The Secrets of Solid Phase Extraction (SPE) for Sample Preparation. Agilent AccuBOND II, AccuBOND II ENV and EVIDEX II SPE

Transcription

1 The Secrets of Solid Phase Extraction (SPE) for Sample Preparation Agilent AccuBOND II, AccuBOND II ENV and EVIDEX II SPE

2 What Is Solid-Phase Extraction (SPE)? Sample preparation technique with principles similar to those of HPLC for selective adsorption of analytes or interferences from complex matrices Used for sample cleanup and analyte concentration preceding LC, GC, ion chromatography and other techniques Cost-effective alternative to/replacement for liquid-liquid extraction (productivity, solvent, waste) CI0126C 2

3 Advantages of SPE vs. Liquid-Liquid Extraction Improved throughput (parallel vs. serial processing) Decreased organic solvent usage and waste generation Higher and more reproducible recoveries Cleaner extracts (contamination, solvent impurities) No emulsions Tunable selectivity ( SPE phase choices, solvent mixtures) Readily automated CI0126C 3

4 Typical Applications of SPE Sample Cleanup Combinatorial reaction cleanup before LC-MS or LC Pharmacokinetic studies, dissolution testing Isolate analytes from complex matrices urine, plasma Remove column killers or major interferences Eliminate late-eluters to allow isocratic analysis Trace Enrichment Environmental analysis Pharmaceutical and Agrochemical applications Desalting Solvent Exchange Sample Preservation and Storage CI0126C 4

5 Application and Importance of SPE is Growing Rapidly Applied in cartridge format for 20+ years Critically important for sample cleanup and trace enrichment (esp. environmental and pharmaceutical labs) SPE usage is accelerating Combinatorial synthesis and high-throughput LC-MS analysis Overall LC-MS usage increases and greater instrument uptime Smaller samples requiring smaller bed masses (tubes and plates) Minimization of organic solvents and waste Ease of automation (cost, reproducibility, throughput) Liquid handling and SPE workstations 96-well plate autosamplers CI0126C 5

6 Typical SPE Formats SPE Cartridge Schematic Schematic Diagram Diagram of a of 96-Well a SPE 96-Well Extraction Plate Extraction Plate System* (LC-GC, S9, May, 1998) CI0126C 6 Single Extraction Disk Manifold System R. E. Majors, LC-GC, 15(4), 318 * 3M (1997) Corp.

7 SPE Modes Digital Chromatography Analyte Adsorption Analyte(s) retained (k >> 1) Matrix unretained (k ~ 0) and/or strongly retained (k >> 1) Preconcentration factor Cleaner extracts Load at 1-3 drops/sec (recovery 1/flow) Capacity issues may be more important Matrix Adsorption Analyte(s) unretained (k ~ 0) Matrix retained (k >> 1) No preconcentration advantage Eluates may not be as clean Sample loading often gravity fed Used less often than analyte adsorption CI0126C 7

8 Fundamental Steps for SPE Adsorption Mode Prewash* Precondition Load Wash Elute Remove contaminants that could elute with analyte Prepare cartridge to accept sample Load sample and rinse reservoir(s) Wash with solvent that won t elute analyte Elute analyte in smallest volume possible 1 2 *Not necessary for Agilent SPE If elution solvent will be stronger than precond. solvent 1. MeOH or ACN 2. Weak solvent (water, buffer) Weakly retained matrix compds elute Analyte and other matrix compds retained Elute analyte leaving highly retained compds CI0126C 8

9 General SPE Method Development Strategy SPE Adsorption Mode: Background Research the Problem Previous SPE and analysis conditions for the analyte and matrix? Characterize the Analyte Structure, pk a, polarity, functional groups Solvent solubility and stability Any restrictions on final solvent and concentration (technique or instrument)? Characterize the Sample Matrix Possible interferences similar functional groups, pk a, etc. ph, ionic strength Solvent solubility and stability Qualitative and quantitative variability CI0126C 9

10 General SPE Method Development Strategy SPE Adsorption Mode: Experimental Develop or apply effective HPLC or GC conditions to monitor progress Assess recovery and eluate cleanliness Select and test sorbents Determine which sorbents provide maximum analyte retention Determine which eluent solvents yield highest recoveries Identify optimum wash solvent Assess eluate cleanliness under conditions of maximum analyte retention Determine strongest wash solvent that will not elute analyte Test blank and fortified matrix Assess eluate cleanliness and recovery using optimum wash and eluent solvents Test real samples and fortified samples Assess eluate cleanliness and recovery for different unfortified and fortified samples CI0126C 10

11 SPE Method Validation Variables to Consider Validate SPE procedure Sorbent (sorbent weight, different cartridges, different lots) Preconditioning (strong solvent, weak solvent) Loading solvent (% organic, ph, ionic strength, volume) Wash solvent (% organic, ph, ionic strength, volume) Eluent (volume, % organic ) Flow rates (loading, wash, elution) Linearity and range (different analyte concs. and matrix loadings) Analyte stability (loading solvent, eluent) Matrix stability (loading solvent) CI0126C 11

12 Design Characteristics for SPE Family Provide a variety of popular chemistries to meet most application needs Provide silica and PS-DVB base materials Provide most popular sizes of cartridges and bulk material for self-packers Pre-clean packing, tubes and frits low organic extractables for high sensitivity work Economical for routine applications Excellent quality assurance program to ensure consistent results from batch-to-batch and year-to-year Specialty phase for drugs of abuse testing CI0126C 12

13 Break Time For Questions and Answers Press *1 on Your Phone to Ask a Question CI0126C 13

14 SPE Products from Agilent AccuBOND II and EVIDEX II Improved AccuBOND II SPE Products Solid Phase Extraction Cartridges Bulk SPE Adsorbents and Accessories Method Development Kits Applications EVIDEX II SPE Specialty Cartridges Drugs of Abuse General Pharmaceutical Extractions Applications CI0126C 14

15 AccuBOND II SPE Phases SPE Mode Phases Available Reversed Phase Normal Phase (polar) Strong Anion Exchange (SAX) Strong Cation Exchange (SCX) C2, C8, C18, Phenyl, ENV PS-DVB* Silica, -CN, Diol, Amino, Alumina (acidic, basic, and neutral), Florisil Quaternary amine Sulfonic acid * Newest phase CI0126C 15

16 AccuBOND II SPE Cartridges Silica and new PS-DVB cartridges available Wide variety of cartridge sizes 100, 200, 500 and 1000 mg 1, 3, and 6 ml tubes 96 well plates in final development Tabless cartridges for automation (i.e. Gilson) Excellent quality assurance program Random testing of cartridges surface characteristics packing parameters Performance certificate in each box Low extractables Pre-washed packings, frits, and tubes Bulk packings available 25 g bottles CI0126C 16

17 AccuBOND II SPE Low Levels of Extractables Tube and Frit Waters Sep-Pak C18 Tube/Frit Only Programmed Temperature GC-FID Analysis Splitless Injection Total = 50 ppm Internal Standard min Agilent SPE AccuBOND II Pre-washed Tube/Frit Only Total = 5 ppm Internal Standard min CI0126C 17

18 AccuBOND II SPE Method Development Kits SPE Method Development Kits groups of phases that separate using the same mechanism Kit A (Reversed Phase) C2, CN, Phenyl, C8 and C18 Kit B (Normal Phase) Si, Amino, Diol and CN Kit C (Ion Exchange) SCX, SAX and Amino Multiple kits provide a wide variety of options for determining the best mechanism and phase type for your analyte and matrix CI0126C 18

19 AccuBOND II SPE Application Notes Application Notes for AccuBOND II Environmental Extraction of Chlorinated Pesticides in Water (SE-1) Extraction of Polynuclear Aromatic Hydrocarbons in Water (SE-2) Extraction of PCB s in Water (SE-3) Extraction of Triazines Using SPE Cartridges Pharmaceutical Anabolic Steroids in Urine and Serum (SC-1) Benzodiazepines in Serum (SC-2) Caffeine and Metabolites in Serum (SC-3) Barbiturates in Serum (SC-4) Available online: CI0126C 19

20 AccuBOND II Example Applications Triazines in Different Matrices Characterize the Analyte Structure, pk a, polarity, functional groups Solvent solubility and stability Any restrictions on final solvent and concentration due to technique or instrument? Characterize the Sample Matrix Solvent solubility and stability ph, ionic strength Possible interferences similar functional groups, pk a, etc. Qualitative and quantitative variability Triazines Three major species simazine, atrazine and propazine all structurally similar Mode of Action: Herbicides Practically insoluble in water Soil large number of charged species-adjust ph to retain triazines and do ion-exchange Muscle tissue large amounts of nonpolar lipids retain these and elute triazines using C18 Corn oil non-polar glycerides and fatty acids weakly retained on diol while triazines are strongly retained N Cl N NH N NH R 1 R 2 CI0126C 20

21 SPE Methods for Triazines in Complex Matrices Soil Muscle Tissue Corn Oil CARTRIDGE SCX ODS Diol EXTRACTION Shaken in acetonitrile Homogenized in methanol None PRE-TREAT Acetic acid Methanol Methanol, hexane LOAD Diluted with acetic acid Diluted with water Diluted with hexane WASH Acetic acid, acetonitrile, water, 0.1 M K 2 HPO 4 Water Hexane ELUTE Acetonitrile/K 2 HPO 4 Methanol Methanol CI0126C 21

22 HPLC Analysis of Triazines Extracted from Different Matrices Triazines from Soil with SCX Triazines from Muscle Tissue with ODS Triazines from Corn Oil with Diol Column: C18, 4.6 x 150 mm, 5 mm Mobile Phase: 50% methanol:50% 0.01M K 2 HPO 4 Flow Rate: 2 ml/min Detection: UV 254 nm Sample: Triazines 1. Simazine 2. Atrazine 3. Propazine CI0126C 22

23 AccuBOND II Example Applications Barbiturates in Serum O R 1 N O Characterize the Analyte Structure, pk a, polarity, functional groups Solvent solubility and stability Any restrictions on final solvent and concentration due to technique or instrument? Characterize the Sample Matrix Solvent solubility and stability ph, ionic strength Possible interferences similar functional groups, pk a, etc. Qualitative and quantitative variability Barbiturates Non-polar side groups make retention on C18 possible but retention not strong Mid ph will eliminate charge and improve retention Soluble in water Final solvent easy to evaporate or compatible with HPLC Serum has many components competing for retention Serum will vary from person to person HN O R 3 R 2 CI0126C 23

24 Analysis of Barbiturates in Serum AccuBOND II SPE C18 Cartridge Solid Phase Extraction Method CARTRIDGE: AccuBOND II ODS (C18) 6 ml/500 mg (P/N /box) Sample Preparation: Add 1mL0.5M K 2 HPO 4 to 4 ml of serum Precondition: 5 ml Acetone 5 ml Deionized Water Load: 5 ml Sample (see sample preparation above) Wash 2.0 ml 95% water:5% acetonitrile Elute: 3.0 ml Acetone Evaporate and Reconstitute: Evaporate Acetone in a nitrogen stream at <45 C Add 200 µl of acetonitrile:water (20/80) CI0126C 24

25 Break Time For Questions and Answers Press *1 on Your Phone to Ask a Question CI0126C 25

26 HPLC Analysis of Barbiturates Extracted from Human Serum Column: C18, 4.6 x 150 mm, 5 mm Mobile Phase: Time ACN Water Flow Rate: 1 ml/min Detection: UV 235 nm Sample: Barbiturates 1. Barbital 2. Allobarbital 3. Aprobarbital 4. Phenobarbital 5. Butabarbital 6. Alphenal 7. Hexobarbital 8. Amobarbital 9. Mephobarbital 10. Secobarbital CI0126C 26

27 Recovery Data for Barbiturates in Serum Compound Barbital Allobarbital Aprobarbital Phenobarbital Butabarbital Butethal Butalbital Alphenal Hexobarbital Pentobarbital Amobarbital Mephobarbital Secobarbital RRT Serum spiked at 1mg/mL n= 4 RRT = retention time relative to barbital Mean Std. Dev CI0126C 27

28 New AccuBOND II ENV for Environmental Analytes CI0126C 28 New PS-DVB SPE material for environmental applications High surface area 600 m 2 /g provides good retention Large particle size for high flow mm High capacity for good retention Capacity of caffeine 320 mg/g Provides high recovery of phenols in water wide range of polarities and solubilities Higher recovery than with silica (phenol> 70%, other phenols>90%) Optimized for the analysis of phenols High sample delivery rate for short extraction time Follow with GC or GC/MS analysis Being evaluated for other environmental analytes - pesticides and PAHs

29 SPE Procedure for Extraction of Phenols Solid Phase Extraction Method CARTRIDGE: AccuBOND II ENV PS-DVB, 1000 mg*, 6 ml (P/N , 30/box) *1000 mg is required for optimal recovery Sample and Recovery Standard Preparation: 1L water Deuterated phenol, 2,4- dibromophenol, and 2,4,6- tribromophenol recovery standards added at 10 ppb level Sample and standards mixed and ph lowered to <2 with 5N HCl Precondition: 9 12 ml Dichloromethane 9 CI0126C ml Methanol Load: 1.0 L water sample at ml/min (see sample preparation above) Elute: 9.0 ml Dichloromethane (dried with anhydrous sodium sulfate) Evaporate and Reconstitute: Evaporate Dichloromethane in nitrogen stream at room temperature and transferred to silanized amber vial Bring to 900 µl of Dichloromethane µl of a solution containing 2,5-dibromotolune and 2,2,5,5 -tetrabromobiphenyl at 0.05 mg/ml in Dichloromethane as internal standards Publication Number of phenol application: EN Available on the Agilent web site:

30 CI0126C 30 GC Analysis of Extractable Phenols

31 AccuBOND II SPE Product Features COA, Packaging and Labeling Certificate of analysis with each box Mass consistency Flow rate consistency Trace metal analysis Extract cleanliness QC chromatogram Packaged in trilayer laminated bag Excludes air and water Ensures clean product Individually printed tubes and plates Easy identification Versatility for method development CI0126C 31

32 Certificate of Performance for AccuBOND II page 1 Description: AccuBOND II C8 100 mg 1 ml cartridge Catalog No: Lot No.: AMC18-0X Run No.: XXXXXXX This AccuBOND product and sorbent have been manufactured, tested and assembled under the control of an ISO 9001 registered quality system. This AccuBOND SPE product has been subjected to the following QC tests: Base Silica Characteristics Bonded Silica Tests Surface Area: 546 m2/g Carbon Loading: 13.0% Average Pore Size: 60Å Surface Coverage: 2.5 µmoles/m2 Surface ph: 7 Extraction Residue: Pass Metal Analysis: Pass Exchange Capacity: N/A Particle Size Data Packed Cartridge Test Particle Shape: Irregular Cartridge Flow Resistance: Pass Average Particle Size (µm): 56 Frit Purity Test (GC): Pass Percentage of Particles <10 µm: 0.00 Material Weight Check: Pass Percentage of Particles <25 µm: 1.36 Percentage of Particles <90 µm: 4.95 CI0126C 32

33 Certificate of Performance for AccuBOND II page 2 Test Condition and Results Conditions: Column: Mobile Phase: Flow R ate: 4.6 x 250 mm 70/30 MeOH/H2O 2.0 m l/m in Specifications: QA K biphenyl = 3.5 to 6.5 K toluene = 1.5 to 3.5 K acetophenone = 0.6 to 1.4 T f u racil = 0.3 to 1.5 Results for Lot AMC8X-XX QC K toluene = 4.5 Peak 1 = Uracil K dimethylaniline = 2.1 Peak 2 = Acetophenone K n itrob enzene = 1.0 P eak 3 = T olu en e T f dim ethylaniline = 1.4 Peak 4 = B iphenyl Notice: M aterial Safety D ata Sheets (M SDS) are available at: /scripts/cag_m sdssearch.asp This product has passed all Agilent Technologies, inc. quality control specifications. CI0126C 33

34 EVIDEX II SPE Cartridges for Drugs of Abuse in Urine Proprietary bonding chemistry Mixed RP and cation-exchange bonded phase Designed for NIDA-5 Drug Classes Amphetamine/Methamphetamine PCP ( Angeldust ) Benzoylecgonine (Cocaine metabolite) Codeine and Morphine THC-COOH (Marijuana metabolite) Tested with actual drugs of abuse Ensures lot-to-lot reproducibility Ensures high recoveries Produces clean extracts with excellent S/N ratios in GC-MS Good general purpose pharmaceutical mixed mode phase CI0126C 34 N O CH 3 O O CH 3 OH NH 2 HO N CH 3 O H CH 3 NH CH 3 Amphetamine Methamphetamine PCP Benzoylecgonine Codeine Morphine O OH H OH O THC-COOH CH 3 H H 3 C O CH 3 H 3 C HO N CH 3 O H N OH

35 EVIDEX II Drugs of Abuse (DOA) Methods EVIDEX II Methods for NIDA drug classes One SPE cartridge type for all analytes and methods Two cartridge configurations 200 mg/3ml and 400 mg/6 ml for different sample sizes Step-by-step instructions Robust procedures minor changes in reagent volumes and concentration do not affect results GC-MS analysis using column specific for DOA (DB-5 MS EVDX GC) Accurate, reproducible results (<5% RSD) CI0126C 35 Bottom Line: Results are defensible in Effect of Varying Method Conditions on Benzoylecgonine SPE Recovery EVIDEX II methods are robust!

36 EVIDEX II SPE Cartridges for Drugs of Abuse Opiates in Urine Solid Phase Extraction Method CARTRIDGE: EVIDEX II 400 mg, 6 ml (P/N , Box of 30) Precondition: 6 ml methanol 6 ml 0.1 M potassium phosphate (ph 6.0) Do not let the phase go dry Load: Add 3 ml 0.1 M potassium phosphate (ph 6.0) to the cartridge Attach an 8 ml reservoir Add the urine sample Rinse: Remove reservoir 3 ml water 3 ml 0.1 M sodium acetate (ph 4.5) 3 ml methanol Elute: Place a collection tube beneath cartridge 3 ml methylene chloride/isopropyl alcohol/ NH 4 OH (78/20/2) Collect the eluate Codeine Clean extracts from a very dirty matrix Morphine Codeine 30 ng Column: DB-5MS EVDX Carrier: Helium at 40 cm/sec Oven: 65 degrees for 1 min, degrees at 20 degrees/min Injector: Splitless, 250 C Detector: MSD, 300 C transfer line Morphine 30 ng CI0126C 36

37 DOA Recoveries are Reproducible Using EVIDEX II SPE Cartridges Recovery Levels (x±%, n = 8) Level in ng/ml Amphetamine 76 ±4 74 ±2 96 ±3 Methamphetamine 85 ±1 71 ±3 98 ±3 Level in ng/ml PCP 90 ±3 91 ±2 88 ±3 Level in ng/ml Benzoylecgonine 98 ±8 93 ±4 97 ±3 Level in ng/ml Codeine 99 ±1 97 ±3 98 ±1 Morphine 96 ±2 96 ±3 93 ±4 Level in ng/ml THC-COOH 92 ±4 96 ±8 94 ±3 CI0126C 37

38 Summary Solid Phase Extraction Probably the most important technique for sample cleanup and concentration today Increases productivity, column lifetime and instrument uptime AccuBOND II Available with both silica and PS-DVB base materials Many bonded phases for every sample type Low extractable levels prewashed tubes, frits, and packing High quality SPE products for any sample type Low extractables from packing, tubes and frits are compatible with sensitive detectors like MS EVIDEX II Accurate, reproducible, robust methods for drugs of abuse and other pharmaceutical applications CI0126C 38

39 Appendix

40 SPE Tips For Improving Recovery and Precision General Keep cartridges in sealed bags until use. Store in zipper-locked bags or in desiccator once opened. When using empty reservoirs attached to cartridges, use long disposable pipets to ensure proper flow from reservoir to cartridge. Use stopcocks to adjust/control flow through individual cartridges. Use mass balance for all fractions to determine fate of analyte (adsorption to surfaces, loading effluent, washes, eluate, etc.) during method development. Residual water can be removed effectively by centrifugation (5000 rpm, 5 min.) compared to drying with vacuum or nitrogen. Cartridge capacity for analytes and matrix is typically about 1-3% of cartridge bed weight (ion-exchange not included). NEVER allow the cartridge to dry out until the elution step. CI0126C 40

41 SPE Tips For Improving Recovery and Precision Prewash Remove all strong prewash solvent for GC (e.g., dichloromethane, hexane, ethyl acetate) before preconditioning and loading. Precondition Make sure ph is correct for ion-suppression (acids) or minimal silanol interactions (bases). Leave ~1-2 mm of preconditioning solvent above sorbent bed to prevent bed from drying. Leave ~1/4 to 1/2 of tube volume above sorbent bed when using empty reservoir above cartridge. CI0126C 41

42 SPE Tips For Improving Recovery and Precision Load Leave ~1/4 to 1/2 tube volume above sorbent bed when using tube reservoir above cartridge. Use drop wise solvent flow when time/throughput is not a major concern. Wash Wipe needles of manifold before elute step to minimize contamination of eluate. Elute When choosing eluent, consider ease of evaporation if reconstitution is needed. Allow cartridge/plate to soak with eluent for min. ( recovery). Sometimes several smaller eluent aliquots can improve recovery. CI0126C 42

43 SPE Extraction and Analysis of Triazines in Several Matrices Triazine pesticides may need to be extracted from many different sample matrices One SPE procedure will not be right for all sample types A variety of SPE bonded phases and mechanisms will be needed for different matrices Reversed-phase Normal phase Ion-exchange AccuBOND II family provides the necessary variety of SPE cartridges for any matrix CI0126C 43

44 Extraction and Separation of PAHs from Water AccuBOND II ODS Solid Phase Extraction Method CARTRIDGE: AccuBOND II, 500 mg, 6 ml (P/N , 30/box) Sample Preparation: Add 2 ml Isopropanol to 20 ml Water Sample Mix thoroughly Precondition: 5 ml Methylene Chloride 5 ml Methanol 5 ml Deionized Water Add ~1 ml Deionized Water to top of bed Load: Attach 24-mL sample reservoir to cartridge Add sample to reservoir. Ensure flow into cartridge with glass pipet. Apply vacuum and keep flow rate <10 ml/min. Slower flow gives better results. Wash: 3 ml 50:50 Acetonitrile/Deionized Water Apply vacuum until 30 sec. after wash has passed through cartridge Centrifuge cartridge at rpm for 5 min. (removes excess water) Elute: 3 ml Methylene Chloride and collect eluent. Evaporate to µl. Do not apply heat or smaller PAHs will be volatilized. Add Methylene Chloride to bring sample to final volume of 200 µl. Inject 2 µl. CI0126C 44 Cl Water Spiked at 50 ppb Avg. Compound Recovery (%) Std. Dev. (%) Naphthalene Chloronaphthalene 78 8 Acenaphthylene 81 9 Fluorene 99 9 Phenanthrene 98 9 Fluoranthene 91 8 Chrysene Benzo(b)-fluoranthene 60 8 Benzo(a)pyrene 55 8 Indene(1,2,3-c,d)pyrene Benzo(g,h,i)perylene 59 10

45 Typical SPE Extraction Protocol for Opiates Sample Preparation: Add 0.5 ml hydrochloric acid to 5 ml urine Cap Heat at 120 C for 20 min Cool to room temperature Add 0.75 ml 10 N NaOH Adjust to ph with ~2.5 ml 0.5 M phosphoric acid Condition: 6 ml methanol 6 ml 0.1 M K 2 HPO 4 (ph 6.0) Do not let the phase go dry Load: Add 3 ml 0.1 M K 2 HPO 4 to the cartridge Attach an 8 ml reservoir Add the urine sample Rinse: Remove reservoir 3 ml water 3 ml 0.1 M sodium acetate (ph 4.5) 3 ml methanol Elute: Place a collection tube beneath cartridge 3 ml methylene chloride/isopropyl alcohol/ NH 4 OH (78/20/2) Collect the eluant Analysis Preparation: Concentrate the eluant to dryness Do not over dry 75 µl ethyl acetate 25 µl BSTFA/1% TMCS Cap Heat at 60 C for 10 min Inject 1-2 µl All flow rates should not exceed 5 ml/min Reagents: 10 N NaOH 40 g sodium hydroxide in 100 ml DI water. 0.1 M K 2 HPO 4 (ph 6.0) 1.74 g potassium phosphate, dibasic (anhydrous) in 100 ml DI water. Adjust to ph 6.0 ± 0.1 with phosphoric acid. 0.1 M sodium acetate (ph 4.5) 0.82 g in 100 ml DI water. Adjust to ph 4.5 ± 0.1 with glacial acetic acid. Methylene chloride/isopropyl alcohol/nh 4 OH (78/20/2) 78 ml methylene chloride, 20 ml isopropyl alcohol, 2 ml ammonium hydroxide. Make fresh daily. BSTFA/1% TMCS 9 ml N,O-bis(trimethylsilyl)trifluoroacetamide, 0.1 ml trimethylchlorosilane, or can be purchased premixed. Other reagents Methanol DI water Ethyl acetate Hydrochloric acid Phosphoric acid CI0126C 45

46 Certificate of Performance for AccuBOND II Test Condition and Results Description: AccuBOND C8 100mg 1mL cartridge Catalog No: Lot No.: AMC18-0X Run No.: XXXXXXX This AccuBOND product and sorbent have been manufactured, tested And assembled under the control of an ISO 9001 registered quality system. This AccuBOND SPE product has been subjected to the following Q.C. tests: Base Silica Characteristics Bonded Silica Tests Surface Area: 546 m 2 /g Carbon Loading: 13.0 % 2 Average Pore Size: 60 Å Surface Coverage: 2.5 µmoles/m Surface ph: 7 Extraction Residue: Pass Metal Analysis: Pass Exchange Capacity: N/A Conditions: Column: Mobile Phase: Flow Rate: 4.6 x 250 mm 70/30 MeOH/H2O 2.0 ml/min Specifications: QA K biphenyl = 3.5 to 6.5 K toluene = 1.5 to 3.5 K acetophenone = 0.6 to 1.4 T f uracil = 0.3 to 1.5 Results for Lot AMC8X-XX QC K toluene = 4.5 Peak 1 = Uracil K dimethylaniline = 2.1 Peak 2 = Acetophenone K nitrobenzene = 1.0 Peak 3 = Toluene T f dimethylaniline = 1.4 Peak 4 = Biphenyl Particle Size Data Particle Shape: Irregular Average Particle Size (µm): 56 Percentage of Particles <10µm: 0.00 Percentage of Particles <25µm: 1.36 Percentage of Particles >90µm: 4.95 Packed Cartridge Test Cartridge Flow Resistance: Pass Cartridge Purity Test (GC): Pass Frit Purity Test (GC): Pass Material Weight Check: Pass Notice: Material Safety Data Sheets (MSDS) are available at: This product has passed all Agilent Technologies, inc. quality control specifications. CI0126C 46

47 HPLC Column Technical Support (phone: US & Canada) * (phone) * * Select option 4, then option (fax) CI0126C 47