The Secrets of Solid Phase Extraction (SPE) for Sample Preparation

Agilent AccuBONDII, AccuBONDII ENV and EVIDEXII SPE

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

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

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

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

Typical SPE Formats

SPE Cartridge

Schematic Diagram of a 96-Well SPE Schematic Diagram of a Extraction Plate System* 96-Well Plate Extraction System
(LC-GC, S9, May, 1998)

Single Extraction Disk Manifold System

R. E. Majors, LC-GC, 15(4), 318 (1997) * 3M Corp.
CI0126C 6

SPE ModesDigital 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

Fundamental Steps for SPE Adsorption Mode

Prewash*
Remove contaminants that could elute with analyte

Precondition
Prepare cartridge to accept sample

Load
Load sample and rinse reservoir(s)

Wash
Wash with solvent that wont elute analyte

Elute
Elute analyte in smallest volume possible

1
aryE s es t SP c neilen t o *N r Ag fo

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

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, pKa, 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, pKa, etc. pH, ionic strength Solvent solubility and stability Qualitative and quantitative variability

CI0126C 9

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

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

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

Break Time

For Questions and Answers Press *1 on Your Phone to Ask a Question

CI0126C 13

SPE Products from Agilent AccuBONDII and EVIDEXII

Improved AccuBONDII SPE Products Solid Phase Extraction Cartridges Bulk SPE Adsorbents and Accessories Method Development Kits Applications EVIDEXII SPE Specialty Cartridges Drugs of Abuse General Pharmaceutical Extractions Applications

CI0126C 14

AccuBONDII SPE Phases SPE Mode

Reversed Phase Normal Phase (polar) Strong Anion Exchange (SAX) Strong Cation Exchange (SCX)
* Newest phase
CI0126C 15

Phases Available
C2, C8, C18, Phenyl, ENV PS-DVB* Silica, -CN, Diol, Amino, Alumina (acidic, basic, and neutral), Florisil Quaternary amine Sulfonic acid

AccuBONDII 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

AccuBONDII SPE Low Levels of Extractables Tube and Frit

Waters Sep-Pak C18 Tube/Frit Only
Total = 50 ppm

Programmed Temperature GC-FID Analysis Splitless Injection

Internal Standard

12

16

min

Agilent SPE AccuBONDII Pre-washed Tube/Frit Only

Total = 5 ppm Internal Standard

0
CI0126C 17

12

16

min

AccuBONDII 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

AccuBONDII SPE Application Notes

Application Notes for AccuBONDII Environmental Extraction of Chlorinated Pesticides in Water (SE-1) Extraction of Polynuclear Aromatic Hydrocarbons in Water (SE-2) Extraction of PCBs 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: www.agilent.com/chem

CI0126C 19

AccuBONDII Example Applications Triazines in Different Matrices Cl

N N N NH R1 NH R2

Characterize the Analyte Structure, pKa, 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 interferencessimilar functional groups, pKa, 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

CI0126C 20

SPE Methods for Triazines in Complex Matrices

Soil CARTRIDGE EXTRACTION PRE-TREAT LOAD SCX Shaken in acetonitrile Acetic acid Diluted with acetic acid Acetic acid, acetonitrile, water, 0.1 M K2HPO4 Acetonitrile/K2HPO4

Muscle Tissue ODS Homogenized in methanol Methanol

Corn Oil Diol None Methanol, hexane

Diluted with water Diluted with hexane

WASH ELUTE

Water Methanol

Hexane Methanol

CI0126C 21

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 K2HPO4 Flow Rate: 2 mL/min Detection: UV 254 nm Sample: Triazines 1. Simazine 2. Atrazine 3. Propazine
CI0126C 22

AccuBONDII Example Applications Barbiturates in Serum

O

R1 N O

Characterize the Analyte Structure, pKa, 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 interferencessimilar functional groups, pKa, etc. Qualitative and quantitative variability

Barbiturates

HN R3 O

R2

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

CI0126C 23

Analysis of Barbiturates in Serum AccuBONDII SPE C18 Cartridge

Solid Phase Extraction Method
CARTRIDGE:
ODS (C18) 6 mL/500 mg (P/N 188-1356 30/box) AccuBONDII

Load:
5 mL Sample (see sample preparation above)

Wash Sample Preparation:

Add 1 mL 0.5M K2HPO4 to 4 mL of serum 2.0 mL 95% water:5% acetonitrile

Precondition:
5 mL Acetone 5 mL Deionized Water

Elute:
3.0 mL Acetone

Evaporate and Reconstitute:

Evaporate Acetone in a nitrogen stream at <45C Add 200 L of acetonitrile:water (20/80)

CI0126C 24

Break Time

For Questions and Answers Press *1 on Your Phone to Ask a Question

CI0126C 25

HPLC Analysis of Barbiturates Extracted from Human Serum

Column: C18, 4.6 x 150 mm, 5 mm Mobile Phase: Time ACN Water 0 20 80 5 20 80 15 40 60 20 40 60 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

Recovery Data for Barbiturates in Serum

Compound Barbital Allobarbital Aprobarbital Phenobarbital Butabarbital Butethal Butalbital Alphenal Hexobarbital Pentobarbital Amobarbital Mephobarbital Secobarbital RRT 1.00 1.99 2.47 2.87 3.06 3.29 3.45 3.46 4.09 4.12 4.20 4.36 4.59 Mean 70 82 92 78 89 88 88 87 92 102 93 93 95 Std. Dev. 11 11 9 8 7 6 14 8 6 5 3 5 8

Serum spiked at 1mg/mL n= 4 RRT = retention time relative to barbital

CI0126C 27

New AccuBONDII ENV for Environmental Analytes

New PS-DVB SPE material for environmental applications High surface area 600 m2/g provides good retention Large particle size for high flow 75 150 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
CI0126C 28

SPE Procedure for Extraction of Phenols

Solid Phase Extraction Method CARTRIDGE:
Load:
1.0 L water sample at 20 25 mL/min (see sample preparation above)

AccuBONDII ENV PS-DVB, 1000 mg*, 6 Elute: mL 9.0 mL Dichloromethane (dried with anhydrous (P/N 188-3060, 30/box) sodium sulfate) *1000 mg is required for optimal recovery Sample and Recovery Standard Preparation: 1L water Deuterated phenol, 2,4dibromophenol, and 2,4,6tribromophenol recovery standards added at 10 ppb level Sample and standards mixed and pH lowered to <2 with 5N HCl Precondition: 9 12 mL Dichloromethane CI0126C 29 mL Methanol 9 12
Evaporate and Reconstitute:
Evaporate Dichloromethane in nitrogen stream at room temperature and transferred to silanized amber vial Bring to 900 L of Dichloromethane + 100 L 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: 59885255EN Available on the Agilent web site: www.agilent.com/chem

GC Analysis of Extractable Phenols

CI0126C 30

AccuBONDII 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

Certificate of Performance for AccuBONDII page 1

Description: AccuBONDII C8 100 mg 1 mL cartridge Catalog No: 188-0310 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 Surface Area: Average Pore Size: Surface pH: Metal Analysis: Bonded Silica Tests Carbon Loading: Surface Coverage: Extraction Residue: Exchange Capacity: Packed Cartridge Test Cartridge Flow Resistance: Frit Purity Test (GC): Material Weight Check:

546 m2/g 60 7 Pass

13.0% 2.5 moles/m2 Pass N/A

Particle Size Data Particle Shape: Irregular Average Particle Size (m): 56 Percentage of Particles <10 m: 0.00 Percentage of Particles <25 m: 1.36 Percentage of Particles <90 m: 4.95
CI0126C 32

Pass Pass Pass

Certificate of Performance for AccuBONDII page 2

T e s t C o n d i t io n a n d R e s u lt s
C o n d itio n s : C o lu m n : M o b ile P h a s e : F lo w R a te : S p e c i f i c a t i o n s : QA K b ip h e n y l K to lu e n e K a c e to p h e n o n e T f u r a c il 4 .6 x 2 5 0 m m 7 0 /3 0 M e O H /H 2 O 2 .0 m l/m in

= = = =

3 .5 1 .5 0 .6 0 .3

to to to to

6 .5 3 .5 1 .4 1 .5

R e s u l t s f o r L o t A M C 8 X - X X QC K to lu e n e = 4 .5 K d im e th y la n ilin e = 2 .1 K n itr o b e n z e n e = 1 .0 T f d im e th y la n ilin e = 1 .4

P eak P eak P eak P eak

1 2 3 4

= = = =

U r a c il A c e to p h e n o n e T o lu e n e B ip h e n y l

N o tic e : M a te ria l S a fe t y D a ta S h e e ts (M S D S ) a re a v a ila b le a t: h t t p : / / w w w .c h e m . a g i l e n t . c o m / s c r i p t s / c a g _ m s d s s e a r c h .a s p T h is p ro d u c t h a s p a s s e d a ll A g ile n t T e c h n o lo g ie s , in c . q u a lity c o n tro l s p e c ific a tio n s .

CI0126C 33

EVIDEXII SPE Cartridges for Drugs of Abuse in Urine

Proprietary bonding chemistry Mixed RP and cation-exchange bonded phase Designed for NIDA-5 Drug Classes
N
CH3 NH2

NH CH3 CH3
N

Amphetamine
CH3 O

Methamphetamine

PCP

Amphetamine/Methamphetamine PCP (Angeldust) Benzoylecgonine (Cocaine metabolite) Codeine and Morphine THC-COOH (Marijuana metabolite)
O O

OH

HO

CH3

HO

OH

H N CH3

H N CH3

Benzoylecgonine
O

Codeine
OH

Morphine

OH H

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

H H3C O H3C CH3

THC-COOH

EVIDEXII Drugs of Abuse (DOA) Methods

EVIDEXII 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

EVIDEXII methods are robust!

EVIDEXII SPE Cartridges for Drugs of Abuse Opiates in Urine

Solid Phase Extraction Method CARTRIDGE: EVIDEXII 400 mg, 6 mL (P/N 188-2946, 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/ NH4OH (78/20/2) Collect the eluate

Codeine

Morphine

Codeine 30 ng

Morphine 30 ng

Clean extracts from a very dirty matrix

Column: Carrier: Oven: Injector: Detector:

DB-5MS EVDX Helium at 40 cm/sec 65 degrees for 1 min, 65-325 degrees at 20 degrees/min Splitless, 250C MSD, 300C transfer line

CI0126C 36

DOA Recoveries are Reproducible Using EVIDEXII SPE Cartridges

Recovery Levels (x%, n = 8) Level in ng/mL Amphetamine Methamphetamine Level in ng/mL PCP Level in ng/mL Benzoylecgonine Level in ng/mL Codeine Morphine Level in ng/mL THC-COOH 1000 76 4 85 1 50 90 3 300 98 8 600 99 1 96 2 30 92 4 1750 74 2 71 3 87.5 91 2 525 93 4 1050 97 3 96 3 52.5 96 8 2500 96 3 98 3 125 88 3 750 97 3 1500 98 1 93 4 75 94 3

CI0126C 37

Summary
Solid Phase Extraction Probably the most important technique for sample cleanup and concentration today Increases productivity, column lifetime and instrument uptime AccuBONDII Available with both silica and PS-DVB base materials Many bonded phases for every sample type Low extractable levelsprewashed 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 EVIDEXII Accurate, reproducible, robust methods for drugs of abuse and other pharmaceutical applications

CI0126C 38

Appendix

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

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

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 0.5 - 1 min. (recovery). Sometimes several smaller eluent aliquots can improve recovery.

CI0126C 42

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 AccuBONDII family provides the necessary variety of SPE cartridges for any matrix

CI0126C 43

Extraction and Separation of PAHs from Water II ODS AccuBOND Solid Phase Extraction Method
CARTRIDGE: AccuBONDII , 500 mg, 6 mL (P/N 188-1356, 30/box)

Sample Preparation:
Add 2 mL Isopropanol to 20 mL Water Sample Mix thoroughly

Cl

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 1000-1500 rpm for 5 min. (removes excess water)

Elute:
3 mL Methylene Chloride and collect eluent. Evaporate to 50-200 L. Do not apply heat or smaller PAHs will be volatilized. Add Methylene Chloride to bring sample to final volume of 200 L. Inject 2 L.
CI0126C 44

Water Spiked at 50 ppb Std. Avg. Compound Recovery Dev. (%) (%) Naphthalene 80 12 2-Chloronaphthalene 78 8 Acenaphthylene 81 9 Fluorene 99 9 Phenanthrene 98 9 Fluoranthene 91 8 Chrysene 97 12 Benzo(b)-fluoranthene 60 8 Benzo(a)pyrene 55 8 Indene(1,2,3-c,d)pyrene 60 10 Benzo(g,h,i)perylene 59 10

Typical SPE Extraction Protocol for Opiates

Sample Preparation: Add 0.5 mL hydrochloric acid to 5 mL urine Cap Heat at 120C for 20 min Cool to room temperature Add 0.75 mL 10 N NaOH Adjust to pH 6.5-7.5 with ~2.5 mL 0.5 M phosphoric acid Condition: 6 mL methanol 6 mL 0.1 M K2HPO4 (pH 6.0) Do not let the phase go dry Load: Add 3 mL 0.1 M K2HPO4 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/ NH4OH (78/20/2) Collect the eluant
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 K2HPO4 (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/NH4OH (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 Analysis Preparation: Concentrate the eluant to dryness Do not over dry 75 L ethyl acetate 25 L BSTFA/1% TMCS Cap Heat at 60C for 10 min Inject 1-2 L

CI0126C 45

Certificate of Performance for AccuBONDII

Test Condition and Results

Conditions: Column: Mobile Phase: Flow Rate: Specifications: QA K biphenyl K toluene K acetophenone Tf uracil 4.6 x 250 mm 70/30 MeOH/H2O 2.0 ml/min

Description: Catalog No: Lot No.: Run No.:

AccuBOND C8 100mg 1mL cartridge 188-0310 AMC18-0X 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 2 Surface Area: 546 m /g Average Pore Size: 60 Surface pH: 7 Metal Analysis: Pass Bonded Silica Tests Carbon Loading: 13.0 % 2 Surface Coverage: 2.5 moles/m Extraction Residue: Pass Exchange Capacity: N/A

= = = =

3.5 to 6.5 1.5 to 3.5 0.6 to 1.4 0.3 to 1.5

Results for Lot AMC8X-XX K toluene = K dimethylaniline = K nitrobenzene = Tf dimethylaniline =

QC 4.5 2.1 1.0 1.4

Peak 1 Peak 2 Peak 3 Peak 4

= = = =

Uracil Acetophenone Toluene Biphenyl

Particle Size Data Particle Shape: Irregular Average Particle Size (m): 56 Percentage of Particles <10m: 0.00 Percentage of Particles <25m: 1.36 Percentage of Particles >90m: 4.95
CI0126C 46

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: http://www.chem.agilent.com/scripts/cag_msdssearch.asp This product has passed all Agilent Technologies, inc. quality control specifications.

HPLC Column Technical Support

800-227-9770 (phone: US & Canada)* 302-993-5304 (phone)* * Select option 4, then option 2. 916-608-1964 (fax) www.agilent.com/chem

CI0126C 47