.4) for those compounds that have labeled analogs. Compute the percent recovery of each pollutant that has no labeled analog by the internal standard method (Section 10.5), using the 3,4,5-trichlorophenol (SMIS) as the internal standard. Compute the percent recovery of the labeled compounds and the SMIS by the internal standard method, using the 2,2'-difluorobiphenyl as the internal standard.

9.6.2.1 For each compound, compare the recovery with the limits for ongoing precision and recovery in Table 5. If all compounds meet the acceptance criteria, system performance is acceptable and analysis of blanks and samples may proceed. If, however, any individual recovery falls outside of the range given, system performance is unacceptable for that compound. In this event, there may be a problem with the GCMS or with the derivatization/extraction/concentration systems.

9.6.2.2 GCMS system: To determine if the failure of the OPR test (Section 9.6.2.1) is due to instrument drift, analyze the current calibration verification extract (Section 7.13.4), calculate the percent recoveries of all compounds, and compare with the OPR recovery limits in Table 5. If all compounds meet these criteria, GCMS performance/stability is verified, and the failure of the OPR analysis is attributed to problems in the derivatization/extraction/concentration of the OPR. In this case, analysis of the sample extracts may proceed. However, failure of any of the recovery criteria in the analysis of a sample extract requires rederivatization of that sample (Sections 13.3.1 and 13.3.2). If, however, the performance/stability of the GCMS is not verified by analysis of the calibration verification extract, the GCMS requires recalibration and all extracts associated with the failed OPR must be reanalyzed.

9.6.3 Add results that pass the specifications in Section 9.6.2.1 to initial and previous ongoing data for each compound. Update QC charts to form a graphic representation of continued laboratory performance. Develop a statement of laboratory accuracy for each pollutant and labeled compound in each matrix type (reagent water, C-stage filtrate, E-stage filtrate, final effluent, etc.) 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 to 105%.

9.7 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 10) and for initial (Section 9.3.2) and ongoing (Section 9.6) precision and recovery should be identical, so that the most precise results will be obtained. The GCMS instrument in particular will provide the most reproducible results if dedicated to the settings and conditions required for the analyses of chlorophenolics by this method.

9.8 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 the internal standard method is used.

10.0 Calibration and Standardization

10.1 Assemble the GCMS and establish the operating conditions in Section 12. Analyze standards per the procedure in Section 12 to demonstrate that the analytical system meets the minimum levels in Table 2, and the mass-intensity criteria in Table 3 for 50 ng DFTPP.

10.2 Mass-spectral libraries: Detection and identification of compounds of interest are dependent upon spectra stored in user-created libraries.

10.2.1 Obtain a mass spectrum of the acetyl derivative of each chlorophenolic compound (pollutant, labeled compound, and the sample matrix internal standard) by derivatizing and analyzing an authentic standard either singly or as part of a mixture in which there is no interference between closely eluting 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.

10.2.2 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 m/z's or introduce other distortion.

10.2.3 The authentic reference spectrum is obtained under DFTPP tuning conditions (Section 10.1 and Table 3) to normalize it to spectra from other instruments.

10.2.4 The spectrum is edited by removing all peaks in the m/z 42 to 45 range, and saving the five most intense mass spectral peaks and all other mass spectral peaks greater than 10% of the base peak (excluding the peaks in the m/z 42 to 45 range). The spectrum may be further edited to remove common interfering m/z's. The spectrum obtained is stored for reverse search and for compound confirmation. 10.3 Minimum level: Demonstrate that the chlorophenolics are detectable at the minimum level (per all criteria in Section 14). The nominal 5 µg/mL calibration standard (Section 7.13) can be used to demonstrate this performance.

10.4 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 m/z (Table 4) extracted ion-current profile (EICP) area for the compound is in the calibration range. Alternative labeled compounds and quantitation m/z's may be used based on availability. If any of the above conditions preclude isotope dilution, the internal standard calibration method (Section 10.5) is used.

10.4.1 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% error limits (dotted lines). Relative response (RR) is determined according to the procedures described below. A minimum of five data points are employed for calibration.

10.4.2 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 the internal standard method.

10.4.3 Capillary columns sometimes separate the pollutant-labeled pair when deuterium labeled compounds are used, with the labeled compound eluted first (Figure 2). For this case,


10.4.4 When the pollutant-labeled pair is not separated (as occurs with carbon-13-labeled compounds), or when another labeled compound with interfering spectral masses overlaps the pollutant (a case which can occur with isomeric compounds), it is necessary to determine the contributions of the pollutant and labeled compound 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 10.4.3 apply. This usually occurs when the height of the valley between the two GC peaks at the same m/z is less than 70 to 90% of the height of the shorter of the two peaks. If significant GC and spectral overlap occur, RR is computed using the following equation:


Where:

Rx is measured as shown in figure 3A,

Ry is measured as shown in figure 3B,

Rm is measured as shown in figure 3C.

For example, Rx = 46100/4780 = 9.644; Ry = 2650/43600 = 0.0608; Rm = 49200/48300 = 1.1019; thus, RR = 1.114. 10.4.5 To calibrate the analytical system by isotope dilution, analyze a 1-µL aliquot of each of the calibration standards (Section 7.13) using the procedure in Section 12. Compute the RR at each concentration.

10.4.6 Linearity: If the ratio of relative response to concentration for any compound is constant (less than 20% coefficient of variation) over the five-point calibration range, an averaged relative response/concentration ratio may be used for that compound; otherwise, the complete calibration curve for that compound shall be used over the five-point calibration range.

10.5 Calibration by internal standard: The method contains two types of internal standards, the sample matrix internal standard (SMIS) and the instrument internal standard (IIS), and they are used for different quantitative purposes. The 3,4,5-trichlorophenol sample matrix internal standard (SMIS) is used for measurement of all pollutants with no labeled analog and when the criteria for isotope dilution (Section 10.4) cannot be met. The 2,2'-difluorobiphenyl instrument internal standard (IIS) is used for determination of the labeled compounds and the SMIS. The results are used for intralaboratory statistics (Sections 9.4.4 and 9.6.3).

10.5.1 Response factors: Calibration requires the determination of response factors (RF) for both the pollutants with no labeled analog and for the labeled compounds and the SMIS. The response factor is defined by the following equation:


Where:

As=the area of the characteristic mass for the compound in the daily standard.

Ais=the area of the characteristic mass for the internal standard.

Cis=the concentration of the internal standard (µg/mL).

Cs=is the concentration of the compound in the calibration standard (µg/mL).

When this equation is used to determine the response factors for pollutant compounds without labeled analogs, use the area (Ais) and concentration (Cis) of 3,4,5-trichlorophenol (SMIS) as the internal standard. When this equation is used to determine the response factors for the labeled analogs and the SMIS, use the area (Ais) and concentration (Cis) of 2,2'-difluorobiphenyl as the internal standard.

10.5.2 The response factor is determined for at least five concentrations appropriate to the response of each compound (Section 7.13); nominally, 5, 10, 20, 50, and 100 µg/mL. The amount of SMIS added to each solution is the same (25 µg/mL) so that Cis remains constant. Likewise, the concentration of IIS is constant in each solution. The area ratio (As/Ais) is plotted versus the concentration ratio (Cs/Cis) for each compound in the standard to produce a calibration curve.

10.5.3 Linearity: If the response factor (RF) for any compound is constant (less than 35% coefficient of variation) over the five-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 five-point range.

10.6 Combined calibration: By using calibration solutions (Section 7.13) containing the pollutants, labeled compounds, and the internal standards, 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 9) by analyzing the OPR standard, or an optional calibration verification (VER) standard. Recalibration is required only if OPR criteria (Section 9.6 and Table 5) cannot be met.

11.0 Sample Derivatization, Extraction, and Concentration

The procedure described in this section uses a stir-bar in a beaker for the derivatization. The extraction procedures applied to samples depend on the type of sample being analyzed. Extraction of samples from in-process wastewaters is performed using a separatory funnel procedure. All calibrations, IPR, OPR, and blank analyses associated with in-process wastewater samples must be performed by the separatory funnel procedure.

Extraction of samples of final effluents and raw water may be performed using either the stir-bar procedure or the separatory funnel procedure. However, all calibrations, IPR, OPR, blank, and sample analyses must be performed using the same procedure. Both procedures are described below.

11.1 Preparation of all sample types for stir-bar derivatization.

11.1.1 Allow sample to warm to room temperature.

11.1.2 Immediately prior to measuring, shake sample vigorously to insure homogeneity.

11.1.3 Measure 1000 mL (±10 mL) of sample into a clean 2000-mL beaker. Label the beaker with the sample number.

11.1.4 Dilute aliquot(s).

11.1.4.1 Complex samples: For samples that are expected to be difficult to derivatize, concentrate, or are expected to overload the GC column or mass spectrometer, measure an additional 100 mL (±1 mL) into a clean 2000-mL beaker and dilute to a final volume of 1000-mL (±50 mL) with reagent water. Label with the sample number and as the dilute aliquot. However, to ensure adequate sensitivity, a 1000-mL aliquot must always be prepared and analyzed.

11.1.4.2 Pulp and paper industry samples: For in-process streams such as E-stage and C-stage filtrates and other in-process wastewaters, it may be necessary to prepare an aliquot at an additional level of dilution. In this case, dilute 10 mL (±0.1 mL) of sample to 1000-mL (±50 mL).

11.1.5 QC aliquots: For a batch of samples of the same type to be extracted at the same time (to a maximum of 20), place two 1000-mL (±10 mL) aliquots of reagent water in clean 2000-mL beakers. Label one beaker as the blank and the other as the ongoing precision and recovery (OPR) aliquot. Because final effluent samples are treated with ascorbic acid and in-process wastewater samples are not (see Section 11.1.6), prepare an OPR aliquot and a blank for the final effluent and a separate pair for the in-process samples. Treat these QC aliquots in the same fashion as the associated samples, adding ascorbic acid to the pair associated with the final effluents, and not adding ascorbic acid to the pair associated with the in-process samples.

11.1.6 Ascorbic acid: Added to stabilize chlorocatechols. However, for pulp and paper industry in-process streams and other in-process wastewaters, the addition of ascorbic acid may convert chloro-o-quinones to catechols if these quinones are present. Separate calibration curves must be prepared with and without the addition of ascorbic acid (Section 7.13.2).

11.1.6.1 Spike 5 to 6 mL of the ascorbic acid solution (Section 7.2.2) into each final effluent sample, and the associated calibration standards, IPR and OPR aliquots, and blank.

11.1.6.2 For pulp and paper industry C-stage filtrates, E-stage filtrates, and untreated effluents, omit the ascorbic acid to prevent the conversion of chloro-o-quinones to catechols. Prepare calibration standards, IPR and OPR aliquots, and blanks associated with these samples without ascorbic acid as well.

11.1.7 Spike 1000 µL of the labeled compound spiking solution (Section 7.8) into the sample and QC aliquots.

11.1.8 Spike 500 µL of the nominal 50 µg/mL calibration solution (Section 7.13.4) into the OPR aliquot.

11.1.9 Adjust the pH of the sample aliquots to between 7.0 and 7.1. For calibration standards, IPR and OPR aliquots, and blanks, pH adjustment is not required.

11.1.10 Equilibrate all sample and QC solutions for approximately 15 minutes, with occasional stirring.

11.2 Derivatization: Because derivatization must proceed rapidly, particularly upon the addition of the K2CO3 buffer, it is necessary to work with one sample at a time until the derivatization step (Section 11.2.3) is complete.

11.2.1 Place a beaker containing a sample or QC aliquot on the magnetic stirrer in a fume hood, drop a clean stirring bar into the beaker, and increase the speed of the stirring bar until the vortex is drawn to the bottom of the beaker.

11.2.2 Measure 25 to 26 mL of K2CO3 buffer into a graduated cylinder or other container and 25 to 26 mL of acetic acid into another.

11.2.3 Add the K2CO3 buffer to the sample or QC aliquot, immediately (within one to three seconds) add the acetic anhydride, and stir for three to five minutes to complete the derivatization.

11.3 Extraction: Two procedures are described below for the extraction of derivatized samples. The choice of extraction procedure will depend on the sample type. For final effluent samples, either of two procedures may be utilized for extraction of derivatized samples. For samples of in-process wastewaters, the separatory funnel extraction procedure must be used.

Note: Whichever procedure is employed, the same extraction procedure must be used for calibration standards, IPR aliquots, OPR aliquots, blanks, and the associated field samples.

11.3.1 Stir-bar extraction of final effluents.

11.3.1.1 Add 200 mL (±20 mL) of hexane to the beaker and stir for three to five minutes, drawing the vortex to the bottom of the beaker.

11.3.1.2 Stop the stirring and drain the hexane and a portion of the water into a 500-to 1000-mL separatory funnel. Allow the layers to separate.

11.3.1.3 Drain the aqueous layer back into the beaker.

11.3.1.4 The formation of emulsions can be expected in any solvent extraction procedure. If an emulsion forms, the laboratory must take steps to break the emulsion before proceeding. Mechanical means of breaking the emulsion include the use of a glass stirring rod, filtration through glass wool, and other techniques. For emulsions that resist these techniques, centrifugation is nearly 100% effective.

If centrifugation is employed to break the emulsion, drain the organic layer into a centrifuge tube, cap the tube, and centrifuge for two to three minutes or until the phases separate. If the emulsion cannot be completely broken, collect as much of the organic phase as possible, and measure and record the volume of the organic phase collected.

If all efforts to break the emulsion fail, including centrifugation, and none of the organic phase can be collected, proceed with the dilute aliquot (Section 11.1.4.2). However, use of the dilute aliquot will sacrifice the sensitivity of the method, and may not be appropriate in all cases.

11.3.1.5 Drain the organic layer into a Kuderna-Danish (K-D) apparatus equipped with a 10-mL concentrator tube. Label the K-D apparatus. It may be necessary to pour the organic layer through a funnel containing anhydrous sodium sulfate to remove any traces of water from the extract.

11.3.1.6 Repeat the extraction (Section 11.3.1.1 through 11.3.1.5) two more times using another 200-mL of hexane for each extraction, combining the extracts in the K-D apparatus.

11.3.1.7 Proceed with concentration of the extract, as described in Section 11.4.

11.3.2 Separatory funnel extraction of either final effluents or in-process wastewaters.

11.3.2.1 Transfer the derivatized sample or QC aliquot to a 2-L separatory funnel.

11.3.2.2 Add 200 mL (±20 mL) of hexane to the separatory funnel. Cap the funnel and extract the sample by shaking the funnel for two to three minutes with periodic venting.

11.3.2.3 Allow the organic layer to separate from the water phase for a minimum of 10 minutes.

11.3.2.4 Drain the lower aqueous layer into the beaker used for derivatization (Section 11.2), or into a second clean 2-L separatory funnel. Transfer the solvent to a 1000-mL K-D flask. It may be necessary to pour the organic layer through a funnel containing anhydrous sodium sulfate to remove any traces of water from the extract.

11.3.2.5 The formation of emulsions can be expected in any solvent extraction procedure. If an emulsion forms, the laboratory must take steps to break the emulsion before proceeding. Mechanical means of breaking the emulsion include the use of a glass stirring rod, filtration through glass wool, and other techniques. For emulsions that resist these techniques, centrifugation may be required.

If centrifugation is employed to break the emulsion, drain the organic layer into a centrifuge tube, cap the tube, and centrifuge for two to three minutes or until the phases separate. If the emulsion cannot be completely broken, collect as much of the organic phase as possible, and measure and record the volume of the organic phase collected. If all efforts to break the emulsion, including centrifugation, fail and none of the organic phase can be collected, proceed with the dilute aliquot (Section 11.1.4.2). However, use of the dilute aliquot will sacrifice the sensitivity of the method, and may not be appropriate in all cases.

11.3.2.6 If drained into a beaker, transfer the aqueous layer to the 2-L separatory funnel (Section 11.3.2.1). Perform a second extraction using another 200 mL of fresh solvent.

11.3.2.7 Transfer the extract to the 1000-mL K-D flask in Section 11.3.2.4.

11.3.2.8 Perform a third extraction in the same fashion as above.

11.3.2.9 Proceed with concentration of the extract, as described in Section 11.4.

11.4 Macro concentration: Concentrate the extracts in separate 1000-mL K-D flasks equipped with 10-mL concentrator tubes. Add one to two clean boiling chips to the flask and attach a three-ball macro-Snyder column. Prewet the column by adding approximately 1 mL of hexane 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 to 2 mL of hexane. A 5-mL syringe is recommended for this operation.

11.5 Micro-concentration: Final concentration of the extracts may be accomplished using either a micro-Snyder column or nitrogen evaporation.

11.5.1 Micro-Snyder column: Add a clean boiling chip and attach a two-ball micro-Snyder column to the concentrator tube. Prewet the column by adding approximately 0.5 mL hexane 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 to 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 approximately 0.2 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 approximately 0.2 mL of hexane. Adjust to a final volume of 0.5 mL.

11.5.2 Nitrogen evaporation: Transfer the concentrator tube to a nitrogen evaporation device and direct a gentle stream of clean dry nitrogen into the concentrator. Rinse the sides of the concentrator tube with small volumes of hexane, and concentrate the extract to a final volume of 0.5 mL.

11.6 Spike each extract with 10 µL of the 2,2'-difluorobiphenyl IIS (Section 7.10) and transfer the concentrated extract to a clean screw-cap vial using hexane to rinse the concentrator tube. Seal the vial with a PTFE-lined lid, and mark the level on the vial. Label with the sample number and store in the dark at -20 to -10 °C until ready for analysis.

12.0 GCMS Analysis

12.1 Establish the following operating conditions:

Carrier gas flow: Helium at 30 cm/sec at 50 °C

Injector temperature: 300 °C

Initial temperature: 50 °C

Temperature program: 8 °C/min to 270 °C

Final hold: Until after 2,6-dichlorosyringaldehyde elutes

Adjust the GC conditions to meet the requirements in Section 9.6.1.1 and Table 2 for analyte separation and sensitivity. Once optimized, the same GC conditions must be used for the analysis of all standards, blanks, IPR and OPR aliquots, and samples.

12.2 Bring the concentrated extract (Section 11.6) or standard (Sections 7.13 and 7.14) to room temperature and verify that any precipitate has redissolved. Verify the level on the extract (Sections 7.13, 7.14, and 11.6) and bring to the mark with solvent if required.

12.3 Inject a 1-µL volume of the standard solution or extract using on-column or splitless injection. For 0.5 mL extracts, this 1-µL injection volume will contain 50 ng of the DFB internal standard. If an injection volume other than 1 µL is used, that volume must contain 50 ng of DFB.

12.4 Start the GC column temperature ramp upon injection. Start MS data collection after the solvent peak elutes. Stop data collection after the 2,6-dichlorosyringaldehyde peak elutes. Return the column to the initial temperature for analysis of the next sample.

13.0 Analysis of Complex Samples

Some samples may contain high levels (>1000 µg/L) of the compounds of interest, interfering compounds, and/or other phenolic materials. Some samples will not concentrate to 0.5 mL (Section 11.5); others will overload the GC column and/or mass spectrometer; others may contain amounts of phenols that may exceed the capacity of the derivatizing agent.

13.1 Analyze the dilute aliquot (Section 11.1.4) when the sample will not concentrate to 0.5 mL. If a dilute aliquot was not extracted, and the sample holding time (Section 8.4) has not been exceeded, dilute an aliquot of sample with reagent water, and derivatize and extract it (Section 11.1.4). Otherwise, dilute the extract (Section 14.7.3) and quantitate it by the internal standard method (Section 14.3).

13.2 Recovery of the 2,2'-difluorobiphenyl instrument internal standard: The EICP area of the internal standard should be within a factor of two of the area in the OPR or VER standard (Section 9.6). If the absolute areas of the labeled compounds and the SMIS are within a factor of two of the respective areas in the OPR or VER standard, and the DFB internal standard area is less than one-half of its respective area, then internal standard loss in the extract has occurred. In this case, analyze the extract from the dilute aliquot (Section 11.1.4).

13.3 Recovery of labeled compounds and the sample matrix internal standard (SMIS): SMIS and labeled compound recovery specifications have been developed for samples with and without the addition of ascorbic acid. Compare the recoveries to the appropriate limits in Table 5.

13.3.1 If SMIS or labeled compound recoveries are outside the limits given in Table 5 and the associated OPR analysis meets the recovery criteria, the extract from the dilute aliquot (Section 11.1.4) is analyzed as in Section 14.7.

13.3.2 If labeled compound or SMIS recovery is outside the limits given in Table 5 and the associated OPR analysis did not meet recovery criteria, a problem in the derivatization/extraction/concentration of the sample is indicated, and the sample must be rederivatized and reanalyzed.

14.0 Data Analysis and Calculations

14.1 Qualitative determination: Identification is accomplished by comparison of data from analysis of a sample or blank with data stored in the mass spectral libraries. Identification of a compound is confirmed when the following criteria are met:

14.1.1 The signals for m/z 43 (to indicate the presence of the acetyl derivative) and all characteristic m/z's stored in the spectral library (Section 10.2.4) shall be present and shall maximize within the same two consecutive scans.

14.1.2 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 m/z's stored in the library.

14.1.3 The relative retention time shall be within the window specified in Table 2.

14.1.4 The m/z's present in the mass spectrum from the component in the sample that are not present in the reference mass spectrum shall be accounted for by contaminant or background ions. If the mass spectrum is contaminated, an experienced spectrometrist (Section 1.4) shall determine the presence or absence of the compound.

14.2 Quantitative determination by isotope dilution: By adding a known amount of a labeled compound to every sample prior to derivatization and extraction, correction for recovery of the pollutant can be made because the pollutant and its labeled analog exhibit the same effects upon derivatization, extraction, concentration, and gas chromatography. Relative response (RR) values for sample mixtures are used in conjunction with calibration curves described in Section 10.4 to determine concentrations directly, so long as labeled compound spiking levels are constant. For the phenol example given in Figure 1 (Section 10.4.1), 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).

14.2.1 Compute the concentration in the extract using the response ratio determined from calibration data (Section 10.4) and the following equation:


Where:

Cex = concentration of the pollutant in the extract.

An = area of the characteristic m/z for the pollutant.

Cl = concentration of the labeled compound in the extract.

Al = area of the characteristic m/z for the labeled compound.

RR = response ratio from the initial calibration.

14.2.2 For the IPR (Section 9.3.2) and OPR (Section 9.6), compute the percent recovery of each pollutant using the equation in Section 14.6. The percent recovery is used for the evaluation of method and laboratory performance, in the form of IPR (Section 9.3.2) and OPR (Section 9.6).

14.3 Quantitative determination by internal standard: Compute the concentration using the response factor determined from calibration data (Section 10.5) and the following equation:


Where:

Cex = concentration of the pollutant in the extract.

As = area of the characteristic m/z for the pollutant.

Cis = concentration of the internal standard in the extract (see note below).

Ais = area of the characteristic m/z for the internal standard.

RF = response factor from the initial calibration.

Note: When this equation is used to compute the extract concentrations of native compounds without labeled analogs, use the area (Ais) and concentration (Cis) of 3,4,5-trichlorophenol (SMIS) as the internal standard.

For the IPR (Section 9.3.2) and OPR (Section 9.6), compute the percent recovery using the equation in Section 14.6.

Note: Separate calibration curves will be required for samples with and without the addition of ascorbic acid, and also for both extraction procedures (stir-bar and separatory funnel) where applicable.

14.4 Compute the concentration of the labeled compounds and the SMIS using the equation in Section 14.3, but using the area and concentration of the 2,2'-difluorobiphenyl as the internal standard, and the area of the labeled compound or SMIS as As.

14.5 Compute the concentration of each pollutant compound in the sample using the following equation:


Where:

Cs = Concentration of the pollutant in the sample.

Cex = Concentration of the pollutant in the extract.

Vex = Volume of the concentrated extract (typically 0.5 mL).

Vo = Volume of the original sample in liters.

14.6 Compute the recovery of each labeled compound and the SMIS as the ratio of concentration (or amount) found to the concentration (or amount) spiked, using the following equation:


These percent recoveries are used to assess method performance according to Sections 9 and 13.

14.7 If the EICP area at the quantitation m/z for any compound exceeds the calibration range of the system, three approaches are used to obtain results within the calibration range.

14.7.1 If the recoveries of all the labeled compounds in the original sample aliquot meet the limits in Table 5, then the extract of the sample may be diluted by a maximum of a factor of 10, and the diluted extract reanalyzed.

14.7.2 If the recovery of any labeled compound is outside its limits in Table 5, or if a tenfold dilution of the extract will not bring the pollutant within the calibration range, then extract and analyze a dilute aliquot of the sample (Section 11). Dilute 100 mL, 10 mL, or an appropriate volume of sample to 1000 mL with reagent water and extract per Section 11.

14.7.3 If the recoveries of all labeled compounds in the original sample aliquot (Section 14.7.1) meet the limits in Table 5, and if the sample holding time has been exceeded, then the original sample extract is diluted by successive factors of 10, the DFB internal standard is added to give a concentration of 50 µg/mL in the diluted extract, and the diluted extract is analyzed. Quantitation of all analytes is performed using the DFB internal standard.

14.7.4 If the recoveries of all labeled compounds in the original sample aliquot (Section 14.7.1) or in the dilute aliquot (Section 14.7.2) (if a dilute aliquot was analyzed) do not meet the limits in Table 5, and if the holding time has been exceeded, re-sampling is required.

14.8 Results are reported for all pollutants, labeled compounds, and the sample matrix internal standard in standards, blanks, and samples, in units of µg/L.

14.8.1 Results for samples which have been diluted are reported at the least dilute level at which the area at the quantitation m/z is within the calibration range (Section 14.7).

14.8.2 For compounds having a labeled analog, results are reported at the least dilute level at which the area at the quantitation m/z is within the calibration range (Section 14.7) and the labeled compound recovery is within the normal range for the method (Section 13.3).

15.0 Method Performance

15.1 Single laboratory performance for this method is detailed in References 1, 2, and 11. Acceptance criteria were established from multiple laboratory use of the draft method.

15.2 A chromatogram of the ongoing precision and recovery standard (Section 7.14) is shown in Figure 4.

16.0 Pollution Prevention

16.1 The solvents used in this method pose little threat to the environment when recycled and managed properly.

16.2 Standards should be prepared in volumes consistent with laboratory use to minimize the volume of expired standards to be disposed.

17.0 Waste Management

17.1 It is the laboratory's responsibility to comply with all federal, state, and local regulations governing waste management, particularly the hazardous waste identification rules and land disposal restrictions, and to protect the air, water, and land by minimizing and controlling all releases from fume hoods and bench operations. Compliance with all sewage discharge permits and regulations is also required.

17.2 Samples preserved with HCl or H2SO4 to pH < 2 are hazardous and must be neutralized before being disposed, or must be handled as hazardous waste.

17.3 For further information on waste management, consult “The Waste Management Manual for Laboratory Personnel”, and “Less is Better: Laboratory Chemical Management for Waste Reduction”, both available from the American Chemical Society's Department of Government Relations and Science Policy, 1155 16th Street N.W., Washington, DC 20036.

18.0 References

18.1 “Chlorinated Phenolics in Water by In Situ Acetylation/GC/MS Determination,” Method CP–86.01, National Council of the Paper Industry for Air and Stream Improvement, Inc., 260 Madison Avenue, New York, NY 10016 (July 1986).

18.2 “6240-Chlorinated Phenolics (Interim Standard),” Draft Version, U.S. Environmental Protection Agency, Manchester Laboratory, Manchester, Washington.

18.3 “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).

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

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

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

18.7 “Interlaboratory Validation of U. S. Environmental Protection Agency Method 1625A, Addendum Report,” SRI International, Prepared for Analysis and Evaluation Division (WH–557), USEPA, 401 M St., SW., Washington, DC 20460 (January 1985).

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

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

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

18.11 “Determination of Chlorophenolics, Special Analytical Services Contract 1047, Episode 1886,” Analytical Technologies, Inc., Prepared for W. A. Telliard, Industrial Technology Division (WH–552), USEPA, 401 M St., SW., Washington, DC 20460 (June 1990).

18.12 “Determination of Chlorophenolics by GCMS, Development of Method 1653,” Analytical Technologies, Inc., Prepared for W. A. Telliard, Industrial Technology Division (WH–552), USEPA, 401 M St., SW., Washington, DC 20460 (May 1991).

19.0 Tables and Figures


Table 1_Chlorophenolic Compounds Determined by GCMS Using Isotope Dilution and Internal Standard Techniques
----------------------------------------------------------------------------------------------------------------
Pollutant Labeled compound
Compound -----------------------------------------------------------------------------
CAS registry EPA-EGD Analog CAS registry EPA-EGD
----------------------------------------------------------------------------------------------------------------
4-chlorophenol.................... 106-48-9 1001
2,4-dichlorophenol................ 120-83-2 1002 d3 93951-74-7 1102
2,6-dichlorophenol................ 87-65-0 1003
2,4,5-trichlorophenol............. 95-95-4 1004
2,4,6-trichlorophenol............. 88-06-2 1005
2,3,4,6-tetrachlorophenol......... 58-90-2 1006
pentachlorophenol................. 87-86-5 1007 \13\C6 85380-74-1 1107
4-chloroguaiacol.................. 16766-30-6 1008 \13\C6 136955-39-0 1108
3,4-dichloroguaiacol.............. 77102-94-4 1009
4,5-dichloroguaiacol.............. 2460-49-3 1010
4,6-dichloroguaiacol.............. 16766-31-7 1011
3,4,5-trichloroguaiacol........... 57057-83-7 1012
3,4,6-trichloroguaiacol........... 60712-44-9 1013
4,5,6-trichloroguaiacol........... 2668-24-8 1014 \13\C6 136955-40-3 1114
tetrachloroguaiacol............... 2539-17-5 1015 \13\C6 136955-41-4 1115
4-chlorocatechol.................. 2138-22-9 1016
3,4-dichlorocatechol.............. 3978-67-4 1017
3,6-dichlorocatechol.............. 3938-16-7 1018
4,5-dichlorocatechol.............. 3428-24-8 1019 \13\C6 136955-42-5 1119
3,4,5-trichlorocatechol........... 56961-20-7 1020
3,4,6-trichlorocatechol........... 32139-72-3 1021
tetrachlorocatechol............... 1198-55-6 1022 \13\C6 136955-43-6 1122
5-chlorovanillin.................. 19463-48-0 1023 \13\C6 136955-44-7 1123
6-chlorovanillin.................. 18268-76-3 1024
5,6-dichlorovanillin.............. 18268-69-4 1025
2-chlorosyringaldehyde............ 76341-69-0 1026
2,6-dichlorosyringaldehyde........ 76330-06-8 1027
trichlorosyringol................. 2539-26-6 1028
Sample matrix internal standard
(SMIS)
3,4,5-trichlorophenol............. 609-19-8 184
Instrument internal standard (IIS)
2,2[prime]-difluorobiphenyl....... 388-82-9 164
----------------------------------------------------------------------------------------------------------------




Table 2_Gas Chromatography and Method Detection Limits for Chlorophenolics
----------------------------------------------------------------------------------------------------------------
Minimum
Retention EGD ref level \4\ MDL \5\
EGD No. \1\ Compound time mean No. RRT window \3\ (µg/ (µg/
(sec) \2\ L) L)
----------------------------------------------------------------------------------------------------------------
1001........................... 4-chlorophenol... 691 184 0.651-0.681 1.25 1.11
1003........................... 2,6- 796 184 0.757-0.779 2.5 1.39
dichlorophenol.
1102........................... 2,4- 818 164 0.986-0.998
dichlorophenol-
d3.
1202........................... 2,4- 819 1102 0.997-1.006 2.5 0.15
dichlorophenol.
164............................ 2,2[prime]- 825 164 1.000
difluorobiphenyl
(I.S.).
1108........................... 4-chloroguaiacol- 900 164 1.077-1.103
\13\C6.
1208........................... 4-chloroguaiacol. 900 1108 0.998-1.002 1.25 0.09
1005........................... 2,4,6- 920 184 0.879-0.895 2.5 0.71
trichlorophenol.
1004........................... 2,4,5- 979 184 0.936-0.952 2.5 0.57
trichlorophenol.
1016........................... 4-chlorocatechol. 1004 184 0.961-0.975 1.25 0.59
1011........................... 4,6- 1021 184 0.979-0.991 2.5 0.45
dichloroguaiacol.
1009........................... 3,4- 1029 184 0.986-0.998 2.5 0.52
dichloroguaiacol.
184............................ 3,4,5- 1037 164 1.242-1.272
trichlorophenol
(I.S.).
1010........................... 4,5- 1071 184 1.026-1.040 2.5 0.52
dichloroguaiacol.
1018........................... 3,6- 1084 184 1.037-1.053 2.5 0.57
dichlorocatechol.
1006........................... 2,3,4,6- 1103 184 1.050-1.078 2.5 0.38
tetrachloropheno
l.
1123........................... 5-chlorovanillin- 1111 164 1.327-1.367
\13\C6.
1223........................... 5-chlorovanillin. 1111 1123 0.998-1.001 2.5 1.01
1013........................... 3,4,6- 1118 184 1.066-1.090 2.5 0.46
trichloroguaiaco
l.
1024........................... 6-chlorovanillin. 1122 184 1.070-1.094 2.5 0.94
1017........................... 3,4- 1136 184 1.083-1.105 2.5 0.60
dichlorocatechol.
1119........................... 4,5- 1158 164 1.384-1.424
dichlorocatechol-
\13\C6.
1219........................... 4,5- 1158 1119 0.998-1.001 2.5 0.24
dichlorocatechol.
1012........................... 3,4,5- 1177 184 1.120-1.160 2.5 0.49
trichloroguaiaco
l.
1114........................... 4,5,6- 1208 164 1.444-1.484
trichloroguaiaco
l-\13\C6.
1214........................... 4,5,6- 1208 1114 0.998-1.002 2.5 0.25
trichloroguaiaco
l.
1021........................... 3,4,6- 1213 184 1.155-1.185 5.0 0.44
trichlorocatecho
l.
1025........................... 5,6- 1246 184 1.182-1.222 5.0 0.80
dichlorovanillin.
1026........................... 2- 1255 184 1.190-1.230 2.5 0.87
chlorosyringalde
hyde.
1107........................... pentachlorophenol- 1267 164 1.511-1.561
\13\C6.
1207........................... pentachlorophenol 1268 1107 0.998-1.002 5.0 0.28
1020........................... 3,4,5- 1268 184 1.208-1.238 5.0 0.53
trichlorocatecho
l.
1115........................... tetrachloroguaiac 1289 164 1.537-1.587
ol-\13\C6.
1215........................... tetrachloroguaiac 1290 1115 0.998-1.002 5.0 0.23
ol.
1028........................... trichlorosyringol 1301 184 1.240-1.270 2.5 0.64
1122........................... tetrachlorocatech 1365 164 1.630-1.690
ol-\13\C6.
1222........................... tetrachlorocatech 1365 1122 0.998-1.002 5.0 0.76
ol.
1027........................... 2,6- 1378 184 1.309-1.349 5.0 1.13
dichlorosyringal
dehyde.
----------------------------------------------------------------------------------------------------------------
\1\ Four digit numbers beginning with 10 indicate a pollutant quantified by the internal standard method; four
digit numbers beginning with 11 indicate a labeled compound quantified by the internal standard method; four
digit numbers beginning with 12 indicate a pollutant quantified by isotope dilution.
\2\ The retention times in this column are based on data from a single laboratory (reference 12), utilizing the
GC conditions in Section 11.
\3\ Relative retention time windows are estimated from EPA Method 1625.
\4\ The minimum level (ML) is defined as the level at which the entire analytical system must give a
recognizable signal and acceptable calibration point for the analyte. It is equivalent to the concentration of
the lowest calibration standard, assuming that all method-specified sample weights, volumes, and cleanup
procedures have been employed.
\5\ 40 CFR Part 136, Appendix B; from reference 2.




Table 3_DFTPP Mass Intensity Specifications \1\
------------------------------------------------------------------------
Mass Intensity required
------------------------------------------------------------------------
51........................... 8 to 82% of m/z 198.
68........................... Less than 2% of m/z 69.
69........................... 11 to 91% of m/z 198.
70........................... Less than 2% of m/z 69.
127.......................... 32 to 59% of m/z 198.
197.......................... Less than 1% of m/z 198.
198.......................... Base peak, 100% abundance.
199.......................... 4 to 9% of m/z 198.
275.......................... 11 to 30% ofm/z 198. (continued)