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LC/MS WORKSHOP Iowa State University K amel H arrata

LC/MS WORKSHOP Iowa State University K amel H arrata. Instrument Description Data Acquisition Data Processing. What is Mass Spectrometry?.

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LC/MS WORKSHOP Iowa State University K amel H arrata

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  1. LC/MS WORKSHOPIowa State UniversityKamelHarrata • Instrument Description • Data Acquisition • Data Processing

  2. What is Mass Spectrometry? • Mass spectrometry is an analytical technique that involves the study in the gas phase of ionized molecules with the aim of one or more of the following: • Structural characterization • Gas phase reactivity study • Qualitative and quantitative analysis of components in a mixture. • Molecular weight determination

  3. LC/MS and GC/MS Mass Spectrometers GC/MS LC/MS Agilent QTOF 6540 Micromass GCT m/z Inlet Ion Source Analyzer Detector Q, QQQ EB, Ion Trap TOF, QTOF FT, Orbitrap Data System Direct Probe Flow Injection GC, LC EI, CI APCI, ESI EM, PM

  4. Applications of Mass Spectrometry • Pharmaceutical analysis • Biomoleculecharacterization • Proteins and peptides • Oligonucleotides • Environmental analysis • Pesticides on foods • Soil and groundwater contamination • Forensic analysis/clinical

  5. TOF equation

  6. Time of flight scan

  7. QTOF 6540 Capabilities • MS and MS/MS • Mass Accuracy of better than 3ppm • Resolution (FWHM) 45,000 for mass 1000 • Rapid switching between positive and negative ion modes

  8. LC System • 4 reservoir bottles A1, A2, B1, B2 • Binary Pump can handle up to 8000 psi • UV detector • LC Column housing • Autosampler

  9. Electrospray Ionization Desolvation + Coulomb Explosions

  10. Agilent Electrospray Ion Source (positive mode)

  11. Electrospray Spectrum of Myoglobin

  12. Relative Applicability of LC/MS Techniques

  13. Advantages of Electrospray • Soft ionization • LC/MS interface with best sensitivity • Extends mass range for multiply charged analytes • Works with a wide range of moderate to high polarity compounds • Low maintenance

  14. ElectrospraySolution Chemistry • - Mobile phase pH has a major effect for analytethat are ions in solution • - Basic pH (>7.0; 9 preferred) for negative ions • - Acidic pH (<7.0; 5 preferred) for positive ions • - Manipulation of pH can enhance performance for analytes that are not normally ionized in solution

  15. QTOF 6540 Ion Path Elements

  16. Mass Spectrometer Type: Mass Accuracy • Quadrupole 200-300 ppm • Linear Time of Flight 200 ppm • Reflectron Time of Flight 25-50 ppm • OR-Time of Flight 1-3 ppm • Fourier Transform MS 0.1-1 ppm

  17. Mass Accuracy-deviation in ppm

  18. Exact mass calculation and deviation

  19. How Much Mass Accuracy is needed? • Elemental composition implication

  20. TOF Resolution Resolution is the ability to distinguish between two mass peaks that are extremely close together Resolution = M / Peak Width (FWHM: full width at half maximum)

  21. LC/MS of SeMix(10 ug/ml) 10 ppm

  22. Qualitative data processingMass Hunter Qual software • Open File • Use default layout: Chromatogram, Mass Specta • Process and print data • Display mass spectrum • Average few scans • Background substraction • Peak integration

  23. Positive ion mode LC/ESI of SeMix

  24. Selected Ions from SeMix

  25. ESI Mass Spectra of Semix

  26. LC/ESI of Surfactine mixtures

  27. 14 Dalton difference between compounds

  28. Positive ion mode APCI of Carotenoids m/z Positive ion mode LC/APCI is the method of choice for analyzing and quantifying Carotenoids

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