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LC-MS intro
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LC-MS
Lecture 7
LC-MS
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
Interface MassAnalyzer DetectorLC
Extraction ofThe analyte fromThe solvent
Ion evaporationor ionization.Fragmentation
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
Historical Moving belt Thermospray Common interfaces Electrospray APCI Special interfaces Particle beam LC/MS Continuous flow FAB Atmospheric pressure photon ionization MALDI
10-5 Torr
Richard B. Cole, Electrospray Ionization Mass Spectrometry, Fundamentals Instrumentation and Applications, Wiley Interscience, 1997
1 Torr10-2 – 10-4 Torr
Formation of gas phase ions from solution phase
760 Torr
Ion optics
10-5 Torr
N
Basic Electrospray Schematic
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• pH of the mobile phase
APCI INTERFACE
APCI IONIZATION CHAMBER
Fragmentation
PROTEIN SPECTRUM (multiple charge)
M/z
(M+1)/(z+1)
(M+2)/(z+2)
MM
M MMi
i ii
1
1
1
MASS ANALYZERS
Quadrupole Ion Trap Time-of-Flight Magnetic Sector ICR (Ion-cyclotron resonance)
Resolution of MA
Comparison of MA
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
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