LCMS Lecture 7 LCMS LC Interface Mass Analyzer

LC-MS LC Interface Mass Analyzer Detector Extraction of Ion evaporation The analyte from or

PROBLEMS IN COMBINING HPLC AND MS · · · HPLC Liquid phase operation 25

Interface w w w Historical Moving belt Thermospray Common interfaces Electrospray APCI Special interfaces

Basic Electrospray Schematic N 760 Torr 10 -2 – 10 -5 -4 Torr 10

ELECTROSPRAY Factors to consider • • • Ionic strength Surface tension of the solvent

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

MASS ANALYZERS w Quadrupole w Ion Trap w Time-of-Flight w Magnetic Sector w ICR

Quadrupole MA – Mass Filter Only ions with the specific M/z could pass between

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LC-MS Lecture 7

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

PROBLEMS IN COMBINING HPLC AND MS · · · 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

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

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

API-Electrospray Interface (HP)

Orthogonal ESI schematic (Waters)

ZQ interface (Waters)

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 p. H of the mobile phase

APCI INTERFACE

APCI IONIZATION CHAMBER

Fragmentation

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

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

Resolution of MA

Comparison of MA

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

Quadrupole MA

Electromagnetic field in Quadrupole

Ion Trap

Ion trajectory in Ion-Trap

Time-of-Flight

Bipolar TOF

Reflectron Time-of-Flight

MS-MS

TRIPLE QUADRUPOLE

MS-MS (triple quad – TOF)

Magnetic Sector

Double focusing Magnetic sector