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Sample Prep Options forGC users
Tina ChambersTechnical Specialist Sample Preparation
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
Matching SPP to Analytical Method
All GC based systems require highest degree of cleanliness, regardless of detector!!
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
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?
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!
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
The SLE Process
Aqueous layer
Organic layer
Dry sorbent
Before ExtractionApply
SampleExtract with
Organic Solvent
layer layer
The Chem Elut Method
Aqueous samplebeing applied
Solid support adsorbs water ontohigh surface area particles
Organic extraction solvent
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).
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
Considerations for Solid Phase Extraction (SPE)
- High degree of versatility (over 40 different chemistries available)
- Concentrates dilute samples
- Method development often necessary
Solid Phase Extraction Triangle
Often, both the the analyte and the matrix are known, it is sorbent choice which is the critical component
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
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
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
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
Silica and Conditioning
Benefits by Design: Sorbent Quality
Bond Elut Plexa Oasis HLB
Bond Elut Plexa PCX Oasis MCX
See Plexa Advantage Note, SI-1935Confidentiality Label
Size Distribution
May 30, 2013Confidentiality Label
Non-Polar Method Development
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
Method Development Considerations
Solubility characteristics of target compound?
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
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
NH3+
Silica baseOH
SO3-
Interactions on Ion Exchange Sorbents
Electrostatic attraction
CO2H
SO3
CO2-
OCON(CH3)2
+N
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!
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)
NH3+
Silica base
OH
SO3-
Interactions on Ion Exchange Sorbents
50%
CO2H
SO3
CO2H
NH2If the pKa=9
and the pH=9
50%
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
NH3+
Silica base
OH
SO3-
Interactions on Ion Exchange Sorbents
100%
CO2H
SO3
CO2H
NH2If the pKa=9
and the pH=7
0%
NH3+
Silica base
OH
SO3-
0%
Interactions on Ion Exchange Sorbents
CO2H
CO2H
NH2If the pKa=9
and the pH=11
100%
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
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
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
Second Step – Dispersive SPE
7) Choose d-SPE kit based on matrix characteristics
8) Transfer 1-8ml alliquot, vortex 1 minute
Page 37
9) Centrifuge
11) Analyze by GC/MS or LC/MS
QuEChERS Applications• Pesticides• PAHs• Hormones• Antibiotics• Acrylamides • Vet Drug Residues
Page 38
• Animal tissue• Fish and Shellfish• Fruits and Vegetables• Food oils• Soil• Baby Food
Questions?