that

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 ug/mL. The amount of SMIS added to each solution is the same (25 ug/mL) so that Cis remains constant. Likewise, the concentration of IIS is constant in each solution. The area ratio (A, A,s) is plotted versus the concentration ratio (C. 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 fivepoint 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.

11.1.4.1 Complex samples: For samples

are expected to be difficult to derivatize, concentrate, or are expected to overload the GC column or mass spectrometer, measure an additional 100 mL (11 mL) into a clean 2000-ml beaker and dilute to a final volume of 1000-mL (150 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 (250 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 1000mL (+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 inprocess 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 Cstage 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 uL of the labeled compound spiking solution (Section 7.8) into the sample and QC aliquots.

11.1.8 Spike 500 uL of the nominal 50 ug/ 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.

11.0 Sample Derivatization, Extraction, and

Concentration The procedure described in this section uses а 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).

11.1.10 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 (120 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 500to 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 KD 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 ertraction, 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 KD 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 1000mL 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 microSnyder 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 uL 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 PTFElined 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

2,6dichlorosyringaldehyde 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-uL volume of the standard solution or extract using on-column or splitless injection. For 0.5 mL extracts, this 1-uL injection volume will contain 50 ng of the DFB internal standard. If an injection volume other than 1 uL 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 da collection

after
the

2,6dichlorosyringaldehyde peak elutes. Return the column to the initial temperature for analysis of the next sample.

13.0 Analysis of Compler Samples Some samples may contain high levels (>1000 ug/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 irr 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, а problem in the derivatization/extraction concentration of the sample is indicated, and the sample must be rederivatized and reanalyzed.

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 ug/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:

Cex (ug/mL) = (A, XC)/(A, RRR)

Where:
Cex concentration of the pollutant in the

extract. An = area of the characteristic miz for the

pollutant. C = concentration of the labeled compound

in the extract. A, = area of the characteristic m/z for the la

beled compound. RR = response ratio from the initial calibra

tion.

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:

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 mz'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 mz'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.

Cex (ug/mL) = (A, Cis)/(Ais XRF)

Where:
Cex = concentration of the pollutant in the

extract. A, = area of the characteristic mz for the

pollutant. Cis = concentration of the internal standard

in the extract (see note below). Ais = area of the characteristic m/z for the in

ternal standard. RF = response factor from the initial calibra

as

area (Ais) and concentration (Cs) of 3,4,5trichlorophenol (SMIS) 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 2,2difluorobiphenyl as the internal standard, and the area of the labeled compound or SMIS as As.

Where:
Cs = Concentration of the pollutant in the

sample. Cex = Concentration of the pollutant in the

extract. Ve = 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 míz 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 ug/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 ug/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.