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©2012 Waters Corporation 1
Sample Preparation Strategies for
Complex Environmental Analysis
SPE for Environmental Samples
©2012 Waters Corporation 2
Outline
Introduction – Why is Sample Prep Done – Why SPE
Strategies
– Traditional approaches o Normal Phase o Reverse Phase o Mixed Modes
– Modern approaches o Mixed Modes o Reverse phase
Summary
©2012 Waters Corporation 3
Sample Preparation Techniques
Sample Preparation- The simplification of sample
matrix and enrichment of target analyte(s)
Types of Sample Prep include:
– Dilution (simple, fast, no cleanup, no enrichment)
– Centrifugation (cleanup, no enrichment)
– Filtration (simple, fast, cleanup, no enrichment)
– Liquid/Liquid Extraction (cleanup, enrichment, cumbersome)
– Solid Phase Extraction (cleanup, fast, enrichment)
©2012 Waters Corporation 4
For high sensitivity analyses, such as those employing LC/MS/MS, proper sample preparation can be critical for minimizing matrix effects and concentrating analytes of interest.
Why is Sample Prep Done?
60% of the work activity and operating cost is spent on sample preparation
for introduction into the analytical system
Three Purposes:
– Removes interferences from sample matrix
– Concentrating analytes of interest
– Present sample matrix not compatible with analytical system
©2012 Waters Corporation 5
Why Use SPE in Environmental Analysis ?
Sample Enrichment
– To achieve low ppt detection limits, usually the sample must be
concentrated 10-1000 times
o using liquid extraction (LE), up to 500 mL of solvent must be
evaporated after cumbersome manual extraction
o using SPE, only 10 mL of solvent must be evaporated after more
user friendly procedure
Sample Cleanup
– Cleanup steps can often be incorporated into the SPE experiment –
this cannot be done using LE
– SPE cartridges can be used to further cleanup extracts obtained from
either SPE or LE
©2012 Waters Corporation 6
SPE Sorbents for Environmental Analysis
Normal-Phase Sorbents
– Silica, Alumina, Florisil®, Aminopropyl silica, PSA, Diol silica,
Reversed-Phase Sorbents
– Oasis® HLB
– C18, C8 etc (alkyl silica's)
– Graphitized carbon and activated carbon
Ion Exchange
– Accell Plus™ CM, QMA
Mixed Mode (ion-exchange/reversed phase)
– Oasis® MAX, Oasis WAX (strong and weak anion-exchange)
– Oasis® MCX, Oasis WCX (strong and weak cation-exchange)
©2012 Waters Corporation 7
Specialty Products for Environmental & Food Analysis
• Sep-Pak® DNPH (air samples, aldehydes and keytones)
• XPoSure Aldehyde Sampler (air samples, large particle size for personal air
monitors)
• Ozone Scrubber (removes ozone interference)
• Sep-Pak® Dry (high capacity desiccant, removes water from normal phase extracts)
• PoraPak RDX (explosives, EPA Method 8330)
• Sep-Pak® PS2 (multi-residue pesticide analysis in water)
• Sep-Pak® AC2 (removes/enriches polar organic molecules in water)
• Carbon Black / Aminopropyl (pesticide clean-up in food)
• Carbon Black / PSA (two layer sorbent bed, pesticide cleanup in food)
• DisQuE™ – dispersive SPE – QuEChERS – multiresidue pesticide analysis
SPE Sorbents for Environmental Analysis
©2012 Waters Corporation 8
Waters SPE History
1978
Sep-Pak
(silica based SPE introduced)
1980s
Sep-Pak
(additional silica
bonded phases)
1994
Oasis HLB
(co-polymeric
resin)
1999
Oasis MCX, MAX
(mixed-mode, strong
ion exchange)
2002
Oasis WCX, WAX
(mixed-mode, weak
ion exchange)
2005
Sep-Pak
(GCB/Amino)
2008
PSA
(Primary-secondary
amine silica)
2008
DisQuE
(Dispersive SPE –
QuEChERS)
©2012 Waters Corporation 9
Outline
Introduction – Why sample Prep – Considerations
o Choices of tools – Why SPE
SPE Strategies – Traditional approaches
o Normal Phase o Reverse Phase o Mixed Modes
– Modern approaches o Mixed Modes o Reverse phase
Summary
©2012 Waters Corporation 10
SPE Strategies
1. Strategy #1
Pass-through cleanup
2. Strategy #2
Retention, cleanup, elution
3. Strategy #3
Dispersion Cleanup (DisQuE™ for QuEChERS)
©2012 Waters Corporation 11
SPE Strategy #1 Pass-Through Cleanup
1. Sample is passed through sorbent and collected
• no sample enrichment
2. Matrix interferences are retained on sorbent
pass through
Blue = Compound of interest
Purple = Sample
Sample is not concentrated
©2012 Waters Corporation 12
1. load 2. wash 3. elute
1. Sample is loaded onto SPE sorbent • Analyte(s) of interest
are retained on sorbent
2. Matrix interferences are washed off sorbent
3. Analytes are eluted from sorbent
SPE Strategy #2 Retention-Cleanup-Elution
Blue = Compound of interest
Purple = Sample
Sample Concentrated
©2012 Waters Corporation 13
SPE Strategy #3 Dispersive SPE
Bulk sorbent is added to sample with agitation
Sample is filtered or centrifuged
Supernatant is collected for analysis
Quicker
Easier
Cheaper
Effective enough in many cases
Accepted for Agro analysis of pesticides – applications for Environmental analysis include soil and water screening
©2012 Waters Corporation 14
Outline
Introduction – Why sample Prep – Considerations
o Choices of tools – Why SPE
SPE Strategies
– Traditional approaches o Normal Phase o Reverse Phase
– Modern approaches o Mixed Modes
Summary
©2012 Waters Corporation 16
Isolation of Aromatic Hydrocarbons Contaminated Soil
Prepare Sample
Condition/Equilibrate 4 mL DCM
10 mL hexane
Load Sample 0.25 mL of pre-extract in hexane
Elute 1 3 mL hexane
Elute 2 2 mL DCM
Evaporate to Final Volume
Sep-Pak® Silica
SPE Protocol
Pre-extraction Soils are mixed with sodium sulfate and
extracted with DCM or DCM/acetone.
The extract is exchanged to hexane (weak
solvent). The extract must be water-free.
Aliphatic hydrocarbons are removed.
This fraction may be collected and
analyzed if desired.
Aromatic hydrocarbons are eluted with
strong solvent.
Conditions for 6 cc 500 mg cartridges
©2012 Waters Corporation 17
Hydrocarbons Recovered from Diesel Contaminated Soil (spiked)
Diesel
Diesel – Elute 1
Aliphatic (hexane) fraction
Diesel – Elute 2
Aromatic (DCM) fraction
©2012 Waters Corporation 18
Aromatic Hydrocarbons Recovered from Diesel Contaminated Soil (GC-FID)
Elute 2:
Sep-Pak® Silica SPE
(aliphatic hydrocarbon
interference eliminated)
Minutes 0 5 10 15 20 25 30 35
naphthalene
2-methylnaphthalene
1-methylnaphthalene
phenanthrene
Diesel:
No SPE
FID
Gas Chromatography Column: RTX-5, 30 m x 0.25 mm (ID) 0.25 µm film 1 mL/min Helium carrier gas 35o C for 1 min, then 8oC/min to 320
©2012 Waters Corporation 19
Normal Phase Pass-Thru Cleanup (additional applications)
Chlorinated Pesticides in Soil or Water
– Liquid extraction
– exchange to hexane
– pass-thru cleanup on Florisil or Alumina
GC-MS Pesticides in fruit and vegetables
– extract with acetonitrile/toluene
– pass-thru cleanup on aminopropyl silica
o often performed with Graphitized carbon
©2012 Waters Corporation 20
Outline
Introduction – Why sample Prep – Considerations
o Choices of tools – Why SPE
SPE Strategies
– Traditional approaches o Normal Phase o Reverse Phase
– Modern approaches o Mixed Modes
Summary
©2012 Waters Corporation 21
Reversed Phase Retention SPE Typical Protocol
Condition cartridge – strongest solvent first (DCM, MTBE,
ethyl acetate)
– intermediate solvent next (methanol)
– weak solvent last (water)
Load Sample – dissolve in or exchange to weak
solvent (water or water/methanol)
Wash Cartridge – use strongest possible solvent without
eluting analyte (methanol/water)
Elute Cartridge with strong solvent* – methanol, IPA,MTBE, DCM
*Oasis HLB – elute solvent should have at
least 5 % methanol or IPA as polar modifier
Prepare Sample
Condition/Equilibrate 1 mL methanol, 1 mL water
Load Sample
Wash 1 mL 5% methanol/water
Elute 2 mL methanol
Evaporate, Reconstitute
Oasis® HLB
SPE Protocol
Conditions for 3 cc 60 mg cartridges
©2012 Waters Corporation 22
Reversed-Phase SPE
Sorbent surface is relatively hydrophobic – Sep-Pak® C18 Silica, other alkyl silica, Oasis® HLB
Sample is prepared for SPE in aqueous or other polar sorbent
Oasis® HLB is better – pH stable from 1-14
– Doesn’t de-wet
Examples:
Isolation and enrichment of organic contaminants from surface
waters
• Simple method for aromatics in groundwater
• Advanced method for emerging contaminants
o Perfluorinated compounds
o Endocrine disruptors
o Pharmaceuticals
©2012 Waters Corporation 23
Advantages of Reversed-Phase SPE
Many pesticides, pharmaceuticals, surfactants, industrial
chemicals and other important compounds of environmental
concern can be retained using reversed-phase chromatography
principles applied to SPE
pH and solvent strength can be manipulated for the load, elute,
and particularly the wash step, to optimize the SPE experiment
©2012 Waters Corporation 24
Oasis HLB: Importance of Wettability
Procainamide
Acetaminophen (polar)
Ranitidine
Propranolol
Doxepin
% S
PE
Recovery
Bouvier, Caparella
Silica C18: pore dewetting results in breakthrough at load step = poor SPE recoveries
Oasis HLB: No breakthrough = good SPE recoveries
C18 (1cc/100mg) HLB (1cc/30mg)
0
20
40
60
80
100
0 4 8 Drying Time (minutes)
0
20
40
60
80
100
0 5 10 Drying Time (minutes)
Acetaminophen
(Polar)
©2012 Waters Corporation 25
Reversed-Phase Retention Map: The Impact of pH on Method Development
pH Range for Silica Particles
pH
0
5
10
15
20
25
30
35
40
0 2 4 6 8 10 12
Ret
enti
on
Fac
tor
(k)
Acid
Base
Neutral
Note: Retention of neutral analytes not affected by pH
Maximum acidic compound retention range
Maximum basic compound retention range
pH Range for Hybrid Particles
©2012 Waters Corporation 26
Simple Reversed-Phase SPE Groundwater Aromatic Hydrocarbons
Prepare Sample pH 2
Condition/Equilibrate 1 mL methanol, 1 mL water
Load Sample 250 mL Groundwater
Wash 1 mL 5% methanol/water
Elute 2 mL 10:90 methanol/DCM
Evaporate to Final Volume Micro K-D
Oasis® HLB
SPE Protocol
Conditions for 3 cc 60 mg cartridges
1 2
3
4
5
6
7 8
9 10 11
1. toluene
2. o-xylene
3. m,p-xylene
4. styrene
5. indan
6. naphthalene (2100 µg/L)
7. 2-methylnaphthalene
8. 1-methylnaphthalene
9. acenaphthylene
10. acenaphthene
11. fluoranthene (43 µg/L)
©2012 Waters Corporation 27
Optimized Reversed-Phase SPE Phenols, Estrogenic
Prepare Sample pH 2
Condition/Equilibrate 3 mL MTBE, 3 mL methanol, 3 mL water
Load Sample 500 mL Groundwater
Wash 1 3 mL 40% methanol in water
Re-Equilibrate 3 mL water
Wash 2 3 mL 10% methanol/2% NH4OH in water
Elute 6 mL 10:90 methanol/MTBE
Evaporate to Final Volume
Oasis® HLB Optimized Protocol
Conditions for 5 cc 200 mg glass cartridges
40 % methanol acidic wash
removes organic
interferences
pH 11 wash
removes humic
interference
©2012 Waters Corporation 28
5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00
minutes
10
100
%
39
100
%
38
100
%
44
100
%
36
100
% m/z = 267
diethylstilbestrol
10.70
m/z = 269
estrone
9.85
m/z = 295
ethynylestradiol
9.63
m/z = 271
estradiol
8.53
m/z = 227
bisphenolA
7.80
electrospray(neg) cone 27 V
5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00
minutes
10
100
%
39
100
%
38
100
%
44
100
%
36
100
% m/z = 267
diethylstilbestrol
10.70
m/z = 269
estrone
9.85
m/z = 295
ethynylestradiol
9.63
m/z = 271
estradiol
8.53
m/z = 227
bisphenolA
7.80
electrospray(neg) cone 27 V
75 % 5 %
87 % 5 %
96 % 12%
93 % 15 %
113 % 11%
XTerra™ MS C18
2.1 x 100 mm (3µm)
Gradient:
A: pH 10.5 NH4OH in water B: acetonitrile
30 % B initial, linear to 65 % B in 8 min,
then to 90 % B in 9 min
0.200 mL/min
Oasis® HLB 5 ng/L (ppt) Estrogenics
(n = 5)
©2012 Waters Corporation 29
Sep-Pak® AC2 and 1,4-dioxane
AC2 is a high quality activated carbon particle Retention is primarily reversed phase Suitable for retention of polar organics in aqueous
samples – acephate – N-nitroso-N,N-dimethylamine(NDMA) – 1,4-dioxane
1,4-dioxane Volatile, water soluble solvent
– useful in organic and polymer chemistry – soluble in polar and non-polar solvents
Persistent organic pollutant – highly water soluble – resistant to biodegradation/natural attenuation
Not amenable to ‘purge and trap’ analysis
O
O
©2012 Waters Corporation 30
Sep-Pak® AC2 SPE Method for 1,4-Dioxane
SPE Protocol
– condition Sep-Pak® AC2 cartridge with 6 mL methylene chloride
(DCM), 2 mL methanol, 2 mL water
– load water sample onto AC2 cartridge at ~ 5 mL /min
– wash with 2 mL water
– air dry 20 seconds
– attach AC2 cartridge atop preconditioned Sep-Pak® Dry cartridge
(sodium sulfate drying cartridge)
– elute with 2.0 mL DCM
For typical groundwater, the maximum sample loading volume was about 60 mL
before breakthrough. For this application, we used 40 mL spiked samples of well
water. Each sample was also spiked with 300 ng/mL d8-dioxane internal standard.
©2012 Waters Corporation 31
1,4-Dioxane 2 µg/L Sample
0
100
200
300
400
500
600
700
800
900
1000
0 200 400 600 800 1000 1200
0
100
200
300
400
500
600
700
800
900
1000
0 200 400 600 800 1000 1200
response
(rela
tive to IS
TD
)
1,4 dioxane µg/L
r2 = 0.99993
Column: ZB5, 60 m x 0.25 mm ID x 0.25 µm film
Instrument: Agilent 5890 Series II GC/5972 MSD
injection temp: 250
1µL injection volume
Oven ramp: Init temp: 35
hold: 2.00 min
ramp 1: 8 /min to 100
ramp2: 30 /min to 325
Pressure program: 30.0 PSI pulse for 1.00 min
then 1.0 mL/min constant flow for rest of run
MS operated in SIM mode
Ions monitored: 58, 88 (1,4-dioxane) and 64, 96 (d8-dioxane)
LOQ level sample
2 µg/L
©2012 Waters Corporation 32
Outline
Introduction – Why sample Prep – Considerations
o Choices of tools – Why SPE
SPE Strategies
– Traditional approaches o Normal Phase o Reverse Phase
– Modern approaches o Mixed Modes
Summary
©2012 Waters Corporation 33
Ion-Exchange and Mixed-Mode
Many compounds of environmental interest are weak acids
(i.e. dinoseb) or weak bases (i. e. aniline).
– weak acids can be ionized at high pH
– weak bases can be ionized at low pH
Some compounds are strong acids (i.e. PFOA) or strong
bases (i.e. chlorhexidine) that are ionic except at extreme
pH values
A few of these compounds are quaternary amines (i.e.
chlormequat), ionic at all pH
Ionizable Compounds
©2012 Waters Corporation 34
Why Mixed-Mode?
Mixed-Mode SPE extends pH range for good retention of acids
or bases
Retention can be by reversed-phase, ion-exchange or both
– Chose retention mode by adjusting pH
– ion-exchange allows for good retention in strong solvent
o acids can be retained by anion-exchange while bases/neutrals
are washed off with strong solvent
o bases can be retained on cation-exchange while acids/neutrals
are washed off with strong solvent
For environmental analysis, mixed-mode SPE allows
simultaneous retention of acids and bases
©2012 Waters Corporation 35
Oasis® Family of Sorbents: Reversed-Phase and Ion Exchange Retention
©2012 Waters Corporation 36
Oasis® 2x4 Method: The Process
Strategies for Isolation and Enrichment of Individual
Compounds or Compound Classes:
Oasis® 2x4SM Method
Characterize your analyte
Select the appropriate Oasis® Sorbent
Apply the starting protocol (1 of 2)
Analyze chromatograms and SPE recoveries
Optimize protocol as necessary
©2012 Waters Corporation 37
Oasis® 2x4 Method Select Sorbents
For Strong Acids
pKa <1.0
Select
Oasis® WAX
For Strong Bases*
pKa >10
Select
Oasis® WCX
For Acids
pKa 2-8
Select
Oasis® MAX
For Bases
pKa 2-10
Select
Oasis® MCX
Characterize your Analyte and Select the Sorbent
Neutrals
are Retained on
all Sorbents
*also quats
©2012 Waters Corporation 38
Oasis® 2x4 Method: Starting Protocols For Acids and Bases
Neutrals
For Bases:
pKa 2-10
Use Oasis® MCX
For Strong Acids
pKa <1.0
Use Oasis® WAX
For Strong Bases
pKa >10
Use Oasis® WCX
For Acids
pKa 2-8
Use Oasis® MAX
Prepare Sample
Condition/Equilibrate
Load Sample
Wash:
5% NH4OH
Elute 1:
100% MeOH
Elute 2:
2% Formic Acid in MeOH
Protocol 2 Prepare Sample
Condition/Equilibrate
Load Sample
Wash:
2% Formic acid
Elute 1:
100% MeOH
Elute 2:
5% NH4OH in MeOH
Protocol 1
Bases Strong
Acids
Strong
Bases Acids
©2012 Waters Corporation 39
Reversed phase vs. mixed-mode SPE
Prepare Sample
Condition/Equilibrate methanol, water
Load Sample
Wash 5% methanol/water
Elute methanol
Evaporate, Reconstitute
Oasis® HLB
SPE Protocol
Prepare Sample
Condition/Equilibrate
Load Sample
Wash:
2% Formic acid
Wash 2:
100% MeOH
Elute:
5% NH4OH in MeOH
Oasis® MCX
SPE Protocol
©2012 Waters Corporation 40
Aggressive washing with mixed-mode SPE
Prepare Sample
Condition/Equilibrate
Load Sample
Wash 1:Water
Wash 2: 0.1 M HCl
Wash 3: 100% MeOH
Wash 4: DCM
Wash 5: 100% MeOH
Wash 6: NH4OH/MeOH/Water
(4/30/66)
Elute:
5% NH4OH in MeOH
Oasis® MCX
SPE Protocol
Example: Selective Serotonin Re-uptake Inhibitors (SSRI) in Fish Tissue
reference: Dr. Metcalfe, Trent University, J Chrom. A 1163 (2007) 112-118
Fluoxetine (Prozac)
Paroxetine (Paxil)
©2012 Waters Corporation 41
F3C
O
OH
F
F
F
F
F
F
F
F
F
FF
F
perfluorooctanoic acid
PFOA
pKa ~ 1
perfluorooctanesulfonate
PFOS
pKa<<1
F
F
F
F
F
F
F
F
F
F F
F
S
O
O
O- F 3 C
F
F
Step 1 – characterize analytes; they are strong acids For Strong Acids
pKa <1.0
Select
Oasis® WAX
PFOA and PFOS are Persistent Organic Pollutants of interest worldwide.
Oasis® 2x4 Method Step 1: Characterize Analytes
©2012 Waters Corporation 42
Oasis® 2x4 Method Step 2: Choose Starting Protocol
The Oasis® WAX
cartridge was chosen for
these analytes
Logic: PFOA pKa ~1
PFOS pKa < 1
For Strong Acids
pKa <1.0
Use Oasis® WAX
Prepare Sample
Condition/Equilibrate
Load Sample
Wash:
2% Formic acid
Elute 1:
100% MeOH
Elute 2:
5% NH4OH in MeOH
Oasis® WAX
Protocol 1
PFOS
PFOA
Oasis® WAX
N O
N
N
N
N
H
H
H +
+
H
pKa ~6
©2012 Waters Corporation 43
UPLCTM Analysis Perfluorinated Acids, 2 µg/L (ppb) in Well Water
Conditions for 3 cc cartridges
Prepare Sample pH 3
Condition 2 mL methanol/2 mL water
Load 25 mL sample
Wash #1 1 mL 2% Formic acid
Elute 1 (Wash #2) 2 mL methanol
Elute 2 2 mL 5% ammonia in MeOH
Oasis® WAX
Protocol 1
MRM of 7 Channels ES-
TIC 1.68e5
0.20 0.60 1.00 1.40 1.80 2.20 2.60 3.00 3.40 3.80
%
0
100
C4
C3
C5
C7 C6
C8
C9
blank
C3 (perfluoropropanoic) 85%
C4 (perfluorobutyric) 92%
C5 (perfluoropentanoic) 70%
C6 (perfluorohexanoic) 110%
C7 (perfluoroheptanoic) 95%
C8 (perfluorooctanoic) 120%
C9 (perfluorononanoic) 80%
ACQUITY UPLC™ BEH C18 2.1 x 50 mm (1.7µm )
Gradient:
A: 2 mM ammonium acetate/water B: acetonitrile
15 % B initial, linear to 100 % B in 8 min,
0.500 mL/min
Waters Quattro Premier™ XE, ESI-, MRM mode
Transitions:
C3 162.7>118.7 C7 362.8>318.8
C4 212.7>168.7 C8 412.8>368.8
C5 262.8>218.7 C9 462.8>418.8
C6 312.8>268.8
©2012 Waters Corporation 44
B#3
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
%
0
100
PFOS_030806 AQC21x50C18_11 Sm (Mn, 1x1); Sm (Mn, 1x1) MRM of 2 Channels ES- TIC
8.16e3
1.62
3.14
3.01
PFOS_030806 AQC21x50C18_18 Sm (Mn, 1x1) MRM of 2 Channels ES- TIC
8.16e3
Conditions for 3 cc cartridges
Prepare Sample pH 3
Condition 2 mL methanol/2 mL water
Load 25 mL sample
Wash #1 1 mL 2% Formic acid
Elute 1 (Wash #2) 2 mL methanol
Elute 2 2 mL 1% ammonia in MeOH
Oasis® WAX Optimized Protocol 1
ACQUITY UPLC™ BEH C18 2.1 x 50 mm (1.7µm dp)
Gradient:
A: 2 mM ammonium acetate/water B: acetonitrile
15 % B initial, linear to 100 % B in 8 min,
0.500 mL/min
Waters Quattro Premier™ XE, ESI-, MRM mode
Transitions:
PFOS
PFBS
UPLCTM Analysis PFOS/PFBS (perfluorobutanesulfonate)
blank
5 µg/L
PFOS
PFBS
©2012 Waters Corporation 45
Oasis® Mixed-Mode Sorbents
Mixed-Mode strong ion-exchange sorbents (Oasis MCX and Oasis
MAX) can simultaneously retain polar acids and bases better than
the best reversed-phase sorbents such as Oasis HLB
– Oasis® MCX, sample adjusted to low pH
o acids/neutrals retained by reversed-phase
o bases retained by mixed-mode cation-exchange
– Oasis® MAX, sample adjusted to high pH
o acids retained by mixed-mode anion-exchange
o bases/neutrals retained by reversed-phase
Strategies for Multi residue Isolation and Enrichment
©2012 Waters Corporation 46
Retention Factor (k’) as function of pH
Oasis® MCX
pH
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
k’
Acids (HA)
Neutral
Bases (BH+)
A-
B
indicates pH range for best retention of both acids and bases
pH
©2012 Waters Corporation 47
Multi residue Analysis Oasis® MCX Method for GC
prepare reagent using
anhydrous ammonia in
methanol
Prepare Sample pH 2
Condition 2 mL DCM, 2 mL methanol, 2 mL water
Load 250 mL sample
Wash 2 mL 5 % MeOH/water
Elute 4 mL of 0.7 M NH4OH in 90:10 DCM/MeOH
Oasis® MCX
Optimized Protocol
Dry over Sodium Sulfate
Evaporate to Final Volume
©2012 Waters Corporation 48
Minutes0 10 20 30 40 50
1
2
3
9
10
11
13
16
17
19 20
2223
2425
26
27
28
29
30
3132
33
4
5
6
7,8
12
14
15
1821
FID
NPD
bases, acids, neutrals
SPE for Base/Neutrals and Acids Oasis® MCX GC Protocol
COMPOUND % RECOVERY RSD % RECOVERY (20 µg/L Tap Water) (20 µg/L River Water)
1. pyridine 61 (17) 40
2. picoline 77 (16) 47
3. aniline 90 (11) 58
4. phenol 65 (14) 61
5. benzyl alcohol 75 (25) 56
6. o-cresol 91 (8.6) 65
7,8. m,p-cresol 91 (8.9) 65
9. o-toluidine 82 (12) 45
10. phentermine 73 (18) 24
11. chloroaniline 82 (11) 49
12. dichlorophenol 57 (6.2) 40
13. phenylenediamine 93 (15) nr
14. 2-methylnaphthalene 81 (8.0) 54
15. trichlorophenol 54 (10) 73
16. 2-nitroaniline 95 (7.2) 74
17. 3-nitroaniline 103 (8.5) 81
18. dibenzofuran 80 (5.4) 62
19. 1-aminonaphthalene 87 (5.1) 77
20. 2-aminonaphthalene 88 (8.5) 58
21. tetrachlorophenol 35 (17) 30
22. 2-methyl-5-nitroaniline 104 (6.2) 91
23. 4-nitroaniline 106 (8.7) 104
24. diphenylamine 93 (4.4) 77
25. phenacetin 85 (7.3) 92
26. aminobiphenyl 105 (4.2) 83
27. dinoseb 90 (7.1) 97
28. nitroquinoline oxide 100 (6.5) 87
29. methapyrilene 105 (5.5) 100
30. dimethylaminoazobenzene 100 (3.9) 98
31. dimethylbenzidine 64 (8.9) 20
32. acetamidofluorene 135 (5.4) 130
33. dichlorobenzidine 111 (6.0) 75
©2012 Waters Corporation 49
Aniline, phenol and benzyl alcohol on reversed-phase SPE
SPE of Acids and Base/Neutrals
N H 2 O H O H
At pH 2: Aniline is cation – not retained
Phenol is neutral – retained
Benzyl alcohol is neutral – retained
At pH 11 Aniline is neutral – retained
Phenol is ionized – not retained
Benzyl alcohol is neutral – retained
©2012 Waters Corporation 50
Aniline, phenol and benzyl alcohol on Mixed-Mode SPE
SPE of Acids and Base/Neutrals
N H 2 O H O H
At pH 2 on Oasis® MCX: Aniline is cation – retained
Phenol is neutral – retained
Benzyl alcohol is neutral – retained
At pH 11 on Oasis® MAX: Aniline is neutral – retained
Phenol is ionized – retained
Benzyl alcohol is neutral – retained
Acids, Based and Neutrals all retained
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Outline
Introduction – Why sample Prep – Considerations
o Choices of tools – Why SPE
SPE Strategies
– Traditional approaches o Normal Phase o Reverse Phase o Mixed Modes
– Modern approaches o Mixed Modes
Summary
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Summary
Sample Preparation is necessary for the best analytical results – cleanup and enrichment
SPE is considered a very versatile and cost efficient sample preparation technique for Environmental samples.
Waters provides strategies which combine sorbents, formats and methodologies resulting in optimal SPE protocols.
Whether for analysis by LCMS or GCMS; Waters analytical solutions, including SPE, cover a wide range of sample matrices and compounds classes for the analysis of Environmental samples.
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Questions?