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

Section 4: Apparatus and Reagents

4.1 Equipment and Supplies

Extraction Apparatus
200-mL Soxhlet body (Dean-Stark moisture trap optional), condenser, 45 x 125-mm glass or cellulose thimble, 500-mL boiling flask, and a temperature-controlled heating mantle

Evaporative Concentrator
Büchi R-110 rotary evaporator or equivalent

Top-loading macro balance, capacity to 1600 g, readability to 0.01 g, for weighing samples and reagents

Electronic or mechanical pipets with capacities from 10 to 1000 μL

Vacuum Oven
Range to 225°C, for conditioning carbon, silica, reagent-coated silica, sodium sulphate, and glass wool

Tube Furnace
Lindberg or equivalent, for activating alumina at 350°C

Pressure/Vacuum with vacuum to 85 kPa (25 in. Hg) for rotary evaporation and suction filtration

Gelman A/E or equivalent, 11 cm diameter

Weighing Paper

Forceps, Spatulas, Scoops

Ultrasonic Bath


Automatic Glassware Washing Equipment

See Section 6

4.2 Glassware

Assorted volumes from 150 to 2000 mL

Buchner Funnels
12 cm diameter

Filtration Flasks
2 L

Separatory Funnels
2 L

Boiling Flasks
250 and 500 mL

Erlenmeyer Flasks
500 mL with glass stopper

Powder Funnels
7 cm diameter

Graduated Cylinders
10, 50, 100, 250 mL

Pasteur Pipets
22 cm, disposable

Petri Dishes
14 cm diameter

Glass Column
Minimum 1 L capacity, Teflon stopcock, for solvent washing of silica, sodium sulphate, and glass wool

Cleanup Columns
Acid/Base, Alumina (see Figure 4)

Alumina Conditioning Column
See Figure 1

Measuring Pipets
5.0 mL and 10.0 mL, graduated at 0.1 mL intervals, for preparing PCDD/PCDF standard mixtures

Sample Extract Vials
1 mL capacity, internally-tapered, screw cap vials with Teflon-faced septa

Assorted capacities from 1.5 to 40 mL, amber glass, screw cap with Teflon-faced septa

4.3 Reagents

Certified native PCDD/PCDF standard solutions, carbon-l3-labelled standards, window defining mixtures and column performance mixtures are available from Cambridge Isotope Laboratories, Wobum, MA and Wellington Laboratories, Guelph, Ontario.

All solvents are distilled-in-glass quality. Those required include hexane, toluene, dichloromethane, and acetone.

Ultra-high purity grade nitrogen and helium

An in-house supply of high purity distilled or deionized water is used (Millipore Milli-Q Water System or equivalent system using a carbon filtration stage).

Silica (Bio-Sil A, 100 to 200 mesh) and basic alumina (AG 10, 100 to 200 mesh) are available from Bio-Rad Laboratories, Richmond, CA. Suitable equivalents may be available from other sources. AX-21 carbon is available from Anderson Development Company, Adrian, MI. All other reagents, including sulphuric acid, sodium hydroxide, silver nitrate, and sodium sulphate are A.C.S. reagent grade or better.

4.4 Glassware and Material Preparation


All reusable glassware must be scrupulously cleaned as soon as possible after use. Glassware is sequentially rinsed with the last solvent used, followed by hexane and acetone. This is followed by washing with hot detergent solution and sequential rinsing with hot water, deionized water, and three portions each of acetone, hexane, and dichloromethane. For severely contaminated glassware, treatment in an ultrasonic bath filled with detergent solution is often beneficial. If a brush is used to scrub glassware, great care must be taken to ensure that glass surfaces are not scratched. Glassware is either air-dried or dried in an oven, and then stored in a contaminant-free area.

A glassware proofing sample must be analyzed before actual test samples are processed. Each piece of glassware to be used with test samples, including Soxhlet bodies and condensers, flasks, funnels, columns, vials and rotary evaporators, must be rinsed with three portions each of hexane and dichloromethane. All rinses are combined into one sample, which is spiked with surrogates, cleaned up (optional) and analyzed by GC/MS to determine potential interferents and any amounts of PCDD/PCDF that may be present. Sample analysis may proceed only if any PCDD/PCDF present is below maximum allowable levels (see Section 8).

Glass Wool

Compress a quantity of glass wool into a large glass column (1 L capacity or larger) and wash sequentially with hexane and dichloromethane. The volume of solvent used for each wash should be twice the estimated volume of glass wool in the column. Transfer the washed glass wool into a large beaker. Loosely cover the mouth of the beaker with hexane and dichloromethane-rinsed aluminum foil, allow the glass wool to air dry in a fume hood, and then condition overnight at 225°C in a vented oven. Store in a clean, wide-mouth, glass-stoppered bottle.

Sodium Sulphate

Wash the granular, anhydrous sodium sulphate in the same column used for preparing glass wool. Sequentially wash the sodium sulphate twice with hexane and twice with dichloromethane. The volume of solvent used for each washing should be twice the estimated volume of sodium sulphate in the column. Transfer to a large beaker, cover the mouth loosely with solvent-rinsed aluminum foil, and oven dry at 50°C for a minimum of one hour before conditioning overnight at 225°C. Store in a dessicator in a clean, screw-capped bottle fitted with a Teflon-lined cap.


Transfer approximately 350 g of Bio-Sil A (enough silica for 35 cleanup columns) to the large glass column used for preparing glass wool and sodium sulphate. Sequentially wash with hexane and dichloromethane as previously described. Oven dry the silica at 50°C for a minimum of one hour in a beaker loosely covered with solvent-rinsed aluminum foil, then condition at 225°C for a minimum of four hours. Store the clean silica in a dessicator, in a clean, screw-capped glass bottle fitted with a Teflon-lined cap.

44% (w/w) H2SO4 on Silica

Add 78.6 g of concentrated H2SO4 in a stepwise manner (5 mL at a time), to 100 g of freshly conditioned silica in a 500-mL glass-stoppered Erlenmeyer flask. After each addition, shake the flask vigorously to remove all clumps. Store in the stoppered flask. This amount of material is sufficient for 40 cleanup columns. Preparation of larger batches is not recommended. (Caution: This material has all the properties of concentrated H2SO4. Handle with care.)

33% (w/w) of 1 M NaOH on Silica

Add 24.6 g of a 1 M NaOH solution, in a stepwise manner, to 50 g of freshly-conditioned silica in a glass-stoppered Erlenmeyer flask. After each addition, shake the flask to remove all clumps. Store in a screw-capped bottle fitted with a Teflon-lined cap.

10% (w/w) AgN03 on Silica

Dissolve 5.6 g of silver nitrate in 21.5 mL of deionized water. Add this solution, in a stepwise manner, to 50 g of freshly conditioned silica in a glass-stoppered Erlenmeyer flask. Between additions, shake the flask until a uniformly-coated, free-flowing powder is produced. When all silver nitrate has been added, allow the material to stand for approximately 30 minutes, cover the mouth of the flask with solvent-rinsed aluminum foil, and place in an oven at 30°C. Over a five-hour period, gradually raise the oven temperature to 120°C, and continue to condition overnight at this temperature. Cool to room temperature and immediately transfer to an amber glass, screw-capped bottle fitted with a Teflon-lined cap. Minimize exposure of this material to light. Store in a dessicator until use.

5% AX-21 Carbon/Silica

Wash 7 g of AX-21 carbon powder by suspending in 100 mL of methanol, then vacuum filter through a glass fibre filter (Gelman A/E or equivalent) fitted in a Buchner funnel. Follow with two 100-mL methanol rinses and continue suction until methanol flow stops completely. Dry the washed AX-21 carbon in a vacuum oven for several hours at room temperature and then maintain at 130°C for a minimum of 72 hours.

Combine 5.0 g of prepared AX-21 carbon with 95 g of prepared silica gel in a wide-mouth bottle with a Teflon-lined screw cap. Blend by shaking until a uniform colour is achieved. Activate the blended AX-21 carbon/silica at 130°C for a minimum of 24 hours, then cap and store in a dessicator. Reactivation of the carbon/silica may be required after several weeks of storage.

Basic Alumina

Weigh out 2 to 3 g more alumina than is required (2.5 g/sample) for the number of samples to be batch-processed at one time. Add the alumina to the conditioning column, shown in Figure 1, and wash with dichloromethane, then hexane (two portions each). The volume of solvent used for each wash should be two to three times the estimated volume of alumina in the conditioning column. After draining, insert a pre-cleaned glass wool plug into the column to immobilize the alumina. Drain as much solvent as possible from the wet alumina by applying suction at the end of the column, then place the column in the tube furnace. Connect the glass jointed end of the column to a cylinder of pre-purified nitrogen. With the furnace off, purge the alumina with nitrogen at 200 to 400 mL/min. for approximately 30 minutes. While maintaining the nitrogen purge, condition the alumina at 350°C for a minimum of two hours. Conditioned alumina should be used immediately after removal from the tube furnace. Do not store for later use. (Caution: Hot glass.)

Figure 1: Alumina Conditioning Column
Alumina Conditioning Column

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