Chromatography 2010/2011. Thermo Scientific. Chromatography Columns and Consumables. Columns and Consumables

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1 North America Asia USA and Canada fax Japan fax China Shanghai: fax Beijing: fax Guangzhou: fax Europe France +33 (0) (0) fax fr.commande@thermofisher.com India (toll-free) fax contact.lpg.in@thermofisher.com Germany fax analyze.de@thermofisher.com Switzerland fax info.ch@thermofisher.com All Other Enquiries United Kingdom fax fsuk.sales@thermofisher.com +44 (0) (0) fax salesorders.columns.uk@thermofisher.com Technical Support North America chromatography.ts@thermofisher.com Outside North America +44 (0) techsupport.columns@thermofisher.com Resources for Chromatographers Beyond this catalog, our Chromatography team shares its extensive expertise through our web-based Chromatography Resource Center and the Separated by Experience enewsletter. The Chromatography Resource Center, accessed at provides technical support, applications, technical tips and literature to help you move your separations forward, quickly and easily. The bi-monthly Separated by Experience enewsletter keeps you up-to-date on the latest technical and product information of interest to chromatographers. Valuable information developed by chromatographers for chromatographers. Subscribe today at Chromatography Columns and Consumables How to Order } 2010/2011 Thermo Scientific Chromatography Columns and Consumables The right tools, separation after separation Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details For more information, please visit our website at CTGSCCHROMCAT 0110 ThermoScientific_CVR_IFC_IBC_BC_FINAL.indd 1 12/7/2009 5:20:15 PM

2 Welcome to the Thermo Scientific Chromatography Columns and Consumables Catalog Notes This year s catalog offers you 540 new pages of innovative and proven chromatography tools. It showcases our commitment of continued investment in all areas of chromatography to develop and expand our outstanding product portfolio using the most advanced materials and technologies to give you confidence, separation after separation. Where accuracy and repeatability are paramount for the delivery of consistently high quality results, we provide a wealth of chromatography consumables to ensure you have the right tools to meet all your separation needs. With a long history of chromatography expertise and established products we now have grown our offering to include trusted names, such as Hypersil, Chromacol and National Scientific. This gives you choice, quality and dependability. Today, our comprehensive Thermo Scientific product portfolio continues to expand and includes many new and unique additions. With an extensive range of products, we place the broadest collection of chromatography consumables at your finger tips, including: Hypersil GOLD HPLC Columns TraceGOLD and TracePLOT GC Columns HyperSep Retain SPE Solutions Reagents and accessories National Scientific and Chromacol vials and closures tips. In recognition of today s stringent regulatory requirements, our investment extends to the quality assurance of our products. We can guarantee consistently accurate research tools. From R&D through to final production, utilizing our own global manufacturing and column packing facilities means our products are of the highest quality. Such is our commitment to chromatography consumables; we are currently expanding our cutting edge research and manufacturing facilities on a global scale. This new Thermo Scientific Chromatography Columns and Consumables catalog is supported by our in-depth scientific applications and knowledge. Our expert technical team, in collaboration with many of our customers, is dedicated to constantly developing new techniques and troubleshooting guides to further improve method development and advance chromatography approaches in line with current and future analytical demands. From sample preparation to separation and analysis, our tools help you achieve repeatable, predictable results separation after separation. We hope that you find the information in this catalog helpful in your product selection and support. In addition, we urge you to visit our website, for additional information. Thank you for your continuing support of our product line. Darren Thomas Product Line Director, Chromatography Consumables CCG_CVR_IFC_IBC_BC_ FINAL.indd 2 12/7/2009 5:11:13 PM

3 Sample Preparation Products Our comprehensive range of sample preparation products have been developed for rapid, effective and economical sample preparation. Thermo Scientific HyperSep SPE products are available in a range of formats including columns, multi-well plates and pipette tips. We also offer the complete line of National Scientific syringes and the innovative WebSeal system. 86

4 Sample Preparation trust HyperSep Retain Thermo Scientific HyperSep Retain is ideal for fast and easy sample preparation and method development. WebSeal Our Chromacol WebSeal products are ideal for high throughput screening, combinational chemistry life science and HPLC. QuEChERS Thermo Scientific HyperSep Dispersive SPE products provide efficient sample preparation and clean-up using the QuEChERS Method. Go to PAGE 94 Go to PAGE 150 Go to PAGE

5 SPE Method Development The following flow chart briefly describes the common steps in SPE method development and optimization. SPE Method Selection Overview 88

6 SPE Method Development 89

7 SPE Column Selection Thermo Scientific HyperSep SPE products have been developed to meet the requirements of today s sample preparation challenges. HyperSep SPE columns and multi-well plates are offered in a range of phases, ideal for use in application areas such as: Pharmaceutical, Biochemical, Environmental, and Food and Beverage. We are committed to ensuring that high quality products are selected for your application. Please refer to the selection guide below to determine the correct product(s) for your particular application. SPE Phase Selection 90

8 SPE Column Selection SPE Phase Selection by Manufacturer Thermo Scientific HyperSep Product Alternative To Thermo Scientific HyperSep Product Alternative To HyperSep C18, page 99 CLEAN-UP C18-U Supelclean ENVI-18 / LC-18 SampliQ C18 BAKERBOND spe Polar Plus Isolute C18 CHROMABOND C18 Bond Elut C18 strata C18-U Sep-Pak C18 HyperSep Florisil, page 107 CLEAN-UP Florisil Supelclean ENVI-Florisil / LC-Florisil SampliQ Florisil BAKERBOND spe Florisil Isolute Florisil CHROMABOND Florisil Bond Elut Florisil strata FL-PR Sep Pak Florisil HyperSep C8, page 100 HyperSep Phenyl, page 101 CLEAN-UP C8 Supelclean ENVI-8 / LC-8 SampliQ C8 BAKERBOND spe Octyl C8 Isolute C8 CHROMABOND C8 Bond Elut C8 strata C8 Sep Pak C8 CLEAN-UP Phenyl Supelclean LC-Ph SampliQ Phenyl BAKERBOND spe Phenyl Isolute Ph Bond Elut Ph strata Phenyl (PH) HyperSep SAX, page 103 HyperSep SCX, page 104 CLEAN-UP QAX Supelclean LC-SAX SampliQ Si-SAX BAKERBOND spe Quaternary amine Isolute SAX CHROMABOND SB Bond Elut SAX strata SAX CLEAN-UP BCX Supelclean LC-SCX SampliQ Si-SCX BAKERBOND spe Aromatic Sulfonic Acid Isolute SCX CHROMABOND SA Bond Elut SCX strata SCX HyperSep Silica, page 102 HyperSep Aminopropyl, page 108 CLEAN-UP Silica Supelclean LC-Si SampliQ Silica BAKERBOND spe Silica Gel Isolute Silica CHROMABOND SiOH Bond Elut Si strata Si-1 Sep Pak Si CLEAN-UP Aminopropyl Supelclean LC-NH 2 SampliQ Amino BAKERBOND spe Amino Isolute NH 2 CHROMABOND NH 2 Bond Elut NH 2 strata NH 2 Sep-Pak NH 2 HyperSep Verify-AX, page 106 HyperSep Verify-CX, page 105 HyperSep Retain PEP, page 94 CLEAN-UP THC Isolute HAX Bond Elut Certify II strata Screen-A CLEAN-UP DAU Discovery DSC-MCAX SampliQ C8/SCX Isolute HCX CHROMABOND Drug Bond Elut Certify I strata Screen-C Oasis HLB (Waters) Strata-X (Phenomenex) FOCUS (Varian) Easy (Macherey Nagel) Isolute ENV+ (Biotage) SampliQ OPT StyreScreen DVB (UCT) H 2 O-philic DVB (JT Baker) HyperSep Retain-CX, page 96 Oasis MCX (Waters) SampliQ SCX Strata-X-C (Phenomenex) StyreScreen DBX (UCT) HyperSep Retain-AX, page 98 Oasis MAX (Waters) SampliQ SAX StyreScreen QAX (UCT) 91

9 evol Dispensing System Allows precise and accurate, operator-independent dispensing for better deployment of laboratory staff Applications: Routine dispensing of volatile solvents, hazardous, corrosive or viscous chemicals } Can be calibrated easily by user to ensure validity of results } Intuitive interface with touch wheel and full-color screen } Complies to GLP and GMP protocols } Improves confidence in reported results } Unifies two precision devices, a digitally controlled electronic drive and an XCHANGE TM analytical syringe } Integrated XCHANGE TM coupling allows syringes to be quickly and easily changed } XCHANGE TM syringes offer exceptional versatility and functionality } Addition of MEPS to XCHANGE TM syringe offers automated sample preparation by SPE in a handheld system evol Dispensing System Type Volume Cat. No. Quantity Sample Dispensing System N/A Each evol TM XCHANGE syringe 5µL Each evol TM XCHANGE syringe 50µL Each evol TM XCHANGE syringe 500µL Each Sample Dispensing System Kit N/A Each evol Stand N/A Each HyperSep Hypercarb Unique material for retention of highly polar compounds } Flat, 100% porous graphitic carbon (PGC) with selectivity for structurally similar compounds, offering separation of compounds with simple solvents } ph stable 0 to 14 } High batch-to-batch reproducibility } Strong retention properties allow use of low bed weights for concentrated extracts } Interaction mechanism with polar molecules HyperSep Hypercarb SPE Columns Bed Weight Column Volume Cat. No. Quantity 25mg 1mL Pack 50mg 1mL Pack 100mg 1mL Pack 200mg 3mL Pack 500mg 6mL Pack 1g 6mL Pack 2g 15mL Pack HyperSep-96 Hypercarb Well Plates and Individual Wells Bed Weight Volume Cat. No. Quantity Individual Wells 10mg 1mL Pack 25mg 1mL Pack 50mg 1mL Pack Well Plates 10mg 1mL Each 25mg 1mL Each 50mg 1mL Each 92

10 Sample Preparation HyperSep Hypercarb SPE Columns Porous Graphitic Carbon (PGC) is a unique stationary phase composed of flat sheets of hexagonally arranged carbon atoms with a satisfied valence, as in a very large polynuclear aromatic molecule. PGC is unlike traditional silica bonded phases in both its structure and retentive properties, allowing for total ph stability and the retention and separation of highly polar species. HyperSep Hypercarb TM is ideal for problem analytes in SPE applications. Thermo Scientific HyperSep Reversed Phase / Polar Retention Effect on Graphite Method (Hypercarb): 1. Analyte Properties Polar, uncharged species can also retain some ionic/charged species 2. Sample Preparation/Application (Polar, aqueous) Add internal standard to the sample if quantification is desired. Optimize sample application by removing particulates if necessary (centrifugation or filtration) and/or diluting viscous matrices with water or buffer to ensure proper ph for desired interactions. This will require some experimentation. 3. Column Conditioning 2-5 column volumes of a strong eluting solvent followed by 2-5 column volumes of water. 4. Column Wash Use water or a weak eluting solvent which will wash most interferences from the sorbent without loss of analytes. Wash ph may greatly affect cleanup and/or recovery. 5. Elution Use a strong eluting solvent to elute the analytes of interest. Addition of 0.1% TFA to the solvent will increase its elution strength or polar analytes. Suggested Elution Solvents MeOH THF* Ethyl Acetate* Dichloromethane* Chloroform* Increasing Elution Strength *Elution strength will depend on the nature of the analytes. Application Example for HyperSep Hypercarb Cyanuric Acid in Drinking Water Cyanuric Acid is an FDA-accepted component of feed-grade biuret, a ruminant feed additive. It is also found in swimming pool water a chlorine stabilizer. Consumer exposure may be through swallowing swimming pool water, through drinking water processed from surface water and through fish which may accumulate this chemical. Cyanuric Acid in Drinking Water Compounds: Cyanuric Acid Part Number: Phase: HyperSep Hypercarb Volume: 6mL Bed Weight: 500mg Sample Adjust water sample to ph 3 Pretreatment: Conditioning: Wash column with 10mL methanol Condition column with 10mL LC-grade water Application: Force/aspirate mL of water sample into column at rate of 5mL/min. Washing: Dry column under vacuum Elution: 20mL methanol evaporate to dryness at 50 C under nitrogen Source: Marie Claire Hennion, ESPCI, Paris Loading Capacity and the Effects of Drying Out of Hypercarb The loading capacity and the effects of drying out of material on recovery were investigated for HyperSep Hypercarb using a 200mg 3mL column (part number ). Here, melamine was used to demonstrate Hypercarb s ability to recover polar compounds. The Effect of Post-Loading or Washing Drying Time The HyperSep Hypercarb SPE cartridges were dried after loading the analytes by drawing air through the packed bed. HyperSep Hypercarb gave a 95% recovery of melamine, irrespective of the drying time. (Figure 1) Loading Capacity The loading capacity of HyperSep Hypercarb was established using an aqueous solution of melamine with a concentration equal to that of 1% of the sorbent bed weight in 1mL. (Figure 2) HyperSep Hypercarb was found to have a loading capacity of approximately 4% of the column bed weight. (Figure 1) (Figure 2) 93

11 HyperSep Retain PEP Generic Procedure for HyperSep Retain PEP: 1. Conditioning: Methanol followed by de-ionised water 2. Sample Loading: Load sample at a rate of 1-2mL/min. 3. Washing: Methanol / de-ionised water (5/95, v/v) 4. Elution: Organic solvent such as methanol or acetonitrile Application Example for HyperSep Retain PEP Thiourea Herbicides in Soil 1. SAMPLE PRE-TREATMENT: Thiourea herbicides extracted from soil (1ppm) in phosphorate buffer solution (ph 2.5) 2. CONDITIONING: 5mL methanol followed by 5mL phosphorate buffer (ph 2.5) 3. SAMPLE LOADING: Load 10mL sample at 1-2mL /min. 4. WASHING: 3mL phosphorate buffer (ph 2.5) 5. ELUTION: 5mL acetonitrile / phosphorate buffer (ph 7.8) (9:1, v/v) Results: Compound Name Nicosulfuron 0.91 Thifensulfuron-methyl 0.89 Metsulfuron-methyl 0.89 Sulfometuron-methyl 0.86 Chlorsulfuron 0.99 Ethametsulfuron-methyl 0.81 Tribenuron 0.15 Bensulfuron-methyl 0.82 Pyrazosulfuron-ethyl 0.93 Chlorimuron-ethyl 1.07 HyperSep Retain PEP Recovery Benefits of using HyperSep Retain PEP A comparison was made between HyperSep Retain PEP & HyperSep C18 SPE Cartridges to demonstrate the benefits of using a polymeric-based material for SPE applications. There are benefits to using HyperSep Retain polymeric SPE materials for SPE applications. Here, a HyperSep Retain PEP 60mg 3mL column (part number ) was compared with a HyperSep C18 200mg 3mL column (part number ). The Effect of Post-Loading or Washing Drying Time The SPE cartridges were dried after loading the analytes by drawing air through the packed bed: The effect of drying time, measured using recovery levels of salicyclic acid and acetaminophen After drying the HyperSep C18 bed for 1 minute, the recovery levels of both salicyclic acid and acetaminophen were significantly reduced. Acetaminophen showed a 20% decrease in recovery, with subsequent drying producing additional losses, eventually demonstrating recoveries of less than 30% after 10 minutes. The experiment was repeated using the HyperSep Retain PEP sorbent and the acetaminophen recovery was sustained at > 95% irrespective of drying time. Loading capacities of HyperSep Retain PEP (60mg) and HyperSep C18 (200mg) as measured using breakthrough of acetaminophen. Loading Capacity The loading capacities of the HyperSep Retain PEP and HyperSep C18 were compared using acetaminophen as the compound of interest. On the HyperSep C18 sorbent, compound breakthrough occurred when the sample load was less than 5% of the packed bed weight. However, HyperSep Retain PEP demonstrated a sample loading capacity up to 15% of the packed bed weight. This effect may be attributed to the higher surface area of the polymeric media. Consequently, the higher capacity of the polymeric material results in a reduced risk of compound breakthrough. A further advantage of the HyperSep Retain PEP over HyperSep C18 is that equal or better recovery can be obtained using a smaller bed weight, leading to smaller volumes of solvent, which has environmental and economic advantages. 94

12 Sample Preparation HyperSep Retain PEP Versatile polymeric material for retention of polar and non-polar analytes Applications: Drugs and metabolites in biological fluids Peptides in serum, plasma or biological fluids Environmental samples } Exceptional recoveries for polar and non-polar analytes } High and consistent recoveries } High capacity material is a high purity, highly porous polystyrene DVB material modified with urea functional groups to give balanced retention of polar and non-polar analytes } Fast and easy sample preparation and method development } ph stable 0 to 14 HyperSep Retain PEP SPE Columns Bed Weight Column Volume Cat. No. Quantity 30mg 1mL Pack 30mg 3mL Pack 60mg 3mL Pack 60mg 6mL Pack 100mg 6mL Pack 150mg 6mL Pack 200mg 3mL Pack 200mg 6mL Pack 500mg 3mL Pack 500mg 6mL Pack 1g 25mL Pack 2g 25mL Pack HyperSep-96 Retain PEP Well Plates and Individual Wells Bed Weight Volume Cat. No. Quantity Individual Wells 5mg 1mL Pack 10mg 1mL Pack 30mg 1mL Pack 60mg 1mL Pack Well Plates 5mg 1mL Each 10mg 1mL Each 30mg 1mL Each 60mg 1mL Each 95

13 HyperSep Retain-CX Versatile polymeric material for retention of basic compounds Applications: Analysis of a wide range of drugs of abuse including basic and neutral drugs } Exceptional recoveries for basic analytes } High and consistent recoveries } High capacity material is a high purity, highly porous polystyrene DVB material partially modified with sulfonic acid functional groups } Fast and easy sample preparation and method development } ph stable 0 to 14 HyperSep Retain CX SPE Columns Bed Weight Column Volume Cat. No. Quantity 30mg 1mL Pack 30mg 3mL Pack 60mg 3mL Pack 60mg 6mL Pack 100mg 6mL Pack 150mg 6mL Pack 200mg 3mL Pack 200mg 6mL Pack 500mg 3mL Pack 500mg 6mL Pack 1g 25mL Pack 2g 25mL Pack HyperSep-96 Retain-CX Well Plates and Individual Wells Bed Weight Column Volume Cat. No. Quantity Individual Wells 5mg 1mL Pack 10mg 1mL Pack 30mg 1mL Pack 60mg 1mL Pack Well Plates 5mg 1mL Each 10mg 1mL Each 30mg 1mL Each 60mg 1mL Each 96

14 Sample Preparation HyperSep Retain-CX Method / Application Example / Retain-CX vs Retain PEP for basic compounds Generic Procedure for HyperSep Retain-CX: 1. Conditioning: Methanol followed by de-ionised water 2. Sample Loading: Load sample at a rate of 1-2mL/min. 3. Washing: Methanol / de-ionised water (5/95, v/v) 4. Elution: Organic solvent such as methanol or acetonitrile Application Example Melamine in Animal Feeds Comparison Acetaminophen in Calf Serum HyperSep Retain-CX offers additional selectivity for basic compounds. A comparison of the recovery levels generated by HyperSep Retain PEP 60mg 3mL column (part number ) and HyperSep Retain-CX 60mg 3mL column (part number ) were compared. The compound analysed was acetaminophen from calf serum. Part Number: Phase: HyperSep Retain-CX Volume: 3 ml Bed Weight: 60 mg Sample To 5 g of animal feed, add 50 ml of 0.1% trichloroacetic acid Pretreatment: aqueous solution. Vortex for 1 min, then add 2 ml of 2% lead acetate aqueous solution. Sonicate for 20 mins, then transfer a portion of the mixture to a 10 ml Centrifuge tube. Centrifuge at 8000 rpm for 10 mins. Conditioning: 3 ml methanol followed by 3 ml deionized water Application: Load 3 ml sample at 1-2 ml/min Washing: 3 ml water followed by 3 ml methanol. Purge column to dryness Elution: 5 ml ammonia in methanol (5:95, v/v). Dry sample under nitrogen stream. Reconstitute with 20% methanol aqueous solution. 1. SAMPLE PRE-TREATMENT: Dilute serum with 10mmol phosphoric acid of the same volume 2. CONDITIONING: 2mL methanol followed by 2mL DI Water 3. SAMPLE LOADING: Load 2mL sample 4. WASHING: 2mL methanol / water (5/95, v/v) 5. ELUTION: 2mL methanol SPE Column Recovery HyperSep Retain PEP 86.9% HyperSep Retain-CX 94.6% HyperSep Retain-CX gave additional selectivity for a basic compound such as acetaminophen when compared with HyperSep Retain PEP. 97

15 HyperSep Retain-AX Versatile polymeric material for retention of acidic compounds Applications: Analysis of THC and its metabolites } Exceptional recoveries for acidic analytes } High and consistent recoveries } High capacity material is a high purity, highly porous polystyrene DVB material partially modified with quaternary amine functional groups } Fast and easy sample preparation and method development } ph stable 0 to 14 HyperSep Retain AX SPE Columns Bed Weight Column Volume Cat. No. Quantity 30mg 1mL Pack 30mg 3mL Pack 60mg 3mL Pack 60mg 6mL Pack 100mg 6mL Pack 150mg 6mL Pack 200mg 3mL Pack 200mg 6mL Pack 500mg 3mL Pack 500mg 6mL Pack 1g 25mL Pack 2g 25mL Pack HyperSep-96 Retain-AX Well Plates and Individual Wells Bed Weight Volume Cat. No. Quantity Individual Wells 5mg 1mL Pack 10mg 1mL Pack 30mg 1mL Pack 60mg 1mL Pack Well Plates 5mg 1mL Each 10mg 1mL Each 30mg 1mL Each 60mg 1mL Each Generic Procedure for HyperSep Retain-AX: 1. Conditioning: Methanol followed by de-ionised water 2. Sample Loading: Load sample at a rate of 1-2mL/min. 3. Washing: Water containing 2% ammonium hydroxide 4. Elution: Methanol containing 2% acetic acid Application Example Nadifloxacin Compounds: Nadifloxacin Part Number: Phase: HyperSep Retain-AX Volume: 3mL Bed Weight: 60mg Sample 5ppm of nadifloxacin sample in Pretreatment: 50mM phosphate ph 7.4 Conditioning: 1mL methanol followed by 1mL of 2M NaOH and 1mL deionized water Application: Load 5mL sample at 1-2mL/min. Washing: 1mL of 5% ammonia aqueous solution followed by 1mL methanol Elution: 3mL of methanol with 4% acetic acid Application Example Methylmalonic Acid Compounds: Methylmalonic acid (MMA) Part Number: Phase: HyperSep Retain-AX Volume: 3mL Bed Weight: 60mg Sample To 250μL serum or plasma add 100μL Pretreatment: D3MMA (2.5μM/L) and 1.5mL water Conditioning 2mL methanol followed by 2mL water Application: Load samples at 1-2mL/min. Washing: 2mL water followed by 2mL methanol with 0.5% acetic acid Elution: 2mL Tert Butyl Methyl Ether (TBME) with 3% formic acid. Evaporate to dryness at <40ºC. Derivatization: Add 50μL acetonitrile and 25μL MTBSTFA + 1% TBDMCl. Heat at 70ºC for 10 mins. Source: Department of Laboratory Medicine, The Hospital of Telemark 98

16 HyperSep C18 Sample Preparation Feature a highly retentive alkyl-bonded phase for non-polar to moderately polar compounds Applications: Retentive for non-polar compounds Retains most organic analytes from aqueous matrices } Retentive for most non-polar compounds } Retains most organic analytes from aqueous matrices } Hydrophobic reversed phase } For nonpolar to moderately polar compounds HyperSep C18 SPE Columns Bed Weight Column Volume Cat. No. Quantity 50mg 1mL Pack 100mg 1mL Pack 200mg 3mL Pack 500mg 3mL Pack 500mg 6mL Pack 1g 6mL Pack 2g 15mL Pack 5g 25mL Pack 10g 75mL Pack HyperSep-96 C18 Well Plates and Individual Wells Bed Weight Volume Cat. No. Quantity Individual Wells 10mg 1mL Pack 25mg 1mL Pack 50mg 1mL Pack 100mg 1mL Pack Well Plates 10mg 1mL Each 25mg 1mL Each 50mg 1mL Each 100mg 1mL Each 99

17 HyperSep C8 Less retentive alternative to C18 for polar and non-polar compounds Applications: Drugs and their metabolites in biological samples Peptides in biological samples } Well-suited for methods requiring less retention than C18 } Excellent for moderately polar analytes } Potential for polar interactions greater than with C18 HyperSep C8 SPE Columns Bed Weight Column Volume Cat. No. Quantity 50mg 1mL Pack 100mg 1mL Pack 200mg 3mL Pack 500mg 3mL Pack 500mg 6mL Pack 1g 6mL Pack 2g 15mL Pack 5g 25mL Pack 10g 75mL Pack HyperSep-96 C8 Well Plates and Individual Wells Bed Weight Volume Cat. No. Quantity Individual Wells 10mg 1mL Pack 25mg 1mL Pack 50mg 1mL Pack 100mg 1mL Pack Well Plates 10mg 1mL Each 25mg 1mL Each 50mg 1mL Each 100mg 1mL Each 100

18 Sample Preparation HyperSep Phenyl Offer alternative selectivity for retention of basic compounds Applications: Extraction of aromatic compounds Extraction of basic analytes } Commonly employed for nonpolar interactions } The electron density of the aromatic ring provides different selectivity than other non-polar sorbents HyperSep Phenyl SPE Columns Bed Weight Column Volume Cat. No. Quantity 50mg 1mL Pack 100mg 1mL Pack 200mg 3mL Pack 500mg 3mL Pack 500mg 6mL Pack 1g 6mL Pack 2g 15mL Pack 5g 25mL Pack 10g 75mL Pack HyperSep-96 Phenyl Well Plates and Individual Wells Bed Weight Volume Cat. No. Quantity Individual Wells 10mg 1mL Pack 25mg 1mL Pack 50mg 1mL Pack 100mg 1mL Pack Well Plates 10mg 1mL Each 25mg 1mL Each 50mg 1mL Each 100mg 1mL Each 101

19 HyperSep Silica A polar sorbent primarily used to retain analytes in nonpolar matrices Applications: Extraction of polar compounds including aldehydes, amines, drugs, pesticides and herbicides Extraction of carotenoids, fat-soluble vitamins, aflatoxins in food matrices Extraction of fatty acids and phospholipids } Effectively separates compounds of very similar structure } Adsorbs analytes from nonpolar solvents like hydrocarbons, less polar esters and ethers } Suitable for use as an intermediate strength cation exchanger in aqueous medium HyperSep Silica SPE Columns Bed weight Column Volume Cat. No. Quantity 50mg 1mL Pack 100mg 1mL Pack 200mg 3mL Pack 500mg 3mL Pack 500mg 6mL Pack 1g 6mL Pack 2g 15mL Pack 5g 25mL Pack 10g 75mL Pack HyperSep-96 Silica Well Plates and Individual Wells Bed Weight Volume Cat. No. Quantity Individual Wells 10mg 1mL Pack 25mg 1mL Pack 50mg 1mL Pack 100mg 1mL Pack Well Plates 10mg 1mL Each 25mg 1mL Each 50mg 1mL Each 100mg 1mL Each 102

20 Sample Preparation HyperSep SAX Strong Anion Exchanger Strong anion exchange sorbent for extraction of weak acids Applications: Isolation of anionic proteins Removal of acidic food pigments Isolation of phenolic compounds Nucleic acids, nucleotides and surfactants } Extracts negatively charged compounds from both aqueous and non-aqueous solutions } Ideally suited for the extraction of weak acids, e.g. carboxylic acids } Amine group masks the effect of the carbon chain of the functional group } Selectivity can be tuned by selection of buffer HyperSep SAX SPE Columns Bed Weight Column Volume Cat. No. Quantity 50mg 1mL Pack 100mg 1mL Pack 200mg 3mL Pack 500mg 3mL Pack 500mg 6mL Pack 1g 6mL Pack 2g 15mL Pack 5g 25mL Pack 10g 75mL Pack Applications: Extraction of antibiotics, drugs, organic bases, amino acids, catecholamines and herbicides HyperSep-96 SAX Well Plates and Individual Wells Bed Weight Volume Cat. No. Quantity Individual Wells 10mg 1mL Pack 25mg 1mL Pack 50mg 1mL Pack 100mg 1mL Pack Well Plates 10mg 1mL Each 25mg 1mL Each 50mg 1mL Each 100mg 1mL Each 103

21 HyperSep SCX Strong Cation Exchanger A strong cation exchange sorbent for extraction of charged basic compounds Applications: } Benzene ring provides potential for non-polar interactions Isolation of anionic proteins } Low pka value sorbent Removal of acidic food pigments Isolation of phenolic compounds Nucleic acids, nucleotides and surfactants HyperSep SCX SPE Columns Bed Weight Column Volume Cat. No. Quantity 50mg 1mL Pack 100mg 1mL Pack 200mg 3mL Pack 500mg 3mL Pack 500mg 6mL Pack 1g 6mL Pack 2g 15mL Pack 5g 25mL Pack 10g 75mL Pack HyperSep-96 SCX Well Plates and Individual Wells Bed Weight Volume Cat. No. Quantity Individual Wells 10mg 1mL Pack 25mg 1mL Pack 50mg 1mL Pack 100mg 1mL Pack Well Plates 10mg 1mL Each 25mg 1mL Each 50mg 1mL Each 100mg 1mL Each 104

22 HyperSep Verify-CX Sample Preparation Features non-polar and anionic characteristics for improved analysis of drugs of abuse Applications: Analysis of a wide range of drugs of abuse from biological matrices, including basic and neutral drugs } Mixed mode sorbent for extraction of basic drugs } Separation based on two functional groups: reversed phase C8 and an ion exchanger, benzene sulfonic acid } Co-polymerized on a rigid, purified silica gel support HyperSep Verify-CX SPE Columns Bed Weight Column Volume Cat. No. Quantity 130mg 1mL Pack 200mg 6mL Pack 300mg 3mL Pack 500mg 3mL Pack 500mg 6mL Pack 1g 6mL Pack HyperSep-96 Verify-CX Well Plates and Individual Wells Bed Weight Volume Cat. No. Quantity Individual Wells 10mg 1mL Pack 25mg 1mL Pack 50mg 1mL Pack 100mg 1mL Pack Well Plates 10mg 1mL Each 25mg 1mL Each 50mg 1mL Each 100mg 1mL Each 105

23 HyperSep Verify-AX Features nonpolar and cationic characteristics for improved analysis of acidic drugs and metabolites Applications: Analysis of THC and its metabolites } Mixed mode sorbent suited for the extraction of acidic drugs and metabolites from biological matrices } Separation based on two functional groups: reversed phase C8 and an ion exchanger, quaternary amine } Rigid, purified silica gel support to which the two functionalities are co-polymerized HyperSep Verify-AX SPE Columns Bed Weight Column Volume Cat. No. Quantity 130mg 1mL Pack 200mg 6mL Pack 300mg 3mL Pack 500mg 3mL Pack 500mg 6mL Pack 1g 6mL Pack HyperSep-96 Verify-AX Well Plates and Individual Wells Bed Weight Volume Cat. No. Quantity Individual Wells 10mg 1mL Pack 25mg 1mL Pack 50mg 1mL Pack 100mg 1mL Pack Well Plates 10mg 1mL Each 25mg 1mL Each 50mg 1mL Each 100mg 1mL Each 106

24 Sample Preparation HyperSep Florisil Ideal for the isolation of polar compounds from non-polar matrices Applications: Extraction of pesticides using official methods Polychlorinated biphenyls in transformer oil Alcohol, aldehydes, amines and drugs } Magnesia-loaded silica gel offers rapid flowrates for large sample volumes } Extremely polar in nature, especially well-suited for separation of chlorinated pesticides HyperSep Florisil SPE Columns Bed Weight Column Volume Cat. No. Quantity 50mg 1mL Pack 100mg 1mL Pack 200mg 3mL Pack 500mg 3mL Pack 500mg 6mL Pack 1g 6mL Pack 2g 15mL Pack 5g 25mL Pack 10g 75mL Pack HyperSep-96 Florisil Well Plates and Individual Wells Bed Weight Volume Cat. No. Quantity Individual Wells 10mg 1mL Pack 25mg 1mL Pack 50mg 1mL Pack 100mg 1mL Pack Well Plates 10mg 1mL Each 25mg 1mL Each 50mg 1mL Each 100mg 1mL Each 107

25 HyperSep Aminopropyl A polar sorbent for both polar and anion exchange interactions Applications: Separation of structural isomers Drugs and metabolites in biological fluids Separation of saccharides, phenols and petroleum products } Weaker anion exchanger than sorbents like SAX } Enhanced retention of strong anions like sulfonic acids } Excellent retention of drugs, metabolites and structural isomer HyperSep Aminopropyl SPE Columns Bed Weight Column Volume Cat. No. Quantity 50mg 1mL Pack 100mg 1mL Pack 200mg 3mL Pack 500mg 3mL Pack 500mg 6mL Pack 1g 6mL Pack 2g 15mL Pack 5g 25mL Pack 10g 75mL Pack HyperSep-96 Aminopropyl Well Plates and Individual Wells Bed Weight Volume Cat. No. Quantity Individual Wells 10mg 1mL Pack 25mg 1mL Pack 50mg 1mL Pack 100mg 1mL Pack Well Plates 10mg 1mL Each 25mg 1mL Each 50mg 1mL Each 100mg 1mL Each 108

26 Sample Preparation HyperSep Cyano Optimized for the retention of polar compounds from non-polar matrices Applications: Retaining polar compounds from hexane and oils Reversed phase extraction of moderately polar compounds } Medium polarity sorbent, less retentive than silica or diol } Suitable for reverse phase extractions of moderately polar compounds HyperSep Cyano SPE Columns Bed Weight Column Volume Cat. No. Quantity 50mg 1mL Pack 100mg 1mL Pack 200mg 3mL Pack 500mg 3mL Pack 500mg 6mL Pack 1g 6mL Pack 2g 15mL Pack 5g 25mL Pack 10g 75mL Pack HyperSep-96 Cyano Well Plates and Individual Wells Bed Weight Volume Cat. No. Quantity Individual Wells 10mg 1mL Pack 25mg 1mL Pack 50mg 1mL Pack 100mg 1mL Pack Well Plates 10mg 1mL Each 25mg 1mL Each 50mg 1mL Each 100mg 1mL Each 109

27 HyperSep Diol Ideal for the extraction of polar compounds Applications: Normal phase extraction of polar compounds Purification of polar compounds } Short alkyl chains with polar functional groups HyperSep-96 Diol SPE Columns Bed Weight Column Volume Cat. No. Quantity 50mg 1mL Pack 100mg 1mL Pack 200mg 3mL Pack 500mg 3mL Pack 500mg 6mL Pack 1g 6mL Pack 2g 15mL Pack 5g 25mL Pack 10g 75mL Pack HyperSep-96 Diol Well Plates and Individual Wells Bed Weight Volume Cat. No. Quantity Individual Wells 10mg 1mL Pack 25mg 1mL Pack 50mg 1mL Pack 100mg 1mL Pack Well Plates 10mg 1mL Each 25mg 1mL Each 50mg 1mL Each 100mg 1mL Each 110

28 Sample Preparation HyperSep Universal Vacuum Manifold } Accommodates both SPE columns and 96-well plates System supplied with: Manifold, base/gauge, flask & stopper, tubing & spigots Universal Vacuum Manifold Universal Manifold Base/Gauge HyperSep Universal Vacuum Manifold Description Cat. No. Quantity Universal Vacuum Manifold Each Vacuum Pump, North American Plug Each Vacuum Pump, European Plug Each Plugs for 24-position Extraction Plate Pack Plugs for 48-position Extraction Plate Pack

29 HyperSep Glass Block Manifolds } 16-Port Vacuum Manifold: Glass Block, Corian Manifold Lid, Cover Gasket, Vacuum Gauge and Valve Assembly, 16 Teflon Tips, Adjustable Collection Rack, Bulkhead Luer Fittings, 16 Plugs and Manifold Safety Tray } 24-Port Vacuum Manifold: Glass Block, Corian Manifold Lid, Cover Gasket, Vacuum Gauge and Valve Assembly, 24 Teflon Tips, Adjustable Collection Rack, Bulkhead Luer Fittings, 24 Plugs and Manifold Safety Tray Manifold Lid Manifold Lid Legs Collection Rack Vacuum Gauge and Valve Assembly Manifold Safety Tray Luer Lock Plugs Retaining Clips for Collection Rack Bulkhead Luer Fittings Vacuum Gauge and Valve Assembly Stopcocks Teflon Tips 112 HyperSep Glass Block Manifolds Description Cat. No. Quantity 16-port Vacuum Manifold Each 24-port Vacuum Manifold Each Vacuum Pump, North American Plug Each Vacuum Pump, European Plug Each Replacement Parts Vacuum Gauge Each Stopcocks for 16-port Vacuum Manifold Pack Stopcocks for 24-port Vacuum Manifold Pack TFE tips for Vacuum Manifold Pack Vacuum Gauge and Valve Assembly Each Lid for 16-port Glass Block Manifold Each Lid for 24-port Glass Block Manifold Each Gasket for 16-port Manifold Each Gasket for 24-port Manifold Each Collection Rack for 16-port Vacuum Manifold Each Collection Rack for 24-port Vacuum Manifold Each Glass Block for 16-port Vacuum Manifold Each Glass Block for 24-port Vacuum Manifold Each Manifold Safety Tray Each Retaining Clips for Collection Rack Pack Bulkhead Luer Fittings Pack Manifold Lid Legs Pack Luer Lock Plugs Pack

30 Sample Preparation HyperSep-96 Well Plate Manifold Included with system: } Base } Lid } Waste collection tray HyperSep-96 Well Plate Manifold Lid Manifold Base HyperSep-96 well plate is not included and needs to be purchased separately HyperSep-96 Well Plate Manifolds Description Cat. No. Quantity HyperSep-96 Vacuum Manifold Each Vacuum Pump, North American Plug Each Vacuum Pump, European Plug Each HyperSep SPE Accessories A range of accessories to complement the HyperSep SPE Manifolds HyperSep SPE Accessories Description Cat. No. Quantity Base Plate for HyperSep-96 Well Plate Each Base Plate for HyperSep-96 Well Plate Pack Sample Collection Plate, 1mL Pack Sample Collection Plate, 2mL Pack Adaptors for 1mL, 3mL and 6mL SPE Columns Pack Empty 1mL Wells Pack Empty 1mL Wells, Fritted Pack 113

31 QuEChERS Dispersive SPE Product Selection The QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) technique is increasingly becoming the technique of choice for extraction and clean-up of pesticide residues. It was developed by the USDA Eastern Regional Research Centre. Please refer to the information below for selection of the most appropriate QuEChERS product for multi-residue pesticide analysis: There are four variations of the QuEChERS method currently being used: The original QuEChERS method. This was introduced in 2003, using sodium chloride to enhance extraction Dispersive AOAC This uses sodium acetate as a buffer, replacing sodium chloride The dual phase variation this method introduces the use of PSA and GCB to remove high levels of chlorophyll and plant sterols in the final extract without the loss of planar pesticides (polar aromatics) using an acetone:toluene solvent mix (3:1) The European version is similar to the AOAC method, except the extraction uses sodium chloride, sodium citrate dihydrate and disodium citrate sesquihydrate instead of sodium acetate Sorbents used in QuEChERS Methods: Material Purpose Typical Matrices MgSO4 Removal of excess water Fruits, vegetables PSA (Primary/Secondary Amine) Removal of organic acids, Fruits fatty acids, sugars C18 Removal of lipids & sterols Milk, meat, fish GCB (Graphatized Carbon Black) Removal of pigments & sterols Wine, green vegetables, carrots Product Selection: Matrix Type Examples Sorbent Requirements General Matrices Apples MgSO4, PSA Cucumber Melon Fatty Matrices Milk MgSO4, PSA, C18 Cereals Fish Pigmented Matrices Lettuce MgSO4, PSA, C18, GCB Carrot Wine High Pigmented Matrices Spinach MgSO4, PSA, C18, GCB Red Peppers Method Part Number Description Original mL Tube with 4g MgSO4, 1g Sodium Chloride 250pk AOAC mL Tube with 6g MgSO4, 1.5g Sodium Acetate 250pk mL Tube with 150mg MgSO4, 50mg PSA 100pk mL Tube with 150mg MgSO4, 50mg PSA, 50mg C18 100pk mL Tube with 900mg MgSO4, 300mg PSA, 150mg GCB 50pk mL Tube with 900mg MgSO4, 300mg PSA, 150mg C18 50pk mL Tube with 150mg MgSO4, 50mg PSA, 50mg C18, 50mg GCB 100pk mL Tube with 1200mg MgSO4, 400mg PSA 50pk mL Tube with 1200mg MgSO4, 400mg PSA, 400mg C18 50pk mL Tube with 1200mg MgSO4, 400mg PSA, 400mg C18, 400mg GCB 50pk European mL Tube with 6g MgSO4, 1.5g NaCl, 1.5g Sodium EN15662 Citrate Tribasic Dihydrate, 0.75g Sodium Citrate Dibasic Sesquihydrate mL Tube with 4g MgSO4, 1g NaCl, 1g Sodium Citrate Tribasic Dihydrate, 0.5g Sodium Citrate Dibasic Sesquihydrate mL Tube with 900mg MgSO4, 150mg PSA, 50pk mL Tube with 900mg MgSO4, 150mg PSA, 45mg GCB 50pk mL Tube with 900mg MgSO4, 150mg PSA, 15mg GCB 50pk mL Tube with 150mg MgSO4, 25mg PSA 100pk mL Tube with 150mg MgSO4, 25mg PSA, 2.5mg GCB 100pk mL Tube with 150mg MgSO4, 25mg PSA, 7.5mg GCB 100pk mL Tube with 900mg MgSO4, 150mg PSA, 150mg C18 50pk Dual Phase mL Column with 200mg GCB on Top, 400mg PSA Method on Bottom, separated by a Frit 30pk mL Column with 250mg GCB on Top, 500mg PSA on Bottom, separated by a Frit 30pk mL Column with 500mg GCB on Top, 500mg PSA on Bottom, separated by a Frit 30pk Other variations of QuEChERS are available please see page 115 for full ordering information 114

32 Sample Preparation QuEChERS Products Convenient and effective approach for determining pesticide residues in fruit, vegetables and other foods The QuEChERS method is a two-step process: extraction followed by cleanup. The extraction step products use MgSO 4 to aid extraction, along with either NaCl sodium citrate, or anhydrous sodium acetate for base-sensitive compounds (e.g., folpet or captan). The extraction step products are supplied in a 50mL polypropylene centrifuge tube for convenient extractions. The clean-up step products contain PSA (primary/secondary amine) for the removal of organic acids and polar pigments among other compounds. Some products couple the PSA with endcapped C18 for the removalof most lipids and sterols, or graphitized carbon black for the removal of sterols and pigments such as chlorophyll. A variety of tube formats and bed weights are available to accommodate large and small sample sizes. } Determine greater number of pesticides than with standard SPE } Easy to use } Available in a number of configurations QuEChERS Extraction Products Description Capacity Cat. No. Quantity 6g anhydrous MgSO 4, 1.5g anhydrous sodium acetate 50mL Pack 4g anhydrous MgSO 4, 1g anhydrous NaCl 50mL Pack 4g anhydrous MgSO 4, 1g sodium chloride, 1g sodium citrate dihydrate, 0.5g disodium citrate sesquihydrate 50mL Pack 6g anhydrous MgSO 4, 1.5g sodium chloride, 1.5g sodium citrate dihydrate, 0.75g disodium citrate sesquihydrate 50mL Pack QuEChERS Clean-Up Products Description Capacity Cat. No. Quantity 150mg anhydrous MgSO 4, 25mg PSA 2mL Pack 150mg anhydrous MgSO 4, 25mg PSA, 25mg C18 2mL Pack 150mg anhydrous MgSO 4, 25mg PSA, 2.5mg graphitized carbon 2mL Pack 150mg anhydrous MgSO 4, 25mg PSA, 7.5mg graphitized carbon 2mL Pack 150mg anhydrous MgSO 4, 50mg PSA, 50mg graphitized carbon 2mL Pack 150mg anhydrous MgSO 4, 50mg PSA 2mL Pack 150mg anhydrous MgSO 4, 50mg PSA, 50mg endcapped C18 2mL Pack 150mg anhydrous MgSO 4,50mg PSA, 50mg endcapped C18, 50mg graphitized carbon 2mL Pack 900mg anhydrous MgSO 4, 300mg PSA, 150mg endcapped C18 15mL Pack 900mg anhydrous MgSO 4, 300mg PSA, 150mg graphitized carbon 15mL Pack 1200mg anhydrous MgSO 4,400mg PSA 15mL Pack 1200mg anhydrous MgSO 4,400mg PSA, 400mg endcapped C18 15mL Pack 1200mg anhydrous MgSO 4,400mg PSA, 400mg endcapped C18, 400mg graphitized carbon 15mL Pack 900mg anhydrous MgSO 4, 150mg PSA, 150mg C18 15mL Pack 150mg anhydrous MgSO 4,50mg PSA, 50mg graphitized carbon 15mL Pack 900mg anhydrous MgSO 4,150mg PSA, 45mg graphitized carbon 15mL Pack 900mg anhydrous MgSO 4,150mg PSA, 15mg graphitized carbon 15mL Pack 900mg anhydrous MgSO 4,150mg PSA 15mL Pack 150mg anhydrous MgSO 4,300mg PSA, 150mg ChloroFiltr 15mL Pack 750mg anhydrous MgSO 4, 250mg PSA, 250mg endcapped C18, 250mg graphitized carbon 15mL Pack 900mg anhydrous MgSO 4, 300mg PSA 15mL Pack 400mg PSA on bottom, 200mg graphitized carbon on top, separated by a Teflon frit 6mL Pack 500mg PSA on bottom, 250mg graphitized carbon on top, separated by a Teflon frit 6mL Pack 500mg PSA on bottom, 500mg graphitized carbon on top, separated by a Teflon frit 6mL Pack ChloroFiltr is a trademark of United Chemical Technologies Inc

33 QuEChERS Methods For non-base sensitive compounds, such as bendiocarb and diuron using the original QuEChERS method } Add 15mL of acetonitrile to QuEChERS centrifuge tube } Shake to mix contents } Add surrogate or internal standards if necessary } Add 15g of homogenised hydrated sample and shake for 1 minute } Centrifuge tube for 1 minute at 3700rcf } Add an aliquot of the supernatant to the appropriate clean-up tube (and shake for 1 minute) } Centrifuge for 1 minute at 3700rcf } Analyze extract For base sensitive compounds such as folpet and captan using the AOAC QuEChERS method } Add 15mL of 1% acetic acid in acetonitrile to QuEChERS centrifuge tube } Shake to mix contents } Add surrogate or internal standards if necessary } Add 15g of homogenised hydrated sample and shake for 1 minute } Centrifuge tube for 1 minute at 3700rcf } Add an aliquot of the supernatant to the appropriate clean-up tube and shake for 1 minute } Centrifuge for 1 minute at 3700rcf } Analyze extract For non-base sensitive compounds using the European EN15662 method } Weigh 15g of homogenized (hydrated at least 80%) sample in a 50mL centrifuge tube } Add 15mL acetonitrile (or 1:1 acetone/hexane, ethyl acetate) and IS } Shake briefly } Add 6g anhydrous magnesium sulfate, 1.5g sodium chloride, 1.5g sodium citrate Tribadic dehydrate, 0.75g sodium citrate dibasic } Shake by hand for 1 minute } Centrifuge at 5,000 rpm for 5 minutes } Transfer a portion of supernatant to a QuEChERS clean up tube } Shake for 30 seconds } Centrifuge for 1 minute at 6,000 rpm For polar aromatic (planar) compounds such as matrix plant pigments using the Schenck method } Pre-rinse the cartridge with 5mL of toluene } Add an aliquot of the supernatant to the cartridge } Start collection } Elute with 6-12mL of 3:1 acetone:toluene } Concentrate for GC/MS analysis - or - } Concentrate to dryness and reconstitute in mobile phase for LC analysis 116

34 Sample Preparation QuEChERS Dispersive SPE Technical Information Considerations in Method Development: 1) Determine the properties of the pesticides of interest: a. Base sensitive b. ph dependent c. Non-base sensitive Product Selection Sample Extraction: Base Sensitive Compounds Use extraction product with sodium acetate Non-Base Sensitive Compounds Use extraction product with sodium chloride or sodium citrate 2) Determine the properties of the sample matrix: General Fatty Pigmented Highly Pigmented Product Selection Sample Cleanup: QuEChERS Troubleshooting: Problem Causes Recommended Solutions Loss of planar Presence of GCB may result Use a product with less GCB pesticides in a loss of planar compounds Use the Dual Phase QuEChERS product Loss of acidic Presence of PSA will extract Use a product containing compounds e.g. 2,4-D acidic compounds from matrix MgSO4 and C18 from starting matrix Loss of compounds Some compounds are Use an analyte protectant e.g. toluene during subsequent unstable and can break down or sorbitol analysis during analysis Addition of sample to Exothermic reaction between Add the sample to the tube, then the QuEChERS Extraction water in sample and MgSO4 solvents, then the sorbent materials tube containing sorbent causes an exothermic reaction Poor recovery of Sample not in appropriate Wrong products used in method pesticide compounds homogenisation state Ensure sample is hydrated to 80% or higher Verify nature of pesticides e.g. are base sensitive compounds present Matrix Type Examples Sorbent Requirements General Matrices Apples MgSO4, PSA Cucumber Melon Fatty Matrices Milk MgSO4, PSA, C18 Cereals Fish Pigmented Matrices Lettuce MgSO4, PSA, C18, GCB Carrot Wine High Pigmented Matrices Spinach MgSO4, PSA, C18, GCB Red Peppers 117

35 Aspire Protein A and Protein G Tips Purify antibodies in minutes } Capture and purify a wide range of monoclonal and polyclonal IgG antibodies from serum, ascites and cell culture supernatants } Embedded with high quality immobilized Thermo Scientific Protein A Plus and Protein G Plus Agarose with the capacity to purity (1mg of human IgG } Spin column capacity for a fraction of the cost } Fast and easy protocol does not compromise purity and yield Aspire Protein A and Protein G Tips Description Cat. No. Quantity Aspire Protein A Tips Pack Aspire Protein G Tips Pack Aspire IMAC Cobalt Tips Quick and clean his-tagged protein purification } Design in a pipette tip format to effectively purify up to 0.5mg of his-tagged proteins from cell protein lysates } Embedded with Thermo Scientific HisPur Cobalt Resin with higher specificity compared to conventional nickel resins } Spin column capacity for a fraction of the cost per sample } Color-coded, multichannel Thermo Scientific Aspire protocol allows parallel sample processing in minutes without the need for centrifugation Aspire IMAC Cobalt Tips Description Cat. No. Quantity Aspire IMAC Cobalt Tips Pack Aspire RP30 Desalting Tips Effectively remove ion-suppressing contaminants from digested complex protein mixtures for LC/MS and LC/MS/MS analysis within 20 minutes Proprietary reversed-phase resin allows superior peptide binding and recovery of digested complex protein mixtures compared to conventional C18 products. } Removes salts, detergents and other ion-suppressing contaminants } Reduced ion-suppression, increase signal-to-noise ratios and sequence coverage } Effective sample cleanup prior to LC/MS and LC/MS/MS analysis } 20-Minute purification protocol features color-coded parallel sample processing } Bind up to 30µg of total peptide without the need for centrifugation Aspire RP30 Desalting Tips Description Cat. No. Quantity Aspire RP30 Desalting Tips Pack 118

36 Sample Preparation HyperSep Microscale Solid Phase Extraction Tips Revolutionary micropipette tip sample preparation Applications: Mass spectrometry Desalting Protein purification MALDI Electrophoresis HPCE HPLC CEC } Faster sample preparation with minimal sample loss } Patented micropipet tip in which the chromatographic material is directly attached to its inner surface } No contamination from the supporting matrix } Separation in volumes as low as 100nL HyperSep Tip Microscale Solid Phase Extraction Tips Material Cat. No. Quantity 1-10µL Capacity BioBasic C Pack BioBasic C Pack BioBasic C Pack Hypercarb Pack Hypercarb and C18 (mix mode) Pack HILIC Pack Trypsin Pack Titanium Dioxide Pack Zirconium Dioxide Pack µL Capacity BioBasic C Pack BioBasic C Pack BioBasic C Pack Hypercarb Pack Hypercarb and C18 (Mix Mode) Pack HILIC Pack Trypsin Pack Titanium Dioxide Pack Zirconium Dioxide Pack 119

37 HyperSep SpinTip Microscale Solid Phase Extraction Tips Revolutionary micropipette tip for sample preparation } Pipet tips with a 1 to 2µm wide slit at bottom that permits the liquid to pass through but retains the chromatographic material (20 to 30µm) } Faster sample preparation with minimal sample loss } No contamination from the supporting matrix } Separation in volumes as low as 100nL HyperSep SpinTip Microscale Solid Phase Extraction Tips Material Cat. No. Quantity 1-10µL Capacity C Pack C Pack C Pack Hypercarb Pack Hypercarb and C18 (mix mode) Pack HILIC Pack Trypsin Pack POROS Weak Anion Exchanger Pack POROS Strong Anion Exchanger Pack POROS Strong Cation Exchanger Pack Titanium Dioxide Pack Zirconium Dioxide Pack µL Capacity C Pack C Pack C Pack Hypercarb Pack Hypercarb and C18 (mix mode) Pack HILIC Pack Trypsin Pack POROS Weak Anion Exchanger Pack POROS Strong Anion Exchanger Pack POROS Strong Cation Exchanger Pack Titanium Dioxide Pack Zirconium Dioxide Pack 120

38 HyperSep Lab Plates Sample Preparation For the purification and sample preparation of proteins, DNA, RNA and other biomolecules Applications: Tissue culture and separation of products Sample concentration Sample cleanup Collection of sample after chromatography } Sample concentration of small-scale samples } Available in a range of chromatographic materials } 96-Well format with media embedded at the bottom of the plate } Can be processed manually or using a liquid-handling robot } Not suitable for use with a vacuum HyperSep Lab Plates Description Cat. No. Quantity Polystyrene C Pack C Pack C Pack Hypercarb Pack Hypercarb and C18 (Mixed Mode) Pack Zirconium Dioxide Pack Titanium Dioxide Pack SCX Pack SAX Pack Polypropylene C Pack C Pack C Pack Hypercarb Pack Hypercarb and C18 (Mixed Mode) Pack Zirconium Dioxide Pack Titanium Dioxide Pack SCX Pack SAX Pack 121

39 HyperSep Filter Plates For effective cleanup of small-scale samples } 96-Well plate for the purification and separation of proteins, peptides, DNA, RNA and other biomolecules } Cleanup of microgram-level samples } Can be used under vacuum } Available in a range of chromatographic materials HyperSep Filter Plates Description Cat. No. Quantity 5-7µL Bed Volume C Each C Each C Each Hypercarb Each Hypercarb and C18 (Mix Mode) Each Ziconium Dioxide Each Titanium Dioxide Each SCX Each SAX Each 40µL Bed Volume C Each C Each C Each Hypercarb Each Hypercarb and C18 (Mix Mode) Each Zirconium Dioxide Each Titanium Dioxide Each SCX Each SAX Each 122

40 HyperSep MEPS Products Sample Preparation Online SPE for GC and LC sample preparation extraction to injection in a single process Save Hours in Sample Preparation } Reduce the time to prepare and inject samples from hours to minutes } Eliminate all extra steps between sample preparation and sample injection } Reduce buffer and solvent volume from Milliliters to Microliters } Reduce the sample volume needed to as little as 3.6µL What is MEPS? MEPS is Micro Extraction by Packed Sorbent and is a new development in the fields of sample preparation and sample handling. MEPS is the miniaturization of conventional SPE packed bed devices from milliliter bed volumes to microliter volumes. The MEPS approach to sample preparation is suitable for reversed phases, normal phases, mixed mode or ion exchange chemistries. MEPS is available in a variety of common SPE phases. The MEPS Barrel Insert and Needle Assembly contains the stationary phase, and is built into the syringe needle. Why use MEPS? Historically, many sample preparation methods used liquidliquid extraction (LLE) which required large volumes of sample, solvents and time. The advantages of SPE over LLE are that SPE takes much less time, can be developed into a fully automated technique, requires much less solvent and offers selectivity. MEPS performs the same functions as SPE the removal of interfering matrix components and the selective isolation and concentration of analytes. MEPS increases the advantages of conventional SPE in the following ways: } Significantly reduces the time needed to prepare and inject samples } Can be combined with LC or GC automation the extraction step and injection step are performed on-line using the same syringe. } Significantly reduces the volume of solvents needed } Ability to work with samples as small as 3.6µL versus several hundred ml for SPE Sample Size and Sensitivity Sample volumes may be as little as 10µL, or by taking multiple aliquots of 100µL or 250µL, samples of 1mL or larger may be concentrated. Automation The capability to extract samples and make injections on-line using a single device reduces both sample processing times and the need for operator intervention. Sorbent Life Typical BIN life for extraction of whole plasma sample is conservatively about 40 to 100 samples. This significantly increases for cleaner samples. Carryover The small quantity of phase in the MEPS products can be easily and effectively washed between samples to reduce the possibility of carryover. This washing process is simply not practical with off-line SPE devices. With automation of MEPS, washing can occur while the previous sample is running. Flexible and easy to use The dimensions of the sorbent bed ensure that the performance remains identical to conventional SPE devices when used for extraction of similar samples. MEPS products can be used for sample volumes as small as 3.6µL making them particularly well suited to on-line use with LC-MS analysis of volume limited samples

41 HyperSep MEPS Syringe Components MEPS Syringes and Components Description Cat. No. Quantity Thermo Scientific, CTC analytics, HTA and Varian 8400 systems 100µL removable needle MEPS syringe Each Replacement plunger assembly for 100µL MEPS syringe Each 250µL Removable Needle MEPS Syringe Each Replacement plunger assembly for 250µL MEPS syringe Each CTC Analytics Only 250µL removable needle MEPS syringe Each Replacement plunger assembly for 250µL CTC-compatible syringe Each HyperSep MEPS for GC Applications: Thermo Scientific, CTC Analytics, HTA and Varian 8400 Systems } For use with 100 and 250µL MEPS syringes MEPS For GC : Thermo Scientific, CTC Analytics, HTA and Varian 8400 Systems Description Cat. No. Quantity HyperSep Retain PEP MEPS Pack HyperSep Retain-CX MEPS Pack HyperSep Retain-AX MEPS Pack HyperSep Hypercarb MEPS Pack HyperSep Verify-CX MEPS Pack HyperSep Verify-AX MEPS Pack HyperSep C18 MEPS Pack HyperSep Silica MEPS Pack MEPS Development Kit for GC Applications Pack HyperSep MEPS Development Kit for GC applications contains 1 each of Retain PEP, Retain-CX, Retain-AX, Hypercarb and C18 HyperSep MEPS for GC Applications: CTC Analytics Using 250µL Syringes 124 MEPS For GC: CTC Analytics using 250µL Syringes Description Cat. No. Quantity HyperSep Retain PEP MEPS Pack HyperSep Retain-CX MEPS Pack HyperSep Retain-AX MEPS Pack HyperSep Hypercarb MEPS Pack HyperSep Verify-CX MEPS Pack HyperSep Verify-AX MEPS Pack HyperSep C18 MEPS Pack HyperSep Silica MEPS Pack MEPS Development Kit for GC Applications Pack HyperSep MEPS Development Kit for GC applications contains 1 each of Retain PEP, Retain-CX, Retain-AX, Hypercarb and C18

42 Sample Preparation HyperSep MEPS For LC Applications: Thermo Scientific, CTC Analytics, HTA and Varian 8400 Systems } For use with 100 and 250µL MEPS syringes MEPS For LC:Thermo Scientific, CTC Analytics, HTA and Varian 8400 Systems Description Cat. No. Quantity HyperSep Retain PEP MEPS Pack HyperSep Retain-CX MEPS Pack HyperSep Retain-AX MEPS Pack HyperSep Hypercarb MEPS Pack HyperSep Verify-CX MEPS Pack HyperSep Verify-AX MEPS Pack HyperSep C18 MEPS Pack HyperSep Silica MEPS Pack MEPS Development Kit for LC applications Pack HyperSep MEPS Development Kit for LC applications contains 1 each of Retain PEP, Retain-CX, Retain-AX, Hypercarb and C18. HyperSep MEPS For LC Applications: CTC Analytics Using 250uL Syringes } For use with 250µL MEPS syringes MEPS For LC Applications: CTC Analytics using 250µL Syringes Description Cat. No. Quantity HyperSep Retain PEP MEPS Pack HyperSep Retain-CX MEPS Pack HyperSep Retain-AX MEPS Pack HyperSep Hypercarb MEPS Pack HyperSep Verify-CX MEPS Pack HyperSep Verify-AX MEPS Pack HyperSep C18 MEPS Pack HyperSep Silica MEPS Pack HyperSep MEPS Development Kit for LC applications Pack HyperSep MEPS Development Kit for LC applications contains 1 each of Retain PEP, Retain-CX, Retain-AX, Hypercarb and C18. HyperSep Protein Precipitation Plate Removal of proteins using the CRASH method } Dual frit design } Hydrophobic/oleophobic frits to enable only precipitation of proteins } Pore size optimized for ideal flowrate } Specially selected polypropylene for low extractables Protein Precipitation Products Description Cat. No. Quantity Protein Precipitation Plate Each 125

43 HyperSep Online SPE Products Retain specific analytes in a sample matrix when used with an appropriate HPLC column } Effective removal of contaminants such as proteins from samples } Compatible with conventional HPLC systems } Fast and effective clean-up and concentration of target compounds } HyperSep Retain PEP: for retention of polar and non-polar analytes } HyperSep Retain-CX: for retention of basic and non-polar analytes } HyperSep Retain-AX: for retention of acidic and non-polar analytes } HyperSep Hypercarb: for retention of extremely polar analytes HyperSep Javelin Direct-Connect Online SPE Columns I.D. Length Retain-PEP Retain-CX Retain-AX Hypercarb Quantity 2.1mm 10mm Pack 3.0mm 10mm Pack HyperSep UNIGUARD Direct-Connect Online SPE Cartridges I.D. Length Retain-PEP Retain-CX Retain-AX Hypercarb Quantity 2.1mm 10mm Pack 3.0mm 10mm Pack HyperSep HPLC Columns for Online SPE I.D. Length Retain-PEP Retain-CX Retain-AX Hypercarb Quantity 2.1mm 20mm Each 3.0mm 20mm Each This diagram shows the typical load and elution positions for the HyperSep Online SPE Setup 126

44 Sample Preparation HyperSep Retain PEP Doxepin in Rat Serum Compounds: Doxepin Part Number: Phase: HyperSep Retain PEP Volume: 3mL Bed Weight: 60mg Sample Mix 10mL of doxepin aqueous Pretreatment: solution (20mg/L) and 30mL rat serum in a 100mL flask and dilute to volume with 0.5% ammonia solution: 2ppm Conditioning: 2mL methanol, followed by 2mL deionized water Application: Load sample at 1-2mL/min. Washing: 2mL 0.5% ammonia solution containing 5% methanol Elution: 2mL 1% acetic acid/methanol Propranolol in Rat Serum Compounds: Propranolol Part Number: Phase: HyperSep Retain PEP Volume: 3mL Bed Weight: 60mg Sample Mix 10 ml of propranolol aqueous Pretreatment: solution (100mg/L) and 30mL rat serum in a 100mL flask and dilute to volume with 0.5% ammonia solution: 10ppm Conditioning: 2mL methanol, followed by 2mL deionized water Application: Load sample at 1-2mL/min. Washing: 2mL 0.5% ammonia solution containing 5% methanol Elution: 2mL 1% acetic acid/methanol Phenols in Tap Water Acetaminophen in Calf Serum Compounds: Acetaminophen Part Number: Phase: HyperSep Retain PEP Volume: 3mL Bed Weight: 60mg Sample Dilute 10 mg acetaminophen in Pretreatment: 100mL water (100 ppm). Mix 25mL of this solution with 25 ml water. In a separate vessel, mix 25mL solution with 25mL serum/1% H 3PO 4. Conditioning: 2mL methanol followed by 2mL water Application: Load 2mL samples Washing: 2mL methanol/water (5:95, v/v) Elution: 2mL methanol Nitro Anilines Compounds: 2-nitroaniline, 4-nitroaniline Part Number: Phase: HyperSep Retain PEP Volume: 3mL Bed Weight: 60mg Conditioning: 3mL methanol, followed by 3mL deionized water Application: Load 3mL of sample at 1-2mL/min. Washing: 1mL of 0.1% ammonia aqueous solution Elution: 3mL methanol/1% formic acid (95:5, v/v). Dry sample under nitrogen and reconstitute using 1mL methanol. Aniline and N,Ndimethylaniline in Dionized Water Compounds: Aniline, N,N-dimethylaniline Part Number: Phase: HyperSep Retain PEP Volume: 3mL Bed Weight: 60mg Conditioning: 3mL methanol, followed by 3mL deionized water Application: Load 3mL of sample at 1-2mL/min. Washing: 1mL of water Elution: 3mL methanol/water (95:5, v/v). Dry sample under nitrogen and reconstitute using 1mL methanol. Compounds: Phenol, 4-nitrophenol, m-methylphenol, 2-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, pentachlorophenol Part Number: Phase: HyperSep Retain PEP Volume: 3mL Bed Weight: 60mg Sample To a ppm sample of phenols in Pretreatment: tap water add formic acid (1%) Conditioning: 2mL methanol followed by 2mL of 1% formic acid Application: Load 10mL of sample at 1-2mL/min. Washing: 1mL of 1% formic acid Elution: 2mL methanol. Dry sample under nitrogen and reconstitute using 1mL methanol/1% formic acid (1:1). Thiourea Herbicides Extracted from Soil in Phosphorate Buffer Solution (ph 2.5) Compounds: Nicosulfuron, thifensulfuron-methyl, metsulfuron-methyl, sulfometuronmethyl, chlor-sulfuron, ethametsulfron methyl, tribenuron, bensulfuronmethyl, pyrazosulfuron-ethyl, chlorimuron-ethyl Part Number: Phase: HyperSep Retain PEP Volume: 3mL Bed Weight: 60mg Sample Add thiourea herbicides extracted Pretreatment: from soil (1ppm) to phosphorate buffer solution (ph 2.5) Conditioning: 5mL methanol followed by 5mL phosphorate buffer (ph 2.5) Application: Load 10mL of sample at 1-2mL/min. Washing: 3mL of phosphorate buffer (ph 2.5) Elution: 5mL ACN/phosphorate buffer (ph 7.8) (9:1, v/v). Dry sample under nitrogen and reconstitute using 1mL methanol. Atrazine in Deionized Water Compounds: Atrazine Part Number: Phase: HyperSep Retain PEP Volume: 3mL Bed Weight: 60mg Sample 10 ppm atrazine in acetic acid (0.2%) Pretreatment: aqueous solution Conditioning: 2mL methanol followed by 2mL deionized water Application: Load 4 ml of 10 ppm atrazine in acetic acid (0.2%) aqueous solution sample at 1-2mL/min. Washing: 1mL of water Elution: 5mL methanol. Dry sample under nitrogen and reconstitute using 0.5mL water. Acephate Compounds: Acephate Part Number: Phase: HyperSep Retain PEP Volume: 3mL Bed Weight: 60mg Sample 10ppm acephate in ammonium sulfate Pretreatment: aqueous solution (20% w/w) Conditioning: 2mL methanol followed by 2mL deionized water Application: Load 4mL of 10 ppm acephate in ammonium sulfate aqueous solution (20% w/w) at 1-2mL/min. Washing: 1mL of water Elution: 5mL methanol. Dry sample under nitrogen and reconstitute using 0.5mL water

45 HyperSep Retain PEP Acephate Compounds: Acephate Part Number: Phase: HyperSep Retain PEP Volume: 3mL Bed Weight: 60mg Sample 10ppm acephate in ammonium sulfate Pretreatment: aqueous solution (20% w/w) Conditioning: 2mL methanol followed by 2mL deionized water Application: Load 4mL of 10 ppm acephate in ammonium sulfate aqueous solution (20% w/w) at 1-2mL/min. Washing: 1mL of water Elution: 5mL methanol. Dry sample under nitrogen and reconstitute using 0.5mL water. Lovastatin Compounds: Lovastatin Part Number: Phase: HyperSep Retain PEP Volume: 3mL Bed Weight: 60mg Sample 2ppm Lovastatin in 10% acetonitrile Pretreatment: aqueous solution Conditioning: 2mL methanol followed by 2mL deionized water Application: Load 10 ml of sample at 1-2mL/min. Dry column under vacuum. Washing: 1mL of deionized water Elution: 4mL of acetonitrile. Dry sample under nitrogen and reconstitute using 1mL methanol. Cefoperazone Compounds: Cefoperazone Part Number: Phase: HyperSep Retain PEP Volume: 3mL Bed Weight: 60mg Sample 5ppm Cefoperazone solution in water Pretreatment: Conditioning: 2mL methanol followed by 2mL deionized water Application: Load 10mL of 5 ppm cefoperazone solution in water at 1-2mL/min. Washing: 1mL of methanol/deionized water (5:95, v/v). Dry column under vacuum. Elution: 6mL methanol. Dry sample under nitrogen and reconstitute using 0.5mL water. Praziquantel Compounds: Praziquantel Part Number: Phase: HyperSep Retain PEP Volume: 3mL Bed Weight: 60mg Sample 20ppm praziquantel in 0.2% acetic Pretreatment: acid and 5% methanol Conditioning: 2mL methanol followed by 2mL deionized water Application: Load 4mL of 20 ppm praziquatel in 0.2% acetic acid and 5% methanol at 1-2mL/min. Washing: Methanol/deionized water (5:95, v/v) Elution: 4mL methanol. Dry sample under nitrogen and reconstitute using 0.5mL water. Acetaminophen Compounds: Acetaminophen Part Number: Phase: HyperSep Retain PEP Volume: 3mL Bed Weight: 60mg Sample 5ppm of acetaminophen in aqueous Pretreatment: solution containing 0.2% ammonium acetate, ph 5 Conditioning: 2mL methanol followed by 2mL deionized water Application: Load 4 ml of sample at 1-2mL/min. Washing: 1mL deionized water Elution: 5mL of methanol. Dry sample under nitrogen and reconstitute using water. 128

46 Sample Preparation HyperSep Retain-CX Acetaminophen in Calf Serum Compounds: Acetaminophen Part Number: Phase: HyperSep Retain-CX Volume: 3mL Bed Weight: 60mg Sample Dilute 10 mg of acetaminophen to Pretreatment: 100 ml with water (100 ppm). Mix 25mL of acetaminophen (100 ppm) solution and 25mL serum/1% H3PO4 in a 50mL flask. Conditioning: 2mL methanol, followed by 2mL deionized water Application: Load samples at 1-2 ml/min. Washing: 2mL 5% methanol/water Elution: Elute acetaminophen with 2mL methanol Clenbuterol Compounds: Clenbuterol Part Number: Phase: HyperSep Retain-CX Volume: 3mL Bed Weight: 60mg Sample 10ppm of clenbuterol in 20 mm Pretreatment: ammonia acetate Conditioning: 2mL methanol followed by 2mL deionized water and 2mL 30 mm HCl Application: Load 1mL sample Washing: 1mL of methanol followed by 1mL deionized water Elution: 2mL methanol with 4% ammonia. Dry sample under nitrogen and reconstitute using 1mL methanol/0.05% phosphoric acid (40:60, v/v). ß-Agonists in Pig Liver Compounds: Cimaterol, sulfamonomethoxine, clenbuterol hydrochloride, salbutamol Part Number: Phase: HyperSep Retain-CX Volume: 3mL Bed Weight: 500mg Sample Extract 20g of pig liver sample using Pretreatment: ACN, then dry and spike with a standard of chloric acid (10 mmol) solution containing the 4 agonist compounds of interest to get sample solutions at 5 different concentrations (1μg/mL, 2μg/mL, 5μg/mL,10μg/mL and 100μg/mL) Conditioning: 5mL methanol, followed by 5mL deionized water and 5mL 30mmol/L chloric acid Application: Load sample at 1-2mL/min. Washing: 5mL deionized water followed by 5mL methanol. Dry column under vacuum Elution: 5mL of methanol containing 4% ammonia. Evaporate to dryness at 50 C using a nitrogen stream. Melamine in Milk Products for HPLC Analysis Compounds: Melamine Part Number: Phase: HyperSep Retain-CX Volume: 3mL Bed Weight: 60mg Sample Weigh 5g of milk powder (or measure Pretreatment: 10mL of milk) into a 250mL flask. Add 50mL of 1% Trichloroacetic acid (TCA). Mix / vortex. Add 2mL of 2% lead acetate / water solution into the mixture then sonicate for 20 minutes. Transfer part of the final mixture into a 10mL centrifuge tube. Centrifuge for 10 minutes at 8,000 rpm. Conditioning 3mL methanol followed by 3mL DI water Application: Load 6mL sample at 1-2mL/min. Washing: 3mL DI water followed by 3mL methanol. Dry column (5 minutes at > 10 inches Hg) Elution: 5mL 5% ammonia / methanol Collect eluate at 1 to 2 ml/min. Evaporate to dryness at <50ºC using nitrogen. Reconstitute sample using 1mL of mobile phase. Melamine in Animal Feeds Part Number: Phase: HyperSep Retain-CX Volume: 3mL Bed Weight: 60mg Sample To 5g of animal feed, add 50mL of Pretreatment: 0.1% trichloroacetic acid aqueous solution. Vortex for 1 min, then add 2mL of 2% lead acetate aqueous solution. Sonicate for 20 mins, then transfer a portion of the mixture to a 10mL Centrifuge tube. Centrifuge at 8000 rpm for 10 mins. Conditioning: 3mL methanol followed by 3mL deionized water Application: Load 3mL sample at 1-2 ml/min. Washing: 3mL water followed by 3mL methanol. Purge column to dryness. Elution: 5 ml ammonia in methanol (5:95, v/v). Dry sample under nitrogen stream. Reconstitute with 20% methanol aqueous solution. Salbutamol Compounds: Salbutamol Part Number: Phase: HyperSep Retain-CX Volume: 3mL Bed Weight: 60mg Sample 10ppm of salbutamol in 20 mm Pretreatment: ammonia acetate Conditioning: 2mL methanol followed by 2mL deionized water and 2mL 30 mm HCl Application: Load 1mL sample Washing: 1mL of methanol followed by 1mL deionized water Elution: 2mL methanol with 4% ammonia. Dry sample under nitrogen and reconstitute using 1mL methanol/0.05% phosphoric acid (40:60, v/v)

47 HyperSep Retain-AX Nadifloxacin Compounds: Nadifloxacin Part Number: Phase: HyperSep Retain-AX Volume: 3mL Bed Weight: 60mg Sample 5ppm of nadifloxacin sample in Pretreatment: 50mM phosphate ph 7.4 Conditioning: 1mL methanol followed by 1mL of 2M NaOH and 1mL deionized water Application: Load 5mL sample at 1-2mL/min. Washing: 1mL of 5% ammonia aqueous solution followed by 1mL methanol Elution: 3mL of methanol with 4% acetic acid Nicosulfuron Compounds: Nicosulfuron Part Number: Phase: HyperSep Retain-AX Volume: 3mL Bed Weight: 60mg Sample 10ppm of nicosulfuron in Pretreatment: deionized water Conditioning: 1mL methanol followed by 1mL deionized water Application: Load 2mL sample Washing: 1mL of 2% ammonia hydroxide followed by 1mL methanol Elution: 2mL methanol with 2% acetic acid Methylmalonic Acid (MMA) from Serum or Plasma for GCMS Analysis Compounds: Methylmalonic acid (MMA) Part Number: Phase: HyperSep Retain-AX Volume: 3mL Bed Weight: 60mg Sample To 250μL serum or plasma add 100μL Pretreatment: D3MMA (2.5μM/L) and 1.5mL water Conditioning 2mL methanol followed by 2mL water Application: Load samples at 1-2mL/min. Washing: 2mL water followed by 2mL methanol with 0.5% acetic acid Elution: 2mL Tert Butyl Methyl Ether (TBME) with 3% formic acid. Evaporate to dryness at <40ºC. Derivatization: Add 50μL acetonitrile and 25μL MTBSTFA + 1% TBDMCl. Heat at 70ºC for 10 mins. Source: Department of Laboratory Medicine, The Hospital of Telemark Ethametsulfuron in Aqueous Solution Compounds: Ethametsulfuron Part Number: Phase: HyperSep Retain-AX Volume: 3mL Bed Weight: 60mg Sample Dilute 1mg ethametsulfuron in Pretreatment: 100mL 2% ammonia aqueous solution (10 ppm) Conditioning: 1mL methanol followed by 1mL deionized water Application: Load 2 ml sample Washing: 1mL of 2% ammonia hydroxide solution followed by 1mL methanol Elution: 2mL methanol with 2% acetic acid Pharmaceutical and Biochemical Cyclosporin from Blood Compounds: Cyclosporin Part Number: Phase: HyperSep C18 Volume: 3mL Bed Weight: 500mg Sample Mix 1mL heparinised blood Pretreatment: with 2mL water/acetonitrile (7:3, v/v). Stir mixture and centrifuge after 5 min. Conditioning: 3mL acetonitrile followed by 3mL water/acetonitrile (8:2, v/v) Application: Force or aspirate the sample slowly through column Washing: 0.5 M acetic acid/acetonitrile (8:2, v/v) followed by 0.5 M acetic acid/acetonitrile (6:4, v/v) Elution: Acetonitrile Gabapentin in Serum, Plasma or Whole Blood Compounds: Gabapentin Part Number: Phase: HyperSep C18 Volume: 1mL Bed Weight: 100mg Sample 500µL sample, calibrator or Pretreatment: control to be placed into a glass test tube. Add 25µL internal standard (5.0mg/L). Add 500µL 20% acetic acid and vortex tube. Conditioning: 3mL CH 3 OH followed by 3mL deionised water and 1mL 100mM HCl Application: Load sample at 1-2mL/min. Washing: 3mL deionized water followed by 3mL ethyl acetate and 3mL hexane. Dry column under vacuum for 30 sec Elution: 1mL 2% NH 4 OH in CH 3 OH Evaporate to dryness at <40 C Ketamine in Urine Compounds: Ketamine Part Number: Phase: HyperSep Verify-CX Volume: 10mL Bed Weight: 200mg Sample To 2mL urine add internal standard Pretreatment: and 1mL 100mM phosphate buffer (ph 6). Mix/vortex. Use 100mM monobasic or dibasic sodium phosphate to ensure sample ph of 6 Conditioning: 3mL CH 3 OH followed by 3mL deionized water and 1mL 100mM phosphate buffer (ph 6) Application: Load at 1mL/min. Washing: 3mL deionized water 1mL 100mM acetic acid 3mL CH 3 OH Dry column (5 min at > 10 inches Hg) Elution: 3mL dichloromethane/isopropanol/ ammonium hydroxide (78:20:2) collect eluents at 1-2mL/min. using minimal vacuum Evaporate to dryness at <40 C 130

48 Sample Preparation Antiepileptics from Serum Compounds: Carbamazepine, dilantin, phenobarbital, primidone Part Number: Phase: HyperSep C18 Volume: 1mL Bed Weight: 100mg Sample Mix 500µL serum with 500µL Pretreatment: 4-methylprimidone in citrate buffer ph 4 (internal standard) Conditioning: 2mL methanol followed by 2mL water Application: Force or aspirate sample slowly through column Washing: 2 column volumes distilled water Elution: 2 x 100µL volumes acetone Antineoplastic Agents from Plasma Compounds: Bisantrene, mitoxantrone Part Number: Phase: HyperSep C18 Volume: 1mL Bed Weight: 100mg Conditioning: 2mL methanol followed by 2mL distilled water Application: Force or aspirate 1-2mL plasma slowly through column Washing: 2mL distilled water Elution: 2 x 200µL volumes 0.5M methanolic HCl Antiarrhythmic Drug Flecainide from Plasma Compounds: Flecainide Part Number: Phase: HyperSep C8 Volume: 1mL Bed Weight: 100mg Sample Mix 1mL plasma with 1mL water Pretreatment: and 200µL 0.2M sodium carbonate solution Conditioning: 2mL methanol followed by 2mL distilled water Application: Force or aspirate sample slowly through column Washing: 2mL distilled water Elution: 500µL methanol, then elute from column after 1 min. Catecholamine Metabolites from Urine Compounds: Vanillylmandelic acid, homovanillic acid Part Number: Phase: HyperSep SAX Volume: 3mL Bed Weight: 500mg Sample Pretreatment: Conditioning: Application: Washing: Elution: Collect 24h urine (preserved with 0.1M HCl). Store at 4 C Dilute sample prior to extraction 1:1 with water. Use 0.5M NaOH to adjust ph to mL methanol followed by 6mL distilled water Force or aspirate pretreated sample through column 6mL distilled water 6mL 1.5M sodium hydroxide solution 131

49 Environmental Cyanuric Acid in Drinking Water Compounds: Cyanuric Acid Part Number: Phase: HyperSep Hypercarb Volume: 6mL Bed Weight: 500mg Sample Adjust water sample to ph 3 Pretreatment: Conditioning: Wash column with 10mL methanol Condition column with 10mL LC-grade water Application: Force/aspirate mL of water sample into column at rate of 5mL/min. Washing: Dry column under vacuum Elution: 20mL methanol evaporate to dryness at 50 C under nitrogen Source: Marie Claire Hennion, ESPCI, Paris Extraction of Tear Gas Compounds: Chloroacetophenone (cs), o-chlorobenzylidenemalonitrile (cn), trans-8-methyl-n-vanillyl-6-nonenamide (oc) Part Number: Phase: HyperSep Verify-CX Volume: 10mL Bed Weight: 200mg Sample Clothing: Cut out portion of sprayed Pretreatment: area and a negative control sample. Extract each into hexane Canisters: Spray onto a Kimwipe and extract sprayed area and a negative control sample into hexane. Conditioning: 3mL CH 3 OH followed by 3mL deionized water and 1mL 100mM phosphate buffer (ph 6) Application: Load at 1mL/min. Washing: 3mL deionized water and 3mL hexane. Dry column for 5 min at >10 inches Hg Elution: 1mL CH 3 OH Evaporate to dryness at <40 C Organochlorine Insecticides from Water Compounds: Aldrin, p,p -DDE, o,p -DDE, o,p -DDT, p,p -DDT, dieldrin, endosulfan I, endosulfan II, endrin, heptachlor, heptachlor epoxide, lindane, p,p -methoxychlor Part Number: Phase: HyperSep C18 Volume: 6mL Bed Weight: 500mg Sample Filter sample if required Pretreatment: Conditioning: 12mL ethyl acetate followed by 6mL methanol and 6mL distilled water Application: Force or aspirate sample slowly through column. Washing: 6mL distilled water dry column under vacuum for 15 min. Elution: 2 x 500µL ethyl acetate Concentrate eluate to 250µL in stream of nitrogen at 40 C. Chlorophenoxy Acid Herbicides in Water for GC or GCMS Analysis Compounds: 2,4-D Acid, 2,4,5-trichloro phenoxy propionic acid (Silvex), Dicamba, Dinitro-sec-butyl phenol Part Number: Phase: HyperSep C18 Volume: 6mL Bed Weight: 1g Sample Adjust ph of 1 litre of water sample Pretreatment: to ph 1.0 with hydrochloric acid. Conditioning 10mL Hexane / Acetone (50:50) 10mL Acidified Methanol (5% HCl in MEOH) 10mL distilled Water Application: Load 1 Litre of ph adjusted water sample at a rate of 8-10mL/min. Washing: 10mL Distilled Water adjusted to ph 1.0 with HCl. Dry column under maximum vacuum pressure for minutes. Elution: 10mL of Hexane / Acetone (50:50) Concentration/ Add 500μL of a keeper solvent (meth- Evaporation: anol, DMF, other). Evaporate to 500μL under a nitrogen stream at room tem perature. Reconstitute with 100μL TCTEF. Inject at 1-2μL onto GC Explosives from Water Compounds: 1,3-dinitrobenzene, 2,6-dinitrotoluene, 2,4-dinitrotoluene, nitrobenzene, RDX (hexahydro-1,3,5-trinitro-s-triazine), tetryl (N-methyl-N,2,4,6-tetranitroaniline), 1,3,5-trinitrobenzene, 2,4, 6-trinitrotoluene Part Number: Phase: HyperSep C18 Volume: 6mL Bed Weight: 500mg Sample Adjust 500mL water sample Pretreatment: to ph 6 Dissolve 150g NaCl in the sample and filter Conditioning: 12mL methanol followed by 12mL water Application: Force or aspirate sample slowly through column Washing: 1mL distilled water. Dry column under vacuum for 5 min. Elution: 2 x 1mL volumes methanol Polynuclear Aromatic Hydrocarbons in Pond Water for GC or GCMS Analysis Compounds: Napthene, Fluorene, Acenapthene, Phenanthrene, Anthracene, Fluoranthene, Pyrene, B(a)anthracene, Chrysene, B(e)pyrene, B(b)fluoranthene, B(k)fluoranthene, B(a)pyrene, D(a,h)anthracene, B(g,hi)perylene, Indeno(1,2,3,-cd)pyrene Part Number: Phase: HyperSep Aminopropyl Volume: 3mL Bed Weight: 500mg Sample Filter water through a 0.5μm filter. Pretreatment: Add 2mL methanol to 200mL of filtered water sample. Mix and de-gas the sample for 2 minutes. Conditioning 15-20mL Methylene Chloride / Trichlorotrifluoroethylene (TCTFE) 15-20mL TCTFE then dry for 5 minutes 15-20mL Methanol 20mL Distilled Water Application: Load 200mL water sample at a rate of 8-10mL/min. Washing: 20mL Distilled Water. Dry column for minutes under maximum vacuum pressure Elution: 20mL TCTFE Concentration/ Evaporate to dryness under a nitrogen Evaporation: stream at room temperature. Reconstitute with 100μL TCTEF. Inject at 1-2μL onto GC 132

50 Sample Preparation Trace Metal Elements from Water Compounds: Bi, Cd, Co, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Ti Part Number: Phase: HyperSep Phenyl Volume: 3mL Bed Weight: 500mg Sample Adjust 500mL water to ph 8-9 Add Pretreatment: 1mL 0.1% aqueous sodium diethyl dithiocarbamate solution Conditioning: 3mL methanol followed by 3mL water Application: Force or aspirate sample through column at rate of 3-4mL/min. Washing: 2mL distilled water. Dry column under vacuum for 3-4min. Elution: 6mL Methanol Pesticides and PAHs from Water Compounds: Pesticides, PAHs Part Number: Phase: HyperSep C18 Volume: 1mL Bed Weight: 100mg Conditioning: 1mL methanol followed by 1mL distilled water Application: Force or aspirate mL water through column Washing: Dry column under vacuum Elution: Pour 500µL ethyl acetate into column. Allow to percolate without vacuum. Collect eluate for subsequent analysis Forensics/Toxicology Carboxy D 9 Tetrahydrocannibinol (THC) in Urine Compounds: Carboxy D 9 THC Part Number: Phase: HyperSep C18 Volume: 3 ml Bed Weight: 500 mg Sample To 5 ml urine add 0.5 ml 10N KOH Pretreatment: Heat at 55 C for 15 min and then cool. Add 1 ml glacial acetic acid. Conditioning: 6 ml methanol followed by 6 ml 0.01N HCl Application: Force or aspirate sample slowly through column Washing: 2 x 500 µl volumes acetonitrile/ 0.01N HCl (60:40, v/v). Evaporate to dryness under vacuum Elution: 2 x 500 µl volumes n-heptane/ethyl acetate (85:15, v/v) Amphetamines, Opiates and Phencyclidine in Oral Fluid Compounds: Amphetamines, opiates, phencyclidine Part Number: Phase: HyperSep Verify-CX Volume: 1mL Bed Weight: 50mg Sample Add µl neat sample to a Pretreatment: clean tube. Add internal standard and leave for 10 min at ambient temperature. Add 800µL 100 mm phosphate buffer ph 6 Mix/vortex for 10 sec. Use 100 mm monobasic or dibasic sodium phosphate to ensure ph 6. Conditioning: 200µL CH 3 OH 200µL deionized water 200µL 100 mm phosphate buffer ph 6 Application: Do not exceed 1 ml/min Washing: 500µL deionized water 500µL 100 mm acetic acid 500µL CH 3 OH. Dry column Elution: 800µL CH 2 Cl 2 /IPA/NH 4 OH (70:26:4) Do not exceed 1mL/min Prepare elution solvent daily Therapeutic and Abused Drugs in Urine for Acid/ Neutral and Basic Drugs Compounds: Barbiturates, ibuprofen, cotinine, amphetamine, codeine, ketamine Part Number: Phase: HyperSep Verify-CX Volume: 10mL Bed Weight: 200mg Sample Pretreatment: Conditioning: Application: Washing: Elution: To 2mL urine add internal standard(s) and 1 ml 100 mm phosphate buffer (ph 6). Mix/vortex. Use 100 mm monobasic or dibasic sodium phosphate to ensure sample ph of 6. 3mL CH 3 OH 3mL deionized water 2mL 100 mm phosphate buffer (ph 6) Aspirate at < 3 inches Hg Load at 1 to 2mL/min 3mL deionized water 1mL 100 mm acetic acid Dry column (5 min at >10 inches Hg) 2mL hexane Acidic and Neutral: 3mL hexane/ethyl acetate (50:50) Collect eluate at < 2mL/min Evaporate to dryness at < 40 C Basic: 3mL CH 2 Cl 2 /IPA/NH 4 OH (78:20:2). Collect eluate at 1-2mL/min Evaporate to dryness at < 40 C using evaporator 133

51 Forensics/Toxicology Beta Agonists in Urine for GC or GC/MS Analysis Compounds: Beta Agonists Part Number: Phase: HyperSep Verify-CX Volume: 10mL Bed Weight: 200mg Sample To 1mL urine add 2mL of 0.1M Pretreatment: Acetate buffer at ph 4.7. Conditioning 3mL methanol then aspirate 3mL DI water then aspirate 1mL 0.1M acetate buffer (ph 4.7). NOTE: Aspirate at < 3 Hg to prevent sorbent drying Application: Load sample at 1-2mL/min. Washing: 2 x 1mL Acetone / Methanol (1:1) then aspirate. Dry column (5 minutes at > 10 Hg) Elution: 1mL CH 2 Cl 2 / IPA / NH 4 OH (82/16/2). Collect the eluate by gravity feed. NOTE: Prepareelution solvent fresh daily. Add IPA / NH 4 OH mix, then add CH 2 Cl 2 (ph 11-12). Evaporate to dryness at <40ºC. Derivatization: Methaneboronic acid at 5mg/mL prepared in dry ethyl acetate (use molecular sieve) stored at -20ºC (freezer conditions) until use. Add 100μL of the methaneboronic acid solution then mix / vortex. React for 15 minutes at 70ºC. Remove from heat source to cool. NOTE: Do not evaporate this solution. Methadone in Urine Compounds: Methadone Part Number: Phase: HyperSep Verify-CX Volume: 10mL Bed Weight: 200mg Sample To 2mL urine add internal Pretreatment: standard(s) and 1mL 100mM phosphate buffer (ph 6). Mix/vortex. Use 100mM monobasic or dibasic sodium phosphate to ensure sample ph of 6. Conditioning: 3mL CH 3 OH 3mL deionized water 2mL 100mM phosphate buffer (ph 6) Aspirate at < 3 Hg Application: Load at 1 to 2mL/min. Washing: 3mL deionized water 1mL 100mM acetic acid 3mL CH 3 OH Dry column (5 min at >10 Hg) Elution: 3mL CH 2 Cl 2 /IPA/NH 4 OH (78:20:2) Collect eluate at 1-2mL/min. Prepare elution solvent daily Benzodiazepines from Serum Compounds: Benzodiazepines Part Number: Phase: HyperSep C18 Volume: 1mL Bed Weight: 100mg Sample Mix 500µL serum with 100µL Pretreatment: 0.1M sodium carbonate solution. Add internal standard if required Conditioning: 2mL methanol followed by 2mL distilled water Application: Force or aspirate sample slowly through column Washing: 2mL distilled water followed by 50µL methanol Elution: 2 x 200 µl volumes methanol Drugs from Blood and Urine Compounds: Amphetamines, barbiturates, opiates Part Number: Phase: HyperSep C18 Volume: 3mL Bed Weight: 500mg Sample Pretreatment: Conditioning: Application: Washing: Elution: Adjust 10mL urine to desired ph value using HCI or ammonia and centrifuge Amphetamines and Opiates: ph 8-9 Active components: ph 7-8 Barbiturates: ph 7 6mL methanol followed by 6mL Distilled water ph7 Force or aspirate sample slowly through column 6mL water. Dry column under vacuum for 5 min. Aspirate 750µL eluent into column. Elute after 1 min. and then flush with 750µL eluent. Eluents: Cannabinoids: acetone. Barbiturates, active components, bases, amphetamines: acetone/chloroform (1:1) Amphetamines in Urine Compounds: Amphetamines Part Number: Phase: HyperSep Verify-CX Volume: 10mL Bed Weight: 200mg Sample To 2mL urine add internal Pretreatment: standard(s), 1mL 100mM phosphate buffer ph 6 and 1mL 0.35M sodium periodate. Mix/vortex and incubate at ambient temperature for 20 min. Use 100mM monobasic or dibasic sodium phosphate to ensure sample ph of 6.0±0.5. Conditioning: 3mL CH 3 OH followed by 3mL deionised water and 1mL 100mM phosphate buffer (ph 6) Aspirate at < 3 Hg to prevent sorbent drying Application: Load at 1 to 2mL/min. Washing: 3mL deionized water followed by 1mL 100 mm acetic acid and 3mL CH 3 OH. Dry column (5 min at >10 Hg) Elution: 3mL CH 2 Cl 2 /IPA/NH 4 OH (78:20:2) Collect eluate at 1 to 2mL/min. Prepare elution solvent daily Concentrate eluate by adding 30µL silation grade DMF to eluate. Evaporate to 30µL at <40 C 134

52 Sample Preparation Opiates in Urine Oxime TMS Procedure for GC or GC/MS Analysis Compounds: Codeine TMS, Morphine TMS, Hydrocodone Oxime TMS, Oxycodone- Oxime TMS, Oxmorphone-Oxime TMS Part Number: Phase: HyperSep Verify-CX Volume: 10mL Bed Weight: 200mg Sample To 2mL urine add internal standard(s)* Pretreatment: and 400μL concentrated HCl. Add 200μL 10% Hydroxylamine Solution. (Acid Mix / Vortex. Hydrolysis of Glucuronides) Heat to 90ºC for 20 min. in a heating block or an autoclave for 15 minutes on a liquid cycle. Cool before proceed ing. Centrifuge for 10 minutes 1t 2,000rpm and discard pellet. Add 500μL 50% Ammonium Hydroxide, then mix/vortex. Adjust sample ph to by drop-wise addition with 50% Ammonium Hydroxide Conditioning: 3mL methanol then aspirate 3mL DI water then aspirate 2mL 0.1M phos phate buffer (ph 6.0) then aspirate NOTE: Aspirate at < 3 Hg to prevent sorbent drying Application: Load sample at 1-2mL/min. Washing: 3mL DI water then aspirate 3mL 0.1M acetate buffer (ph 4.5) then aspirate 3ml methanol then aspirate Dry column (5 minutes at >10 Hg) Elution: 3mL CH2Cl2/IPA/NH4OH (78/20/2); collect eluate at 1-2ml/min. NOTE: Prepare elution solvent daily. Add IPA/NH4OH, them mix, then add CH2Cl2 (ph 11-12). Evaporate to dryness at <40ºC. Derivatization: Add 100μL ethyl acetate and 50μL MSTFA (Part number TS-38831) Overlayer with N2 and cap. Mix/vortex. React 20 minutes at 85ºC in a heat block. Remove from heat source to cool. NOTE: Do not evaporate MSTFA solution Abused Drugs in Equine Urine Compounds: Aminocaproic acid (6-aminohexanoic acid) Part Number: Phase: HyperSep SCX Volume: 1mL Bed Weight: 100mg Sample Mix 1mL urine with 1mL phosphoric Pretreatment: acid (7mM). Use concentrated H 3 PO 4 to adjust ph to 2 Conditioning: 1mL methanol followed by 1mL distilled water and 1mL 7mM H 3 PO 4 Application: Force or aspirate sample slowly through column Dry column under vacuum for 30 sec. Washing: 1mL 7mM phosphoric acid 0.5mL 0.1M acetic acid 1mL methanol Dry column under vacuum for 30 sec. Elution: 2 x 1 ml ammoniacal methanol (1%) Nicotine and Cotinine in Urine or Serum for GC or GC/MS Analysis Compounds: Beta Agonists Part Number: Phase: HyperSep Verify-CX Volume: 10mL Bed Weight: 200mg Sample To 2mL urine or serum add internal Pretreatment: standard(s) and 2mL of 0.1M phos phate buffer (ph 6.0). Mix / vortex. Conditioning 3mL methanol then aspirate 3mL DI water then aspirate 2mL 0.1M phosphate buffer (ph 6.0) then aspirate. NOTE: Aspirate at < 3 Hg to prevent sorbent drying Application: Load sample at 1mL/min. Washing: 3mL DI Water then aspirate 1mL 0.1M acetic acid then aspirate Dry column (5 minutes at >10 Hg). 2mL Hexane then aspirate Elution 3mL Hexane / Ethyl Acetate (50:50), (Cotinine): collect at 1-2mL/min. Washing: Remove rack of collection tubes to re-wash columns 3mL Methanol then aspirate Dry column, 5 minutes at >10 Hg. Elution Replace rack of collection tubes (Nicotine): 3mL CH 2 Cl 2 / IPA / NH 4 OH (78/20/2); collect eluate at 1mL/minute. NOTE: Prepare elution solvent fresh daily. Add IPA / NH 4 OH mix, then add CH 2 Cl 2 (ph 11-12). Evaporate to dryness at <40ºC. Take care not to over-heat or over-evaporate. Derivatization: 100μL Methanol Inject 1-2μL onto the GC

53 Flunitrazepam & Metabolites in Urine for GC or GC/MS Analysis Compounds: Flunitrazepam, 7-aminoflunitrazepam, desmethylflunitrazepam Part Number: Phase: HyperSep Verify-CX Volume: 10mL Bed Weight: 200mg Sample To 2mL urine add internal standard(s)* Pretreatment: and 1mL ß-glucuronidase solution. ß-glucuronidase solution contains 5,000 F units/ml Patella Vulgata in 0.1M acetate buffer (ph = 5.0). Mix / vortex. Hydrolyze for 3 hours at 65ºC. Centrifuge for 10 minutes at 2,000 rpm and discard pellet. Cool before proceeding. Conditioning: 3mL methanol then aspirate 3mL DI water then aspirate 1mL 0.1M phosphate buffer (ph 6.0). NOTE: Aspirate at < 3 Hg to prevent sorbent drying Application: Load sample at 1-2mL/min. Washing: 2mL DI water then aspirate 2mL 20% acetonitrile in 0.1M phos phate buffer (ph 6.0) then aspirate Dry column (5 minutes at > 10 Hg). 2mL hexane then aspirate Elution: 3mL ethyl acetate with 2% NH4OH; collect eluate at 1 to 2mL/minute. Prepare fresh daily. Evaporate to dry ness at <40ºC. Derivatization: Add 50μL ethyl acetate and 50μL MTBSTFA (with 1% TBDMCS) (Part number TS-48927) Overlayer with N2 and cap. Mix/vortex. React 20 minutes at 70ºC. Remove from heat source to cool. NOTE: Do not evaporate MTBSTFA solution. Phencyclidine in Urine for GC or GC/MS Analysis Compounds: Phencyclidine Part Number: Phase: HyperSep Verify-CX Volume: 10mL Bed Weight: 200mg Sample To 2mL urine add internal standard(s) Pretreatment: and 1mL of 0.1M phosphate buffer (ph 6.0). Mix / vortex. Sample ph should be 6.0±0.5. Adjust ph accord ingly with 0.1M monobasic or dibasic sodium phosphate. Conditioning 3mL methanol then aspirate 3mL DI water then aspirate 1mL 0.1M phosphate buffer (ph 6.0) then aspirate NOTE: Aspirate at <3 Hg to prevent sorbent drying. Application: Load sample at 1-2mL/min. Washing: 3mL DI Water then aspirate. 1mL 0.1M acetic acid then aspirate 3mL Methanol then aspirate Dry column (5 minutes at >10 Hg). Elution: 1mL CH 2 Cl 2 / IPA / NH 4 OH (78/20/2). Collect the eluate by gravity feed. NOTE: Prepare elution solvent fresh daily. Add IPA / NH 4 OH mix, then add CH 2 Cl 2 (ph 11-12). Evaporate to dryness at <40ºC. Remove immediately upon completion. Derivatization: 100μL ethyl acetate. Inject 1-2μL onto the GC. 4 4-Methylenedianaline in Serum for LC or LCMS Analysis Compounds: 4 4-Methylenedianaline Part Number: Phase: HyperSep C18 Volume: 1mL Bed Weight: 100mg Sample None Pretreatment: Conditioning 3mL methanol 3mL DI Water Application: Load sample at 1mL/min. Washing: 1mL DI Water Elution: 0.25mL Methanol containing 1M ammonium hydroxide Injection / Inject 10μL onto the HPLC system analysis: 4 4-Methylenedianaline extracted 100μg/mL sample Retention time = minutes Mobile phase: Methanol / water (50:50) C18 column Flow rate = 1.2mL/min. Inj. Volume = 10μL Wavelength = 254nm Methadone in Urine for GC or GC/MS Analysis Compounds: Methadone Part Number: Phase: HyperSep Verify-CX Volume: 10mL Bed Weight: 200mg Sample To 2mL urine add internal standard(s) Pretreatment: and 1mL of 0.1M phosphate buffer (ph 6.0). Mix / vortex. Sample ph should be 6.0±0.5. Adjust ph accordingly with 0.1M monobasic or dibasic sodium phosphate. Conditioning: 3mL methanol then aspirate 3mL DI water then aspirate 1mL 0.1M phosphate buffer (ph 6.0) then aspirate NOTE: Aspirate at <3 Hg to prevent sorbent drying. Application: Load sample at 1-2mL/min. Washing: 3mL DI Water then aspirate. 1mL 0.1M acetic acid then aspirate 3mL Methanol then aspirate Dry column (5 minutes at > 10 Hg). Elution: 1mL CH 2 Cl 2 / IPA / NH 4 OH (78/20/2). Collect eluants at 1-2mL/min. using minimal vacuum NOTE: Prepare elution solvent fresh daily. Add IPA / NH 4 OH mix, then add CH 2 Cl 2 (ph 11-12). Derivatization: 100μL ethyl methanol Inject 1-2μL onto the GC. 136

54 Sample Preparation Food Safety Aflatoxin M1 Mycotoxin from Milk Compounds: Aflatoxin M1 Part Number: Phase: HyperSep C18 Volume: 6mL Bed Weight: 500mg Sample Dilute 20 ml milk with 30mL Pretreatment: distilled water Conditioning: 10mL methanol followed by 10mL distilled water Application: Force or aspirate sample slowly through column Washing: 10mL distilled water followed by 10mL n-hexane then dry column at 50 C for min or at ambient temp overnight Elution: 3mL dichloromethane/ acetone (4:1, v/v) Water Soluble Vitamins from Aqueous Solutions Compounds: Niacinamide, pyridoxine, riboflavin, thiamine Part Number: Phase: HyperSep C18 Volume: 3mL Bed Weight: 500mg Sample Pretreatment: Conditioning: Application: Washing: Elution: Use an amber glass bottle. Mix 50mL sample with riboflavin content < 6mg with 0.5mL acetic acid and 0.1g heptane-1-sulphonic acid sodium salt. Flush bottle with nitrogen, heat to 55 C, shake. Cool down rapidly. 3mL Methanol followed by 3mL of a solution of 0.5mL acetic acid and 0.1g heptane-1-sulfonic acid sodium salt in 50mL water (to be prepared daily) 2mL sample solution to be forced or aspirated through column 2 x 250µL of a solution of 0.5mL acetic acid and 0.1g heptane-1-sulphonic acid sodium salt in 50mL water 3 x 500µL methanol Immediate analysis required Anthocyan Dyes from Red Wine Compounds: Anthrocyan dye Part Number: Phase: HyperSep C8 Volume: 3mL Bed Weight: 500mg Conditioning: 3mL methanol followed by 3mL distilled water Application: Force or aspirate wine sample slowly through column Washing: 1.5mL distilled water Elution: Small volume methanolic HCl Folic Acid from Food Compounds: Folic acid Part Number: Phase: HyperSep SAX Volume: 3mL Bed Weight: 500mg Sample Homogenize 10 g food sample Pretreatment: in 100 ml 0.01 M phosphate buffer ph 7.4. Filter Conditioning: 6mL n-hexane followed by 6mL methanol and 6mL distilled water Application: Force or aspirate 10mL filtrate through column Washing: 6mL distilled water Elution: 5mL 10% NaCl in 0.1M sodium acetate buffer 137

55 Other Considerations for SPE Method Development Determine appropriate column volume The column volume referred to in the ordering information is the maximum volume of sample or solvent that can be applied to the SPE tube or well per aliquot. The most appropriate column volume for a SPE Troubleshooting specific method will allow for easy application of the sample and will closely match the necessary volume of wash solvent for the method. Determine the appropriate bed weight The bed weight defines the maximum amount of analyte a sorbent can retain. Bed weights should be chosen based on the sample size, taking into consideration the approximate amount of contaminants present and the separation mode. For normal and reversed phase modes, typical bed capacity is 1-5% of the bed weight, up to 100mg/g packing for strongly retained compounds. For example, a 500mg bed weight can retain up to 25mg of solute mass. For less retained compounds, if the bed weight is too small relative to the sample matrix volume, breakthrough (early elution) can occur. For ion exchange sorbents, the number of charged sites available for interaction with the analyte determines the capacity. This ionic capacity of an ion exchange sorbent is given in meq/g. The mean ionic capacity values range from to 0.320mEq/g. Problem Causes Recommended Solutions Poor Recovery of Analytes Improper conditioning. Condition column according to phase: Reversed phase: use methanol, acetonitrile or isopropanol followed by solvent used for sample solution. Ion exchange: use methanol or isopropanol followed by a buffer at a ph which charges both sorbent and analyte. Analytes have a greater affinity for the sample solution Choose a column having a greater selectivity for analytes. than for the column. Change ph of sample to increase affinity of analytes to sorbent. Change polarity of sample solvent to produce lower affinity for the analytes. Improper washing. Wash column using appropriate wash solution. Poor elution. Increase eluent volume, increase eluent strength. Decrease elution strength of washing solvent. Analytes Not Eluted from Column Analytes have stronger interaction with column Choose a column that is less retentive for the analytes. sorbent than with eluting solvent. Elution volume is too low. Increase the elution volume. Elution solvent is too weak to disrupt analyte interaction with the column. Change ph of eluting solvent to ensure greater affinity for the analytes. Change polarity of eluting solvent to ensure greater affinity for the analytes. Inconsistent Extraction Column dries before sample added. Re-condition column. Capacity of column is exceeded. Decrease sample volume. Use a column with a larger amount of sorbent. Sample loading flow rate is too high. Decrease flow rate. Elution flow rate is too fast. Allow elution solvent to seep into column before aspirating or forcing through column. Apply in two separate aliquots versus a single aliquot. Using too strong a wash solution to remove interferences. Reduce strength of wash solvent. Elution volume is too small. Increase volume of elution solvent. Interferences Eluted with Analytes Interferences are extracted at the same time as analytes. Selectively wash interferences from column prior to elution. Use a column which retains the analytes more than the interferences. Interference is due to leachables from column. Wash column with eluting solvent prior to conditioning. Slow Column Flowrate Excessive particulate matter in sample. Filter or centrifuge sample. Sample solution is too viscous. Dilute sample with a weak solvent. Inadequate vacuum. Increase vacuum. Large Volume of Sample Required Sample volumes larger than extraction column capacity. Use larger extraction column volumes. Low Throughput Improper methodology. Consider a different extraction mechanism sorbent or format. 138

56 Sample Preparation SPE Technical Section The Role of ph in SPE: Reversed Phase Applications: If trapping of the analyte in the tube is desired, the ph of the conditioning solvent should be adjusted If the compound of interest is acidic or basic use ph at which compound is not charged The retention of neutral compounds is not usually affected by ph Normal Phase Applications: ph is not normally an issue in normal phase applications Solvents used are typically non-polar organic solvents, rather than water There is no need to verify sample application ph Ion Exchange Applications: ph and pka are important considerations here Acidic compounds are extracted by an anion-exchange columne from a sample solution 1-2 ph units above the pka of the analyte Basic compounds are extracted by a cation-exchange column from a sample solution 1-2 ph units below the pka of the analyte Key Terms used in SPE: Term Analyte Bed Volume Breakthrough Capacity Eluent Interference Matrix Sorbent Surface Area Definition Compound to be isolated & measured in the SPE system The sum of the interstitial volume plus the pore volume of the sorbent within the SPE column Lack of analyte retention which occurs when the total mass of the solutes (analytes & interferences) exceeds the capacity of the sorbent Total quantity of compounds (analytes & interferences) which can be retained from a specific sample matrix solution by a given mass of sorbent Solvent used for elution in SPE to remove the analytes from the solid phase Substance in the sample of separation system which may influence the retention of the analyte on the sorbent or may co-elute with the analyte and influence analytical determination All components of the sample including the solvent, but excluding the analytes Bonded phase silica or adsorbent used as the stationary phase in SPE Sum of the external surface area of a sorbent particle plus the accessible surface area within the pores (m 2 /g) Adjust ph accordingly using appropriate buffers Recommended Solvents: Non-polar solvents such as THF are strong solvents in reversed phase interactions Polar solvents such as methanol and water are strong solvents in normal phase interactions The choice of solvent is dependent upon application and analyte properties Solvent Hexane Isooctane Petroleum ether Cyclohexane Carbon tetrachloride Chloroform Methylene chloride Tetrahydrofuran Diethyl ether Ethyl acetate Acetone Acetonitrile Isopropanol Methanol Water Acetic acid Polarity Non-polar Polar 139

57 National Scientific Target Syringe Filters Syringe Filter Membrane Selection Guide Choose a filter or membrane based on: 1. Chemical compatibility of the membrane and housing with your sample matrix 2. Size and amount of particulates in the sample 3. Potential interactions (binding) between the membrane and sample components 4. Special considerations such as requirement for pre-filter or inorganic ion certification Target Syringe Filter Housings Target Syringe filter housings are manufactured from solvent-resistant, low-extractable polypropylene resins specifically selected for wide compatibility with common HPLC sample matrices. Solutions at temperatures up to 100 C can be filtered using Target syringe filters. Target syringe filters can be sterilized by autoclave at 125 C for 15 minutes. The inlet connection is an enhanced female Luer-Lok fitting designed for extra security when attached to a Luer-Lok syringe. The outlet fitting is a standard size male Luer-slip fitting for ease of filtrate collection. Target polypropylene syringe filter housings meet the requirements of 21 CFR This table offers general guidelines for membrane characteristics and compatible applications. Membrane Type Membrane Characteristics Applications Cellulose Acetate Low protein binding, ideal for aqueous-based samples; high protein recovery from Tissue Culture media filtration, sensitive filtrate; lower protein binding compared to PVDF biological samples Glass MicroFiber Larger porosity; able to remove large particulates without clogging Dissolution testing, general filtration Nylon Most frequently selected membrane; broad compatibility with aqueous and organic General laboratory filtration; filtration for most samples; naturally hydrophilic membrane; extremely low in extractables; excellent HPLC samples. NOTE: Nylon binds protein, flowrate with most sample matrices; not compatible with strong acids or bases do not use when high protein recovery is desired Polyethersulfone High flowrates with good throughput volume; low protein binding; compatible with high PES is certified for Ion Chromatography; temperature liquids; mechanically strong membrane low in inorganic extractable ions Tissue Culture filtration; filtration of proteins and nucleic acids Polypropylene Hydrophilic membrane has wide chemical compatibility with organic solvents; Filtration of biological samples; filtration of low nonspecific protein binding aggressive organic solutions PTFE Hydrophobic membrane is resistant to nearly all solvents, acids, and bases; membrane is Filtration of aggressive organic, highly basic or mechanically strong and will withstand exposure to high temperature liquids; hot solutions, ideal for transducer protectors low in extractables; PTFE blocks water vapor; can be used to filter aqueous solutions after prewetting with an alcohol PVDF Hydrophilic membrane with good solvent resistance; low UV absorbing extractables General biological filtration; filtration of and low nonspecific binding samples where high protein recovery is desired Regenerated Cellulose Hydrophilic membrane with good solvent resistance, extremely low nonspecific binding; Membrane of choice for low nonspecific compatible with nearly all common HPLC solvents; tolerates aqueous samples in binding applications; Tissue Culture media ph range of 3 to 12 filtration and general biological sample filtration Go to: page 8 See our range of Certified Vials 140

58 Nylon Syringe Filters Sample Preparation Naturally hydrophilic membrane offers broad compatibiity with aqueous and organic samples Applications: General laboratory filtration Filtration for most HPLC samples Solvent-resistant, low-extractable polypropylene housing. Syringe filters can be sterilized by autoclave at 125 for 15 minutes. } Membrane: HPLC Certified Nylon } Prefilter: binder-free glass microfiber } Connections: enhanced female Luer-Lok inlet, male slip outlet } Max. operating temperature: 100 C } Max. operating pressure: 4mm - 75psi, 17mm - 115psi, 30mm - 90psi } Retention volumes: 4mm - <15µL, 17mm - <29µL, 30mm - <137µL National Scientific Target Nylon Syringe Filters Outside Dia. Pore Size Prefilter Cat. No. Quantity 4mm 0.45µm No F Pack 4mm 0.2µm No F Pack 17mm 0.45µm No F Pack 17mm 0.2µm No F Pack 30mm 0.45µm No F Pack 30mm 0.2µm No F Pack 30mm 1.5µm No F Pack 30mm 5.0µm No F Pack 30mm 0.45µm Yes F Pack 30mm 0.2µm Yes F Pack 141

59 PTFE Syringe Filters Strong, hydrophobic membrane is resistant to nearly all solvents, acids and bases Applications: Filtration of aggressive organic, highly basic or hot solutions, transducer protectors Filter aqueous solutions after prewetting with an alcohol Solvent-resistant, low-extractable polypropylene housing. Syringe filters can be sterilized by autoclave at 125 for 15 minutes. } Membrane: HPLC certified PTFE, with polypropylene support } Housing: medical grade, solvent-resistant, virgin polypropylene } Prefilter: binder-free glass microfiber } Connections: enhanced female Luer-Lok inlet, male Luer slip outlet } Max. operating temperature: 100 C } Max. operating pressure: 4mm - 75psi, 17mm - 115psi, 30mm - 90psi } Retention volumes: 4mm - <15µL, 17mm - <29µL, 30mm - <137µL National Scientific Target PTFE Syringe Filters Outside Dia. Pore Size Prefilter Cat. No. Quantity 4mm 0.45µm No F Pack 4mm 0.2µm No F Pack 17mm 0.45µm No F Pack 17mm 0.2µm No F Pack 30mm 0.45µm No F Pack 30mm 0.2µm No F Pack 30mm 1.0µm No F Pack 30mm 0.45µm Yes F Pack PVDF Syringe Filters Perfect for general biological filtration and filtration of samples where high protein recovery is desired Applications: General biological filtration Filtration of samples where high protein recovery is desired Solvent-resistant, low-extractable polypropylene housing. Syringe filters can be sterilized by autoclave at 125 for 15 minutes. } Membrane: HPLC-certified PVDF } Hydrophilic membrane with good solvent resistance } Housing: medical grade, virgin polypropylene } Prefilter: binder-free glass microfiber prefilter } Connections: enhanced female Luer-Lok inlet, male Luer slip outlet } Max. operating temperature: 110 C } Max. operating pressure: 4mm - 75psi, 17mm - 115psi, 30mm - 9psi } Retention volumes: 4mm - <15µL, 17mm - <29µL, 30mm - <137µL National Scientific Target PVDF Syringe Filters Outside Dia. Pore Size Prefilter Cat. No. Quantity 4mm 0.45µm No F Pack 4mm 0.2µm No F Pack 17mm 0.45µm No F Pack 17mm 0.2µm No F Pack 30mm 0.45µm No F Pack 30mm 0.2µm No F Pack 142

60 Regenerated Cellulose Syringe Filters Ideal for low nonspecific binding applications; tissue culture media filtration and general biological sample filtration Sample Preparation Applications: Low nonspecific binding applications Tissue culture media filtration and general biological sample filtration Solvent-resistant, low-extractable polypropylene housing. Syringe filters can be sterilized by autoclave at 125 for 15 minutes. } Membrane: HPLC Certified Regenerated Cellulose } Hydrophilic membrane with good solvent resistance; compatible with nearly all common HPLC solvents } Housing: medical grade, virgin polypropylene } Protein binding: <5µg/cm 2 } Connections: enhanced female Luer-Lok inlet, male slip outlet } Max. operating temperature: 110 C } Max. operating pressure: 4mm - 75psi, 17mm - 115psi, 30mm - 90psi } Retention volumes: 4mm - <15µL, 17mm - <29µL, 30mm - <137µL National Scientific Target Regenerated Cellulose Syringe Filters Outside Dia. Pore Size Prefilter Cat. No. Quantity 4mm 0.45µm No F Pack 4mm 0.2µm No F Pack 17mm 0.45µm No F Pack 17mm 0.2µm No F Pack 30mm 0.45µm No F Pack 30mm 0.2µm Yes F Pack PES (Polyethersulfone) Syringe Filters Low protein binding, PES membrane provides high flowrates with good throughput volume Applications: Ion chromatography Tissue culture filtration, filtration of proteins and nucleic acids High-temperature liquids Solvent-resistant, low-extractable polypropylene housing. Syringe filters can be sterilized by autoclave at 125 for 15 minutes. } Membrane: ICP Certified PES (Polyethersulfone) } Housing: medical grade, virgin polypropylene } Connections: enhanced female Luer-Lok inlet, male slip outlet } Max. operating temperature: 100 C } Max. operating pressure: 4mm - 75psi, 17mm - 115psi, 30mm - 90psi } Retention volumes: 4mm - <15µL, 17mm - <29µL, 30mm - <137µL National Scientific Target PES (Polyethersulfone) Syringe Filters Outside Dia. Pore Size Prefilter Cat. No. Quantity 17mm 0.45µm No F Pack 17mm 0.2µm No F Pack 30mm 0.45µm No F Pack 30mm 0.2µm No F Pack 143

61 GMF (Glass MicroFiber) Syringe Filters Larger porosity membrane; able to remove large particulates without clogging Applications: Dissolution testing General filtration Solvent-resistant, low-extractable polypropylene housing. Syringe filters can be sterilized by autoclave at 125 for 15 minutes. } Membrane: Binder-free Glass Microfiber } Housing: medical grade, virgin polypropylene } Connections: enhanced female Luer-Lok inlet, male Luer slip outlet } Max. operating temperature: 110 C } Max. operating pressure: 30mm - 90psi } Retention volumes: 30mm - <137µL National Scientific Target GMF (Glass MicroFiber) Syringe Filters Outside Dia. Pore Size Prefilter Cat. No. Quantity 30mm 0.7µm No F Pack 30mm 1.2µm No F Pack 30mm 3.1µm No F Pack Polypropylene Syringe Filters Polypropylene membrane offers wide chemical compatibility with organic solvents Applications: Filtration of biological samples Filtration of aggressive organic solutions Solvent-resistant, low-extractable polypropylene housing. Syringe filters can be sterilized by autoclave at 125 for 15 minutes. } Membrane: Hydrophilic Polypropylene } Low nonspecific protein binding } Housing: medical grade, virgin polypropylene } Connections: enhanced female Luer-Lok inlet, male slip outlet } Max. operating temperature: 110 C } Max. operating pressure: 17mm - 115psi, 30mm - 90psi } Retention volumes: 17mm - <29µL, 30mm - <137µL National Scientific Target Polypropylene Syringe Filters Outside Dia. Pore Size Prefilter Cat. No. Quantity 4mm 0.45µm No F Pack 4mm 0.2µm No F Pack 17mm 0.45µm No F Pack 17mm 0.2µm No F Pack 30mm 0.45µm No F Pack 30mm 0.2µm No F Pack 30mm 0.45µm Yes F Pack 144

62 Sample Preparation Cellulose Acetate Syringe Filters Low protein binding membrane ideal for aqueous-based samples. Applications: Tissue culture media filtration, sensitive biological samples Solvent-resistant, low-extractable polypropylene housing. Syringe filters can be sterilized by autoclave at 125 for 15 minutes. } Membrane: HPLC Certified Cellulose Acetate } Housing: medical grade, virgin polypropylene } Protein binding: <24µg/cm 2 } Connections: enhanced female Luer-Lok inlet, male Luer slip outlet } Max. operating temperature: 110 C } Max. operating pressure: 4mm - 75psi, 17mm - 115psi, 30mm - 90psi } Retention volumes: 4mm - <15µL, 17mm - <29µL, 30mm - <137µL National Scientific Target Cellulose Acetate Syringe Filters Outside Dia. Pore Size Prefilter Cat. No. Quantity 4mm 0.45µm No F Pack 4mm 0.2µm No F Pack 17mm 0.45µm No F Pack 17mm 0.2µm No F Pack 30mm 0.45µm No F Pack 30mm 0.2µm No F Pack 145

63 All-Plastic Disposable Syringes Disposable syringes with polyethylene barrels and polypropylene plungers; use for all syringe filter applications } Two-part, all-plastic construction eliminates the need for rubber or synthetic plunger gaskets } No silicone or oil lubricant is required in the barrel } Choose Luer-Slip or Luer-Lok syringes, in capacities ranging from 1 to 50mL National Scientific Target Target All-Plastic Disposable Syringes Capacity Cat. No. Quantity Luer-Slip Syringes 1mL S Pack 3mL S Pack 5mL S Pack 10mL S Pack 20mL S Pack 30mL S Pack 50mL S Pack Luer-Lok Syringes 3mL S Pack 5mL S Pack 10mL S Pack 20mL S Pack Nonsterile; packed in bulk. 750µL Micro-Centrifugal Filters, Nonsterile Filter volumes as low as 50µL with low hold-up volume } Filter volumes as low as 50µL up to 750µL with low hold-up volume } Use with any laboratory microcentrifuge } Virgin polypropylene filter housing with tapered 2mL, capped receiver tube } 10,000xG maximum centrifugal force National Scientific 750µL Micro-Centrifugal Filters, Nonsterile Material [Membrane] Pore Size Cat. No. Quantity Cellulose Acetate 0.22µm F Pack Cellulose Acetate 0.45µm F Pack Nylon 0.2µm F Pack Nylon 0.45µm F Pack PVDF 0.2µm F Pack PVDF 0.45µm F Pack Regenerated Cellulose 0.2µm F Pack Regenerated Cellulose 0.45µm F Pack PTFE 0.2µm F Pack PTFE 0.45µm F Pack 146

64 Sample Preparation 2mL Centrifugal Filters, Nonsterile } Filter sample volumes up to 2mL } Virgin polypropylene filter housing with tapered 5mL, capped receiver tube } Use with benchtop or floor model centrifuges } 5,000xG maximum centrifugal force National Scientific 2mL Centrifugal Filters, Nonsterile Material Pore Size Cat. No. Quantity Cellulose Acetate 0.22µm F Pack Cellulose Acetate 0.45µm F Pack Nylon 0.2µm F Pack Nylon 0.45µm F Pack PVDF 0.2µm F Pack PVDF 0.45µm F Pack PTFE 0.2µm F Pack PTFE 0.45µm F Pack 25mL Centrifugal Filters, Nonsterile } Filter sample volumes up to 25mL } Virgin polypropylene filter housing with conical receiver } Use with benchtop or floor model centrifuges } 2,500xG maximum centrifugal force National Scientific 25mL Centrifugal Filters, Nonsterile Material Pore Size Cat. No. Quantity Cellulose Acetate 0.22µm F Pack Cellulose Acetate 0.45µm F Pack Nylon 0.22µm F Pack Nylon 0.45µm F Pack PVDF 0.22µm F Pack PVDF 0.45µm F Pack 147

65 Syringe Filter Membrane Compatibility Chart Use the information in this table to determine the ability of a specific syringe filter membrane to withstand exposure to a solvent. All concentrations are 100% unless noted. LEGEND C = Compatible LC = Limited Compatibility (Membrane may swell and shrink) IC = Incompatible (Not Recommended) ND = No Compatibility Data Currently Available PTFE = Polytetrafluoroethylene (Teflone) PVDF = Polyvinylidene PES = Polyethersulfone CA = Cellulose Acetate RC = Regenerated Cellulose PP = Polypropylene GMF = Glass MicroFiber Chemical Nylon PTFE PVDF PES CA RC PP GMF ACIDS Acetic, Glacial LC C C C IC C C C Acetic, 25% C C C C CA C C C Hydrochloric, Concentrated IC C C C IC IC C C Hydrochloric, 25% IC C C C IC IC C C Sulfuric, Concentrated IC C IC IC IC IC C C Sulfuric, 25% IC C C C IC LC C C Nitric, Concentrated IC C C IC IC IC C LC Nitric, 25% IC C C C IC IC C LC Phosphoric, 25% IC C ND ND C LC C ND Formic, 25% IC C ND ND LC C C C Trichloroacetic, 10% IC C ND ND C C C ND ALCOHOLS Methanol, 98% C C C C C C C C Ethanol, 98% C C C C C C C C Ethanol, 70% LC C C C C C C C Isopropanol C C C C C C C C n-propanol C C C C C C C C Amyl Alcohol (Butanol) C C C C C C C C Benzyl Alcohol C C C ND LC C C IC Ethylene Glycol C C C C C C C C Propylene Glycol C C C C LC C C C Glycerol C C C C C C C C ALKALIES Ammonium Hydroxide, 25% C C LC C C LC C C Sodium Hydroxide, 3N C C C C IC LC C IC AMINES AND AMIDES Dimethyl Formamide LC C IC IC IC LC C C Diethylacetamide C C ND ND IC C ND C Triethanolamine C C ND ND C C ND ND Aniline ND C ND ND IC C ND ND Pyridine C C IC IC IC C IC C Acetonitrile C C C LC IC C C C 148

66 Sample Preparation Chemical Nylon PTFE PVDF PES CA RC PP GMF ESTERS Ethyl Acetate/Methyl Acetate C C C IC IC C LC C Amyl Acetate/Butyl Acetate C C IC IC LC C LC C Propyl Acetate C C IC IC LC C LC ND Propylene Glycol Acetate ND C ND IC IC C C ND 2-Ethoxyethyl Acetate ND C ND IC LC C ND ND Methyl Cellusolve ND C ND IC IC C C C Benzyl Benzoate C C ND IC C C ND ND Isopropyl Myristate C C ND IC C C ND ND Tricresyl Phosphate ND C ND IC C C ND ND HALOGENATED HYDROCARBONS Methylene Chloride LC C C IC IC C LC C Chloroform C C C IC IC C LC C Trichloroethylene C C C IC C C C C Chlorobenzene C C C LC C C C C Freon C C C LC C C C C Carbon Tetrachloride C C C IC LC C LC C HYDROCARBONS Hexane/Xylene C C C IC C C IC C Toluene/Benzene C C C IC C C IC C Kerosene/Gasoline C C C LC C C LC ND Tetralin/Decalin ND C C ND C C ND ND KETONES Acetone C C IC IC IC C C C Cyclohexanone C C IC IC IC C C C Methyl Ethyl Ketone C C LC IC LC C LC C Isopropylacetone C C IC IC C C ND C Methyl Isobutyl Ketone ND C LC IC ND C LC C ORGANIC OXIDES Ethyl Ether C C C C C C LC ND Dioxane C C LC IC LC C C C Tetrahydrofuran C C LC IC LC C C C Triethanolamine C C ND ND C C ND ND Dimethylsulfoxide (DMSO) C C IC IC LC C C C Isopropyl Ether ND C C C C C C ND MISCELLANEOUS Phenol, Aqueous Solution, 10% ND C LC IC IC IC C C Formaldehyde Aqueous Solution, 30% C C C C C LC C C Hydrogen Peroxide, 30% C C ND ND C C ND ND Silicone Oil/Mineral Oil ND C C C C C C C 149

67 Chromacol WebSeal Product Selection: The Chromacol WebSeal system is a comprehensive range of 96-well titer plates with glass inserts and a silicone/ptfe coated sealing mat. This system provides the analyst with 96 completely inert chambers and reduces the risk of cross-contamination from well to well when removing the cover. The products are ideal for High Throughput Screening, Combinatorial Chemistry, Life Science applications and HPLC. WebSeal mats are typically blue or clear silicone rubber in nature. The clear mats have no PTFE coating, making them ideal for use with aqueous mobile phases. Blue mats have a thin protective film of sprayed PTFE, making them ideal for use with organic solvents. Mats are available in standard and pre-cut versions for delicate autosampler needles. The addition of vials to the 96-well plates allows the use of aggressive solvents in the WebSeal system. Products are available in volumes from 500µL to 1.5mL. Product Selection according to Manufacturer Type: Manufacturer 96 Round 96 Round 96 Square 96 Square 384 Square 384 Square Silicone Silicone/PTFE Silicone Silicone/PTFE Silicone/PTFE Abgene WSM-2E WSM-2 CoStar WSM-2FBE WSM-2FB WSM-5E WSM-5 WSM-2 Greiner WSM-2FBE WSM-2FB WSM-3SXE WSM-3S WSM-5E WSM-5 WSM-2E WSM-2 Matrix WSM-2FBE WSM-2FB WSM-3SXE WSM-3S WSM-2E WSM-2 NUNC WSM-7E WSM-3SXE WSM-3S WSM-5E WSM-5 Porvair WSM-2FBE WSM-2FB WSM-3SXE WSM-3S WSM-5E WSM-5 WSM-2E WSM-2 Whatman WSM-2FBE WSM-2 WSM-3SXE WSM-3S WSM-2E 150

68 WebSeal 500µL Kit and Accessories Mid-depth polypropylene 96-well microtiter plate, pre-inserted 500µL glass vials and WebSeal silicone/ptfe mats Sample Preparation } Manufactured to a standard 96-Well footprint and compatible with all well-plate autosamplers Chromacol WebSeal 500µL Kit Description Cat. No. Quantity 96-well microtiter plate with 500µL glass vials and sealing mats 05-MTPVC-96 5 Pack Accessories 96-well mid-depth polypropylene plate for 500µL vials 05-MTP-96 5 Pack 500µL clear glass replacement vials for kit 05-MTV Pack 96 round well silicone/ptfe sealing mat WSM-1 5 Pack 96 round well silicone/ptfe sealing mat, pre-slit WSM-1X 5 Pack WebSeal 700µL Kit and Accessories 700µL kits available with clear glass, amber glass and PTFE vials pre-inserted into 96-Well polypropylene microtiter plates } Cutting tool included for vial removal Chromacol WebSeal 700µL Kit Description Cat. No. Quantity 96-well plate, 700µL clear glass vials, sealing mat and cutting tool MTPVC-96 5 Pack 96-well plate, 700µL amber glass vials, sealing mat and cutting tool MTPVCA-96 5 Pack 96-well plate, 700µL PTFE vials, sealing mat and cutting tool MTPTC-96 1 Each Accessories 700µL clear glass vials for kit 1-MTV Pack 700µL amber glass vials for kit 1-MTV(A) Pack 700µL PTFE vials for kit 1-MTTV Pack 96-well polypropylene plate for 700µL vials MTP-96 5 Pack 96 round well silicone/ptfe sealing mat WSM-1 5 Pack 96 round well silicone/ptfe sealing mat, pre-slit WSM-1X 5 Pack Cutting tool MTPC-1 1 Each 151