fundamentals of spe - waters corporationthe new spe “textbook”. part #: 715003405. 212 pages,...
TRANSCRIPT
![Page 1: Fundamentals of SPE - Waters CorporationThe New SPE “Textbook”. Part #: 715003405. 212 pages, paperback. Size: 8.25 x 11” >150 full color figures and diagrams. Chapter Titles](https://reader035.vdocument.in/reader035/viewer/2022071421/611b0a11b14887010a516d7a/html5/thumbnails/1.jpg)
©2013 Waters Corporation 1
Fundamentals of Solid-Phase Extraction (SPE):
Principles and Practical Tips for Developing
Sample Preparation Methods
WELCOME!
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©2013 Waters Corporation 2
Agenda
The Importance of Solid Phase Extraction
How SPE Works
Device Design and Tips for Processing Samples
Chromatographic Sorbent Choices
Typical SPE Strategies and Starting Protocols
Was My SPE Method Successful?
Appendix
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©2013 Waters Corporation 3
The New SPE “Textbook”
Part #: 715003405
212 pages, paperback
Size: 8.25 x 11”
>150 full color figures and diagrams
Chapter Titles
– Benefits of SPE in Sample Preparation
– SPE is LC
– Key Terms and Calculations
– In the Lab
– Method Development
– Troubleshooting
– Appendix: Glossary of SPE and LC Terms
– Appendix: Oasis Sorbent Technology for SPE
– Appendix: Applications
– Appendix: Additional Reference Materials
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©2013 Waters Corporation 4
Why do Sample Preparation?
Issue: Plugging
Cause: solid particulates
Impact: may limit instrument/system up-time
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©2013 Waters Corporation 5
Sample Prep Tools
For solid particulates:
– Filters
– Centrifuge
For sample matrix components:
– Precipitation
– Liquid-Liquid Extraction (LLE)
– Solid-Phase Extraction (SPE)
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©2013 Waters Corporation 6
Why do Sample Prep?
Issue: Complicated analytical results / too much variability and
potential resolution issues
Cause: analytes contained in a complex sample matrix
Impact:
Quantitation errors: slight changes might resolute in loss of
resolution of critical pairs
May reduce instrument/system up-time
AU
0.00
0.02
0.04
0.06
0.08
Minutes
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
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©2013 Waters Corporation 7
Improve Analytical Results: Example #1
AU
0.00
0.02
0.04
0.06
0.08
Minutes
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
AU
0.00
0.02
0.04
0.06
0.08
Minutes
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
Only need to analyze these peaks
Improvement: Remove unnecessary peaks
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©2013 Waters Corporation 8
Improve Analytical Results: Example #2
PD
A (0
.01 A
UFS)
1 2
Sample Prep
0 10 5
1
2
Sample
Minutes
Improvement: Remove Baseline interferences
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©2013 Waters Corporation 9
Improve Analytical Results: Example #3
Spiked Sample
Blank Sample Matrix
1 2
3 0.004 AU
10 8 6 4 2 0 min.
0.0
0
0.0
0
2.0
0
2.0
0
4.0
0
4.0
0
6.0
0
6.0
0
8.0
0
8.0
0
10.0
0
10.0
0
Min
ute
s
Min
ute
s
Spiked Sample
Blank Sample Matrix
1
3
2 0.004 AU
Peak 1 has baseline contamination from sample matrix
Peaks 2 and 3 are clean
Peak 1 is clean
With SPE Clean-up
Improvement: Remove Baseline interferences
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©2013 Waters Corporation 10
Why do Sample Prep?
Issue: not enough sensitivity
Cause:
Analyte concentration in original sample matrix
TOO LOW to measure by instrument
Impact:
Quantitation errors
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©2013 Waters Corporation 11
Improve Analytical Results: Example #4
Concentration of Analyte in Original Sample
TOO LOW (difficult to quantitate)
Time 0 1 2 3 4 5
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©2013 Waters Corporation 12
Improve Analytical Results: Example #4
Concentration of Analyte in
Original Sample TOO LOW
(difficult to quantitate )
Time 0 1 2 3 4 5
Utilize SPE Chromatographic Bed to Trace Concentrate the Original Sample
for that Analyte - Obtain Good Response
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©2013 Waters Corporation 13
Why do Sample Prep?
Issue:
– Not enough sensitivity
Cause:
– For MS detection: minimize ion suppression/enhancement
Impact:
– Quantitation Errors
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©2013 Waters Corporation 14
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
591.7
354.4 518.5
472.6 485.5 609.6
Scan ES+ 354.4
260.2
291.3
609.6
485.6472.6
411.5591.6
50/50 Water/ACN + human plasma supernatant
50/50 Water/ACN
260.2 - 97 %
291.2 - 96 %
354.4 - 86 %
411.4 - 93 %
472.6 - 93 %
485.6 - 95 %
591.6 - 89 %
609.5 - 93 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
591.7
354.4 518.5
472.6 485.5 609.6
Scan ES+ 354.4
260.2
291.3
609.6
485.6472.6
411.5591.6
50/50 Water/ACN + human plasma supernatant
50/50 Water/ACN
260 280 300 320 340 360 380 400 420 440 460 480260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
591.7
354.4 518.5
472.6 485.5 609.6
Scan ES+ 354.4
260.2
291.3
609.6
485.6472.6
411.5591.6
50/50 Water/ACN + human plasma supernatant
50/50 Water/ACN
260.2 - 97 %
291.2 - 96 %
354.4 - 86 %
411.4 - 93 %
472.6 - 93 %
485.6 - 95 %
591.6 - 89 %
609.5 - 93 %
Analytes in human plasma with only Protein Precipitation Standards in aqueous solution give acceptable response
What is Ion Suppression?
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©2013 Waters Corporation 15
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
591.7
354.4 518.5
472.6 485.5 609.6
Scan ES+ 354.4
260.2
291.3
609.6
485.6472.6
411.5591.6
50/50 Water/ACN + human plasma supernatant
50/50 Water/ACN
260.2 - 97 %
291.2 - 96 %
354.4 - 86 %
411.4 - 93 %
472.6 - 93 %
485.6 - 95 %
591.6 - 89 %
609.5 - 93 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
591.7
354.4 518.5
472.6 485.5 609.6
Scan ES+ 354.4
260.2
291.3
609.6
485.6472.6
411.5591.6
50/50 Water/ACN + human plasma supernatant
50/50 Water/ACN
260 280 300 320 340 360 380 400 420 440 460 480260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
591.7
354.4 518.5
472.6 485.5 609.6
Scan ES+ 354.4
260.2
291.3
609.6
485.6472.6
411.5591.6
50/50 Water/ACN + human plasma supernatant
50/50 Water/ACN
260.2 - 97 %
291.2 - 96 %
354.4 - 86 %
411.4 - 93 %
472.6 - 93 %
485.6 - 95 %
591.6 - 89 %
609.5 - 93 %
Analytes in human plasma with only Protein Precipitation
Analyte standards in aqueous solution
% Loss
Complex Sample Matrix: Ion Suppression
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©2013 Waters Corporation 16
Do all Sample Preparation Techniques Give the Same Result?
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
%
0
100 MRM
472.2 > 436.4 1.27e6
1.89
MRM 472.2 > 436.4
1.27e6 1.91
80% ion suppression
Minimal ion suppression B (SPE) (Oasis® MCX)
A (PPT)
Note: These samples are dried and reconstituted. Gradient time = 1.5 min
No loss in signal observed for analytes when the interferences, which cause the suppression,
are removed by SPE.
MRM for Terfenadine
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©2013 Waters Corporation 17
The Ultimate Goal is to make your analytical lab more productive
- Lower Limits of Detection
- Run more samples with less time
- Minimize costs in manpower and equipment maintenance
Goals of Sample Preparation: Summary
To remove interferences for
– Better chromatography
– More confident analytical results
– Longer column lifetime
– Less instrument downtime
To enrich sample for
– Higher detection sensitivity
To make sample more compatible for separation and detection
– Matching solvent strength
– Eliminating ion-suppression in LC/MS analysis
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©2013 Waters Corporation 18
Agenda
The Importance of Solid Phase Extraction
How SPE Works
Device Design and Tips for Processing Samples
Chromatographic Sorbent Choices
Typical SPE Strategies and Starting Protocols
Was My SPE Method Successful?
Appendix
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©2013 Waters Corporation 19
x
Yellow is the earliest eluting analyte “band”, it “likes” the mobile phase,
has very low ‘k” (little retention)
Blue is well retained, it “likes” the particles, has high “k” (high
retention - “captured”)
Mix Yellow, Red and Blue Dyes together to create what appears,
to our eyes as a “Black” Sample
Food Dyes
How a Chromatographic Column Works -- “BANDS”
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©2013 Waters Corporation 20
Yellow is the earliest eluting analyte “band” - it “likes” the mobile phase,
has very low ‘k” (little retention)
Blue is well retained - it “likes” the particles,
has high “k” (high retention - “captured”)
How a Chromatographic Column Works -- “BANDS”
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©2013 Waters Corporation 21
Band Migration on an SPE Cartridge
SPE follows the same principles as LC
Blue Red
Yellow
Sample
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©2013 Waters Corporation 22
Agenda
The Importance of Solid Phase Extraction
How SPE Works
Device Design and Tips for Processing Samples
Chromatographic Sorbent Choices
Typical SPE Strategies and Starting Protocols
Was My SPE Method Successful?
Appendix
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©2013 Waters Corporation 23
SPE Configurations from Waters
Syringe Barrel Cartridge
Standard 96-Well Plate
On-line SPE Devices
melution 96-Well Plate
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©2013 Waters Corporation 24
Sample Processing: Gravity
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©2013 Waters Corporation 25
Stop Cock flow control valves- to prevent drying – out effect
Male-male Adapter
Syringe Adapter
Vacuum Manifold
Sample Processing: Glass Vacuum Manifold
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©2013 Waters Corporation 26
Vacuum Manifold
Sample Processing: Large Liquid Volumes
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©2013 Waters Corporation 27
Sample Processing: 96-well Plate
Set-up for typical 96-well plates Under vacuum, a properly seated well-plate should move down and the tips will extend into the wells of the collection plate.
Collection Plate
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©2013 Waters Corporation 28
Sample Processing: Positive Pressure
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©2013 Waters Corporation 29
Tip: Cartridge Stacking
Multiple cartridges can be stacked for a mixed-mode approach
2 of the SAME for More Capacity
2 Different Sorbents
for 2 Dimensional
Selectivity
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©2013 Waters Corporation 30
Sample Processing: Positive Pressure Manifold
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©2013 Waters Corporation 31
Agenda
The Importance of Solid Phase Extraction
How SPE Works
Device Design and Tips for Processing Samples
Chromatographic Sorbent Choices
Typical SPE Strategies
Was My SPE Method Successful?
Appendix
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©2013 Waters Corporation 32
SPE Terminology
Sorbent -- Chromatographic packing material (Stationary Phase)
Retention Mechanisms: 1. Reversed Phase (RP) 2. Normal Phase (NP) 3. Ion-Exchange (IEX)
Syringe Barrel Style
Sorbent
Filter/Frit
Filter/Frit
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©2013 Waters Corporation 33
Sorbents for SEP-PAK® Products Silica and Alumina Based
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©2013 Waters Corporation 34
Sorbents for SEP-PAK® Products Reversed-Phase
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©2013 Waters Corporation 35
Sorbents for SEP-PAK® Products Normal Phase
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©2013 Waters Corporation 36
Sorbents for SEP-PAK® Products Ion-Exchange (Silica Based)
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©2013 Waters Corporation 37
Sorbents for SEP-PAK® Products Specialty Phases
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©2013 Waters Corporation 38
Hydrophilic
monomer
Lipophilic
monomer
N O
Reversed-phase Retention
Hydrophilic-Lipophilic Balanced Copolymer
Polymeric SPE Sorbent Chemistry: Oasis® HLB
• Water wettable
• Polar retention
• Stable across pH 1-14
• No silanol interactions
• High recoveries for acids, bases and neutrals
Retention of Polars
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©2013 Waters Corporation 39
Polymeric SPE Sorbent Chemistries: Oasis® Mixed-mode Ion Exchangers
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©2013 Waters Corporation 40
Agenda
The Importance of Solid Phase Extraction
How SPE Works
Device Design and Tips for Processing Samples
Chromatographic Sorbent Choices
Typical SPE Strategies and Starting Protocols
Was My SPE Method Successful?
Appendix
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©2013 Waters Corporation 41
SPE Strategies
Typical SPE Strategies and Protocols –Pass Through
–Clean-up (retention of analytes of interest)
–Fractionation
–Trace Concentration
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©2013 Waters Corporation 42
SPE Strategy #1: Pass Through Purification
Pass Through Approach:
Load: want k = high for interferences (max retention)
want k = 0 for analytes -- no retention
Advantages: clean-up is easy, fast, with little or no method
development
Disadvantages: no concentration of analytes; no solvent switch
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©2013 Waters Corporation 43
Analyte is a
Purple Compound
Pass Through SPE: Load and Collect
Same Sample Solvent
k=0 for analyte
k=0
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©2013 Waters Corporation 44
Ostro™ Plate Technology Pass Through Strategy
Specifically Designed for
Phospholipid Removal which, when present,
results in Significant
Ion-Suppression
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©2013 Waters Corporation 45
Methodology
Precipitate
Proteins
Held up by
Filters
Phospholipids
Retained
by SPE
Sorbent Bed,
Analytes
Pass Through
Ostro™ Plate Technology Pass Through Strategy
It is possible to work with lower sample volumes (such as
25µL). When doing so you will need a higher
organic solvent to sample ratio, such as
10:1 or 20:1.
The well volume is 1.9 mL, however in order to mix by aspiration, the
maximum volume is 1.4 mL. This translates to a maximum sample size
of 350µL.
Place Ostro plate onto collection plate
Pipette 50-200µL of plasma into wells
Forcefully add 2% formic acid in acetonitrile,
3:1 solvent:plasma
(methanol not recommended)
Mix thoroughly by aspirating 3x with pipette
Filter samples using vacuum manifold or
positive pressure manifold
Analyze samples
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©2013 Waters Corporation 46
Competitive Techniques: Phospholipids Remaining
MRM of m/z 184-184
Time 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
%
0
100
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
%
0
100
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
%
0
100
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
%
0
100 184.4 > 184.4 (Lipid 184) 2.00e8
2.88 2.29 2.21
2.10
1.90
2.60 2.78 2.72
184.4 > 184.4 (Lipid 184) 2.00e8
2.80 2.27
1.90
2.62 2.56 2.68
184.4 > 184.4 (Lipid 184) 2.00e8
1.96 1.90 1.77
184.4 > 184.4 (Lipid 184) 2.00e8
2.84 2.21 1.96 1.75 1.42 1.38
1.32
1.63 1.51
PPT
Ostro™
LLE with MTBE
LLE with 5% NH4OH in MTBE
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©2013 Waters Corporation 47
SPE Strategy #2: Capture Retain Analytes of Interest
Capture Approach:
Load: want k = high for analyte (max retention – capture)
Wash: want k = 0 for interferences -- no retention
want k= high for analyte so we don’t lose to waste
Elution: want k = 0 for analyte (no retention - strong solvent) to
elute and collect
Advantages: clean-up, desalting, concentration, solvent switching
= clean extracts
Disadvantages: requires method development
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©2013 Waters Corporation 48
Capture SPE Method (RP): Conditioning
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©2013 Waters Corporation 49
Capture SPE Method (RP): Equilibration
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©2013 Waters Corporation 50
Proper Wetting Conditioning
and Equilibration
Good Capture
Capture SPE Method (RP): Load
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©2013 Waters Corporation 51
Capture SPE Method (RP): Wash
Wash Solvent
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©2013 Waters Corporation 52
Capture SPE Method (RP): Elution
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©2013 Waters Corporation 53
Capture SPE Method (RP): Dry down and reconstitute
These steps are performed for some applications where the mobile phase conditions, for the actual analytical
separation, will require a change in the sample solvent. Typically, the strong
elution solvent from the SPE Protocol is carefully evaporated away and the analytes
are reconstituted with the mobile phase used in the instrument.
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©2013 Waters Corporation 54
Optimizing Peak Shape Poor Peak Shape Sample Solvent TOO Strong
Tip: Solvent Strength
Use the correct solvent strength to optimize peak shape
Sample: 80% Organic Mobile Phase: 40% Result: Poor Peak Shapes
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©2013 Waters Corporation 55
Same Column, Same Mobile Phase Weaker Sample Solvent
Tip: Solvent Strength
Sample: 0% Organic Mobile Phase: 40%
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©2013 Waters Corporation 56
Use slower flow rate to increase
contact time
Tip: Contact Time
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©2013 Waters Corporation 57
Tip: Solvent Volume
Sorbent per
Well
Maximum Mass
Capacity
Typical Sample
Volume (Pre-
Dilution)
Typical Elution
Volume
2 mg 0.03 to 0.05 mg 5 to 200 µL ≤ 50 µL
5 mg 0.15 to 1 mg 10 to 200 µL ≤ 150 µL
10 mg 0.35 to 2 mg 50 to 400 µL ≤ 250 µL
30 mg 1 to 5 mg 100 µL to 1 mL > 400 µL
60 mg 2 to 10 mg 200 µL to 2 mL > 800 µL
Solvent volume is directly proportional to bed mass
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©2013 Waters Corporation 58
SPE Protocol: Reversed-Phase
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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©2013 Waters Corporation 59
SPE Protocol: Reversed-Phase vs. Mixed-Mode IEX
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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©2013 Waters Corporation 60
Polymeric SPE Sorbent Chemistries: Oasis® Mixed-mode Ion Exchangers
Strong Cation
+ RP
Strong Anion
+ RP
Weak Cation
+ RP
Weak Anion
+ RP
Reversed Phase
RP
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©2013 Waters Corporation 61
Oasis® 2x4 Method: 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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©2013 Waters Corporation 62
Oasis® 2x4 Method: Neutrals
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
Reversed-phase Backbone
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©2013 Waters Corporation 63
SPE Strategy #3: Fractionation of Analytes
Capture and Fractionation:
Load: k = high for analytes (max retention)
Elution 1: k = 0 for one analyte class only [slightly stronger solvent]
(remaining k’s = high)
Elution 2: k = 0 for second analyte class only [slightly stronger solvent]
(remaining k’s = high for remaining)
Elution 3: etc…
Advantages: separates classes of compounds, if they have
different polarities and hydrophobicities
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©2013 Waters Corporation 64
Grape Kool-Aid®
Sample Matrix
Polar Compounds Polar Water
Red Dye # 40 8%IPA
Blue Dye # 1 35%IPA
Non-Polar Oils Non-Polar 70%IPA
{Reversed-Phase Conditions}
Polarity Elution
Solvent
Solution LOOKS Purple
Dry Powder Fruit Flavored
Mix – add Water
Capture and Fractionation
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©2013 Waters Corporation 65
Capture and Fractionation: Load
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©2013 Waters Corporation 66
Capture and Fractionation: Elute #1
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©2013 Waters Corporation 67
Capture and Fractionation: Elute #2
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©2013 Waters Corporation 68
Capture and Fractionation: Elute #3
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©2013 Waters Corporation 69
Capture and Fractionation: Elute #4
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©2013 Waters Corporation 70
Capture and Fractionate: Summary
Polars Red Dye Blue Dye Non- Polars
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©2013 Waters Corporation 71
SPE Strategy #4: Trace Enrichment
Trace Concentration/Enrichment:
Load: k = high for analytes (max retention)
and load large sample volume
Elution: k = 0 for analytes
Advantages: concentration of very low level analytes
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©2013 Waters Corporation 72
Trace Enrichment: Load
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©2013 Waters Corporation 73
Trace Enrichment: Continue Loading
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©2013 Waters Corporation 74
Trace Enrichment: Elution
Elute the analtyes, which now have been concentrated, by using a stronger solvent to
release them from the device (k = 0)
Determine the concentration of
THIS NEW sample -- and then calculate the
ORIGINAL Sample Concentration by dividing by the volume of
ORIGINAL sample processed by the
SPE Device
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©2013 Waters Corporation 75
Reverse Flow
Direction
Adapter Tube
Elute Band in Less Solvent
More Concentrated
Trace Enrichment: Elution (Reverse Cartridge)
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©2013 Waters Corporation 76
Reverse Flow
Direction
Adapter Tube
Elute Band in
Less Solvent
More
Concentrated
Greatly Enhanced Sensitivity
Trace Enrichment: Elution (Reverse Cartridge)
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©2013 Waters Corporation 77
Agenda
The Importance of Solid Phase Extraction
How SPE Works
Device Design and Tips for Processing Samples
Chromatographic Sorbent Choices
Typical SPE Strategies and Starting Protocols
Was My SPE Method Successful?
Appendix
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©2013 Waters Corporation 78
Determining % Recovery: Calculation
Analytical Result
= 9.5 ng/mL
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©2013 Waters Corporation 79
Determining % Matrix Effect: Calculation
Analytical Result =10 ng/mL
Analytical Result =10 ng/mL
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©2013 Waters Corporation 80
Importance of % Recovery and % Matrix Effect
% Analyte Recovery in plasma
% Matrix Effects in plasma
-100
-80
-60
-40
-20
0
20
40
PPT PLR Plate
LLE RP SPE
Oxycodone
Oxycodone d6
0
10
20
30
40
50
60
70
80
90
100
PPT PLR Plate LLE RP SPE
Oxycodone
Oxycodone d6
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©2013 Waters Corporation 81
Conclusions
SPE is a powerful sample preparation technique that
utilizes extraction selectivity with the tangible
benefits of:
– Clean-up: the simplification of a complex sample matrix
– Reduce ion suppression (or enhancements) for MS
applications
– Fractionate to analyze distinct classes of compounds
– Trace concentration (enrichment) of samples with very low
concentration levels
– Solvent switch
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©2013 Waters Corporation 82
Additional SPE Seminar Topics
Food Compound Specific Analysis of Food
Food Safety Screening
Environmental Environmental Sample Preparation
BioAnalytical Small Molecule Fit for Purpose Bioanalytical Method Development
2x4 Mixed Mode SPE: Troubleshooting and Optimization
BioAnalytical Large Molecule Intro to peptide Bioanalysis
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©2013 Waters Corporation 84
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©2013 Waters Corporation 85
Agenda
The Importance of Solid Phase Extraction
How SPE Works
Device Design and Tips for Processing Samples
Chromatographic Sorbent Choices
Typical SPE Strategies and Protocols
Was My SPE Method Successful?
Appendix
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©2013 Waters Corporation 86
Appendix
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Technology Capability Literature: Primers
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Search Applications
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Applications 65,993 Results
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Specific Topic
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Amyloid Beta Peptides
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Application Note
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SPE Protocol Analytical UPLC® Method
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Pesticides in Fruit Juice
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Journal Listings
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SPE Mosquito Heads
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