Sample Prep Options for GC users
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1 Sample Prep Options for GC users Tina Chambers Technical Specialist Sample Preparation
2 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
3 Matching SPP to Analytical Method All GC based systems require highest degree of cleanliness, regardless of detector!!
4 Sample Preparation Options Dilute and shoot Protein precipitation Liquid-liquid extraction/solid supported liquid extraction Solid phase extraction QuEChERS
5 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?
6 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!
7 Sample Preparation Supported LLE (SLE) Hydromatrix - diatomaceous earth sorbent Composed of fossilized diatoms Purified at high temperatures High surface area for water adsorption Very polar surface Chem Elut - pre-assembled cartridges with Hydromatrix Combilut - 96-well plate filled with Hydromatrix
8 The SLE Process Before Extraction Apply Sample Extract with Organic Solvent Dry sorbent Aqueous layer Organic layer
9 The Chem Elut Method Aqueous sample being applied Solid support adsorbs water onto high surface area particles Organic extraction solvent
10 Multi-Residue Confirmation of Pesticides in Honey using SLE App note SI Recovery comparison of pesticides between solid supported liquid-liquid extraction (SLE) on Chem Elut and classical liquid-liquid extraction (LLE).
11 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
12 Considerations for Solid Phase Extraction (SPE) - High degree of versatility (over 40 different chemistries available) - Concentrates dilute samples - Method development often necessary
13 Solid Phase Extraction Triangle Often, both the the analyte and the matrix are known, it is sorbent choice which is the critical component
14 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 addition Washing: Elimination of undesired interferences Elution: Selective desorption and collection of desired analytes from the sorbent
15 The Four Steps of SPE Selective Elution Green = Blue and Yellow Blue is more non polar than yellow Blue is retained Sample loading Retention Rinsing Elution
16
17 Silica VS. Polymer True polar/ion exchange possible Wide range of chemistries Wide range of established methods Can be more selective Conditioning is crucial Inherent hydrophobicity (conditioning) Higher capacity (sorbent mass/flow) Polarity gradient in Plexa
18 Polymers and Conditioning 450 ng/ml, LCUV 120% 100% 80% % Recovery 60% Methanol Aqueous Non-Conditioned 40% 20% 0% Atenolol Ranitidine Pseudoephedrine Quinidine Brompheniramine Mianserin Fluoxetine
19 Silica and Conditioning
20 Benefits by Design: Sorbent Quality Bond Elut Plexa Oasis HLB Bond Elut Plexa PCX Oasis MCX See Plexa Advantage Note, SI-1935 Confidentiality Label
21 Size Distribution Confidentiality Label May 30, 2013
22 Non-Polar Method Development
23 Method Development Considerations What is the partition coefficient (log P) of the un-ionized compound? log P octanol/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 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
24 Method Development Considerations Solubility characteristics of target compound?
25 Method Development Considerations Select suitable solvents Prepare 0%-100% concentrations Plot recoveries Elution Profile Elution Profile 0% MeOH 10% MeOH 20% MeOH 30% MeOH 40% MeOH 50% MeOH 60% MeOH 70% MeOH 80% MeOH 90% MeOH 100% MeOH
26 Method Development Consideration Highest % organic with low recoveries for wash Lowest % organic with high recoveries for elution Try acid/base modifiers Elution Profile 0% MeOH 10% MeOH 20% MeOH 30% MeOH 40% MeOH 50% MeOH 60% MeOH 70% MeOH 80% MeOH 90% MeOH 100% Elution Profile
27 Interactions on Ion Exchange Sorbents Silica base OH + N SO 3 - SO 3 NH 3 + CO 2 H CO 2 - Electrostatic attraction OCON(CH 3 ) 2
28 Ion Exchange Nomenclature STRONG: Ionic group is always charged (+ or -) WEAK: Ionic group is variably charged (+ or -) CATIONS: (+) Found in basic compounds ANIONS: (-) Found in acidic compounds Extract weak ions with strong exchangers and strong ions with weak exchangers!
29 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 SAX Phosphates, sulfates and other STRONG acids should be processed with weak anion exchangers (Bond Elut NH2, PSA)
30 Interactions on Ion Exchange Sorbents OH Silica base SO 3 - SO 3 50% NH 3 + CO 2 H If the pka=9 and the ph=9 50% NH 2 CO 2 H
31 Method Development Considerations What is the pka of your compound? pk a = -log K a and K a = [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
32 Interactions on Ion Exchange Sorbents OH Silica base SO 3 - SO 3 100% NH 3 + CO 2 H If the pka=9 and the ph=7 0% NH 2 CO 2 H
33 Interactions on Ion Exchange Sorbents OH Silica base 0% SO 3 - NH 3 + CO 2 H If the pka=9 and the ph=11 100% NH 2 CO 2 H
34 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 solvent Some organic should be present even with silica based ion exchangers because of carbon linkers
35 QuEChERS (Pronounced catchers ) Quick, Easy, Cheap, Effective, Rugged, and Safe Portmanteau: blend of 2 or more words Introduced in 2003: M. Anastassiades, S.J. Lehotay, D. Stajnbaher, and F.J. Schenck, J. AOAC Int 86 (2003) 412 Validated in 2005, with subsequent modification in 2007 AOAC and European Method EN Streamlined approach that makes it easier and less expensive to examine pesticide residues in food 5/30/2013
36 QuEChERS First step extraction 1) Weigh sample, add water if needed, spike 2) Add 10ml ACN 3) Vortex 4) Add salt packet 5) Shake 1 minute 6) Centrifuge at 4,000 rpm for 5 minutes
37 Second Step Dispersive SPE 7) Choose d-spe kit based on matrix characteristics 8) Transfer 1-8ml alliquot, vortex 1 minute 9) Centrifuge 11) Analyze by GC/MS or LC/MS Page 37
38 QuEChERS Applications Pesticides PAHs Hormones Antibiotics Acrylamides Vet Drug Residues Animal tissue Fish and Shellfish Fruits and Vegetables Food oils Soil Baby Food Page 38
39 Questions?
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