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