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

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. Basic Electrospray SchematicN
Formation of gas phase ions from solution phase
760 Torr
10-2 –
10-4 Torr
10-5 Torr
10-5 Torr
1 Torr
1. Preformed sample ions in mobile phase leaving HPLC and entering the ESI/MS detector through this needle
2. Eluent is sprayed into charged droplets in the casing where a high potential field was applied to stabilize and the electrostatic charges from neutralization.
3. With the help of desolvation and nebuilizing gases, evaporation of solvent takes place
4. Gas phase ions are formed in this chamber
5. they are accelerated by a voltage drop along the capillary to supersonic speed entering the first vacuum area where gas phase reactions such as formation of clusters with the eluent component / fragmentation happen.
6. Ions are guided by series of ion optics thru the skimmer into the quadropole
where fragmentation and mass selection occurs under high vacuum
Ionized samples can be analyzed in +ve or -ve ion mode depends on whether we are looking at +ve charged or -ve charged ion. MS manufacturers have different design and arrangement, but general mechanism stays the same.
Next, let’s take closer look at how droplets and gas phase ions are formed in this region
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