Instructions for the Supelco Dioxin Prep System: Multi-Layer Silica Gel Column and Dual-Layer Carbon Reversible Column

T704007A©2004 Sigma-Aldrich Co.SUPELCOBellefonte, PAThis Data Sheet Contains Important Information About The Product.Instructions for Supelco Multi-Layer Silica Gel Columnand Dual-Layer Carbon Reversible ColumnP000958The Supelco Dioxin Prep SystemThe Supelco Dioxin Prep system provides a highly efficientmeans of extracting and isolating dioxins, furans, and coplanarPCBs from stack gases, wastewater, soil, food, blood, andmilk. The prep system design reduces solvent usage, de-creases prep time by 1-2 days, and results in extractionrecoveries greater than 85%.The convenient multi-layer silica gel column is key to theextraction process. Seven layers of treated silica oxidize,reduce, and separate polar interferents. For very dirty samples,bulk treated silica gels and empty glass tubes are available tocustomize packings to meet individual sample needs.A unique dual-layer carbon reversible tube isolates the PCBs,dioxins, and furan groups from other non-polar interferences.Isolation and separation is based on the two layers of carbonhaving different affinities for such compounds.An integrated glassware and hardware design makes it conve-nient for analysts to select a few pieces or the entire prepsystem for their extraction needs. A vacuum manifold and avacuum adapter provide the option of running a single sampleor multiple samples at one time, using vacuum or gravity feed.ContentsThe Supelco Dioxin Prep System -------------------------------- 1How to Use the Multi-Layer Silica Gel Column ---------------- 2Multi-Layer Silica Gel Column ConditioningWith the Vacuum Manifold ---------------------------------- 2With the Vacuum Adapter ----------------------------------- 3With Gravity Feed --------------------------------------------- 3Dual-Layer Carbon Column Overview --------------------------- 4Carbon Column ConditioningWith the Vacuum Manifold ---------------------------------- 4With the Vacuum Adapter ----------------------------------- 5With the Syringe Luer Adapter ----------------------------- 6Sample ProcessingThe Two Step Procedure ------------------------------------ 7The Single Step Procedure --------------------------------- 7Ordering Information ------------------------------------------------- 8How to Use the Multi-Layer Silica Gel Column**The multi-layer silica gel column was developed with the assistance of Mr. Masaaki Maeoka at JQA.The Supelco multi-layer silica gel column is designed to meet the requirements of Japanese Industrial Standard Methods K-0311 andK-0312. The column has a 15 mm internal diameter and is 35 cm in length. It contains 7 layers of treated silica gels as described inthe JIS methods under dry packing conditions. The design of the column allows for easy connection to various components includingstopcock valves and separatory flasks through the use of commercially available connectors.Figure 1. Vacuum ManifoldG001788 & G001787Top 250mL Flask(Cat. No. 28449-U)?24/24mmPP Viton Connector(Cat. No. 28432-U)?Multi-Layer Dioxin Column(Cat. No. 28397-U)?6.35/10mmReducing Union(Cat. No. 28398-U)?Vacuum Manifold for Dioxin(Cat. No. 28403-U) Customer SuppliedSeparatory Flask withClear Seal Joint24mm ScrewTop Adapter(Cat. No. 21002-U)*Using top adapter, you canuse separator flask with clearseal joint on your handinstead of top flask.?Clear Seal Joint19/22?2P000958Conditioning the ColumnPrior to sample addition, the column is rinsed with 200mL of n-hexane. This rinse is designed to:1. remove air trapped between and within the particles ofsilica, allowing the sample solution to contact thesurfaces of the various coated silica gels and thusremove any interferences more efficiently,2. establish a steady and consistent flow of n-hexane byremoving air bubbles in the column, and3. ensure the cleanliness of the column packing andremove background contamination.After conditioning with n-hexane, the column should allow aflow of about 2.0-2.5mL/min using gravity feed. Two optionaldevices, the vacuum manifold (Cat. No. 28403-U) (Figure1) orthe vacuum adapter (Cat. No. 28408-U) (Figure 2) are avail-able to perform this conditioning quickly and more effectively.The flow after vacuum assisted conditioning will be about 3mL/min.The column is then ready to accept an n-hexane extract of thesample. The analytes (coplanar PCB/PCDD/PCDFs) in theextract will pass through the column with minimal retentionwhile interferences and carry-over contamination from theextraction will be trapped and retained on the column. Theanalytes can then either be collected in the n-hexane eluate foradditional processing by a rotary evaporator or Kuderna-Danish concentrator, or trapped and desorbed with minimumsolvent using the dual-layer Carbon column, or another suit-able concentration method.Column ConditioningUsing Vacuum ManifoldPreferred method (see Figure 1)Position the manifold beakers to retain the eluted n-hexaneinside the vacuum manifold. Connect the multi-layer columnsto the manifold stopcocks with 6.35/10mm Reducing Union(Cat. No. 28398-U) and to a support with clamps. Turn thestopcock valves to the open position. Place the specifiedamount of clean anhydrous sodium sulfate into the top of eachcolumn and tap the columns to settle the particles. Attach a topflask (Cat. No. 28449-U) or a top adapter (Cat. No. 21002-U)with a customer-supplied clear seal ground joint flask to the topof each multi-layer column with a 24/24mm connector (Cat. No.28432-U). Attach a vacuum source to the manifold and adjustthe amount of vacuum to 100-400mm Hg (0.013-0.053 MPa).Add the specified amount of n-hexane to each top flask orseparatory flask, and allow the n-hexane to flow through thecolumns under vacuum. When the n-hexane level has droppedbut is still above the layer of sodium sulfate, turn the stopcocksto the closed position to stop the flow of n-hexane and to keepair out of the column layers. The column is now ready for use.Top Adapter(Cat. No. 21002-U)?Column ConditioningUsing the Vacuum AdapterSecondary method (see Figure 2)Attach a round bottom flask with clear seal joint 24/40 (Cat. No.21269-U) or other similar container and a stopcock to thevacuum adapter. Connect a multi-layer column to the stopcockwith a 6.35/10mm Reducing Union (Cat. No. 28398-U) and toa support with clamps. Turn the stopcock valve to the openposition. Place the specified amount of clean anhydroussodium sulfate into the top of the column and tap the column tosettle the particles. Attach a top flask (Cat. No. 21001-U) or atop adapter (Cat. No. 21002-U) with a customer-supplied clearseal ground joint flask to the top of the multi-layer column witha 24/24 mm connector (Cat. No. 28432-U). Attach a vacuumsource to the adapter and adjust the amount of vacuum to100-400mm Hg (0.013-0.053 MPa). Add the specified amountof n-hexane to each top flask or separatory flask, and allow then-hexane to flow through the columns under vacuum. When then-hexane level has dropped but is still above the layer of sodiumsulfate, turn the stopcock to the closed position to stop the flowof hexane and to keep air out of the column layers. The columnis now ready for use.Figure 2. Vacuum AdapterColumn ConditioningUsing Gravity FeedLeast preferred methodConnect the multi-layer column to a support with a clamp.Attach a stopcock valve to the column with a 6.35/10mmReducing Union (Cat. No. 28398-U). Turn the valve to the openposition. Place a container under the stopcock to retain theeluted hexane. Place the specified amount of clean anhydroussodium sulfate into the top of the column and tap the column tosettle the particles. Attach a top flask (Cat. No. 21001-U) or topadapter (Cat. No. 21002-U) with a customer-supplied clearseal ground joint flask to the top of multi-layer column with a 24/24 mm connector (Cat. No. 28432-U). Add the specifiedamount of n-hexane to the flask and allow the n-hexane to flowthrough the column and stopcock into the container below.When the n-hexane level has dropped but is still above thelayer of sodium sulfate, turn the stopcock to the closed positionto stop the flow of n-hexane and to keep air out of the columnlayers. The column is now ready for use.3P000963fMulti-LayerDioxin Column?6.35/10mmReducing Union(Cat. No. 28398-U)The Dual-Layer Carbon Column*The Dual-Layer carbon column is composed of two 100mg carbon layers,Carboxen 1016 (Surface area 75m2/g) and Carboxen 1000 (Surface area1200m2/g). The carbon layers are held in place with wire screens and fritsbetween each layer and at the ends of the column.The direction of the initial sample flow has the small 6.35mm end of the columnpointing down. The top bed of the dual-layer column contains Carboxen 1016and the lower bed Carboxen 1000.The sample should flow from the 10mm end to the 6.35mm end of the column.The retained analytex are eluted from the column by a flow in the reversedirection, into the 6.35mm end and out the 10mm end of the column.*The Dual-Layer carbon column was developed with Kawajyu Techno Service, with theassistance of Mr. Yukihiro Nishimura and Mr. Kouji Takayama.How to Use the Dual-Layer Carbon ColumnCaution. Please note the following warnings:Exercise extreme care when removing the dual-layer carbon column from itspackage. As you unscrew the green storage caps on the dual-column ends,do not drop or bump the column as the glass frit may be expelled or the packingbeds may be disturbed.Before connecting dual-layer carbon column to other parts, i.e. stopcock, Lueradapter, etc., inspect each end of the column. Remove any silica gel particles,if present, with a piece of clean lab paper, Q-tip, or with a rinse of n-hexanefrom a squeeze bottle.ConditioningThe purpose of conditioning is to remove pockets of air between and within theparticles of carbon and allow a consistent solvent flow. This will also wet thesurface of the carbons, allowing the analytes to achieve maximum contactwith the surface of the packing material, and will remove background contami-nation that may be present in the packing and glass column.For this conditioning, three techniques are available: the vacuum manifold,the vacuum adapter, and the syringe Luer adapter with syringe (syringe notsupplied). (see below).4P000999Carboxen 1016Carboxen 1000Frit ??Frit?Frit ?Screen?Screen??Figure 3.G0022885Using the Vacuum Manifold(See Figure 3)Place an empty beaker inside the manifold to retain the elutedtoluene in this procedure. Attach a 10/10mm union (Cat. No.28412-U) to the 10mm stopcock (Cat. No. 28425-U). Insert the10mm end of the dual-layer carbon column into the other sideof the union and tighten snugly. Attach the 6.35mm end of thedual-layer carbon column to one side of a 6.35mm union (Cat.No. 28411-U) and tighten snugly. To the other side attach anempty dioxin tube (Cat. No. 28404-U or Cat. No. 28409-U) or acustomer-supplied solvent reservoir and tighten snugly. It isadvisable to support the empty dioxin tube or customer-sup-plied reservoir with a clamp and stand.Add a small amount of toluene to the empty tube or reservoir,turn on and adjust the vacuum to about 100-400mm Hg (0.013-0.053 MPa). Check for leaks as the toluene flows through thecolumn. Tighten the union connections if necessary. Do notovertighten. Add 40mL of toluene and elute the solvent throughthe dual-layer carbon column.Discard this toluene flush. Next, add 50mL of n-hexane andelute through the column to remove any residual toluene.Discard the n-hexane rinse. Repeat this step a second time anddiscard the second rinse. Remove the 10mm union and con-nect the stopcock and the dual-layer column with a reducingunion. Connect the column inlet to a solvent reservoir with areducing union. Cap the solvent reservoir after adding a smallamount of n-hexane above the bed of the reversible column.Capping the reservoir and closing the stopcock will minimizeevaporation and keep the column bed wetted.Note: The beds of the dual-layer column should remain wettedwith the non-polar solvent after conditioning.With the Vacuum Adapter(See Figure 4)Attach a 250mL round bottom flask (Cat. No. 21296-U) or othersuitable vacuum vessel to the vacuum adapter (see Figure 4)to retain the eluted toluene in this procedure. Attach a 10/10mmunion (Cat. No. 28412-U) to the adapter and to the 10mm endof dual-layer column and tighten snugly. Attach the 6.35mmend of the dual-layer column to an empty dioxin tube (Cat. No.28404-U or Cat. No. 28409-U) with a 6.35mm union (Cat. No.28411-U) and tighten snugly. Add a small amount of toluene tothe empty tube, turn on and adjust the vacuum to about 100-400mm Hg (0.013-0.053 MPa). Check for leaks as you draw thetoluene through the reversible dual-layer column. It is advisableto support the empty column with a clamp and stand. Add 40mLof toluene and elute the solvent through the dual-layer carboncolumn.Discard this toluene flush. Next, add 50mL of n-hexane andelute through the column to remove any residual toluene.Discard the n-hexane rinse. Repeat this step a second time anddiscard the second n-hexane rinse.Note: The beds of the dual-layer column should remain wettedwith the non-polar solvent after conditioning.Closing the stopcock and capping the empty tube or column willprevent the evaporation of the solvent from the column. Thecolumn may be reversed and a small amount of hexane placedat the head of the column to keep the column bed wetted. Whenreversing the direction of the column, disconnect the unionsattached to the reversible column. Attach two reducing unionsat both ends of the column and a solvent reservoir to the topunion. Capping the reservoir and the column outlet will mini-mize evaporation.Dioxin Sample Vacuum ManifoldFigure 4.Figure 5.P00092150mLGlassSyringe?Syringe Luer Adapter(Cat. No. 28405-U)?10/10mm Union(Cat. No. 28412-U)P000962P000961G001788, G001787250mL Round Bottom Flask with ClearSeal Joint 24/40(Cat. No. 21269-U)6.35/10mmReducing Union(Cat. No. 28398-U)Vacuum Adapter withClear Seal Joint 24/40(Cat. No. 28408-U)???24mm ScrewTop Adapter(Cat. No. 21002-U)*Using top adapter, you canuse separator flask withclear seal joint on the ringstand instead of top flask.?Clear Seal Joint19/22?Customer SuppliedSeparatory Flaskwith Clear Seal Joint6Top 250mL Flask(Cat. No. 28449-U)?24/24mmPP Viton Connector(Cat. No. 28432-U)?Empty Column(Cat. No. 28404-U)??Dual-LayerCarbon Column?Vacuum Adapter withClear Seal Joint 24/40(Cat. No. 28408-U)250mL Round Bottom Flask with ClearSeal Joint 24/40(Cat. No. 21269-U)?With the Syringe Luer Adapter(See Figure 5)Connect a syringe Luer Adapter to the bottom end of a duallayer column with a 6.35/10mm Reducing Union (Cat. No.28398-U) and tighten snugly. Connect the 10mm end of thecolumn to a stopcock (Cat. No. 28402-U) with a 10mm union(Cat. No. 28412-U) and tighten snugly. Using a clean glasssyringe, elute 40mL of toluene through the column, followed by100mL of n-hexane.Check for leaks during this procedure. Discard the eluate.Note: The beds of the dual-layer column should remain wettedwith the non-polar solvent after conditioning. Closing the stop-cock and capping the column will prevent the evaporation of thesolvent from the dual-layer carbon column. Be certain you loadthe sample onto the reversible dual-layer carbon column fromthe 10mm end.6.35/10mmReducing Union(Cat. No. 28398-U)??Sample Processing with the Multi-Layer Silica Geland Dual-Layer Carbon Reversible ColumnsThe extraction of dioxins from the sample is performed according to the method parameters. The extract is routinely concentrated and/or reconstituted into a non-polar solvent for cleanup, elution, and isolation of dioxins using the Multi-Layer Silica Gel and the Dual-LayerCarbon Columns. This may be done in two steps or in a single step procedure.7The Two Step ProcedureThis procedure consists of two steps. In the first step, the non-polar solution is passed through the multi-layer column into asuitable collection vessel. This eluate may be concentrated. Inthe second step, the concentrated eluate is passed through thedual-layer carbon column to trap the analytes of interest. Theanalytes are then recovered from the dual-layer column with aminimum of solvent.First Step: Dioxin analytes elute and contaminants are trappedon multi-layer column.Uncap the conditioned multi-layer column. Place an appropri-ate collection vessel at the bottom of the multi-layer column.Add the sample solution to the column. Attach solvent reservoirto top of column. Add appropriate amount of the method elutionsolvent. Open the stopcock and allow the solvent to flowcompletely through the column into the collection vessel.After the sample extraction solution is collected from the multi-layer column, check the dual-layer carbon column direction andverify that the 6.35mm end is pointing down.Second Step: Pass the solvent solution through a pre-condi-tioned dual-layer carbon column to trap the dioxins.Attach the sample reservoir to the dual-layer column. Take thecollected eluate from the multi-layer column and add this to thedual-layer column reservoir. Turn the stopcock so the collectedsolvent solution flows through the dual-layer column.Note: If the sample extraction solution was concentrated beforeapplication to the dual-layer column it may be advisable to rinsethe dual-layer column with another solvent mixture in the samedirection as when loading with sample to remove possibleinterferences from the column. A solution of n-hexane contain-ing 3.3% methylene chloride has been found to be useful for thispurpose. Pass about 30mL of this solvent through the dual layercolumn.Now reverse the dual-layer column and pass 40 to 100 mL oftoluene through the column to recover the dioxins from thecolumn. Collect the eluate containing the dioxins in a suitablecontainer. This eluate solution may be concentrated and/orreconstituted before GC analysis.Alternately, the dual-layer carbon column may be attacheddirectly to the multi-layer column and the trapping may beperformed in a single step.The Single Step Procedure*(See Figure 6)Precondition the multi-layer and the dual-layer columns sepa-rately. After conditioning, attach the dual-layer column to thebottom of the multi-layer column using a 6.35/10mm reducingunion (Cat. No. 28398-U). Uncap the conditioned multi-layercolumn. Add a small amount of the method elution solvent.Open the stopcock and watch the solution level as it dropstoward the bed of sodium sulfate. Check for leaks. Close thestopcock. Add the extracted sample solution. Attach a solventreservoir to the top of the column. Open the stopcock and watchthe solvent level drop close to the bed of sodium sulfate. Addthe remainder of the method elution solvent. Close the stop-cock to stop the flow of solvent through the column when thesolvent level approaches the sodium sulfate bed. Disconnectsolvent reservoir and the multi-layer column from the dual-layercolumn. Depending upon the sample matrix it may be advisableto rinse the dual-layer column with a solvent mixture to removepossible interferences. A solution of n-hexane containing 3.3%methylene chloride has been found to be useful for this pur-pose. If so, the use of the wash step and the strength of thesolvent needed must be determined by experiment. If it isdetermined to be necessary attach a solvent reservoir to thedual-layer carbon column and pass about 30mL or so of thissolvent through the dual-layer column. Then close the stopcockand reverse the dual-layer column and pass 40 to 100mL oftoluene through the column to recover the PCBs and dioxinsfrom the column. Collect the eluate in a suitable container.*The One-Step Method using dual-layer carbon column andmulti-layer silica gel column in series was developed with theassistance of Mr. Masaaki Maeoka at Japan Quality AssuranceOrganization.Figure 6.Reference11th Symposium on Environmental Chemistry Programs and Abstracts, 2002June, page 298-299Study on short time pre-treatment for analysis of dioxin, Masaaki Maeoka,Itaru Inoue, Hisano Shimono, Nobumasa Morita (Japan Quality AssuranceOrganization)TrademarkTeflon - E.I. du Pont de Nemours & Co., Inc.P000958ConditionedMulti-LayerSilica GelColumn?ConditionedDual-LayerCarbon ColumnTheSIGMA-ALDRICHFamilyBiochemicals andReagents for LifeScience ResearchOrganics andInorganics forChemical SynthesisSpecialty Chemicalsand AnalyticalReagents for ResearchLaboratory Chemicalsand Reagents forResearch and Analysis© 2002 Sigma-Aldrich Co. Printed in USA. Supelco brand products are sold through Sigma-Aldrich, Inc. Sigma-Aldrich, Inc. warrants that its products conform to the informationcontained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions mayapply. Please see reverse side of the invoice or packing slip.SUPELCO ????? 595 North Harrison Road, Bellefonte, PA 16823-0048 ????? 800-247-6628 ????? www.sigma-aldrich.com?Ordering InformationDescription Cat. No.Dioxin Sample Preparation Kit 28423-UKit includes all glassware and connectors (as listed below).NOTE: Requires, but does not include, multi-layer silica gel columns and dual-layer carbonreversible columns.Required Consumables6.35mm Multi-Layer Silica Gel Column, pk. of 5 28397-U6.6.35/10mm Dual-Layer Carbon Reversible Column, pk. of 10 28399-UReplacement Kit PartsGlasswareTTop 250mL Flask 28449-U6.35mm Empty Dioxin Tube, pk. of 5 28404-U6.35mm Syringe Luer Adapter, pk. of 3 28405-U10mm Vacuum Adapter 28408-UBeaker, 300mL, pk. of 3 21266-U250mL Round Flask, pk. of 3 21269-UVacuum Manifold (includes Stopcocks) 28403-UConnectors6.35mm/10mm Reducing Union, pk. of 3 28398-U10mm/10mm Union, pk. of 3 28412-U24mm/24mm PP Viton Connector, pk. of 6 28432-U6.35mm Union, pk. of 3 28411-UOptional ComponentsGlasswareTop Adapter, pk. of 3 21002-U10mm Short Stem Stopcock, pk. of 3 28402-U10mm Longstem Stopcock, pk. of 3 28425-U6.35mm Empty 20cm Glass Tube w/o frit, pk. of 5 28409-UBulk Treated Silica Gels and Sulfate22% KOH Coated Silica Gel, 100g 21318-U10% Silver Nitrate Coated Silica Gel, 100g 21319-U44% H2SO4 Coated Silica Gel, 100g 21334-U22% H2SO4 Coated Silica Gel, 100g 21341-UWashed Silica Gel, 250g 21342-USodium Sulfate Granular, 500g 239313-5006ChromatographyProducts for Analysisand PurificationStable IsotopeProducts for Researchand Discovery