[ Care and Use Manual ]

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1 Certified Sep-Pak CartridgeS Contents I. INTRODUCTION II. USING CERTIFIED SEP-PAK CARTIDGES III. STRATEGIES FOR SOLID-PHASE EXTRACTION IV. FACTORS AFFECTING SEPARATION EFFICIENCY V. STORAGE AND DISPOSAL OF USED CARTRIDGES VI. Ordering information VII. ADDITIONAL INFORMATION VII. LITERATURE REFERENCES I. INTRODUCTION Waters Certified Sep-Pak cartridges are convenient, reproducible, disposable solid-phase extraction (SPE) devices for sample preparation. Certified Sep-Pak cartridges are manufactured in a Waters ISO and ISO 9002-registered facility in compliance with cgmp guidelines for the U.S. Food and Drug Administration for Class 1 Medical Devices. Certified Sep-Pak cartridges are available in many styles to accommodate manual and automated solid-phase extraction and are available in 3 cc and 6 cc formats in the following chemistries: Reversed-Phase: C 18 Normal-Phase: Silica, Alumina A, Alumina B, Alumina N, Florisil Certified Sep-Pak Cartridges 1

2 II. USING CERTIFIED SEP-PAK CARTRIDGES Below is a five step guideline for using Certified Sep-Pak cartridges. Each separation is different and not all steps may be required for your application. 1. Prepare sample: The sample can be applied to the Certified Sep-Pak cartridge in either liquid or a gas phase. If the sample is a solid, it must be dissolved or extracted prior to loading. 2. Condition/equilibrate cartridge: A conditioning step is required for reversed-phase sorbents (e.g., C 18 ). Use a strong solvent to wet the stationary phase. Conditioning is followed by an equilibration step using a weak solvent. 3. Load sample: The sample is loaded onto the cartridge. 4. Wash: The wash step removes interferences while retaining the analyte. If weakly retained interferences are present in the sample matrix, wash with an appropriate weak solvent. 5. Elute: Use an appropriate stronger solvent to elute the analyte while retaining more strongly bound interferences. III. STRATEGIES FOR SOLID-PHASE EXTRACTION Purification: Use as a chemical filter to retain the component of interest while interferences elute, or to retain the interferences while the analyte passes through unretained. a. Retention-Cleanup-Elution Strategy As the sample is loaded onto the cartridge, the analytes of interest are retained by the sorbent. If needed, an optimized series of washes are used to remove matrix interference from the cartridge. A strong solvent is used to elute the analytes from the cartridge. Sample enrichment results when the final elution volume is smaller than the load volume. Stationary Phase Particles Load Sample (Black) Step Elute 1 Step Elute 2 Step Elute 3 One cartridge can separate all three dyes b. Pass-Through Cleanup Strategy NOTE: Different strength solvents can be used to separate the dyes. Pass-through cleanup methods optimize matrix retention while the analytes of interest pass-through the cartridge unretained. No sample enrichment occurs during the solid-phase extraction (SPE) step. 1. Sample is passed through sorbent and collected No sample enrichment 2. Matrix interferences are retained on sorbent Trace enrichment or concentration: Use to concentrate an analyte from a dilute solution. This is done when the analyte concentration is below the detection limit of the analytical method. Fractionation: Use with a step gradient of increasing solvent strength to selectivity elute and isolate analytes based on differences in polarity. Solvent exchange: Use to adsorb the analyte and elute with a desirable solvent. This is helpful of the sample is dissolved in a solvent that is incompatible with the analytical method. Pass-Through Certified Sep-Pak Cartridges 2

3 c. SPE Procedure Steps The following section describes the steps involved in a complete solid-phase extraction procedure: 1. Pretreatment Solid samples (soil, tissue, etc.) Shake, sonicate, or use soxhlet extraction. - Extract sample with polar organic solvent (methanol, acetonitrile) for polar analytes. - Extract sample with organic solvent and drying agent (dichloromethane, acetone) for non-polar analytes and multi-residue extraction. Non-Aqueous liquid If the sample is soluble in water, dilute it with water for reversed-phase SPE. If the sample is soluble in hexane, dilute it with (or exchange to) hexane for SPE. Wastewater Filter or centrifuge as necessary. 2. Condition For reversed-phase sorbents, preconditioning of the sorbent with an organic solvent, such as methanol, acetonitrile, isopropanol, or tetrahydrofuran, is usually necessary to obtain reproducible results. Without this step, a highly aqueous solvent cannot penetrate the hydrophobic surface and wet the sorbent. Thus, only a small fraction of the sorbent surface area would be available for interaction with the analyte. For the same reason, it is important not to let silica-based SPE cartridges dry out between the solvation step and the addition of the sample. A complete preconditioning of a reversed-phase cartridge includes the solvation step and an equilibration with a low-strength solvent such as water or buffer. 3. Load When the analytes of interest are not retained by the sorbent, this is called analyte breakthrough. For some methods, such as pass-through cleanup, analyte breakthrough is desirable and is maximized for those specific methods. However, in all other cases, analyte breakthrough is unwanted and contributes to poor recovery and method reproducibility. Breakthrough occurs when: There is too high an organic concentration in the load solution for very polar analytes. Dilute sample at least 1:1 with water or buffer prior to loading. The analytes are bound to proteins, they may pass through the sorbent. Ensure that analytes are not bound to proteins by acidifying or basifying the sample. Sorbent is overloaded by the matrix component. Therefore, it is important to choose the correct sorbent mass (see table 1). The flow rate of the load step is too fast. There is not enough contact time between the analytes and the sorbent. Look at the drops and adjust the vacuum so that you see discrete droplets, not a stream of liquid. Table 1. Choice of Cartridges Based on Sample Size Sample Size Cartridge ml 3 cc/200 mg or 6 cc/500 mg ml 3 cc/200 mg or 6 cc/500 mg ml 6 cc/500 mg (LP) or 6 cc/1 g 4. Wash The wash steps are designed to remove unwanted matrix components that remain from the loading step. The ideal wash solvent removes only the matrix while keeping the analytes bound to the sorbent. For complex samples this is impossible, so the wash steps are optimized using ph, solvent strength, and solvent polarity to remove as much matrix as possible while maintaining acceptable analyte recovery. Certified Sep-Pak Cartridges 3

4 5. Elute Once the interferences are washed off the cartridge, a strong solvent is introduced to elute the analytes of interest. The volume and flow rate of the eluting solvents should be precisely controlled as in the load step to ensure reproducible results. Table 2. Guidelines on the Various Types of Separation Mechanisms Analyte Reversed Phase Normal Phase Ion Exchange Low to moderate polarity/ hydrophobic Moderate to highly polar/uncharged Charged or ionized Matrix Aqueous Non-polar organic solvent Aqueous/low ionic strength Condition/Equilibrate 1. Solvate polar organic 2. Water Non-polar organic Low ionic strength buffer Wash Aqueous/buffer Non-polar Low ionic strength buffer Elute Increase polar organic content in steps Increase moderate to high polarity organic content in steps Stronger buffers ionic strength or ph to neutralize the charge IV. FACTORS AFFECTING SEPARATION EFFICIENCY Separation efficiency varies with flow rate and sample load. Here are some guidelines to consider when optimizing your separation. a. Flow Rate If the sample flow rate is too high, components may not interact sufficiently with he sorbent. The result is loss of resolution, analyte breakthrough, and poor recovery. To condition the cartridge, use a flow rate of 5-10 ml/min. To load and elute Certified Sep-Pak Vac 3 cc 200 mg, and 500 mg cartridges, use a flow rate or ml/min. To load and elute all other cartridges, use a flow rate or 2-10 ml/min. b. Sample Load V. STORAGE AND DISPOSAL OF USED CARTRIDGES Cartridges stored in their original sealed pouch remain stable for long periods. Once opened, special care must be taken to ensure that the SPE cartridges are not exposed to the laboratory environment for long periods. The highly active sorbents will readily accumulate background contaminants from the environment and will affect the quality of the device. Waters recommends that you open and use only those cartridges that will be needed. If this is not possible, store the exposed cartridges in clean contaminant free container such as a desiccator. Dispose of used cartridges safely in accordance with applicable government or local regulations. Cartridge capacity depends upon the mass of all components in the sample, volume of solvent, solvent strength, sorbent type, sorbent quality, and retention factor (k) of components. Overloading results in sample breakthrough which leads to variability in sample recovery. Certified Sep-Pak Cartridges 4

5 VI. ORDERING INFORMATION Description Part Number Alumina A Certified Sep-Pak Vac, 3 cc, 500 mg, 50/box Certified Sep-Pak Vac, 6 cc, 500 mg, 30/box Certified Sep-Pak Vac, 6 cc, 1 g, 30/box Alumina B Certified Sep-Pak Vac, 3 cc, 500 mg, 50/box Certified Sep-Pak Vac, 6 cc, 500 mg, 30/box Certified Sep-Pak Vac, 6 cc, 1 g, 30/box Alumina N Certified Sep-Pak Vac, 3 cc, 500 mg, 50/box Certified Sep-Pak Vac, 6 cc, 500 mg, 30/box Certified Sep-Pak Vac, 6 cc, 1 g, 30/box Florisil Certified Sep-Pak Vac, 3 cc, 500 mg, 50/box Certified Sep-Pak Vac, 6 cc, 500 mg, 30/box Certified Sep-Pak Vac, 6 cc, 1 g, 30/box Silica Certified Sep-Pak Vac, 3 cc, 200 mg, 50/box Certified Sep-Pak Vac, 3 cc, 500 mg, 50/box Certified Sep-Pak Vac, 6 cc, 500 mg, 30/box Certified Sep-Pak Vac, 6 cc, 1 g, 30/box C18 Certified Sep-Pak Vac, 3 cc, 200 mg, 50/box Certified Sep-Pak Vac, 3 cc, 500 mg, 50/box Certified Sep-Pak Vac, 6 cc, 500 mg, 30/box Certified Sep-Pak Vac, 6 cc, 1 g, 30/box VII. ADDITIONAL INFORMATION Sep-Pak accessories are available to help with your solid-phase extraction. Ordering information for these products is below. Sep-Pak Cartridge Accessories Description Qty. Part No. 30 cc Reservoir 48/pkg WAT cc Reservoir 12/pkg WAT Reservoir Adapters for 3, and 6 cc VAC 12/pkg WAT Male-Male Adapter 100/pkg WAT Male Luer Plugs WAT Female Luer Plugs WAT Extraction Manifolds 20 position extraction manifold without rack WAT position extraction manifold with 13 x 75 mm test tube rack WAT position extraction manifold with 13 x 100 mm test tube rack WAT Description Qty. Part No. 20 position extraction manifold with 16 x 75 mm test tube rack WAT position extraction manifold with 16 x 100 mm test tube rack WAT Accessories and Spare Parts for the Waters Extraction Manifolds Needle valves, 20/pkg (required when using silicabased SPE cartridges) WAT Needle tips, 20/pkg WAT Cover, 20 position without gauge assembly WAT Gauge assembly, vacuum WAT Reservoir, glass with outlet valve WAT Outlet valve kit WAT Gasket for cover WAT Ejector tool WAT Luer Plugs 25/pkg WAT Rubber ball ring (for vacuum gauge assembly) WAT Reversible Vial Rack for 1 ml or 4 ml autosampler vials WAT x 75 mm test tube rack WAT x 100 mm test tube rack WAT x 75 mm test tube rack WAT x 100 mm test tube rack WAT Adapter (to attach reservoir to 1, 3, and 6 cc Sep-Pak Vac cartridges) 100/pkg WAT Adapter (to attach reservoir to 12, 20, and 35 cc Sep-Pak Vac cartridges) 12/pkg WAT Vacuum pump (110 V, 60 Hz) 10/pkg WAT Vacuum pump (220 V, 50 Hz) WAT Vacuum pump (220 V, 50 Hz) WAT For more detailed information, consult the Chromatography Columns and Supplies Catalog by visiting (literature code EN), or contact your Waters representative. If you require technical assistance, or have any questions regarding sample preparation, visit or call us at VIII. LITERATURE REFERENCES Description Certified Sep-Pak Brochure Sorbent Selection Guide Wall Chart A Sample Preparation Primer and Guide to Solid-Phase Extraction Methods Development Sample Preparation Solutions Brochure Sep-Pak Sample Extraction Products Brochure Food Safety Application Notebook Environmental Chromatography Methods Guide Literature Code EN EN WA EN EN EN EN Certified Sep-Pak Cartridges 5

6 Sales Offices Austria and European Export (Central South Eastern Europe, CIS and Middle East) Australia Belgium Brazil Canada x2205 China CIS/Russia / Czech Republic Denmark Finland France Germany Hong Kong The Netherlands Norway Poland Puerto Rico Singapore Spain Sweden Switzerland Taiwan United Kingdom All other countries: Waters Corporation U.S.A Hungary India and India Subcontinent Ireland Italy Japan Korea Mexico Waters Corporation. Waters, The Science of What s Possible, and Sep-Pak are trademarks of Waters Corporation. Florisil is a trademark of U.S. Silica. Sep-Pak Cartridges are covered by U.S. Patents # 4,211,658 and/or # 4,250,035 and several foreign patents. March EN Rev A VW-PDF Waters Corporation 34 Maple Street Milford, MA U.S.A. T: F: Certified Sep-Pak Cartridges 6