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Michelangelo Anastassiades

Matrix Effects in GC and LC. Michelangelo Anastassiades. GC-Injection - Split/Splitless. GC-Injection – A Critical Step. By Hans Mol. GC-Injection – A Critical Step. By Hans Mol. Overload. Liner volume.

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Michelangelo Anastassiades

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  1. Matrix Effects in GC and LC Michelangelo Anastassiades

  2. GC-Injection - Split/Splitless

  3. GC-Injection – A Critical Step By Hans Mol

  4. GC-Injection – A Critical Step By Hans Mol Overload

  5. Liner volume • The gas volume of the sample injected should be smaller than the internal volume of the liner. • If not, the liner is overloaded and backflash of the sample into various parts of the injector may occur. • Poor injection profiles • Bad peak-shapes • Adsorption onto various parts of inlet and possible carry over. • Typical liners for split/splitless injection are ~80 x 4mm = ~1ml volume • NOTE: Effective vol. available is lower as part of the liner will be filled with carrier gas.

  6. GC-Injection Expansion-Volumes of Solvents boiling point density MW C g/ml μl vapor/μl injection water 100 1 1491 18 41 acetonitrile 82 0.78 511 58 acetone 56 0.79 366 88 ethyl acetate 77 0.9 275 toluene 111 0.87 254 92 hexane 69 0.66 206 86 isooctane 114 99 0.69 162 GC: 30 m x 0.25 mm, 70C, 60kPa

  7. Large Volume Injections By Hans Mol

  8. PTV for Large Volume Injections By Hans Mol

  9. Requirements for robust GC analysis • Avoid water • - activates liner, retention gap, column • - damages filament • - has large expansion volume (upon evaporation) • Note: Water can be conveniently evaporated using PTV Azeotrop: MeCN:H2O = ~84:16- if H2O content is lower it will concentrate to 16% and then evaporate together w. MeCN; - if content is higher water will remain in the system • Avoid non-volatile co-extractants (incl. Fat=triglycerides) • - contaminate liner/retain less volatile analytes • Avoid too high concentrations of volatile co-extractants • - interfere in the chromatograms

  10. Interdependence of analytical steps High Selectivity in Instrumental Analysis gives more flexibility in sample preparathion GC analysis Sample preparation GC-FID GC-ECD GC-NPD GC-FPD GC-MS (quad/IT/TOF) GCxGC-MS, GC-MS/MS, GC-hrMS GCxGC-hrMS, GC-QTOF By Hans Mol MIPs/Immunoaffinity Column chromatography Solid Phase Extraction Dispersive SPE Selective extraction solvent Generic extraction solvent Increasing selectivity Increasing selectivity  Combination should be fit-for-purpose

  11. Decisions Regarding Instrumental Analysis Questions: What are the target analytes? What is the matrix? What is the desired LOQ? What instrumentation is available in your laboratory? Analytes amenable to LC-MS/MS? no yes GC analysis LC-MS/MS available? no yes use LC-MS/MS (+ GC for confirmatory purposes if possible) By Hans Mol

  12. Dealing with Matrix-Effects in GCwith the help of Analyte Protectants (APs)

  13. WHAT ARE MATRIX- EFFECTS ???

  14. GC-Injector schematically GC-Injection Syringe Pneumatics GC-liner Heater z.B. 250°C GC-capillary Sample injection

  15. Analyte: Atrazine ; Matrix: Strawberry 30000 25000 20000 15000 10000 5000 0 8.70 8.75 8.80 8.85 8.90 8.95 9.00 9.05 9.10 9.15 9.20 9.25 9.30 9.35 9.40 9.45 RT= 8,80 min WITH Matrix co-extractives (Strawberry-Extract) WITHOUT Matrix co-extractives (in pure solvent) e.g. Calibration standard RT= 8,92 min • Stronger Tailing • Apex-Shift towards longer RTs! • Ratios: • Peak-Areas: ~ 1,5:1 • Peak-Heights: ~ 4:1 • Peak-Width (at half height): ~ 1:3 Matrix-Induced Peak Enhancement • OVERESTIMATION OF RESULTS!!

  16. „Matrix-Induced Peak Enhancement Effect“ GC-Liner GC-Capillary Active Sites (on Surface of GC-Liner & Column )(Siloxanes & deposited non-volatile matrix-co-extractives) • Analytes (interact with Active Sites) • Unwanted Retention/Tailing • Quasi-catalysed degradation (susceptible compounds) • Matrix-Components (in Excess) • Bloc active sites and protect analytes

  17. Factors influencing in matrix-effects • Number and type of active sites in the inlet and GC column • Chemical structure of the analytes:  H-bondingability, thermolability, volatility • Analyte concentration most pronounced at trace level • Injection temperature • Interaction time function of: analyte volatility (), inlet temperature (), flow rate, gas pressure, injection volume, solvent expansion volume, column diamensions • Matrix type and concentration

  18. Influence of Analyte Concentration 100 % 100 % START 95 % 10 % FINISH

  19. An additional effect… „Response Diminishment” Accumulation of non-volatile matrix compounds on the surface of liner and a first part of GC column Formation of new active sites Increasing number of injections of matrix-containing extracts Gradual decrease in analyte responses and/or increase in peak-tailing

  20. Matrix Effects Response enhancement Response diminishment

  21. Elimination orCompensation for Matrix Effects? ELIMINATION Impossible in practice Completely remove active sites in GC system Completely remove matrix compounds COMPENSATION Standard addition Matrix-matched standards Isotopically labeled internal standards Impractical for routine Practical Analyte protectants

  22. Compensation of matrix effects (1) • Method of standard additions • extra effort • possible inaccuracies due to : • concentration dependence of matrix effects • non-linear response of detector • deterioration of the system as the samples are injected

  23. Method of standard additions Withdraw from extract multiple aliquots of same volume and spike them with increasing amounts of standard No spiking here Peak Ratio Analyte/ISTD Good linearity is paramount because of extrapolation y-intercept ׀x׀ = Slope In case of violations: Standardaddition x Added amount of analyte to the aliquot x: absolute amount of analyte in aliquot before spiking (y=0)

  24. Compensation of matrix effects (2) • Use of matrix-matched standardisation •  need for enough blank matrix (ideally exactly the same as the samples) and its long-term storage •  extra time, labour, and expense for preparation of the blank extracts for calibration standards •  greater amount of matrix material injected onto the column in a sequence  greater GC maintenance •  potentially greater potential for analyte degradation in the matrix solution

  25. Compensation of matrix effects (3) • Isotopically labelled internal standards • Generally not available for all pesticides (or expensive if available) • Restriction in the use of MS techniques • Occupy Measurement time (theoretical consideration) • Obviate the need for Matrix-Matching

  26. Use of isotopically labelled ISTD in MS Chlormequat Chlormequat D4 MW 122.6 Formula: C5H13ClN MW 126.6 Formula C5H9D4ClN Different mass, but exactly same behaviour during extraction, cleanup and chromatography!

  27. Use of isotopically labelled ISTD in MS Co-elution Co-elution Co-elution Co-elution Different mass, but exactly same behaviour during extraction, cleanup and chromatography!

  28. Use of isotopically labelled ISTD in MS Folpet Folpet D4 MW 296.6 Formula C9H4Cl3NO2S MW 300.6 Formula C9D4Cl3NO2S Different mass, but exactly same behaviour during extraction, cleanup and chromatography!

  29. FOLPET Phthalimide CAPTAN Tetrahydrophthalimide Degradation of Folpet and Captan • Degradation at • High pH • High temperatures • e.g.: • During sample prep. • In final extract • In GC-inlet Similar behaviour: • Dicofol, Captafol, Tolylfluanid, Dichlofluanid, Pyridate

  30. Alternative: Isotopically Labelled ISTDs Folpet in Papaya(MRL=0.01 mg/kg) Captan in Blueberries(MRL=0.01 mg/kg) Only via Area Only via Area ISTDs added to extract Unacceptable R2 Should be at least 0.995 0.019 mg/kg 0.030 mg/kg R2 =0.9436 R2 =0.8318 via ISTD (PCB 138) via ISTD (PCB 138) Violation 0.033 mg/kg 0.022 mg/kg R2 =0.9585 R2 =0.7960 via ISTD (Captan D6) via ISTD (Folpet D4) 0.030 mg/kg 0.018 mg/kg No violation R2 =0.9979 R2 =0.9979 Dicofol is the next to check using isotopically labelled ISTD

  31. THE USE OF ANALYTE PROTECTANTS

  32. WHAT ARE ANALYTE PROTECTANTS? PROTECTANT ? ANALYTE

  33. PROTECTANT LORD ANALYTE ?

  34. DANGER ? ANALYTE PROTECTANT

  35. PROTECTANT ANALYTE ?

  36. PROTECTANT ? ANALYTE

  37. Dispersive SPE –Removal of Co-extractives Solutions:  Addition of Acids (see later)  Addition of Analyte Protectants • Drawbacks: • pH rises (degradation risk) • Matrix-Induced Analyte Protection reduced

  38. Analyte Protectants Principle Cleaned-up Extract + AP Standard + AP Addition of „Analyte Protectants“ (AP) Raw Extract PSA cleanup Cleaned up Extract „ Protection“ Standard in Pure Solvent Analyte Protectants help to reduce analyte Interactions with Active Sites and thus Errors related to Matrix-Induced Peak Enhancement in GC

  39.  (no peak in solvent) Use of Analyte Protectants to improve GC-Analysis • Extract acidification improves GC-behaviour of Folpet, Captan and Dicofol • But better results by addition of ANALYTE PROTECTANTS-MIX to extracts • Folpet, Captan work perfectly  • Dicofol still not fully satisfactory  Overestimations if quantified against solvent-based calibr. std

  40. Analyte Protectants-Reduction of Matrix Induced Enhancement Errors Response in Matrix Response in Solvent 1,0 no AP with AP Errors eliminated if: Response in Matrix/Response in Solvent ~ 1 Overestimations when using Standards in Solvent AP was added to both :Sample Extract and Calibration Standard (in pure Solvent)

  41. Analyte Protectants – Examples • Various Compounds Tested for “Protective Potential”. • Best Protection : Polyhydroxy-Compounds (sugars, ~derivatives) Examples: Ethylglycerol δ-Gulonolactone Sorbitol APs typically give broadly eluting peaks For protection over a broad volatility range use AP-Mixtures as each covers a different volatility range

  42. GCGC/TOF-MS of Analyte Protectants By J. Hajslova Degradation products of L-Gulonic acid γ-lactone D-Sorbitol 3-Ethoxy-1,2- propandiol (end of the tail) Second dimension (BPX-50) Elution of pesticides First dimension (DB5-ms)

  43. Comparison of Generated Data Accurate data over a broad analyte spectrum ! By J. Hajslova 3-ethoxy-1,2-propandiol (4 mg/mL) L-gulonic acid γ-lactone (1 mg/mL) D-glucitol (1 mg/mL) 1450% Baby food 0.015 mg/kg 1340%  Without analyte protectants With analyte protectants

  44. Analyte Protectants- Desirable properties • Strong interactions with active sites (H-Bond activity) • Similar volatility to analytes to be protected (so that protection extents during entire run) • Soluble in sample extract (not in non-polar solvents) • Not accumulating in GC-system • Not reactive with analytes (not inducing their degradation) • Minimal interference with analyte detection (small m/z) • Not deteriorating GC-column separation performance • Cheap and not hazardous

  45. Active Site masked by AP Active Sites Aktive Stelle AP- Elution-Band Pestizide Pesticides Pestizid

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