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LC-MS

LC-MS. Lecture 7. LC-MS. LC. Interface. Mass Analyzer. Detector. Extraction of The analyte from The solvent. Ion evaporation or ionization. Fragmentation. LC – Separation of the mixture of analytes Interface – Separation of the analyte from the solvent

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LC-MS

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  1. LC-MS Lecture 7

  2. LC-MS LC Interface Mass Analyzer Detector Extraction of The analyte from The solvent Ion evaporation or ionization. Fragmentation • LC – Separation of the mixture of analytes • Interface – Separation of the analyte from the solvent • MA (mass analyzer) – separation of the analyte molecular ion and fragments according to their mass to charge ratio

  3. HPLC Liquid phase operation 25 - 50 deg. C No mass range limitations Inorganic buffers 1 ml/min eluent flow is equivalent to 500 ml/min of gas MS Vacuum operation 200 - 300 deg. C Up to 4000 Da for quadrupole MS Requires volatile buffers Accepts 10 ml/min gas flow PROBLEMS IN COMBINING HPLC AND MS

  4. Interface • Historical • Moving belt • Thermospray • Common interfaces • Electrospray • APCI • Special interfaces • Particle beam LC/MS • Continuous flow FAB • Atmospheric pressure photon ionization • MALDI

  5. Formation of gas phase ions from solution phase Basic Electrospray Schematic N 760 Torr 10-5 Torr 10-2 – 10-4 Torr 10-5 Torr 1 Torr Ion optics Richard B. Cole, Electrospray Ionization Mass Spectrometry, Fundamentals Instrumentation and Applications, Wiley Interscience, 1997

  6. API-Electrospray Interface (HP)

  7. Orthogonal ESI schematic (Waters)

  8. ZQ interface (Waters)

  9. ELECTROSPRAY • Factors to consider • Ionic strength • Surface tension of the solvent • Volatility of the solvent • Character of the analyte ions in solution: solvated, ion paired, etc • Mobile phase composition and amount of water • pH of the mobile phase

  10. APCI INTERFACE

  11. APCI IONIZATION CHAMBER

  12. Fragmentation

  13. PROTEIN SPECTRUM (multiple charge) (M+2)/(z+2) (M+1)/(z+1) M/z

  14. MASS ANALYZERS • Quadrupole • Ion Trap • Time-of-Flight • Magnetic Sector • ICR (Ion-cyclotron resonance)

  15. Resolution of MA

  16. Comparison of MA

  17. Quadrupole MA Quadrupole MA – Mass Filter Only ions with the specific M/z could pass between rods at set values of DC and RF voltages

  18. Quadrupole MA

  19. Electromagnetic field in Quadrupole

  20. Ion Trap

  21. Ion trajectory in Ion-Trap

  22. Time-of-Flight

  23. Bipolar TOF

  24. Reflectron Time-of-Flight

  25. MS-MS

  26. TRIPLE QUADRUPOLE

  27. MS-MS (triple quad – TOF)

  28. Magnetic Sector

  29. Double focusing Magnetic sector

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