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Reference Method for the Determination of Polychlorinated Dibenzoparadioxins and Polychlorinated Dibenzofurans in Pulp and Paper Mill Effluents

Section 5: Sample Handling and Workup Procedures

5.1 Sample Handling and Custody

Upon arrival at the laboratory, samples must be inspected immediately for their physical condition and to ensure proper labelling. Inform the client of any potential problem concerning the integrity of samples. After logging and labelling with laboratory code numbers, samples should be processed as soon as possible. Any sample-tracking report sheets submitted with samples are completed and signed by authorized lab personnel and retained for auditing. The laboratory's analysis-tracking documents must also be available for auditing.

Samples must be maintained at 1 to 5°C from the time of receipt until extraction. (Caution: Prevent from freezing.)

5.2 Method Performance Tests

Before sample analysis, the laboratory must demonstrate the ability to achieve acceptable precision and accuracy by conducting method performance tests.

Three matrix blanks (1 L of high purity water), spiked with known amounts of native standards and labelled surrogates, must be prepared, processed and analyzed according to the procedures used for actual samples. The native congener spiking solution must contain all 2,3,7,8-substituted dioxin and furan congeners at concentrations equal to those shown for the CS2 calibration standard in Table 4. The composition of the surrogate spiking solution is shown in Table 1. The required spike volume is 50 μL of each solution. Both the native and surrogate standard solutions are diluted with acetone immediately before spiking, as described in Subsection 5.3.

For each of the three tests, recovery for each of the native PCDD/PCDF congeners (corrected for surrogate recovery) must be within the range of 80 to 120% of the spiked value (i.e., accuracy of ± 20%). All labelled surrogate recoveries must be within the range of 40 to 120%.

No field sample shall be processed until the method performance tests yield acceptable results. These tests must be repeated whenever extraction or cleanup procedures are modified in any way, whenever a different reagent lot number is used, or if performance tests have not been conducted in the preceding three-month period.

Table 1: Composition of Surrogate Solution
SurrogateStock Solution
in Toluene
Spike Solution
in Acetone
13C12-OCDF is not used because it may interfere with OCDD analysis

5.3 Sample Preparation

For each batch of 1-L samples processed, prepare a fresh dilution of the PCDD/PCDF surrogate solution as shown in Table 1 by adding 50 μL of this stock solution to 950 μL of acetone (1×20 dilution) for each sample being prepared.

Weigh the nominal 1-L sample, in its bottle, to the nearest gram on a top-loading balance. Sample weight is determined later by subtracting the weight of the dried bottle. Net sample weight must be between 900 g and 1100 g. Within these limits, weight is converted to volume by assuming a sample density of 1.0 g/mL.

Spike the sample (in the bottle) with 1.0 mL of the freshly diluted surrogate solution (Table 1). Carefully shake the bottle several times or mix the solution with a magnetic stirrer during the next hour to allow the surrogates to equilibrate within the raw sample matrix.

After one hour, suction filter the sample through a pre-weighed Gelman A/E glass fibre filter into a 2-L filtration flask. Complete the transfer with several portions of deionized water to remove any remaining particulate. Retain the sample bottle for later use as described in Subsection 5.4.1. Continue suction until the flow of filtrate stops completely. Transfer the filter and any particulate still adhering to the walls of the Buchner funnel to a Petri dish and allow the solids to dry in a dessicator. Weigh the dried filter containing the solid portion. This fraction of the sample is extracted in a Soxhlet apparatus. The filtrate is extracted in a separatory funnel.

5.4 Sample Extraction

Extraction procedures are shown in Figure 2. Detailed descriptions are provided in the following subsections.

Figure 2: Extraction Schematic for Pulp and Paper Mill Effluents
Extraction Schematic for Pulp and Paper Mill Effluents

5.4.1 Extraction of Filtrate

Quantitatively transfer the filtrate from the flask to a clean 2-L separatory funnel using three rinses of deionized water. Add three, 60-mL rinses of dichloromethane to the retained empty sample bottle, shaking each rinse in the bottle before transferring to the separatory funnel. Extract the filtrate with these rinses by shaking the separatory funnel vigorously for two minutes, releasing pressure buildup as required. Allow the sample to sit for at least ten minutes to ensure separation of the aqueous and organic layers. Drain the organic layer into a 500-mL round bottom flask. Persistent emulsions may be drained off into a clean, large beaker and broken using mechanical means, such as passage through loosely packed glass wool.

Repeat these extraction procedures two more times using 100 mL of dichloromethane each time. Rinse the separatory funnel with an additional 30 mL of dichloromethane. If any water or emulsion is observed in the collection flask at this point, the extract must be passed through a bed of dichloromethane-rinsed sodium sulphate held in a powder funnel positioned over a second 500-ml, flask. Concentrate the combined extracts and rinses to 3 to 5 mL by rotary evaporation at 30°C before combining with the extract from the particulate fraction of the sample.

5.4.2 Extraction of Particulate Matter

Place a pre-extracted thimble in a clean glass beaker and transfer the filter, containing the dried particulate, into the thimble. Rinse any small amounts of solids remaining in the Petri dish into the thimble with toluene, then cover the sample with a layer of clean glass wool. Place the thimble into the Soxhlet body. Fill the flask with approximately 350 ml, of toluene. Connect the flask to the Soxhlet body. Rinse the beaker three times with toluene and add the rinsings into the Soxhlet body. Connect the Soxhlet to the water-cooled condenser and wrap the Soxhlet body with aluminum foil for heat insulation. Reflux the sample at a rate of three to four cycles per hour.

After a minimum of 16 hours of extraction, rinse the inner surface of the condenser with toluene and allow the apparatus to cool. Siphon the solvent from the Soxhlet body into the extract flask. Rinse the Soxhlet body three times with toluene, siphoning each rinse into the flask.

Add the concentrated filtrate extract to the particulate extract with three hexane rinsings of the flask. Concentrate the combined sample to 1 to 2 ml, by rotary evaporation at 72°C or lower. Exchange the solvent to hexane by adding approximately 100 ml, of hexane to the sample flask and repeating the concentration step.

Dry the sample by passing it through hexane-rinsed sodium sulphate held in a powder funnel positioned over a 250-mL boiling flask. Complete the transfer by rinsing first the extraction flask, then the sodium sulphate, with three 5-mL portions of hexane. Concentrate the sample to 3 to 5 mL by rotary evaporation at 30°C. The sample is now ready for cleanup.

5.5 Sample Cleanup

The analyst may use the following cleanup procedures, or any other procedures that yield a sample extract fraction of sufficient quality to satisfy all method performance criteria for target analyte determination.

The basic cleanup procedures recommended for sample extracts are shown in Figure 3. Cleanup columns required in this method are shown in Figure 4 (Lamparski and Nestrick, 1980; EC, 1989).

Figure 3: Cleanup and Analysis Schematic for PCDDs/PCDFs in Pulp and Paper Mill Effluents
Cleanup and Analysis Schematic for PCDDs/PCDFs in Pulp and Paper Mill Effluents

Figure 4: Cleanup Columns
Cleanup Columns

Acid/Base/Silver Nitrate /Silica Column

Plug the tip of the column with glass wool and add the following in the order specified: 1.5 g of 10% AgNO3/silica (bottom layer); 1 g of silica; 2 g of the 33% 1 M NaOH/silica; l g of silica; 4 g of 44% H2SO4/silica; 2 g of silica; and approximately 1 g of sodium sulphate to top off the column. After each reagent has been added, gently tap the column to ensure even layering.

Pre-wash the prepared column with 30 mL of 2% dichloromethane in hexane (v/v). Just as the solvent reaches the top of the sodium sulphate layer, place a 250-mL flask under the column. Via Pasteur pipet, transfer the concentrated raw extract onto the column, followed by three 5-mL 2% dichloromethane in hexane (v/v) rinsings of the sample flask, using the same pipet each time.

When the third rinse has drained to the top of the sodium sulphate layer, pour an additional 50 mL of 2% dichloromethane in hexane (v/v) into the column. When the solvent has drained, assess the column for saturation of the acid/silica layer and the silver nitrate/silica layer. This is indicated by an appearance of colour throughout the reagent layer.

Saturation of the acid/silica layer suggests that the sample extract (in hexane) should be washed with concentrated sulphuric acid in a separatory funnel, repeating the washes (maximum of four washings) with fresh acid until no colour is observed in the acid layer. Wash the extract sequentially with deionized water, l M NaOH and a final wash with deionized water. Dry the extract by passing it through sodium sulphate, as previously described.

Saturation of the silver nitrate/silica layer requires passage of the concentrated sample extract through an additional column containing 2.5 g of 10% silver nitrate/silica. Elute the sample extract through the pre-washed column with 30 mL of 2% dichloromethane in hexane.

Add 100 mL of hexane to the column eluate and concentrate to 1 to 2 mL by rotary evaporation at 30°C.

Alumina Column Chromatography

Plug the tip of the alumina column with glass wool and add 2.5 g of freshly prepared basic alumina (see Subsection 4.4). Top off the column with 0.5 cm of sodium sulphate. Add 15 mL of hexane to pre-wash the column. When the hexane has drained just to the top of the sodium sulphate layer, immediately place a 250-mL boiling flask, labelled Fraction 1, under the column tip. Via Pasteur pipet, transfer the concentrated extract from the acid/base/silver nitrate/silica column onto the alumina column, followed, just as the sample extract reaches the sodium sulphate layer, by three 5-mL hexane rinsings of the sample flask. Add an additional 30 mL of hexane (2 × 15 mL) to the column just as the flask rinsings reach the top of the sodium sulphate layer. When this solvent has drained to the top of the sodium sulphate layer, add 20 mL of freshly prepared 1.5% dichloromethane in hexane to the column. This combined fraction (Fraction 1) is archived. When the solvent level in the column again just reaches the top of the sodium sulphate layer, exchange the Fraction 1 flask for a 250-mL flask, labelled Fraction 2.

Add 30 mL of 50% dichloromethane in hexane (v/v) to the column and allow the column to drain completely. This fraction contains the PCDDs/PCDFs. Concentrate this fraction to 1 to 2 mL and exchange the solvent to hexane by adding approximately 100 mL of hexane to the sample flask and repeating the concentration step.

Prepare another alumina column (optional) as just described. Then, following the same procedure, apply the sample (Fraction 2) to the column and collect Fraction I in the flask containing that eluate from the first column. Fraction 2 is also collected in its original flask. Concentrate both fractions to approximately 1 mL by rotary evaporation at 30°C. Transfer Fraction 1 to a 1.5 mL, amber vial and archive. This fraction may be assessed for PCDDs/PCDFs if poor surrogate recovery is observed in Fraction 2.

Fraction 2, containing the PCDDs/PCDFs, is transferred via Pasteur pipet to a 1-mL conical sample vial (as specified in Section 4.2) and concentrated under a gentle stream of pre-purified nitrogen. Complete the sample transfer using the same pipet to take up three, 0.5-mL hexane rinses of the flask, transferring each rinse into the vial. Concentrate the extract to a small volume (approximately 100 μL) and store the sample vial at 5°C or lower in a refrigerator until analysis.

Immediately before GC/MS analysis, blow down the sample just to dryness under a gentle stream of pre-purified nitrogen. Add 20 μL of the recovery standard solution, containing 50 pg/μL each of 13C12-1,2,3,4-TCDD and 13C12-1,2,3,7,8,9-H6CDD in toluene, to the sample vial. Sonicate the capped vial for one minute or allow to sit for a minimum of one hour before analysis.

AX-21 Carbon/Silica Column (optional)

Prepare a 8 × 260 mm glass column by cutting both ends off a disposable 10-mL serological pipet. Pack the column in the following sequence: glass wool plug; 1 g of activated carbon/silica; 1 cm of silica; glass wool plug. Pre-rinse the column with 5 mL of 1:1 cyclohexane/dichloromethane with the silica plug at the bottom. Invert the column and rinse with a second 5 mL of 1:1 cyclohexane/dichloromethane. The sample, in approximately 1 mL of hexane, is applied onto the column with the silica plug at the top. Complete the transfer of sample onto the column using 2 × 2 mL of 1:1 cyclohexaneldichloromethane to rinse the sample flask. Elute with 6 mL of 1:1 cyclohexane/dichloromethane, followed by 20 mL of 75:20:5 dichloromethane/ methanol/toluene. Discard all eluates up to this point. Invert the column so that the silica plug is at the bottom, and elute with 30 mL of toluene. Collect the eluate in a 250-mL flask. Concentrate the sample using rotary evaporation at 72°C and transfer to a reacti-vial as previously described.

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