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10.2 Ion Abundance Ratios, Minimum Levels, Signal-to-Noise Ratios, and Absolute Retention Times—Choose an injection volume of either 1 µL or 2 µL, consistent with the capability of the HRGC/HRMS instrument. Inject a 1 µL or 2 µL aliquot of the CS1 calibration solution (Table 4) using the GC conditions from Section 10.1.1. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, the operating conditions and specifications below apply to analysis of those compounds only.

10.2.1 Measure the SICP areas for each analyte, and compute the ion abundance ratios at the exact m/z's specified in Table 8. Compare the computed ratio to the theoretical ratio given in Table 9.

10.2.1.1 The exact m/z's to be monitored in each descriptor are shown in Table 8. Each group or descriptor shall be monitored in succession as a function of GC retention time to ensure that all CDDs/CDFs are detected. Additional m/z's may be monitored in each descriptor, and the m/z's may be divided among more than the five descriptors listed in Table 8, provided that the laboratory is able to monitor the m/z's of all the CDDs/CDFs that may elute from the GC in a given retention-time window. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, the descriptors may be modified to include only the exact m/z's for the tetra-and penta-isomers, the diphenyl ethers, and the lock m/z's.

10.2.1.2 The mass spectrometer shall be operated in a mass-drift correction mode, using perfluorokerosene (PFK) to provide lock m/z's. The lock-mass for each group of m/z's is shown in Table 8. Each lock mass shall be monitored and shall not vary by more than ±20% throughout its respective retention time window. Variations of the lock mass by more than 20% indicate the presence of coeluting interferences that may significantly reduce the sensitivity of the mass spectrometer. Reinjection of another aliquot of the sample extract will not resolve the problem. Additional cleanup of the extract may be required to remove the interferences.

10.2.2 All CDDs/CDFs and labeled compounds in the CS1 standard shall be within the QC limits in Table 9 for their respective ion abundance ratios; otherwise, the mass spectrometer shall be adjusted and this test repeated until the m/z ratios fall within the limits specified. If the adjustment alters the resolution of the mass spectrometer, resolution shall be verified (Section 10.1.2) prior to repeat of the test.

10.2.3 Verify that the HRGC/HRMS instrument meets the minimum levels in Table 2. The peaks representing the CDDs/CDFs and labeled compounds in the CS1 calibration standard must have signal-to-noise ratios (S/N) greater than or equal to 10.0. Otherwise, the mass spectrometer shall be adjusted and this test repeated until the minimum levels in Table 2 are met.

10.2.4 The absolute retention time of 13C12-1,2,3,4–TCDD (Section 7.12) shall exceed 25.0 minutes on the DB–5 column, and the retention time of 13C12-1,2,3,4–TCDD shall exceed 15.0 minutes on the DB–225 column; otherwise, the GC temperature program shall be adjusted and this test repeated until the above-stated minimum retention time criteria are met.

2010.3 Retention-Time Windows—Analyze the window defining mixtures (Section 7.15) using the optimized temperature program in Section 10.1. Table 5 gives the elution order (first/last) of the window-defining compounds. If 2,3,7,8-TCDD and 2,3,7,8-TCDF only are to be analyzed, this test is not required.

10.4 Isomer Specificity.

10.4.1 Analyze the isomer specificity test standards (Section 7.15) using the procedure in Section 14 and the optimized conditions for sample analysis (Section 10.1.1).

10.4.2 Compute the percent valley between the GC peaks that elute most closely to the 2,3,7,8-TCDD and TCDF isomers, on their respective columns, per Figures 6 and 7.

10.4.3 Verify that the height of the valley between the most closely eluted isomers and the 2,3,7,8-substituted isomers is less than 25% (computed as 100 x/y in Figures 6 and 7). If the valley exceeds 25%, adjust the analytical conditions and repeat the test or replace the GC column and recalibrate (Sections 10.1.2 through 10.7).

10.5 Calibration by Isotope Dilution—Isotope dilution calibration is used for the 15 2,3,7,8-substituted CDDs/CDFs for which labeled compounds are added to samples prior to extraction. The reference compound for each CDD/CDF compound is shown in Table 2.

10.5.1 A calibration curve encompassing the concentration range is prepared for each compound to be determined. The relative response (RR) (labeled to native) vs. concentration in standard solutions is plotted or computed using a linear regression. Relative response is determined according to the procedures described below. Five calibration points are employed.

10.5.2 The response of each CDD/CDF relative to its labeled analog is determined using the area responses of both the primary and secondary exact m/z's specified in Table 8, for each calibration standard, as follows:


where:

A1n and A2n = The areas of the primary and secondary m/z's for the CDD/CDF.

A1l and A2l = The areas of the primary and secondary m/z's for the labeled compound.

Cl = The concentration of the labeled compound in the calibration standard (Table 4).

Cn = The concentration of the native compound in the calibration standard (Table 4).

10.5.3 To calibrate the analytical system by isotope dilution, inject a volume of calibration standards CS1 through CS5 (Section 7.13 and Table 4) identical to the volume chosen in Section 10.2, using the procedure in Section 14 and the conditions in Section 10.1.1 and Table 2. Compute the relative response (RR) at each concentration.

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

10.6 Calibration by Internal Standard—The internal standard method is applied to determination of 1,2,3,7,8,9-HxCDD (Section 17.1.2), OCDF (Section 17.1.1), the non 2,3,7,8-substituted compounds, and to the determination of labeled compounds for intralaboratory statistics (Sections 9.4 and 15.5.4).

10.6.1 Response factors—Calibration requires the determination of response factors (RF) defined by the following equation:


where:

A1s and A2s = The areas of the primary and secondary m/z's for the CDD/CDF.

A1is and A2is = The areas of the primary and secondary m/z's for the internal standard.

Cis = The concentration of the internal standard (Table 4).

Cs = The concentration of the compound in the calibration standard (Table 4).

Note: There is only one m/z for 37Cl4-2,3,7,8-TCDD. See Table 8.

10.6.2 To calibrate the analytical system by internal standard, inject 1.0 µL or 2.0 µL of calibration standards CS1 through CS5 (Section 7.13 and Table 4) using the procedure in Section 14 and the conditions in Section 10.1.1 and Table 2. Compute the response factor (RF) at each concentration.

10.6.3 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.

10.7 Combined Calibration—By using calibration solutions (Section 7.13 and Table 4) containing the CDDs/CDFs and 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 15.3) by analyzing the calibration verification standard (VER, Table 4). Recalibration is required if any of the calibration verification criteria (Section 15.3) cannot be met.

10.8 Data Storage—MS data shall be collected, recorded, and stored.

10.8.1 Data acquisition—The signal at each exact m/z shall be collected repetitively throughout the monitoring period and stored on a mass storage device.

10.8.2 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. Computations of relative standard deviation (coefficient of variation) shall be used to test calibration linearity. Statistics on initial performance (Section 9.2) and ongoing performance (Section 15.5) should be computed and maintained, either on the instrument data system, or on a separate computer system.

11.0 Sample Preparation

11.1 Sample preparation involves modifying the physical form of the sample so that the CDDs/CDFs can be extracted efficiently. In general, the samples must be in a liquid form or in the form of finely divided solids in order for efficient extraction to take place. Table 10 lists the phases and suggested quantities for extraction of various sample matrices.

For samples known or expected to contain high levels of the CDDs/CDFs, the smallest sample size representative of the entire sample should be used (see Section 17.5).

For all samples, the blank and IPR/OPR aliquots must be processed through the same steps as the sample to check for contamination and losses in the preparation processes.

11.1.1 For samples that contain particles, percent solids and particle size are determined using the procedures in Sections 11.2 and 11.3, respectively.

11.1.2 Aqueous samples—Because CDDs/CDFs may be bound to suspended particles, the preparation of aqueous samples is dependent on the solids content of the sample.

11.1.2.1 Aqueous samples visibly absent particles are prepared per Section 11.4 and extracted directly using the separatory funnel or SPE techniques in Sections 12.1 or 12.2, respectively.

11.1.2.2 Aqueous samples containing visible particles and containing one percent suspended solids or less are prepared using the procedure in Section 11.4. After preparation, the sample is extracted directly using the SPE technique in 12.2 or filtered per Section 11.4.3. After filtration, the particles and filter are extracted using the SDS procedure in Section 12.3 and the filtrate is extracted using the separatory funnel procedure in Section 12.1.

11.1.2.3 For aqueous samples containing greater than one percent solids, a sample aliquot sufficient to provide 10 g of dry solids is used, as described in Section 11.5.

11.1.3 Solid samples are prepared using the procedure described in Section 11.5 followed by extraction via the SDS procedure in Section 12.3.

11.1.4 Multiphase samples—The phase(s) containing the CDDs/CDFs is separated from the non-CDD/CDF phase using pressure filtration and centrifugation, as described in Section 11.6. The CDDs/CDFs will be in the organic phase in a multiphase sample in which an organic phase exists.

11.1.5 Procedures for grinding, homogenization, and blending of various sample phases are given in Section 11.7.

11.1.6 Tissue samples—Preparation procedures for fish and other tissues are given in Section 11.8.

11.2 Determination of Percent Suspended Solids.

Note: This aliquot is used for determining the solids content of the sample, not for determination of CDDs/CDFs.

11.2.1 Aqueous liquids and multi-phase samples consisting of mainly an aqueous phase.

11.2.1.1 Dessicate and weigh a GF/D filter (Section 6.5.3) to three significant figures.

11.2.1.2 Filter 10.0 ±0.02 mL of well-mixed sample through the filter.

11.2.1.3 Dry the filter a minimum of 12 hours at 110 ±5 °C and cool in a dessicator.

11.2.1.4 Calculate percent solids as follows:


11.2.2 Non-aqueous liquids, solids, semi-solid samples, and multi-phase samples in which the main phase is not aqueous; but not tissues.

11.2.2.1 Weigh 5–10 g of sample to three significant figures in a tared beaker.

11.2.2.2 Dry a minimum of 12 hours at 110 ±5 °C, and cool in a dessicator.

11.2.2.3 Calculate percent solids as follows:


11.3 Determination of Particle Size.

11.3.1 Spread the dried sample from Section 11.2.2.2 on a piece of filter paper or aluminum foil in a fume hood or glove box.

11.3.2 Estimate the size of the particles in the sample. If the size of the largest particles is greater than 1 mm, the particle size must be reduced to 1 mm or less prior to extraction using the procedures in Section 11.7.

11.4 Preparation of Aqueous Samples Containing 1% Suspended Solids or Less.

11.4.1 Aqueous samples visibly absent particles are prepared per the procedure below and extracted directly using the separatory funnel or SPE techniques in Sections 12.1 or 12.2, respectively. Aqueous samples containing visible particles and one percent suspended solids or less are prepared using the procedure below and extracted using either the SPE technique in Section 12.2 or further prepared using the filtration procedure in Section 11.4.3. The filtration procedure is followed by SDS extraction of the filter and particles (Section 12.3) and separatory funnel extraction of the filtrate (Section 12.1). The SPE procedure is followed by SDS extraction of the filter and disk.

11.4.2 Preparation of sample and QC aliquots.

11.4.2.1 Mark the original level of the sample on the sample bottle for reference. Weigh the sample plus bottle to ±1.

11.4.2.2 Spike 1.0 mL of the diluted labeled-compound spiking solution (Section 7.10.3) into the sample bottle. Cap the bottle and mix the sample by careful shaking. Allow the sample to equilibrate for one to two hours, with occasional shaking.

11.4.2.3 For each sample or sample batch (to a maximum of 20 samples) to be extracted during the same 12-hour shift, place two 1.0 L aliquots of reagent water in clean sample bottles or flasks.

11.4.2.4 Spike 1.0 mL of the diluted labeled-compound spiking solution (Section 7.10.3) into both reagent water aliquots. One of these aliquots will serve as the method blank.

11.4.2.5 Spike 1.0 mL of the PAR standard (Section 7.14) into the remaining reagent water aliquot. This aliquot will serve as the OPR (Section 15.5).

11.4.2.6 If SPE is to be used, add 5 mL of methanol to the sample, cap and shake the sample to mix thoroughly, and proceed to Section 12.2 for extraction. If SPE is not to be used, and the sample is visibly absent particles, proceed to Section 12.1 for extraction. If SPE is not to be used and the sample contains visible particles, proceed to the following section for filtration of particles.

11.4.3 Filtration of particles.

11.4.3.1 Assemble a Buchner funnel (Section 6.5.5) on top of a clean filtration flask. Apply vacuum to the flask, and pour the entire contents of the sample bottle through a glass-fiber filter (Section 6.5.6) in the Buchner funnel, swirling the sample remaining in the bottle to suspend any particles.

11.4.3.2 Rinse the sample bottle twice with approximately 5 mL portions of reagent water to transfer any remaining particles onto the filter.

11.4.3.3 Rinse any particles off the sides of the Buchner funnel with small quantities of reagent water.

11.4.3.4 Weigh the empty sample bottle to ±1 g. Determine the weight of the sample by difference. Save the bottle for further use.

11.4.3.5 Extract the filtrate using the separatory funnel procedure in Section 12.1.

11.4.3.6 Extract the filter containing the particles using the SDS procedure in Section 12.3.

11.5 Preparation of Samples Containing Greater Than 1% Solids.

11.5.1 Weigh a well-mixed aliquot of each sample (of the same matrix type) sufficient to provide 10 g of dry solids (based on the solids determination in Section 11.2) into a clean beaker or glass jar.

11.5.2 Spike 1.0 mL of the diluted labeled compound spiking solution (Section 7.10.3) into the sample.

11.5.3 For each sample or sample batch (to a maximum of 20 samples) to be extracted during the same 12-hour shift, weigh two 10 g aliquots of the appropriate reference matrix (Section 7.6) into clean beakers or glass jars.

11.5.4 Spike 1.0 mL of the diluted labeled compound spiking solution (Section 7.10.3) into each reference matrix aliquot. One aliquot will serve as the method blank. Spike 1.0 mL of the PAR standard (Section 7.14) into the other reference matrix aliquot. This aliquot will serve as the OPR (Section 15.5).

11.5.5 Stir or tumble and equilibrate the aliquots for one to two hours.

11.5.6 Decant excess water. If necessary to remove water, filter the sample through a glass-fiber filter and discard the aqueous liquid.

11.5.7 If particles >1mm are present in the sample (as determined in Section 11.3.2), spread the sample on clean aluminum foil in a hood. After the sample is dry, grind to reduce the particle size (Section 11.7).

11.5.8 Extract the sample and QC aliquots using the SDS procedure in Section 12.3.

11.6 Multiphase Samples.

11.6.1 Using the percent solids determined in Section 11.2.1 or 11.2.2, determine the volume of sample that will provide 10 g of solids, up to 1 L of sample.

11.6.2 Pressure filter the amount of sample determined in Section 11.6.1 through Whatman GF/D glass-fiber filter paper (Section 6.5.3). Pressure filter the blank and OPR aliquots through GF/D papers also. If necessary to separate the phases and/or settle the solids, centrifuge these aliquots prior to filtration.

11.6.3 Discard any aqueous phase (if present). Remove any non-aqueous liquid present and reserve the maximum amount filtered from the sample (Section 11.6.1) or 10 g, whichever is less, for combination with the solid phase (Section 12.3.5).

11.6.4 If particles >1mm are present in the sample (as determined in Section 11.3.2) and the sample is capable of being dried, spread the sample and QC aliquots on clean aluminum foil in a hood. After the aliquots are dry or if the sample cannot be dried, reduce the particle size using the procedures in Section 11.7 and extract the reduced particles using the SDS procedure in Section 12.3. If particles >1mm are not present, extract the particles and filter in the sample and QC aliquots directly using the SDS procedure in Section 12.3.

11.7 Sample grinding, homogenization, or blending—Samples with particle sizes greater than 1 mm (as determined in Section 11.3.2) are subjected to grinding, homogenization, or blending. The method of reducing particle size to less than 1 mm is matrix-dependent. In general, hard particles can be reduced by grinding with a mortar and pestle. Softer particles can be reduced by grinding in a Wiley mill or meat grinder, by homogenization, or in a blender.

11.7.1 Each size-reducing preparation procedure on each matrix shall be verified by running the tests in Section 9.2 before the procedure is employed routinely.

11.7.2 The grinding, homogenization, or blending procedures shall be carried out in a glove box or fume hood to prevent particles from contaminating the work environment.

11.7.3 Grinding—Certain papers and pulps, slurries, and amorphous solids can be ground in a Wiley mill or heavy duty meat grinder. In some cases, reducing the temperature of the sample to freezing or to dry ice or liquid nitrogen temperatures can aid in the grinding process. Grind the sample aliquots from Section 11.5.7 or 11.6.4 in a clean grinder. Do not allow the sample temperature to exceed 50 °C. Grind the blank and reference matrix aliquots using a clean grinder.

11.7.4 Homogenization or blending—Particles that are not ground effectively, or particles greater than 1 mm in size after grinding, can often be reduced in size by high speed homogenization or blending. Homogenize and/or blend the particles or filter from Section 11.5.7 or 11.6.4 for the sample, blank, and OPR aliquots.

11.7.5 Extract the aliquots using the SDS procedure in Section 12.3.

11.8 Fish and Other Tissues—Prior to processing tissue samples, the laboratory must determine the exact tissue to be analyzed. Common requests for analysis of fish tissue include whole fish—skin on, whole fish—skin removed, edible fish fillets (filleted in the field or by the laboratory), specific organs, and other portions. Once the appropriate tissue has been determined, the sample must be homogenized.

11.8.1 Homogenization.

11.8.1.1 Samples are homogenized while still frozen, where practical. If the laboratory must dissect the whole fish to obtain the appropriate tissue for analysis, the unused tissues may be rapidly refrozen and stored in a clean glass jar for subsequent use.

11.8.1.2 Each analysis requires 10 g of tissue (wet weight). Therefore, the laboratory should homogenize at least 20 g of tissue to allow for re-extraction of a second aliquot of the same homogenized sample, if re-analysis is required. When whole fish analysis is necessary, the entire fish is homogenized.

11.8.1.3 Homogenize the sample in a tissue homogenizer (Section 6.3.3) or grind in a meat grinder (Section 6.3.4). Cut tissue too large to feed into the grinder into smaller pieces. To assure homogeneity, grind three times.

11.8.1.4 Transfer approximately 10 g (wet weight) of homogenized tissue to a clean, tared, 400–500 mL beaker. For the alternate HCl digestion/extraction, transfer the tissue to a clean, tared 500–600 mL wide-mouth bottle. Record the weight to the nearest 10 mg.

11.8.1.5 Transfer the remaining homogenized tissue to a clean jar with a fluoropolymer-lined lid. Seal the jar and store the tissue at <-10 °C. Return any tissue that was not homogenized to its original container and store at <-10 °C.

11.8.2 QC aliquots.

11.8.2.1 Prepare a method blank by adding approximately 10 g of the oily liquid reference matrix (Section 7.6.4) to a 400–500 mL beaker. For the alternate HCl digestion/extraction, add the reference matrix to a 500–600 mL wide-mouth bottle. Record the weight to the nearest 10 mg.

11.8.2.2 Prepare a precision and recovery aliquot by adding approximately 10 g of the oily liquid reference matrix (Section 7.6.4) to a separate 400–500 mL beaker or wide-mouth bottle, depending on the extraction procedure to be used. Record the weight to the nearest 10 mg. If the initial precision and recovery test is to be performed, use four aliquots; if the ongoing precision and recovery test is to be performed, use a single aliquot.

11.8.3 Spiking

11.8.3.1 Spike 1.0 mL of the labeled compound spiking solution (Section 7.10.3) into the sample, blank, and OPR aliquot.

11.8.3.2 Spike 1.0 mL of the PAR standard (Section 7.14) into the OPR aliquot.

11.8.4 Extract the aliquots using the procedures in Section 12.4.

12.0 Extraction and Concentration

Extraction procedures include separatory funnel (Section 12.1) and solid phase (Section 12.2) for aqueous liquids; Soxhlet/Dean-Stark (Section 12.3) for solids, filters, and SPE disks; and Soxhlet extraction (Section 12.4.1) and HCl digestion (Section 12.4.2) for tissues. Acid/base back-extraction (Section 12.5) is used for initial cleanup of extracts.

Macro-concentration procedures include rotary evaporation (Section 12.6.1), heating mantle (Section 12.6.2), and Kuderna-Danish (K-D) evaporation (Section 12.6.3). Micro-concentration uses nitrogen blowdown (Section 12.7).

12.1 Separatory funnel extraction of filtrates and of aqueous samples visibly absent particles.

12.1.1 Pour the spiked sample (Section 11.4.2.2) or filtrate (Section 11.4.3.5) into a 2 L separatory funnel. Rinse the bottle or flask twice with 5 mL of reagent water and add these rinses to the separatory funnel.

12.1.2 Add 60 mL methylene chloride to the empty sample bottle (Section 12.1.1), seal, and shake 60 seconds to rinse the inner surface. Transfer the solvent to the separatory funnel, and extract the sample by shaking the funnel for two minutes with periodic venting. Allow the organic layer to separate from the aqueous phase for a minimum of 10 minutes. If an emulsion forms and is more than one-third the volume of the solvent layer, employ mechanical techniques to complete the phase separation (see note below). Drain the methylene chloride extract through a solvent-rinsed glass funnel approximately one-half full of granular anhydrous sodium sulfate (Section 7.2.1) supported on clean glass-fiber paper into a solvent-rinsed concentration device (Section 12.6).

Note: If an emulsion forms, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample, but may include stirring, filtration through glass wool, use of phase separation paper, centrifugation, use of an ultrasonic bath with ice, addition of NaCl, or other physical methods. Alternatively, solid-phase or other extraction techniques may be used to prevent emulsion formation. Any alternative technique is acceptable so long as the requirements in Section 9 are met.

Experience with aqueous samples high in dissolved organic materials (e.g., paper mill effluents) has shown that acidification of the sample prior to extraction may reduce the formation of emulsions. Paper industry methods suggest that the addition of up to 400 mL of ethanol to a 1 L effluent sample may also reduce emulsion formation. However, studies by EPA suggest that the effect may be a result of sample dilution, and that the addition of reagent water may serve the same function. Mechanical techniques may still be necessary to complete the phase separation. If either acidification or addition of ethanol is utilized, the laboratory must perform the startup tests described in Section 9.2 using the same techniques.

12.1.3 Extract the water sample two more times with 60 mL portions of methylene chloride. Drain each portion through the sodium sulfate into the concentrator. After the third extraction, rinse the separatory funnel with at least 20 mL of methylene chloride, and drain this rinse through the sodium sulfate into the concentrator. Repeat this rinse at least twice. Set aside the funnel with sodium sulfate if the extract is to be combined with the extract from the particles.

12.1.4 Concentrate the extract using one of the macro-concentration procedures in Section 12.6.

12.1.4.1 If the extract is from a sample visibly absent particles (Section 11.1.2.1), adjust the final volume of the concentrated extract to approximately 10 mL with hexane, transfer to a 250 mL separatory funnel, and back-extract using the procedure in Section 12.5.

12.1.4.2 If the extract is from the aqueous filtrate (Section 11.4.3.5), set aside the concentration apparatus for addition of the SDS extract from the particles (Section 12.3.9.1.2).

12.2 SPE of Samples Containing Less Than 1% Solids (References 19–20).

12.2.1 Disk preparation.

12.2.1.1 Place an SPE disk on the base of the filter holder (Figure 4) and wet with toluene. While holding a GMF 150 filter above the SPE disk with tweezers, wet the filter with toluene and lay the filter on the SPE disk, making sure that air is not trapped between the filter and disk. Clamp the filter and SPE disk between the 1 L glass reservoir and the vacuum filtration flask.

12.2.1.2 Rinse the sides of the filtration flask with approx 15 mL of toluene using a squeeze bottle or syringe. Apply vacuum momentarily until a few drops appear at the drip tip. Release the vacuum and allow the filter/disk to soak for approx one minute. Apply vacuum and draw all of the toluene through the filter/disk. Repeat the wash step with approx 15 mL of acetone and allow the filter/disk to air dry.

12.2.1.3 Re-wet the filter/disk with approximately 15 mL of methanol, allowing the filter/disk to soak for approximately one minute. Pull the methanol through the filter/disk using the vacuum, but retain a layer of methanol approximately 1 mm thick on the filter. Do not allow the disk to go dry from this point until the end of the extraction.

12.2.1.4 Rinse the filter/disk with two 50-mL portions of reagent water by adding the water to the reservoir and pulling most through, leaving a layer of water on the surface of the filter.

12.2.2 Extraction.

12.2.2.1 Pour the spiked sample (Section 11.4.2.2), blank (Section 11.4.2.4), or IPR/OPR aliquot (Section 11.4.2.5) into the reservoir and turn on the vacuum to begin the extraction. Adjust the vacuum to complete the extraction in no less than 10 minutes. For samples containing a high concentration of particles (suspended solids), filtration times may be eight hours or longer.

12.2.2.2 Before all of the sample has been pulled through the filter/disk, rinse the sample bottle with approximately 50 mL of reagent water to remove any solids, and pour into the reservoir. Pull through the filter/disk. Use additional reagent water rinses until all visible solids are removed.

12.2.2.3 Before all of the sample and rinses have been pulled through the filter/disk, rinse the sides of the reservoir with small portions of reagent water.

12.2.2.4 Allow the filter/disk to dry, then remove the filter and disk and place in a glass Petri dish. Extract the filter and disk per Section 12.3.

12.3 SDS Extraction of Samples Containing Particles, and of Filters and/or Disks.

12.3.1 Charge a clean extraction thimble (Section 6.4.2.2) with 5.0 g of 100/200 mesh silica (Section 7.5.1.1) topped with 100 g of quartz sand (Section 7.3.2).

Note: Do not disturb the silica layer throughout the extraction process.

12.3.2 Place the thimble in a clean extractor. Place 30–40 mL of toluene in the receiver and 200–250 mL of toluene in the flask.

12.3.3 Pre-extract the glassware by heating the flask until the toluene is boiling. When properly adjusted, one to two drops of toluene will fall per second from the condenser tip into the receiver. Extract the apparatus for a minimum of three hours.

12.3.4 After pre-extraction, cool and disassemble the apparatus. Rinse the thimble with toluene and allow to air dry.

12.3.5 Load the wet sample, filter, and/or disk from Section 11.4.3.6, 11.5.8, 11.6.4, 11.7.3, 11.7.4, or 12.2.2.4 and any nonaqueous liquid from Section 11.6.3 into the thimble and manually mix into the sand layer with a clean metal spatula, carefully breaking up any large lumps of sample.

12.3.6 Reassemble the pre-extracted SDS apparatus, and add a fresh charge of toluene to the receiver and reflux flask. Apply power to the heating mantle to begin refluxing. Adjust the reflux rate to match the rate of percolation through the sand and silica beds until water removal lessens the restriction to toluene flow. Frequently check the apparatus for foaming during the first two hours of extraction. If foaming occurs, reduce the reflux rate until foaming subsides.

12.3.7 Drain the water from the receiver at one to two hours and eight to nine hours, or sooner if the receiver fills with water. Reflux the sample for a total of 16–24 hours. Cool and disassemble the apparatus. Record the total volume of water collected.

12.3.8 Remove the distilling flask. Drain the water from the Dean-Stark receiver and add any toluene in the receiver to the extract in the flask.

12.3.9 Concentrate the extract using one of the macro-concentration procedures in Section 12.6 per the following:

12.3.9.1 Extracts from the particles in an aqueous sample containing less than 1% solids (Section 11.4.3.6).

12.3.9.1.1 Concentrate the extract to approximately 5 mL using the rotary evaporator or heating mantle procedures in Section 12.6.1 or 12.6.2.

12.3.9.1.2 Quantitatively transfer the extract through the sodium sulfate (Section 12.1.3) into the apparatus that was set aside (Section 12.1.4.2) and reconcentrate to the level of the toluene.

12.3.9.1.3 Adjust to approximately 10 mL with hexane, transfer to a 250 mL separatory funnel, and proceed with back-extraction (Section 12.5).

12.3.9.2 Extracts from particles (Sections 11.5 through 11.6) or from the SPE filter and disk (Section 12.2.2.4)—Concentrate to approximately 10 mL using the rotary evaporator or heating mantle (Section 12.6.1 or 12.6.2), transfer to a 250 mL separatory funnel, and proceed with back-extraction (Section 12.5).

12.4 Extraction of Tissue—Two procedures are provided for tissue extraction.

12.4.1 Soxhlet extraction (Reference 21).

12.4.1.1 Add 30–40 g of powdered anhydrous sodium sulfate to each of the beakers (Section 11.8.4) and mix thoroughly. Cover the beakers with aluminum foil and allow to equilibrate for 12–24 hours. Remix prior to extraction to prevent clumping.

12.4.1.2 Assemble and pre-extract the Soxhlet apparatus per Sections 12.3.1 through 12.3.4, except use the methylene chloride:hexane (1:1) mixture for the pre-extraction and rinsing and omit the quartz sand. The Dean-Stark moisture trap may also be omitted, if desired.

12.4.1.3 Reassemble the pre-extracted Soxhlet apparatus and add a fresh charge of methylene chloride:hexane to the reflux flask.

12.4.1.4 Transfer the sample/sodium sulfate mixture (Section 12.4.1.1) to the Soxhlet thimble, and install the thimble in the Soxhlet apparatus.

12.4.1.5 Rinse the beaker with several portions of solvent mixture and add to the thimble. Fill the thimble/receiver with solvent. Extract for 18–24 hours.

12.4.1.6 After extraction, cool and disassemble the apparatus.

12.4.1.7 Quantitatively transfer the extract to a macro-concentration device (Section 12.6), and concentrate to near dryness. Set aside the concentration apparatus for re-use.

12.4.1.8 Complete the removal of the solvent using the nitrogen blowdown procedure (Section 12.7) and a water bath temperature of 60 °C. Weigh the receiver, record the weight, and return the receiver to the blowdown apparatus, concentrating the residue until a constant weight is obtained.

12.4.1.9 Percent lipid determination—The lipid content is determined by extraction of tissue with the same solvent system (methylene chloride:hexane) that was used in EPA's National Dioxin Study (Reference 22) so that lipid contents are consistent with that study.

12.4.1.9.1 Redissolve the residue in the receiver in hexane and spike 1.0 mL of the cleanup standard (Section 7.11) into the solution.

12.4.1.9.2 Transfer the residue/hexane to the anthropogenic isolation column (Section 13.7.1) or bottle for the acidified silica gel batch cleanup (Section 13.7.2), retaining the boiling chips in the concentration apparatus. Use several rinses to assure that all material is transferred. If necessary, sonicate or heat the receiver slightly to assure that all material is re-dissolved. Allow the receiver to dry. Weigh the receiver and boiling chips.

12.4.1.9.3 Calculate the lipid content to the nearest three significant figures as follows:


12.4.1.9.4 It is not necessary to determine the lipid content of the blank, IPR, or OPR aliquots.

12.4.2 HCl digestion/extraction and concentration (References 23–26).

12.4.2.1 Add 200 mL of 6 N HCl and 200 mL of methylene chloride:hexane (1:1) to the sample and QC aliquots (Section 11.8.4).

12.4.2.2 Cap and shake each bottle one to three times. Loosen the cap in a hood to vent excess pressure. Shake each bottle for 10–30 seconds and vent.

12.4.2.3 Tightly cap and place on shaker. Adjust the shaker action and speed so that the acid, solvent, and tissue are in constant motion. However, take care to avoid such violent action that the bottle may be dislodged from the shaker. Shake for 12–24 hours.

12.4.2.4 After digestion, remove the bottles from the shaker. Allow the bottles to stand so that the solvent and acid layers separate.

12.4.2.5 Decant the solvent through a glass funnel with glass-fiber filter (Sections 6.5.2 through 6.5.3) containing approximately 10 g of granular anhydrous sodium sulfate (Section 7.2.1) into a macro-concentration apparatus (Section 12.6). Rinse the contents of the bottle with two 25 mL portions of hexane and pour through the sodium sulfate into the apparatus.

12.4.2.6 Concentrate the solvent to near dryness using a macro-concentration procedure (Section 12.6).

12.4.2.7 Complete the removal of the solvent using the nitrogen blowdown apparatus (Section 12.7) and a water bath temperature of 60 °C. Weigh the receiver, record the weight, and return the receiver to the blowdown apparatus, concentrating the residue until a constant weight is obtained.

12.4.2.8 Percent lipid determination—The lipid content is determined in the same solvent system [methylene chloride:hexane (1:1)] that was used in EPA's National Dioxin Study (Reference 22) so that lipid contents are consistent with that study.

12.4.2.8.1 Redissolve the residue in the receiver in hexane and spike 1.0 mL of the cleanup standard (Section 7.11) into the solution.

12.4.2.8.2 Transfer the residue/hexane to the narrow-mouth 100–200 mL bottle retaining the boiling chips in the receiver. Use several rinses to assure that all material is transferred, to a maximum hexane volume of approximately 70 mL. Allow the receiver to dry. Weigh the receiver and boiling chips.

12.4.2.8.3 Calculate the percent lipid per Section 12.4.1.9.3. It is not necessary to determine the lipid content of the blank, IPR, or OPR aliquots.

12.4.2.9 Clean up the extract per Section 13.7.3.

12.5 Back-Extraction with Base and Acid.

12.5.1 Spike 1.0 mL of the cleanup standard (Section 7.11) into the separatory funnels containing the sample and QC extracts from Section 12.1.4.1, 12.3.9.1.3, or 12.3.9.2.

12.5.2 Partition the extract against 50 mL of potassium hydroxide solution (Section 7.1.1). Shake for two minutes with periodic venting into a hood. Remove and discard the aqueous layer. Repeat the base washing until no color is visible in the aqueous layer, to a maximum of four washings. Minimize contact time between the extract and the base to prevent degradation of the CDDs/CDFs. Stronger potassium hydroxide solutions may be employed for back-extraction, provided that the laboratory meets the specifications for labeled compound recovery and demonstrates acceptable performance using the procedure in Section 9.2.

12.5.3 Partition the extract against 50 mL of sodium chloride solution (Section 7.1.4) in the same way as with base. Discard the aqueous layer.

12.5.4 Partition the extract against 50 mL of sulfuric acid (Section 7.1.2) in the same way as with base. Repeat the acid washing until no color is visible in the aqueous layer, to a maximum of four washings.

12.5.5 Repeat the partitioning against sodium chloride solution and discard the aqueous layer.

12.5.6 Pour each extract through a drying column containing 7–10 cm of granular anhydrous sodium sulfate (Section 7.2.1). Rinse the separatory funnel with 30–50 mL of solvent, and pour through the drying column. Collect each extract in a round-bottom flask. Re-concentrate the sample and QC aliquots per Sections 12.6 through 12.7, and clean up the samples and QC aliquots per Section 13.

12.6 Macro-Concentration—Extracts in toluene are concentrated using a rotary evaporator or a heating mantle; extracts in methylene chloride or hexane are concentrated using a rotary evaporator, heating mantle, or Kuderna-Danish apparatus.

12.6.1 Rotary evaporation—Concentrate the extracts in separate round-bottom flasks.

12.6.1.1 Assemble the rotary evaporator according to manufacturer's instructions, and warm the water bath to 45 °C. On a daily basis, preclean the rotary evaporator by concentrating 100 mL of clean extraction solvent through the system. Archive both the concentrated solvent and the solvent in the catch flask for a contamination check if necessary. Between samples, three 2–3 mL aliquots of solvent should be rinsed down the feed tube into a waste beaker.

12.6.1.2 Attach the round-bottom flask containing the sample extract to the rotary evaporator. Slowly apply vacuum to the system, and begin rotating the sample flask.

12.6.1.3 Lower the flask into the water bath, and adjust the speed of rotation and the temperature as required to complete concentration in 15–20 minutes. At the proper rate of concentration, the flow of solvent into the receiving flask will be steady, but no bumping or visible boiling of the extract will occur.

Note: If the rate of concentration is too fast, analyte loss may occur.

12.6.1.4 When the liquid in the concentration flask has reached an apparent volume of approximately 2 mL, remove the flask from the water bath and stop the rotation. Slowly and carefully admit air into the system. Be sure not to open the valve so quickly that the sample is blown out of the flask. Rinse the feed tube with approximately 2 mL of solvent.

12.6.1.5 Proceed to Section 12.6.4 for preparation for back-extraction or micro-concentration and solvent exchange.

12.6.2 Heating mantle—Concentrate the extracts in separate round-bottom flasks.

12.6.2.1 Add one or two clean boiling chips to the round-bottom flask, and attach a three-ball macro Snyder column. Prewet the column by adding approximately 1 mL of solvent through the top. Place the round-bottom flask in a heating mantle, and apply heat as required to complete the concentration in 15–20 minutes. At the proper rate of distillation, the balls of the column will actively chatter, but the chambers will not flood.

12.6.2.2 When the liquid has reached an apparent volume of approximately 10 mL, remove the round-bottom flask from the heating mantle and allow the solvent to drain and cool for at least 10 minutes. Remove the Snyder column and rinse the glass joint into the receiver with small portions of solvent.

12.6.2.3 Proceed to Section 12.6.4 for preparation for back-extraction or micro-concentration and solvent exchange.

12.6.3 Kuderna-Danish (K-D)—Concentrate the extracts in separate 500 mL K-D flasks equipped with 10 mL concentrator tubes. The K-D technique is used for solvents such as methylene chloride and hexane. Toluene is difficult to concentrate using the K-D technique unless a water bath fed by a steam generator is used.

12.6.3.1 Add one to two clean boiling chips to the receiver. Attach a three-ball macro Snyder column. Prewet the column by adding approximately 1 mL of solvent 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.

12.6.3.2 Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 15–20 minutes. At the proper rate of distillation, the balls of the column will actively chatter but the chambers will not flood.

12.6.3.3 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 solvent. A 5 mL syringe is recommended for this operation.

12.6.3.4 Remove the three-ball Snyder column, add a fresh boiling chip, and attach a two-ball micro Snyder column to the concentrator tube. Prewet the column by adding approximately 0.5 mL of solvent through the top. Place the apparatus in the hot water bath.

12.6.3.5 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.

12.6.3.6 When the liquid reaches an apparent volume of 0.5 mL, remove the apparatus from the water bath and allow to drain and cool for at least 10 minutes.

12.6.3.7 Proceed to 12.6.4 for preparation for back-extraction or micro-concentration and solvent exchange.

12.6.4 Preparation for back-extraction or micro-concentration and solvent exchange.

12.6.4.1 For back-extraction (Section 12.5), transfer the extract to a 250 mL separatory funnel. Rinse the concentration vessel with small portions of hexane, adjust the hexane volume in the separatory funnel to 10–20 mL, and proceed to back-extraction (Section 12.5).

12.6.4.2 For determination of the weight of residue in the extract, or for clean-up procedures other than back-extraction, transfer the extract to a blowdown vial using two to three rinses of solvent. Proceed with micro-concentration and solvent exchange (Section 12.7).

12.7 Micro-Concentration and Solvent Exchange.

12.7.1 Extracts to be subjected to GPC or HPLC cleanup are exchanged into methylene chloride. Extracts to be cleaned up using silica gel, alumina, carbon, and/or Florisil are exchanged into hexane.

12.7.2 Transfer the vial containing the sample extract to a nitrogen blowdown device. Adjust the flow of nitrogen so that the surface of the solvent is just visibly disturbed.

Note: A large vortex in the solvent may cause analyte loss.

12.7.3 Lower the vial into a 45 °C water bath and continue concentrating.

12.7.3.1 If the extract is to be concentrated to dryness for weight determination (Sections 12.4.1.8, 12.4.2.7, and 13.7.1.4), blow dry until a constant weight is obtained.

12.7.3.2 If the extract is to be concentrated for injection into the GC/MS or the solvent is to be exchanged for extract cleanup, proceed as follows:

12.7.4 When the volume of the liquid is approximately 100 L, add 2–3 mL of the desired solvent (methylene chloride for GPC and HPLC, or hexane for the other cleanups) and continue concentration to approximately 100 µL. Repeat the addition of solvent and concentrate once more.

12.7.5 If the extract is to be cleaned up by GPC, adjust the volume of the extract to 5.0 mL with methylene chloride. If the extract is to be cleaned up by HPLC, further concentrate the extract to 30 µL. Proceed with GPC or HPLC cleanup (Section 13.2 or 13.6, respectively).

12.7.6 If the extract is to be cleaned up by column chromatography (alumina, silica gel, Carbopak/Celite, or Florisil), bring the final volume to 1.0 mL with hexane. Proceed with column cleanups (Sections 13.3 through 13.5 and 13.8).

12.7.7 If the extract is to be concentrated for injection into the GC/MS (Section 14), quantitatively transfer the extract to a 0.3 mL conical vial for final concentration, rinsing the larger vial with hexane and adding the rinse to the conical vial. Reduce the volume to approximately 100 µL. Add 10 µL of nonane to the vial, and evaporate the solvent to the level of the nonane. Seal the vial and label with the sample number. Store in the dark at room temperature until ready for GC/MS analysis. If GC/MS analysis will not be performed on the same day, store the vial at <-10 °C.

13.0 Extract Cleanup

13.1 Cleanup may not be necessary for relatively clean samples (e.g., treated effluents, groundwater, drinking water). If particular circumstances require the use of a cleanup procedure, the analyst may use any or all of the procedures below or any other appropriate procedure. Before using a cleanup procedure, the analyst must demonstrate that the requirements of Section 9.2 can be met using the cleanup procedure. If only 2,3,7,8-TCDD and 2,3,7,8-TCDF are to be determined, the cleanup procedures may be optimized for isolation of these two compounds.

13.1.1 Gel permeation chromatography (Section 13.2) removes high molecular weight interferences that cause GC column performance to degrade. It should be used for all soil and sediment extracts and may be used for water extracts that are expected to contain high molecular weight organic compounds (e.g., polymeric materials, humic acids).

13.1.2 Acid, neutral, and basic silica gel (Section 13.3), alumina (Section 13.4), and Florisil (Section 13.8) are used to remove nonpolar and polar interferences. Alumina and Florisil are used to remove chlorodiphenyl ethers.

13.1.3 Carbopak/Celite (Section 13.5) is used to remove nonpolar interferences.

13.1.4 HPLC (Section 13.6) is used to provide specificity for the 2,3,7,8-substituted and other CDD and CDF isomers.

13.1.5 The anthropogenic isolation column (Section 13.7.1), acidified silica gel batch adsorption procedure (Section 13.7.2), and sulfuric acid and base back-extraction (Section 13.7.3) are used for removal of lipids from tissue samples.

13.2 Gel Permeation Chromatography (GPC).

13.2.1 Column packing.

13.2.1.1 Place 70–75 g of SX–3 Bio-beads (Section 6.7.1.1) in a 400–500 mL beaker.

13.2.1.2 Cover the beads with methylene chloride and allow to swell overnight (a minimum of 12 hours).

13.2.1.3 Transfer the swelled beads to the column (Section 6.7.1.1) and pump solvent through the column, from bottom to top, at 4.5–5.5 mL/minute prior to connecting the column to the detector.

13.2.1.4 After purging the column with solvent for one to two hours, adjust the column head pressure to 7–10 psig and purge for four to five hours to remove air. Maintain a head pressure of 7–10 psig. Connect the column to the detector (Section 6.7.1.4).

13.2.2 Column calibration.

13.2.2.1 Load 5 mL of the calibration solution (Section 7.4) into the sample loop.

13.2.2.2 Inject the calibration solution and record the signal from the detector. The elution pattern will be corn oil, bis(2-ethyl hexyl)phthalate, pentachlorophenol, perylene, and sulfur.

13.2.2.3 Set the “dump time” to allow >85% removal of the corn oil and >85% collection of the phthalate.

13.2.2.4 Set the “collect time” to the peak minimum between perylene and sulfur.

13.2.2.5 Verify the calibration with the calibration solution after every 20 extracts. Calibration is verified if the recovery of the pentachlorophenol is greater than 85%. If calibration is not verified, the system shall be recalibrated using the calibration solution, and the previous 20 samples shall be re-extracted and cleaned up using the calibrated GPC system.

13.2.3 Extract cleanup—GPC requires that the column not be overloaded. The column specified in this method is designed to handle a maximum of 0.5 g of high molecular weight material in a 5 mL extract. If the extract is known or expected to contain more than 0.5 g, the extract is split into aliquots for GPC, and the aliquots are combined after elution from the column. The residue content of the extract may be obtained gravimetrically by evaporating the solvent from a 50 µL aliquot. (continued)