1. business and industrial
2. chemicals industry
3. plastics and polymers

Sample Preparation Strategies
for Water Analysis
Hannah White
Waters Business Development Manager
©2007 Waters Corporation
Outline
ƒ
Introduction
ƒ
Strategies
ƒ
ƒ
Summary
Appendix
— Why sample Prep
— Considerations
o Choices of tools
— Why SPE
o Pre-Treatment
— Traditional approaches
— Modern approaches
o Mixed Mode
o Reverse phase
©2007 Waters Corporation
2
Why Sample Prep?
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
– Improving analytical system performances
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.
©2007 Waters Corporation
3
Sample Preparation Techniques
ƒ Sample Preparation- The simplification of sample matrix and
enrichment of target analyte(s)
ƒ Types of Sample Prep include:
— Dilution
— Centrifugation
— Filtration
— Liquid/Liquid Extraction
— Solid Phase Extraction
©2007 Waters Corporation
4
Some Considerations
ƒ
Solid samples
— usually start with organic or aqueous extract of tissue or soil
— initial extract is adjusted for optimal SPE enrichment and/or cleanup
o pH adjustment
o solvent adjustment
• acetone/acetonitrile/IPA – suitable for aqueous dilution, Reversed-Phase and
Mixed-Mode SPE
• ethyl acetate/DCM/MTBE – can be exchanged to hexane for normal-phase SPE
ƒ
Aqueous samples (water, beverage, plasma/urine)
— pretreatment may be appropriate
o pH adjustment
o filtration/centrifugation
o protein precipitation
— Usually, aqueous samples can be analyzed using Oasis® ReversedPhase or Mixed-Mode SPE
©2007 Waters Corporation
5
Pre-Treatment Prior to SPE
Pre treatment:
ƒ Solid samples (soil, tissue, etc.)
— shake, sonicate or soxhlet
o extract with polar organic solvent (methanol, acetonitrile); polars
o extract with organic solvent + drying agent (DCM, acetone); nonpolars, multi-residue
ƒ Non aqueous Liquid
o if water soluble, dilute with water for reversed-phase (or mixedmode) SPE
o if hexane soluble, dilute with or exchange to hexane for NP-SPE
ƒ Wastewater
— filter or centrifuge as necessary
o filtered solids and filter may require analysis as solids
©2007 Waters Corporation
6
Why Solid Phase Extraction
ƒ Isolation of the analyte(s) of interest from the matrix
ƒ Sample Cleanup
— removal of matrix interference
— Increased sensitivity
o Increased system uptime
o Longer column lifetime
ƒ Enrichment of analyte(s) of interest
o Increased sensitivity
ƒ Exchange to LC or GC compatible solvent
SPE is also faster and more suitable for automation compared with
liquid-liquid extraction
©2007 Waters Corporation
7
Short List of Sorbent Types
for SPE
ƒ Normal-Phase Sorbents (polar sorbents)
— Silica, Alumina, Florisil®, Aminopropyl silica, Diol silica, GCB
ƒ Reversed-Phase Sorbents (non-polar sorbents)
— Oasis® HLB
— C18, C8 etc (alkyl silica's)
— Carbon based sorbents
ƒ Ion Exchange
— Accell Plus™ CM, QMA
ƒ Mixed Mode (ion-exchange/reversed phase)
— Oasis® MAX, Oasis WAX (strong and weak anion-exchange)
— Oasis® MCX, Oasis WCX (strong and weak cation-exchange)
©2007 Waters Corporation
8
Outline
ƒ
Introduction
ƒ
Strategies
ƒ
ƒ
Summary
Appendix
— Why sample Prep
— Considerations
o Choices of tools
— Why SPE
o Pre-Treatment
— Traditional approaches
— Modern approaches
o Mixed Mode
o Reverse phase
©2007 Waters Corporation
9
SPE Strategies
1. Approach #1
Retention, cleanup, elution
2. Approach #2
Pass-through
3. Approach #3
Dispersion
©2007 Waters Corporation
10
SPE Strategy 1
Retention-Cleanup-Elution
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
1. load
2. wash
3. elute
©2007 Waters Corporation
11
SPE Strategy 2
Pass-Through Cleanup
1. Sample is passed
through sorbent
and collected
•
no sample
enrichment
2. Matrix
interferences are
retained on
sorbent
pass through
©2007 Waters Corporation
12
SPE Strategy 3
Dispersion Cleanup
ƒ Bulk sorbent is added to sample with agitation
ƒ Sample is filtered or centrifuged
ƒ Supernatant is collected for analysis
This is similar to pass-through cleanup, but less effective
- Dispersion SPE is a one stage (one theoretical plate) cleanup
- Pass-through SPE is a multi-stage cleanup
©2007 Waters Corporation
13
Outline
ƒ
Introduction
ƒ
Strategies
ƒ
ƒ
Summary
Appendix
— Why sample Prep
— Considerations
o Choices of tools
— Why SPE
o Pre-Treatment
— Traditional approaches
— Modern approaches
o Mixed Mode
o Reverse phase
©2007 Waters Corporation
14
Ion-Exchange and Mixed-Mode
Ionizable Compounds
ƒ 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
ƒ Some compounds are strong acids (i.e. PFOA) or strong
bases (i.e. chlorhexidine) that are ionic except at extreme
pH values
ƒ A few of these compounds are quaternary amines (i.e.
paraquat), ionic at all pH
©2007 Waters Corporation
15
Why Mixed-Mode?
ƒ Mixed-Mode SPE extends pH range for good retention of
acids or bases
ƒ 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
ƒ For environmental analysis, mixed-mode SPE allows
simultaneous retention of acids and bases
©2007 Waters Corporation
16
Oasis® Family of Mixed-Mode
Sorbents:
Reversed-Phase Retention and Ion Exchange
©2007 Waters Corporation
17
Oasis Mixed-Mode Sorbents
Strategies for Isolation and Enrichment
of Individual Compounds or Compound
Classes
Oasis® 2x4 method
ƒ PFOS, PFOA (perfluoroacids and related compounds)
— Oasis WAX
ƒ Acidic Herbicides
— Oasis MAX
ƒ Quats
— Oasis WCX
ƒ Pharmaceuticals/pesticides (organic bases)
— Oasis MCX
©2007 Waters Corporation
18
Introduction
ƒ Perfluorinated compounds (PFCs) such as
perfluorooctanesulfonate and perfluorooctanoic acid are
persistent organic pollutants (POPs)
ƒ PFCs have been identified in environmental samples
worldwide
— PFOS can be detected at low PPT levels in most humans
— PFOS commonly found in arctic fauna
ƒ There is need for reliable analytical methods for PFCs in
food, drinking water, tissue, plasma and blood
ƒ In this presentation we will discuss sample preparation for
UPLC-MS determination of PFCs in water and tissue
samples
©2007 Waters Corporation
19
UPLC-MS-MS System
ƒ ACQUITY Ultra Performance LC™
— Using 1.7μm particles, and at elevated pressures up to 15,000
psi
¾ Shorter Analysis Time
¾ Higher Resolution
¾ Broad selectivity options
ƒ Quattro Premier™ XE
— Fast acquisition rates
— Sensitive detection
ƒ Oasis sorbents
— Cleaner samples
©2007 Waters Corporation
20
Goals
ƒ Develop an Acquity UPLCTM separation based on a recently
published method*
ƒ Adapt or modify the SPE protocol for UPLC
— River Water sample
— Chicken Liver tissue sample
ƒ Lower the quantification limits to under 1 ppb in Chicken
Liver tissue, and Low ppt level in River Water sample
*S. Taniyasu et. al.
J. Chrom. A., 1093 (2005) pp89-97
©2007 Waters Corporation
21
Structures of PFOS and PFOA
PFOA and PFOS are Persistent Organic Pollutants of high interest
worldwide.
F F F F F F
O
F F F F F F
OH
F3C
perfluorooctanoic acid
PFOA
pKa ~ 1
F
F
F
F
F
F
F O
S O-
F3C
F
F
F
F
F
F
F O
perfluorooctanesulfonate
PFOS
pKa<<1
©2007 Waters Corporation
22
Oasis® 2x4 Method
For Acids, Bases, and 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
Protocol 1
Protocol 2
Prepare Sample
Prepare Sample
Condition/Equilibrate
Load Sample
Condition/Equilibrate
Load Sample
Wash:
2% Formic acid
Wash:
5% NH4OH
Elute 1:
100% MeOH
Elute 2:
5% NH4OH in MeOH
Bases
Strong
Acids
Neutrals
Elute 1:
100% MeOH
Elute 2:
2% Formic Acid in MeOH
Strong
Bases
Acids
©2007 Waters Corporation
23
Optimized SPE Protocol
for River Water
Oasis® WAX sorbent was
selected for these analytes
Conditions for Oasis® WAX 3 cc 60mg cartridges
Oasis® WAX
Optimized Protocol 1
Logic:
PFOA pKa ~1
PFOS pKa < 1
Prepare Sample
pH 3
Condition
2 mL methanol/2 mL water
Load
Oasis® WAX
200 mL
N
H
N+
N
H
N+
H H
N
O
Wash #1
1 mL 2% Formic acid
Elute 1 (Wash #2)
2 mL methanol
Elute 2
2 mL 1% conc. ammonia in
10:90 methanol/MTBE
mixed-mode weak anion-exchange
pKa ~6
Samples were evaporated and
reconstituted in 0.15 mL mobile phase
©2007 Waters Corporation
24
SPE Protocol
Oasis® WAX
Optimized Protocol 1
Prepare Sample
pH 3
@ pH 3 Sorbent, and analytes are fully charged
(assures mixed-mode retention)
Condition
2 mL methanol/2 mL water
Load
200 mL
Wash #1
1 mL 2% formic acid
Wash #2
2 mL methanol
Maximum load for good recovery
of C3, C4 and PFBS
Assures sorbent is charged
Removes neutrals and bases retained
by reversed-phase
Elute 2
2 mL 1% conc. ammonia in
10:90 methanol/MTBE
MTBE based eluent minimizes elution of
any retained humic material
©2007 Waters Corporation
25
PFBS/PFOS in River Water
100 ng/L (ppt)
200mL river water 6cc WAX _200uL recon _BK
PFOS_082306AQC21x50C18_3 Sm (SG, 1x1)
2: MRM of 3 Channels ESTIC
1.34e4
Blank
%
100
0
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
4.75
5.00
5.25
PFOS_082306AQC21x50C18_4 Sm (SG, 1x1)
5.50
5.75
2: MRM of 3 Channels ESTIC
1.34e4
100
Spiked River Water
PFOS
%
PFBS
0
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
4.75
5.00
5.25
5.50
5.75
©2007 Waters Corporation
26
C3-C7 in River Water
100 ng/L (ppt)
200mL river water 6cc WAX _200uL recon _BK
PFOS_082306AQC21x50C18_3 Sm (SG, 1x1)
1: MRM of 5 Channels ESTIC
9.38e4
100
%
Blank
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
PFOS_082306AQC21x50C18_4 Sm (SG, 1x1)
4.20
C7
Spiked River Water
%
4.00
3: MRM of 5 Channels ESTIC
9.38e4
100
C5
3.80
C6
C4
C3
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80
3.00
3.20
3.40
3.60
3.80
4.00
4.20
©2007 Waters Corporation
27
C8-C12 in River Water
100 ng/L (ppt)
200mL river water 6cc WAX _200uL recon _BK
PFOS_082306AQC21x50C18_3
1: MRM of 5 Channels ESTIC
1.61e5
100
%
Blank
0
2.50
3.00
PFOS_082306AQC21x50C18_4
3.50
4.00
4.50
100
5.50
6.50
7.00
C11
C9
C8
6.00
C10
7.50
8.00
1: MRM of 5 Channels ESTIC
1.61e5
%
Spiked River Water
5.00
C12
0
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
©2007 Waters Corporation
28
River Water Recoveries
Spike Level
μg/L
PFBS
0.10
0.30
0.70
1.0
4.0
10
122
110
102
113
104
104
PFOS C3
109
117
98
94
86
100
108
95
91
128
101
98
C4
C5
C6 C7 C8 C9 C10 C11 C12
119 97 154 107 83
132 105 110 119 126
107 93 118 100 78
106 98 130 100 88
99 99 102 102 92
101 100 87 89 82
121
137
103
100
115
103
101
118
126
110
99
99
101 100
94 95
119 121
117 87
84 68
101 66
©2007 Waters Corporation
29
Observations/Recommendations
ƒ Fluorocarbon parts, tubing, etc. are potential sources of
interferences
— UPLC fluidic lines were conditioned with 2% TFA in propanol
followed with 4% conc. ammonia in water (4 hours each step)
ƒ Polypropylene (PP) lab ware may be best for sample prep
— Do not use Teflon!! (possible positive interference)
— Analytes may adsorb to glass (possible negative interference)
ƒ
C3-C5 analytes are highly volatile
— Evaporative losses are possible, much more so at very low pH
ƒ Samples in glass vials may show loss of some analytes with
time
— Analyze within 24 hrs of sample prep
©2007 Waters Corporation
30
Conclusions
• Oasis® WAX SPE method is effective for isolation and
enrichment of C4-C8 perfluorosulfonic acids and C3-C12
perfluorocarboxylic acids from water and tissue
• Acquity UPLC™ provides significantly reduced analysis time
and improved chromatographic behavior for these
compounds compared with traditional HPLC
ƒ The Quattro Premier XE™ API mass spectrometer, operated
in MRM mode, provides outstanding sensitivity and
selectivity for these compounds
©2007 Waters Corporation
31
Acidic Herbicides
OCH2COOH
Cl
Cl
2,4-D
Step 1 – characterize analytes
they are acids pKa 3-6
For Acids
pKa 2-8
Select
Oasis® MAX
These herbicides, such as
2,4-D, are used in cultivated
agriculture, in pasture and
rangeland applications,
forest management and
home and garden. Also in
aquatic applications.
©2007 Waters Corporation
32
Oasis® 2x4 Method:
Starting Protocols For Acids and Bases
For Bases:
pKa 2-10
Use Oasis® MCX
For Strong Acids
pKa <1.0
Use Oasis® WAX
For Acids
For Strong Bases
pKa 2-8
pKa >10
Use Oasis® WCX Use Oasis® MAX
Protocol 1
Protocol 2
Prepare Sample
Prepare Sample
Condition/Equilibrate
Load Sample
Condition/Equilibrate
Load Sample
Wash:
2% Formic acid
Wash:
5% NH4OH
Elute 1:
100% MeOH
Elute 2:
5% NH4OH in MeOH
Bases
Strong
Acids
Neutrals
Elute 1:
100% MeOH
Elute 2:
2% Formic Acid in MeOH
Strong
Bases
Acids
©2007 Waters Corporation
33
Oasis® 2x4SM Method
Choose Starting Protocol
For Acids
pKa 2-8
Use Oasis® MAX
OCH2COOH
Protocol 2
Prepare Sample
Cl
Condition/Equilibrate
Load Sample
Cl
2,4-D
Wash:
5% NH4OH*
The Oasis MAX cartridge
was chosen for retention
of acid herbicides
Logic:
2,4-D and other
acid herbicides
pKa 3-6
Elute 1:
100% MeOH
Elute 2:
1% Formic Acid in MeOH
Acids
©2007 Waters Corporation
34
Oasis® MAX SPE Method
Acidic Herbicides 1µg/kg in River Water
3
Oasis MAX
Protocol 2
Waters XTerra™MS C18, 2.1 x 100 mm
A: 15mM ammonium formate (pH 3.5),
B: acetonitrile
25% B to 60% B in 9 min, hold 5 min,
to 90% B in 16 min
Prepare Sample
Waters ZQ, ESI-, SIR mode
Condition
15
3 mL methanol/ 3 mL water
4
Load
300 mL sample
Wash #1
3 mL 5% NH4OH
1
6
2
7,8
9
12
10
13
5
14
11
Elute 1 (Wash #2)
3 mL methanol
20 min
1 ppb in river water
Elute 2
4 mL 2% Formic Acid in MeOH
Evaporate and Reconstitute
Conditions for 6 cc cartridges
1. picloram
2. chloramben
3. 4-nitrophenol
4. bentazon
5. 2,4-D
6. MCPA
7. dichlorprop
8. 2,4,5-T
9. MCPP
10. DCB
11. acifluorfen
12. 2,4,5-TP
13. 2,4-DB
14. dinoseb
15. pentachlorophenol
©2007 Waters Corporation
35
Paraquat/Diquat
The Oasis WCX cartridge was
chosen for these analytes
For Quats
Logic:
Select
Oasis® WCX
quats are
cationic at all pH
values
quats can be eluted
from Oasis WCX with
acidic solvent
+
+
N+
N CH3
CH3 N
N+
paraquat
diquat
©2007 Waters Corporation
36
Retention and Elution of Paraquat
on Mixed-Mode Sorbents
Retention Factor (k’)
Retention
Oasis® MCX
Oasis® WCX
0
1
2
3
4
5
% Eluted
Elution
6 7
pH
8
9 10 11 12 13 14
note: quats are eluted from
Oasis WCX at low pH
Oasis® WCX
Oasis® MCX
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14
pH of elution solvent (80:20 acetonitrile/water)
©2007 Waters Corporation
37
Paraquat/Diquat
Optimized Oasis MCX Protocol
Protocol 2
For Quats
Prepare Sample
Select
Oasis® WCX
Condition/Equilibrate
Load Sample
Wash1:
5% NH4OH in water
Wash2:
100% MeOH
optimized elution solvent
acetonitrile/water/TFA
Elute :
1.5 mL ACN/water/TFA 84:14:2
©2007 Waters Corporation
38
Optimized SPE Protocol
Paraquat/Diquat
Oasis® WCX SPE Method
Paraquat/Diquat
Conditions for 3 cc cartridges
Prepare Sample
adjust to pH 7
Condition
1mL methanol/ 1 mL water
Load
up to 25 mL sample
Wash
1 mL pH 7 buffer/1mL methanol
Elute
1.5 mL ACN/water/TFA 84:14:2
Evaporate and Reconstitute
0.5 mL mobile phase
50:1 sample enrichment
©2007 Waters Corporation
39
LC-MS Conditions
paraquat/diquat
MS Conditions
Instrument: Waters Quattro micro API™
Paraquat:
cone 40 V
MRM
cone 15 V
MRM
Diquat:
cone 40 V
MRM
cone 15 V
MRM
171 → 77 (CID 35 eV)
171 → 155 (CID 35 eV
93* → 77 (CID 30 eV)
LC Conditions
Column: Waters Atlantis™ HILIC, 2.1 x 150 mm
Flow: 0.4 mL/min
Mobile Phase: 40% acetonitrile
60% aqueous buffer pH 3.7
(200 mM ammonium formate)
o
Column Temp: 30 C
Sample Temp: 5 oC
Injection: 10 µL
0.20 µg/L Spiked Sample
183 → 157 (CID 30 eV)
183 → 168 (CID 35 eV)
100
92* → 85 (CID 30 eV)
paraquat
1
100
1
1
diquat
2
3
4
5
6
7
8
©2007 Waters Corporation
40
Validation
ƒ
Performance was demonstrated
from 0.1 to 5 µg/L using 20 mL
samples of Sudbury River
water.
0.9
0.8
Paraquat Intraday Results (1 µg/L)
Day 1 1.08 µg/L (8.1% RSD)
Day 4 1.10 µg/L (8.0% RSD)
Day 5 0.95 µg/L (7.1% RSD)
0.7
0.6
0.5
0.4
r2 = 0.998
0.3
0.2
Overall (n=15) 1.04 µg/L (9.8% RSD)
0.1
0
0
2
4
6
©2007 Waters Corporation
41
Advantage of Oasis® WCX
for Paraquat/Diquat
ƒ No Salts required for elution
— Eluent can be evaporated and reconstituted in
mimimal volume
— Method is more compatible with API mass
spectrometry
— Method is more compatible with ion-pair
chromatography
— Method is more compatible with on-line SPE
©2007 Waters Corporation
42
Oasis® 2x4 Method:
Starting Protocols For Acids and Bases
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
Protocol 1
Protocol 2
Prepare Sample
Prepare Sample
Condition/Equilibrate
Load Sample
Condition/Equilibrate
Load Sample
Wash:
2% Formic acid
Wash:
5% NH4OH
Elute 1:
100% MeOH
Elute 2:
5% NH4OH in MeOH
Bases
Strong
Acids
Neutrals
Elute 1:
100% MeOH
Elute 2:
2% Formic Acid in MeOH
Strong
Bases
Acids
©2007 Waters Corporation
43
Pharmaceuticals/Pesticides/Industrial
Chemicals
(Organic Bases, pKa 2-10)
Example: Aniline (pKa ~ 4)
+
NH3
NH2
pH 2
Protocol 1
For Bases
pKa 2-10
Select
Oasis® MCX
Prepare Sample
Condition/Equilibrate
Load Sample
Wash:
2% Formic acid
Elute 1:
100% MeOH
Elute :
5% NH4OH in MeOH
To recover acids and neutrals,
analyze Elute 1
for GC, use 90:10
MTBE/methanolic ammonia
for elute 2
©2007 Waters Corporation
44
Pharmaceuticals/Pesticides/Industrial
Chemicals
(Organic Bases, pKa 2-10)
GC-NPD Conditions
COMPOUND
Agilent 5890 series II
30 m x 0.25 mm (ID) RTX 5 (0.25 µm)
EPA 8270C bases, 20 ug/L
200 mL tap water/Oasis MCX protocol
2 uL inject
(20 µg/L Tap Water)
3
29
16
1
17
2
9
22
23
11
10
13
19 20 24
10
20
Minutes
30
NPD
30
25
26
28
31
27
0
% RECOVERY± RSD
40
1. pyridine
2. picoline
3. aniline
9. o-toluidine
10. phentermine
11. chloroaniline
13. phenylenediamine
61 (17)
77 (16)
90 (11)
82 (12)
73 (18)
82 (11)
93 (15)
16. 2 -nitroaniline
17. 3 -nitroaniline
19. 1 -aminonaphthalene
20. 2 -aminonaphthalene
22. 2 -methyl - 5-nitroaniline
23. 4 -nitroaniline
24. diphenylamine
95 (7.2)
103 (8.5)
87 (5.1)
88 (8.5)
104 (6.2)
106 (8.7)
93 (4.4)
26. aminobiphenyl
30. dimethylaminoazobenzene
31. dimethylbenzidine
33. dichlorobenzidine
105 (4.2)
100 (3.9)
64 (8.9)
111 (6.0)
33
32
50
©2007 Waters Corporation
45
Summary
ƒ Sample Preparation is necessary to obtain the best
analytical results
ƒ SPE is a very versatile and cost efficient sample preparation
technique for environmental samples.
ƒ Waters provides strategies which combine sorbents, formats
and methodologies resulting in optimal SPE protocols.
ƒ Whether for analysis by LCMS or GCMS; Waters analytical
solutions, including SPE, cover a wide range of sample
matrices and compounds classes
©2007 Waters Corporation
46
Oasis® Mixed-Mode Sorbents
Strategies for Multiresidue Isolation and Enrichment
(acids, bases and neutrals together)
ƒ 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
©2007 Waters Corporation
47
SPE of Acids and Base/Neutrals
Reversed-Phase Logic
Consider:
Aniline, phenol and benzyl alcohol on Reversed-Phase SPE
NH2
OH
OH
At pH 2: Aniline is cation – not retained
Phenol is protonated – retained
Benzyl alcohol is neutral – retained
At pH 11 Aniline is neutral – retained
Phenol is ionized – not retained
Benzyl alcohol is neutral – retained
©2007 Waters Corporation
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SPE of Acids and Base/Neutrals
Mixed-Mode Logic
Consider:
Aniline, phenol and benzyl alcohol on Mixed-Mode SPE
NH2
OH
OH
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 anion – retained
Benzyl alcohol is neutral – retained
©2007 Waters Corporation
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Multi residue Analysis
Oasis® MCX Method for GC
Oasis® MCX
Optimized Protocol
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
Dry over Sodium Sulfate
Evaporate to Final Volume
prepare reagent using
anhydrous ammonia in
methanol (Aldrich)
Micro K-D
©2007 Waters Corporation
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SPE for Base/Neutrals and Acids
Oasis® MCX GC Protocol
COMPOUND
% RECOVERY ±RSD
(20 µg/L Tap Water)
7,8
6
1.
2.
3.
4.
5.
6.
12
FID
4
5
14
18
21
15
3
29
16
1
17
2
9
22
23
11
10
13
19 20 24
25
26
28
31
27
0
10
20
Minutes
NPD
30
30
bases, acids, neutrals
40
33
pyridine
picoline
aniline
phenol
benzyl alcohol
o-cresol
61
77
90
65
75
91
(17)
(16)
(11)
(14)
(25)
(8.6)
7,8. m,p-cresol
9. o-toluidine
10. phentermine
11. chloroaniline
12. dichlorophenol
13. phenylenediamine
91
82
73
82
57
93
(8.9)
(12)
(18)
(11)
(6.2)
(15)
14.
15.
16.
17.
18.
19.
2-methylnaphthalene
trichlorophenol
2-nitroanili ne
3-nitroanili ne
dibenzofuran
1-ami nonaphthalene
81 (8.0)
54 (10)
95 (7.2)
103 (8.5)
80 (5.4)
87 (5.1)
20.
21.
22.
23.
24.
25.
2-ami nonaphthalene
tetrachlorophenol
2-methyl-5-nitroaniline
4-nitroanili ne
diphenylamine
phenacetin
88 (8.5)
35 (17)
104 (6.2)
106 (8.7)
93 (4.4)
85 (7.3)
26.
27.
28.
29.
30.
31.
aminobi phenyl
dinoseb
nitroqui noline oxide
methapyril ene
dimethylaminoazobenzene
dimethylbenzidine
105 (4.2)
90 (7.1)
100 (6.5)
105 (5.5)
100 (3.9)
64 (8.9)
32
50
32. acetamidofluorene
33. dichlorobenzidi ne
135 (5.4)
111 (6.0)
©2007 Waters Corporation
51