Accelerated Solvent Extraction (ASE) Nutraceutical Seminar Dave Knowles May 31, 2012

2 Proprietary & Confidential Presentation Outline Sample Preparation Overview Accelerated Solvent Extraction (ASE ® ) for Solid Samples Principles and Instrumentation Applications Utilizing in-cell clean-up AutoTrace ® 280 for Liquid Samples Instrumentation Applications Rocket Evaporator Conclusions

3 Proprietary & Confidential Key Benefits of ASE Automation Samples can be run all day and all night-unattended Reduced Solvent Usage Compared to Soxhlet up to 80-90% less solvent used Decreased cost to dispose of solvent waste Increased Throughput Faster extractions More extractions completed Higher throughput-more money!

4 Proprietary & Confidential It is interesting that a technique first described in 1879 for sample preparation is so widely used along with modern and sophisticated instrumentation In the Beginning, There Was Soxhlet …

5 Proprietary & Confidential Sample Preparation Issues Sample handling is the single biggest source of errors Sample prep is the main bottleneck in most analytical processes Long time to process samples All laboratories are being asked to do more with fewer resources Costs of solvent purchase and disposal are increasing The data are only as good as the sample preparation High-price chromatography and data systems will not improve the quality of poorly-prepared samples ASE ® and AutoTrace ® were developed to address these issues

6 Proprietary & Confidential Dionex ASE ® 150 and ASE 350 Instruments

7 Proprietary & Confidential ASE ® Overview An automated extraction technique that uses liquid solvents and solvent mixtures Extracts solid or semisolid samples Uses elevated temperatures (40–200 °C) and pressures (1500 psi) to accelerate the extraction process Requires small quantities of solvent and short periods of time 15 mL and 15 min for 10-g samples Versus several hours and hundreds of mL of solvent (Soxhlet) Supports a wide range of sample sizes (mg–100 g) Widely used by government agencies and laboratories worldwide

8 Proprietary & Confidential ASE ® Process Schematic Extract Total (min) ready 12–20 Cell loading Static extraction 0 to 99 Purge with 1–2 nitrogen Rinse with 0.5 fresh solvent cycle dynamic extraction static extraction Fill, heat, (min) equilibrate 5 to 9 dynamic extraction

9 Proprietary & Confidential ASE ® 150 Features Single bottle, premix solvents Single extraction cell operation Accepts 60- and 250-mL collection bottles pH-hardened pathway with Dionium ™ components Accommodates all cell sizes (1, 5, 10, 22, 34, 66, and 100 mL) No software control

10 Proprietary & Confidential Accommodates multiple configurations of cells and collection vessels Up to 24 cells (1-100 mL) and up to 33 collection vessels pH hardened pathway with Dionium™ components Integrated Solvent Controller Automatic mixing of solvents for fractionation, method optimization and rinsing ASE ® 350 Features

11 Proprietary & Confidential ASE ® 350 Features (continued) Solvent saver modes Reduce solvent consumption 2-5 times less than standard ASE Front panel control Software control via PC optional Method control parameters (temperature, solvent volume, etc.) are automatically adjusted for cell volumes

12 Proprietary & Confidential Tools and Techniques for Sample Preparation Sample and Dispersant Adsorbents added to cell to retain interferences Extraction Flow In-Cell Cleanup with ASE ® for Selective Extraction

13 Proprietary & Confidential Features and Benefits of ASE ® Instruments FeatureBenefit/Value Reduce solvent consumptionLower operating costs; shorter ROI Reduced extraction timesHigher productivity, shorter turn around times Unattended automated operationHigher productivity, lower labor costs, better precision Superior temperature controlConsistent results; high confidence in data Flow-through operationIn-line filtration, in-cell clean-up; combining multiple steps; reducing time and complexity pH Hardened pathwayAccommodates more types of samples Exhaustive extractionComplete analyte recover; save time with no re-extractions needed as with manual methods

14 Proprietary & Confidential Industries Using ASE ® Technology Environmental research & contract labs Petrochemicals & polymer producers Food, dairy, and agriculture laboratories University & government analytical labs Producers of chemicals, pesticides and consumer products Pharmaceutical producers Forensic laboratories Natural products/herbal materials Alternate energy research laboratories

15 Proprietary & Confidential ASE ® Standard Methods EPA Method 3545A (OCP, OPP, BNA, TPH, PCDD, herbicides and semi-volatiles) NIST (National Institute of Standards and Technology) uses two extraction techniques to certify all standard reference materials (SRMs) for organics: Soxhlet and ASE Accepted under CLP SOW OLM04.2 Method 6860/6850: perchlorate from solid waste; ASE for extraction and clean-up NOAA Method NWFS-NWFSC-59 for hydrocarbon and chlorinated hydrocarbon contamination in marine animal tissues, soils and sediments Chinese Method GB/T for 405 pesticides in grains and grain products German Method L (extended and revised version of DFG Method S 19) for pesticides in foodstuffs ASTM Standard Practice D-7210 for additives in polymers and D-7567 for gel content of polyolefins Mexican National Standard NMX-AA-146-SCFI-2008 for PAHs in soils and sediments

16 Proprietary & Confidential ASE ® Environmental Applications US EPA method 3545A Pesticides and herbicides PAHs and semivolatiles PCBs Dioxins and furans TPH (Total Petroleum Hydrocarbons) Explosives Air samples PUF (polyurethane foam) cartridges Quartz filters XAD and charcoal PBDEs in flora, fauna, sediments and soils Method 6860 Perchlorate in soils, plants, and animal tissues

17 Proprietary & Confidential Summary of EPA Equivalency Study for ASE ® Compound ClassComparison TechniqueRelative Recovery Organochlorine pesticides (OCP)Automated Soxhlet97.3% Organophosphorus pesticides (OPP)Automated Soxhlet99.2% Semivolatiles (BNA)Soxhlet98.6% Chlorinated herbicidesShake Method112.9% Polychlorinated biphenyls (PCB)Various Reference Materials98.2% Polycyclic aromatic hydrocarbons (PAH) Various Reference Materials104.8%

18 Proprietary & Confidential ASE ® Food Applications Contaminant analysis Pesticides Herbicides PCBs, dioxins, etc. Component analysis Lipid content Flavors, aromas, etc. Antioxidants

19 Proprietary & Confidential Contaminants in Food Matrices Evaluated with ASE ® Pesticides in fruits and vegetables Antibiotics in animal tissue Dioxins and PCBs in animal tissue and other foods Pesticides and herbicides in wheat Mycotoxins in grain PAH in smoked meats Nitrate and nitrite in bacon Acrylamide in food PAH in seafood

20 Proprietary & Confidential Tools and Techniques for Sample Preparation Selective extractions Temperature or solvent selection Selective extractions Blueberry extracts using ASE ® Same sample extracted with different solvents Fraction of analytes Elimination of interferences Hexane, DCM, Ethyl Acetate, Acetonitrile, Ethanol

21 Proprietary & Confidential Extracts With and Without In-Cell Cleanup of Fish Tissue Using Alumina, Silica Gel, and Acidic Silica Gel (40% H 2 SO 4 ) In-Cell Clean-Up with Salmon Samples using ASE ®

22 Proprietary & Confidential ASE ® Applications—PCBs by Selective Extraction 1- to 5 gram samples of animal feed, fish meal, fish oil, and pork fat Sulfuric acid impregnated silica used as sorbent in extraction cell to retain lipids Heptane at 100 °C, 1500 psi (10.34 MPa) 18 min total time, 15–40 mL total solvent GC with ECD for analysis No additional sample prep after concentration Björklund, et al. J. Chromatogr. A, 2004, 1040, 155–166

23 Proprietary & Confidential Selective ASE ® for PCBs—Results Matrix Total PCBs (% of Certified Value) Cattle Feed110 Poultry Feed98 Fish Meal93 Fish Oil85 Pork Fat87 <0.3 mg fat collected in vial after ASE

24 Proprietary & Confidential Selective ASE Extraction: PAHs from Mussel Tissue Mussel samples purchased at local market Spiked at 10, and 50 ppb with EPA 610 PAH mixture Sample Preparation: 5-g mussel tissue mixed with 10-g hydromatrix In-Cell Adsorbents: Alumina (neutral, acidic), silica gel, etc Solvents: DCM, hexane, acetone Temperature: 100 °C, 125 °C Extraction time: 20min GC/MS for analysis

25 Proprietary & Confidential Selective ASE Extraction: PAHs from Mussel Tissue % Recovery CompoundAcidic Alumina/DCMNeutral Alumina/DCM Pyrene Benzo(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Benzo(g,h,i)perylene Dibenzo[a,h]anthracene Indeno[1,2,3-cd]pyrene

26 Proprietary & Confidential PAHs in Mussel Tissue: Acidic Alumina and DCM

27 Proprietary & Confidential Lipid Determination Using ASE ® Hydrolyze sample (8 M HCl, 30 min at 80 °C, or NH 3 OH) Mix liquid sample with Dionex absorbent (ASE Prep CR) to neutralize acid or base Transfer mixture to Dionium cells Perform solvent extraction: Hexane at 100 ºC Evaporate solvent to dryness and dry in an oven at 100 ºC Weigh vial and calculate % fat residue or derivatize and analyze by GC or GC-MS Total time: approximately 20 min/sample Mojonnier L-L requires over 60 min/sample and no automation

28 Proprietary & Confidential Lipid Determination Using ASE ® 350 with Acid Hydrolysis Comparison between Mojonnier and ASE Average values are weight percent from GC/MS after conversion to fatty acid methyl esters. Vanilla WaferAve.SDRSD Mojonnier n=3 ASE n=6

29 Proprietary & Confidential Food ASE ® Method- Resin Lipid (%) Mojonnier Method Lipid (%) Corn Chips AVG RSD1.1%1.2% Mayonnaise AVG RSD0.58%1.2% Parmesan Cheese AVG RSD0.84%1.1% Bologna AVG RSD1.3%0.97% Shortcake AVG RSD0.32%0.24% Acid Hydrolysis Results (n=3); GC-MS Data

30 Proprietary & Confidential Base Hydrolysis Results (n=3); GC-MS Data Food ASE ® Method- Resin Lipid (%) Standard Method Lipid (%) Butter AVG RSD0.78%1.2% Cream Cheese AVG RSD1.2%1.5% Whipping CreamAVG RSD1.2%1.3%

31 Proprietary & Confidential Total Workflow with Fast Simplified Sample Prep ASE (food, feed, soil) Rocket Evaporator AutoTrace SPE (water) Integrated workflow solutions for sample preparation GC-MS system