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©2012 Waters Corporation 1 Sample Preparation Strategies for Complex Environmental Analysis SPE for Environmental Samples
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Page 1: SPE for Environmental Samples -  · PDF fileSPE for Environmental Samples ... –extract with acetonitrile/toluene ... 1. toluene 2. o-xylene 3. m,p-xylene 4. styrene 5. indan

©2012 Waters Corporation 1

Sample Preparation Strategies for

Complex Environmental Analysis

SPE for Environmental Samples

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©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

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©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)

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©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

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©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

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©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)

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©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

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©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)

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©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

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©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)

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©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

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©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

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©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

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©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

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©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

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©2012 Waters Corporation 17

Hydrocarbons Recovered from Diesel Contaminated Soil (spiked)

Diesel

Diesel – Elute 1

Aliphatic (hexane) fraction

Diesel – Elute 2

Aromatic (DCM) fraction

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©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

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©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

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©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

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©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

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©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

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©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

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©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)

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©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

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©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)

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©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

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©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)

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©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

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©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.

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©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

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©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

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©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

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©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

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©2012 Waters Corporation 35

Oasis® Family of Sorbents: Reversed-Phase and Ion Exchange Retention

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©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

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©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

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©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

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©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

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©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)

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©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

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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

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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

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©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

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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

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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

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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

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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

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©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

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©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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©2012 Waters Corporation 51

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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©2012 Waters Corporation 52

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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©2012 Waters Corporation 53

Questions?


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