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Schematic Diagram of a 96-Well SPE Schematic Diagram of a Extraction Plate System* 96-Well Plate Extraction System
(LC-GC, S9, May, 1998)
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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
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Break Time
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Phases Available
C2, C8, C18, Phenyl, ENV PS-DVB* Silica, -CN, Diol, Amino, Alumina (acidic, basic, and neutral), Florisil Quaternary amine Sulfonic acid
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12
16
min
0
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12
16
min
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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
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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
WASH ELUTE
Water Methanol
Hexane Methanol
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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
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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
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Load:
5 mL Sample (see sample preparation above)
Precondition:
5 mL Acetone 5 mL Deionized Water
Elute:
3.0 mL Acetone
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Break Time
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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
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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:
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
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= = = =
3 .5 1 .5 0 .6 0 .3
to to to to
6 .5 3 .5 1 .4 1 .5
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
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
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THC-COOH
Codeine
Morphine
Codeine 30 ng
Morphine 30 ng
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
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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
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Appendix
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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.
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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.
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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
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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
= = = =
= = = =
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
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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.
800-227-9770 (phone: US & Canada)* 302-993-5304 (phone)* * Select option 4, then option 2. 916-608-1964 (fax) www.agilent.com/chem
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