PROCEDURE FOR THE EXTRACTION AND PURIFICATION OF PCDD/PCDF AND DL-PCBs IN SEDIMENT AND SOIL SAMPLES

Transcription

1 Page 1 of 10 PROCEDURE FOR THE PURIFICATION OF POLYCHLORINATED DIBENZO-p-DIOXINS (PCDD), POLYCHLORINATED DIBENZOFURANS (PCDF) AND DIOXIN-LIKE POLYCHLORINATED BIPHENYLS (DL-PCBs) IN SEDIMENT 1. Introduction 2. Objective 3. Scope 4. Implementation 4.1 Extraction 4.2 Purification 4.3 Fractionation 4.4 Transfer to vial 4.5 Addition of the internal or syringe standard 5. Annexes 5.1 Neutral silica clean-up 5.2 Preparation of basic silica (33%) 5.3 Preparation of acid silica (44%) 5.4 Preparation of the multi-layer silica column 5.5 Florisil conditioning 5.6 Preparation of the Florisil column 5.7 Alumina conditioning 5.8 Preparation of the alumina column 6. Change history Prepared by: Revised by: Approved by: Signed: M G. Martrat LABORATORY OF DIOXINS IDÆA-CSIC Date: 6/04/2011 Signed: E. Abad LABORATORY OF DIOXINS IDÆA-CSIC Date: 11/04/2011 Signed: H. Fiedler UNEP Chemicals Branch Date: 15/04/2011

2 Page 2 of INTRODUCTION This procedure has been prepared in the Laboratory of Dioxins of the Department of Environmental Chemistry of the Institute of Environmental Assessment and Water Research (IDÆA) of the Spanish Council for Scientific Research (CSIC) in Barcelona (Spain), in collaboration with the United Nations Environment Programme (UNEP) to support the Implementation of the Global Monitoring Plan for Persistent Organic Pollutants (POPs) under the Stockholm Convention. The programme includes countries from Latin America and the Caribbean participating in laboratory training projects funded by the Global Environment Facility (GEF), as well as by the Strategic Approach to International Chemicals Management (SAICM). The following people assisted in preparing this document: 2. OBJECTIVE Dr. Esteban Abad Holgado Dr. Heidelore Fiedler Prof. Josep Rivera Aranda Dr. Manuela Ábalos Navarro Ms. Laura Morales Pérez Mr. Jordi Sauló Dalmau Ms. Mª Generosa Martrat Castellví Mr. Jordi Parera Costa Mr. Miquel Àngel Adrados León Mr. Karell Martínez Guijarro Mr. Joan Rivera Austrui The objective of the Global Monitoring Plan (GMP) is to support the effectiveness evaluation of the Stockholm Convention with regards to environmental background concentrations in media with a high potential for comparability. The Conference of the Parties has decided that air monitoring and exposure through breast milk or human blood will be used as core media for the first evaluation. This procedure aims to describe the process for the extraction and purification of PCDD/PCDF and dl-pcbs in sediment and soil samples. 3. SCOPE This procedure describes the process for the extraction and purification of PCDD/PCDF and dl-pcbs in sediment and soil samples.

3 Page 3 of IMPLEMENTATION 4.1. EXTRACTION Weigh approximately 7 g of dry sediment and 5 g of copper powder or 25 g of soil and 10 of copper in a glass Soxhlet thimble with a precision of 0.1 g. Place the thimble in a 250 ml Soxhlet body and attach the apparatus to a 500 ml roundbottom flask previously identified with the sample reference, and add several granules of porous porcelain Fortification (only for the isotope dilution method): By weighing: Remove the vial with the labeled standard from the refrigerator and allow it to warm to room temperature Take a 1 ml vial. Tare on an analytical balance and, using a micropipette or a glass syringe, measure the volume of standard required for the fortification. Weigh the vial with the added standard on an analytical balance Measure approximately 10 ml of toluene. With a Pasteur pipette, transfer the standard contained in the vial into the sample. With a Pasteur pipette, transfer part of the 10 ml of solvent into the vial that contained the standard. With a Pasteur pipette, empty the vial into the sample. Repeat the previous steps until no solvent is left. Cover the top of the Soxhlet body with aluminum foil and let it stand for 2 h before starting the Soxhlet extraction By volume: Using a micropipette or glass syringe, measure the volume of standard required for the fortification. Add the standard to the sample. Cover the top of the Soxhlet body with aluminum foil and let it stand for 2 h before starting the Soxhlet extraction Remove the aluminum foil and cover the glass Soxhlet thimble with a small amount of washed glass wool Add 400 ml of toluene, measured with a graduated cylinder, to the Soxhlet body, trying to wet the whole sample in the thimble. Induce reflux so that almost the whole volume of solvent is contained in the flask Place the Soxhlet apparatus and the flask on a heating mantle and attach a condenser on top of the Soxhlet body. Secure the whole apparatus in place to maintain its stability Check that the cooling water is turned on and adjust the power of the heating mantle ensuring an approximate measure of 3-4 refluxes per hour Allow the sample to extract for 24 h. Turn the heating mantle off and let it cool to

4 Page 4 of 10 room temperature If there is any solvent left in the Soxhlet body, induce reflux so that the whole volume is contained in the flask and allow the thimble to air dry placed on top of the Soxhlet body. Separate the flask from the rest of the apparatus If a precipitate is observed in the flask, the following procedure should be followed: Concentrate the extract up to approximately 5-10 ml, keeping the bath temperature below 40 C, to avoid any evaporation loss of our compounds Using a filtration funnel on which a glass fiber filter is placed, filter the extract and collect it in a 250 ml round-bottom flask previously identified with the sample reference. Add, approximately 2 or 3 times, 2 ml of toluene to the original flask to clean it up, trying to collect any remaining precipitate. Filter the cleaning solvent through the same filter Place the filter in a 250 ml Soxhlet body and attach the apparatus to the 500 ml round-bottom flask of the initial extraction, adding several granules of porous porcelain if necessary Proceed to the filter extraction following steps 4.1.6, and Concentrate the extract from the filter extraction, keeping the bath temperature below 40 C. Collect the initial extract and that obtained in the filter extraction in only one of the used flasks with a Pasteur pipette. Add, approximately 2 or 3 times, 2 ml of toluene to the discarded flask to clean it up and transfer the cleaning solvent to the other flask Concentrate the extract, keeping the bath temperature below 40 C, up to an approximate volume of 2 ml. Add 100 ml of hexane to the flask, measured with a graduated cylinder, and concentrate the extract to an approximate volume of 2 ml PURIFICATION Once the multi-layer silica column has been prepared according to section 5.4, place a 1 L round-bottom flask previously identified with the sample reference below the column and make sure the column stopcock is closed Measure 250 ml of hexane with a graduated cylinder, dissolve the sample residue contained in the flask. In the smallest volume of hexane possible from the graduated cylinder, transfer the sample with a Pasteur pipette to the top of the column. Wash the flask containing the sample with a few milliliters of hexane (from the 250 ml graduated cylinder) and transfer them to the top of the column. Wash the flask at least six times. The final volume of hexane to elute all the analytes of the column shall be 500 ml Open the stopcock to start the elution of the column.

5 Page 5 of When the hexane reaches the top of the acid silica column (44%), close the stopcock Concentrate the extract, keeping the bath temperature below 40 C, up to an approximate volume of 2 ml FRACTIONATION Florisil column Once the Florisil column has been prepared according to section 5.6, place below the column a 500 ml round-bottom flask previously identified with the sample reference, with the note fraction of hexane, and make sure the column stopcock is closed Measure 250 ml of hexane with a graduated cylinder, dissolve the sample residue contained in the flask. In the smallest volume of hexane possible from the graduated cylinder, transfer the sample with a Pasteur pipette to the top of the column. Wash the flask containing the sample with a few milliliters of hexane (from the 250 ml graduated cylinder) and transfer them to the top of the column. Wash the flask at least six times. The final volume of hexane to elute all the analytes of the column shall be 250 ml Open the stopcock to start the elution of the column When the hexane reaches the top of the sodium sulfate column, close the stopcock Remove the 500 ml flask and place another flask of 250 ml labeled with the sample name and the note fraction toluene-diethyl ether that will identify it as the fraction of toluene-ether from the Florisil column Concentrate the extract of the flask with the note fraction of hexane, keeping the bath temperature below 40 C, up to an approximate volume of 2 ml. Dl- PCBs are found in this fraction Measure 98 ml of toluene with a 100 ml graduated cylinder and measure 2 ml of diethyl ether with a 10 ml graduated cylinder, mix it in a bottle labeled toluene-diethyl ether 2%, and repeat this process as many times as necessary. Add 170 ml of the solution of toluene-diethyl ether (2%) to the top of the Florisil column. Open the stopcock to start the Florisil elution. The Florisil column is considered to be finished when all the solvent has eluted through the column Alumina column The alumina column is usually prepared in a subsequent step to the Florisil column.

6 Page 6 of Once the column has been prepared according to section 5.8, add the sample to the column. To do so, the extract to be added should be in hexane. If the sample is from the Florisil, reconcentrate to 1 ml, and change to hexane (100 ml) to reconcentrate it and get to 1-2 ml The sample is placed on the top of the alumina column with 25 ml of hexane, measured with a 25 ml graduated cylinder. Wash the flask containing the extract with those 25 ml. Place a 50 ml flask below the column to collect this fraction. Label it with the sample name and the note fraction of hexane Once the 25 ml of hexane have eluted, add the second fraction of the elution of the alumina. It is prepared with a solution of hexane-methylene chloride (2%). Measure 98 ml of hexane with a 100 ml graduated cylinder and measure 2 ml of methylene chloride with a 10 ml graduated cylinder, mix it in a bottle labeled hexane-dcm 2%, and repeat this process as many times as necessary. The mixture has to be prepared again each day the analysis is conducted, since its expiry date is daily. Measure 20 ml of the solution with a 25 ml graduated cylinder, and add it to the top of the column. Place a 50 ml flask below the column to collect this fraction. Label it with the sample name and the note Fr. 2% Once the 20 ml of hexane-methylene chloride (2%) have eluted, add the third fraction of elution of the alumina. It is prepared with a solution of hexanemethylene chloride (50%). Measure 50 ml of hexane with a 100 ml graduated cylinder and measure 50 ml of methylene chloride with a 100 ml graduated cylinder, mix it in a bottle labeled hexane-dcm 50%, and repeat this process as many times as necessary. The mixture has to be prepared again each day the analysis is conducted, since its expiry date is daily. Measure 50 ml of the solution with a 100 ml graduated cylinder, and add it to the top of the column. Place a 100 ml flask below the column to collect this fraction. Label it with the sample name and the note Fr. 50%. PCDD/PCDF are eluted in this fraction When the elution of the third fraction of the alumina is finished, pass another fraction of methylene chloride (50 ml), for security reasons. Place a 100 ml flask below the column to collect this fraction. Label it with the sample name and the note Fr. 100% Concentrate the extract of the round-bottom flask with the note fraction toluene-diethyl ether 50%, keeping the bath temperature below 40 C, up to an approximate volume of 2 ml TRANSFER TO VIAL With a Pasteur pipette, transfer the extract contained in the flask from the Florisil and alumina columns to two 2 ml vials. Concentrate the extract in each vial under a stream of nitrogen. When the volume contained in the vial reaches 20 µl, washing is carried out in the flask which contained the sample. With a Pasteur pipette,

7 Page 7 of 10 transfer 2 ml of hexane to the flask which contained the sample. With a Pasteur pipette, transfer the flask content to the vial. Repeat the previous steps 4 more times. Finally, leave the vial under the stream of nitrogen until dryness ADDITION OF THE INTERNAL OR SYRINGE STANDARD By weighing: Remove the vial with the labeled standard from the refrigerator and allow it to warm to room temperature Take the vial containing the sample. Tare on an analytical balance and, using a micropipette or a glass syringe, measure the volume of internal or syringe standard required and add it to the sample vial. Weigh the vial with the added standard on an analytical balance By volume: Using a micropipette or glass syringe, measure the volume of internal or syringe standard required and add it to the sample vial. 5. ANNEXES 5.1. NEUTRAL SILICA CLEAN-UP Take the neutral silica With a spatula, add the neutral silica to a 1000 ml beaker Add methylene chloride until the neutral silica is soaked and place it in an ultrasonic bath for 15 minutes Decant the methylene chloride and discard it Repeat steps and twice Allow the neutral silica to dry in the beaker, covering it with aluminum foil with ventilation holes. Allow it to dry to room temperature in the desiccator Once dry, store it in a properly labeled glass bottle PREPARATION OF BASIC SILICA (33%) After washing the silica as described in section 5.1, weigh 53.0 g of neutral silica in a 250 ml Erlenmeyer flask with a ground neck Weigh 26.4 g of a dilution of 1 N NaOH prepared by weighing 0.4 g of sodium hydroxide (NaOH) in a toploading balance and dissolving it in 100 ml of Milli-Q water with the help of an ultrasonic bath.

8 Page 8 of Add 2 ml of NaOH dropwise with a Pasteur pipette to the neutral silica in the Erlenmeyer flask Close with a ground-glass stopper and stir until the NaOH is absorbed in the silica Repeat steps and until the weighed NaOH runs out Store in a labeled glass bottle PREPARATION OF ACID SILICA (44%) After washing the silica as described in section 5.1, weigh 78.4 g of neutral silica in a 250 ml Erlenmeyer flask with a ground neck Weigh 61.6 g of concentrated sulfuric acid in a 100 ml graduated cylinder Add 2 ml of sulfuric acid dropwise with a Pasteur pipette to the neutral silica in the Erlenmeyer flask Close with a ground-glass stopper and stir until the sulfuric acid is absorbed in the silica Repeat the two previous steps until the sulfuric acid runs out Store in a labeled glass bottle PREPARATION OF THE MULTI-LAYER SILICA COLUMN Take a glass column 38 cm long with a diameter of 5 cm, and place a base of glass wool Weigh and add to the column 30 g of silica modified with sodium hydroxide (33%), prepared as described in section 5.2 of this procedure Weigh and add to the column 5 g of neutral silica prepared as described in section 5.1 of this procedure Weigh and add to the column 60 g of silica modified with sulfuric acid (44%), prepared as described in section 5.3 of this procedure Weigh and add to the column 2 g of sodium sulfate After each addition, lightly tap the column to avoid the formation of preferential flow paths Elute the column with 100 ml of hexane Before it dries, close the stopcock FLORISIL CONDITIONING

9 Page 9 of Weigh the corresponding amount of Florisil according to the number of samples to be purified in a porcelain crucible Place the crucible in the muffle furnace Set the temperature in the muffle furnace indicator for the activation of Florisil (650 C) Turn it on and leave the Florisil in activation for at least 16 h When the minimum activation time has passed, turn the muffle off and allow it to cool The Florisil should be used within one day from its activation or it should be activated again PREPARATION OF THE FLORISIL COLUMN Take a glass column 30 cm long with a diameter of 1.5 cm, and place a base of glass wool Weigh and add to the column 5 g of Florisil, for PCDD/PCDF, or 10 g of Florisil, for marker PCBs, activated as described in section 5.5 of this procedure Weigh and add to the column 2 g of sodium sulfate After each addition, lightly tap the column to avoid the formation of preferential flow paths Attach the addition flask Elute the column with 50 ml of hexane Before it dries, close the stopcock ALUMINA CONDITIONING Weigh the corresponding amount of alumina according to the number of samples to be analyzed in a porcelain crucible Place the crucible in the muffle furnace Set the temperature in the muffle furnace indicator for the activation of alumina (300 C) Turn it on and leave the alumina in activation for at least 16 h When the minimum activation time has passed, turn the muffle off and allow it to cool The alumina should be used on the same day it was activated or it should be discarded PREPARATION OF THE ALUMINA COLUMN

10 Page 10 of Take a glass column 30 cm long with a diameter of 1.5 cm, and place a base of glass wool Weigh and add to the column 6 g of alumina, activated as described in section 5.7 of this procedure Weigh and add to the column 2 g of sodium sulfate After each addition, lightly tap the column to avoid the formation of preferential flow paths Attach the addition flask Elute the column with 50 ml of hexane Before it dries, close the stopcock. 6. CHANGE HISTORY CHANGE DATE REFERENCE OF CHANGES MADE REVISION NO. 24/May/2012 First Edition 1