Sample Prep Options - Agilent Prep... · sample, add water if needed, spike 2) Add 10ml ACN 3)...
Transcript of Sample Prep Options - Agilent Prep... · sample, add water if needed, spike 2) Add 10ml ACN 3)...
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Sample Prep Options forGC users
Tina ChambersTechnical Specialist Sample Preparation
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Why Bother?
• Removal of interferences which would otherwise affect detection of analyte
•Removal of interferences which would otherwise affect instrumentation and/or columns
• Concentration of an analyte to a detectable levels
• Solvent switching into an analytically more compatible solvent
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Matching SPP to Analytical Method
All GC based systems require highest degree of cleanliness, regardless of detector!!
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Sample Preparation Options
•Dilute and shoot
•Protein precipitation
•Liquid-liquid extraction/solid supported liquid extraction•Liquid-liquid extraction/solid supported liquid extraction
•Solid phase extraction
•QuEChERS
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Considerations for LLE/SLE
- Polarity of the analyte – soluble in non-miscible organic?
- Nature of interfering compounds – presence of lipids?
- LODs/LOQs – is concentration necessary?
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TOXI-TUBES A & B ®
• Contain solvents and salts ideally proportioned for Clinical/Forensic drug analysis • Ultimate in ease-of-use: just add sample, mix and centrifuge• Can be used with challenging matrices: post-mortem blood, gastric content, liver tissue etc.• Absolutely no method development required!
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Sample Preparation – Supported LLE (SLE)
Hydromatrix™ - diatomaceous earth sorbent
• Composed of fossilized diatoms• Purified at high temperatures• Purified at high temperatures• High surface area for water adsorption• Very polar surface
Chem Elut™ - pre-assembled cartridges with HydromatrixCombilut ™- 96-well plate filled with Hydromatrix
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The SLE Process
Aqueous layer
Organic layer
Dry sorbent
Before ExtractionApply
SampleExtract with
Organic Solvent
layer layer
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The Chem Elut Method
Aqueous samplebeing applied
Solid support adsorbs water ontohigh surface area particles
Organic extraction solvent
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Multi-Residue Confirmation of Pesticides in Honey using SLE
App note SI-01002
Recovery comparison of pesticides between solid supported liquid-liquid extraction (SLE) on Chem Elut extraction (SLE) on Chem Elut and classical liquid-liquid extraction (LLE).
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LLE/SLE summary
- Polarity of compound and nature of interferences dictate whether this is suitable
- pH adjustments and/or addition of salts can be helpful
- LLE/SLE solvents are often a good choice for GC analysis
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Considerations for Solid Phase Extraction (SPE)
- High degree of versatility (over 40 different chemistries available)
- Concentrates dilute samples
- Method development often necessary
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Solid Phase Extraction Triangle
Often, both the the analyte and the matrix are known, it is sorbent choice which is the critical component
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Four Steps of SPE
• Conditioning: Preparation of the sorbent prior to sample addition
• Retention: Analytes of interest and other interferences adsorb onto the surface of the sorbent during sample additionaddition
• Washing: Elimination of undesired interferences
• Elution: Selective desorption and collection of desired analytes from the sorbent
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The Four Steps of SPE – Selective Elution
Sample loading Retention Rinsing Elution
Green = Blue and Yellow
Blue is more non polar than yellow
Blue is retained
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Silica VS. Polymer
• “True” polar/ion exchangepossible
• Wide range of chemistries• Inherent hydrophobicity
(conditioning)• Wide range of chemistries• Wide range of established
methods• Can be more selective• Conditioning is crucial
(conditioning)• Higher capacity
(sorbent mass/flow)• Polarity gradient in Plexa
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Polymers and Conditioning
450 ng/mL, LCUV
60%
80%
100%
120%
% R
ecov
ery
MethanolAqueous
Non-Conditioned
0%
20%
40%
Atenolol Ranitidine Pseudoephedrine Quinidine Brompheniramine Mianserin Fluoxetine
% R
ecov
ery
Non-Conditioned
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Silica and Conditioning
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Benefits by Design: Sorbent Quality
Bond Elut Plexa Oasis HLB
Bond Elut Plexa PCX Oasis MCX
See Plexa Advantage Note, SI-1935Confidentiality Label
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Size Distribution
May 30, 2013Confidentiality Label
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Non-Polar Method Development
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Method Development Considerations
• What is the partition coefficient (log P) of the un-ionized compound?
log Poctanol/water = log ([solute]octanol/[solute]water)
• If log P > 1.5, good candidate for hydrophobic method
• If log P unknown, “rule of thumb”: four covalently bound • If log P unknown, “rule of thumb”: four covalently bound carbon atoms or benzene ring minimum requirement
• If compound is ionizable, determine pKa• Load acids at pH<pKa by 2 units minimum• Load bases at pH>pKa by 2 units maximum
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Method Development Considerations
Solubility characteristics of target compound?
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Method Development Considerations
• Select suitable solvents
• Prepare 0%-100% concentrations
• Plot recoveries
80000100000120000
Elution Profile
020000400006000080000
0% M
eOH
10%
MeO
H20
% M
eOH
30%
MeO
H40
% M
eOH
50%
MeO
H60
% M
eOH
70%
MeO
H80
% M
eOH
90%
MeO
H10
0% M
eOH
Elution Profile
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Method Development Consideration
100000120000
Elution Profile
• Highest % organic with low recoveries for wash
• Lowest % organic with high recoveries for elution
• Try acid/base modifiers
020000400006000080000
100000
0% M
eOH
10%
MeO
H20
% M
eOH
30%
MeO
H40
% M
eOH
50%
MeO
H60
% M
eOH
70%
MeO
H80
% M
eOH
90%
MeO
H10
0% … Elution Profile
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NH3+
Silica baseOH
SO3-
Interactions on Ion Exchange Sorbents
Electrostatic attraction
CO2H
SO3
CO2-
OCON(CH3)2
+N
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STRONG: Ionic group is always charged (+ or -)
WEAK: Ionic group is variably charged (+ or -)
CATIONS: (+) Found in basic compounds
ANIONS: (-) Found in acidic compounds
Ion Exchange Nomenclature
ANIONS: (-) Found in acidic compounds
Extract weak ions with strong exchangers and strong ions with weak exchangers!
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Ion Exchange Sorbent Selection
• All bases except quarternary amines can be processed on Plexa PCX or Bond Elut SCX
• Only carboxylic acids can be processed on Plexa PAX or Bond Elut SAXBond Elut SAX
• Phosphates, sulfates and other STRONG acids should be processed with weak anion exchangers (Bond Elut NH2, PSA)
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NH3+
Silica base
OH
SO3-
Interactions on Ion Exchange Sorbents
50%
CO2H
SO3
CO2H
NH2If the pKa=9
and the pH=9
50%
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Method Development Considerations
What is the pKa of your compound?
pKa= -log Ka
and
Ka = [A-][H+]/[HA]
• If pH=pKa, 50% of the compound is ionized and 50% is neutral
• To ensure full charge or full neutralization, employ the rule of 2
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NH3+
Silica base
OH
SO3-
Interactions on Ion Exchange Sorbents
100%
CO2H
SO3
CO2H
NH2If the pKa=9
and the pH=7
0%
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NH3+
Silica base
OH
SO3-
0%
Interactions on Ion Exchange Sorbents
CO2H
CO2H
NH2If the pKa=9
and the pH=11
100%
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Important Consideration for Ion Exchange
• Reduce ionic strength of “salty” matrices by dilution
• Consider competitive binding when choosing bed mass
• Remember that ALL polymeric exchangers are mixed-mode, elute in organic solventmode, elute in organic solvent
• Some organic should be present even with silica based ion exchangers because of carbon linkers
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QuEChERS(Pronounced “catchers”)• Quick, Easy, Cheap, Effective, Rugged, and Safe
• Portmanteau: blend of 2 or more words
• www.quechers.com
• Introduced in 2003: M. Anastassiades, S.J. Lehotay, D. Stajnbaher, and F.J. Schenck, J. AOAC Int 86 (2003) 412F.J. Schenck, J. AOAC Int 86 (2003) 412
• Validated in 2005, with subsequent modification in 2007
• AOAC 2007.01 and European Method EN 15662
• Streamlined approach that makes it easier and less expensive to examine pesticide residues in food
5/30/2013
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QuEChERS First step extraction
1) Weigh sample,
add water if needed, spike
2) Add 10ml ACN
3) Vortex3) Vortex
4) Add salt packet
5) Shake 1 minute
6) Centrifuge at 4,000 rpm for 5 minutes
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Second Step – Dispersive SPE
7) Choose d-SPE kit based on matrix characteristics
8) Transfer 1-8ml alliquot, vortex 1 minute
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9) Centrifuge
11) Analyze by GC/MS or LC/MS
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QuEChERS Applications• Pesticides• PAHs• Hormones• Antibiotics• Acrylamides • Vet Drug Residues
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• Animal tissue• Fish and Shellfish• Fruits and Vegetables• Food oils• Soil• Baby Food
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Questions?