Download Document 7858626

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 aof96-Well
SPE
Schematic
Diagram
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 Modes—”Digital Chromatography”
Analyte Adsorption
• Analyte(s) retained
(k >> 1)
• Matrix unretained
(k ~ 0)
and/or strongly retained (k >> 1)
Matrix Adsorption
• Analyte(s) unretained (k ~ 0)
• Matrix retained
(k >> 1)
• Preconcentration factor
• Cleaner extracts
• Load at 1-3 drops/sec (recovery ∝
1/flow)
• Capacity issues may be more
important
•
•
•
•
CI0126C 7
No preconcentration advantage
Eluates may not be as clean
Sample loading often gravity fed
Used less often than analyte
adsorption
Fundamental Steps for SPE Adsorption Mode
Prewash*
Remove contaminants that
could elute with analyte
Precondition
Load
Wash
Elute
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
Weakly retained
matrix compds elute
Analyte and other
matrix compds
retained
Elute analyte
leaving highly
retained compds
1
2
aryE
s
es t SP
c
neilen
t
o
*N r Ag
fo
If elution solvent
will be stronger
than precond. solvent
CI0126C 8
1. MeOH or ACN
2. Weak solvent
(water, buffer)
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)
Phases Available
C2, C8, C18, Phenyl,
ENV PS-DVB*
Silica, -CN, Diol, Amino,
Alumina (acidic, basic, and
neutral), Florisil
Strong Anion Exchange
(SAX)
Quaternary amine
Strong Cation Exchange
(SCX)
Sulfonic acid
* Newest phase
CI0126C 15
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
Programmed Temperature
GC-FID Analysis
Splitless Injection
Waters’ Sep-Pak C18
Tube/Frit Only
Total = 50 ppm
0
4
8
Internal Standard
12
16
min
Agilent SPE AccuBONDII
Pre-washed Tube/Frit Only
Internal Standard
Total = 5 ppm
0
CI0126C 17
4
8
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 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:
www.agilent.com/chem
CI0126C 19
AccuBONDII Example Applications
Triazines in Different Matrices Cl
N
•
•
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 interferences—similar
functional groups, pKa, etc.
•
Qualitative and quantitative
variability
CI0126C 20
Triazines
N
NH
R1
•
•
•
•
•
•
N
NH
R2
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
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
WASH
ELUTE
CI0126C 21
Diluted with acetic
acid
Acetic acid,
acetonitrile, water,
0.1 M K2HPO4
Acetonitrile/K2HPO4
Diluted with water Diluted with hexane
Water
Hexane
Methanol
Methanol
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
•
•
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 interferences—similar
functional groups, pKa, etc.
•
Qualitative and quantitative
variability
CI0126C 23
Barbiturates
R1
N
O
R2
HN
R3
O
•
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
Analysis of Barbiturates in Serum
AccuBONDII SPE C18 Cartridge
Solid Phase Extraction Method
Load:
CARTRIDGE:
5 mL Sample (see sample preparation
above)
AccuBONDII
ODS (C18) 6 mL/500 mg
(P/N 188-1356 30/box)
Wash
Sample Preparation:
2.0 mL 95% water:5% acetonitrile
Add 1 mL 0.5M K2HPO4 to 4 mL of serum
Elute:
Precondition:
5 mL Acetone
5 mL Deionized Water
CI0126C 24
3.0 mL Acetone
Evaporate and Reconstitute:
Evaporate Acetone in a nitrogen stream at
<45°C
Add 200 µL of acetonitrile:water (20/80)
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
Serum spiked at 1mg/mL
n= 4
RRT = retention time relative to barbital
CI0126C 27
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
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
Load:
CARTRIDGE:
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
29 mL Methanol
9CI0126C
– 12
Evaporate and Reconstitute:
Evaporate Dichloromethane in nitrogen stream
at room temperature and transferred to silanized
amber vial
Bring to 900 µL of Dichloromethane + 100 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: 5988–5255EN
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:
546 m2/g
60Å
7
Pass
Particle Size Data
Particle Shape:
Irregular
Average Particle Size (µm):
56
Percentage of Particles <10 µm: 0.00
Percentage of Particles <25 µm: 1.36
Percentage of Particles <90 µm: 4.95
CI0126C 32
Bonded Silica Tests
Carbon Loading:
Surface Coverage:
Extraction Residue:
Exchange Capacity:
13.0%
2.5 µmoles/m2
Pass
N/A
Packed Cartridge Test
Cartridge Flow Resistance:
Frit Purity Test (GC):
Material Weight Check:
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
CH3
CH3
Amphetamine
• Designed for NIDA-5 Drug Classes
N
CH3
— Amphetamine/Methamphetamine
— Codeine and Morphine
N
CH3
Methamphetamine
PCP
O
OH
— PCP (“Angeldust”)
— Benzoylecgonine (Cocaine
metabolite)
NH
NH2
HO
O
O
O
CH3
HO
H
H
O
Benzoylecgonine
N
N
CH3
CH3
Codeine
O
Morphine
OH
— THC-COOH (Marijuana metabolite)
OH
H
• Tested with actual drugs of abuse
H
H3C
O
H3C
— 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
O
THC-COOH
CH3
OH
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)
35
•CI0126C
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
CI0126C 36
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, 250°C
MSD, 300°C transfer line
DOA Recoveries are Reproducible
Using EVIDEXII SPE Cartridges
Recovery Levels (x±%, n = 8)
CI0126C 37
Level in ng/mL
Amphetamine
Methamphetamine
1000
76 ±4
85 ±1
1750
74 ±2
71 ±3
2500
96 ±3
98 ±3
Level in ng/mL
PCP
50
90 ±3
87.5
91 ±2
125
88 ±3
Level in ng/mL
Benzoylecgonine
300
98 ±8
525
93 ±4
750
97 ±3
Level in ng/mL
Codeine
Morphine
600
99 ±1
96 ±2
1050
97 ±3
96 ±3
1500
98 ±1
93 ±4
Level in ng/mL
THC-COOH
30
92 ±4
52.5
96 ±8
75
94 ±3
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 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
• 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:
Cl
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 1000-1500 rpm for 5 min. (removes excess
water)
Elute:
3 mL Methylene Chloride and collect eluent.
Evaporate to 50-200 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
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
Analysis Preparation:
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 6.5-7.5 with ~2.5 mL 0.5 M phosphoric acid
•
•
•
•
•
•
•
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
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
CI0126C 45
Certificate of Performance for AccuBONDII
Test Condition and Results
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.
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
=
=
=
=
3.5 to 6.5
1.5 to 3.5
0.6 to 1.4
0.3 to 1.5
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 Å
Surface pH:
7
Metal Analysis:
Pass
Bonded Silica Tests
Carbon Loading: 13.0 %
2
Surface Coverage: 2.5 µmoles/m
Extraction Residue: Pass
Exchange Capacity: N/A
Particle Size Data
Packed Cartridge Test
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
Cartridge Flow Resistance: Pass
Cartridge Purity Test (GC): Pass
Frit Purity Test (GC): Pass
Material Weight Check: Pass
CI0126C 46
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
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