1. Mass Spectrometry
Obaid M. Shaikh

2. “Mass spectrometry is the branch of science dealing with all aspects of mass spectroscopes and the results obtained with these instruments.’’
1. It measures mass better than any other technique.
2. It can give information about chemical structures.
To identify, verify, and quantify: metabolites, recombinant proteins, proteins isolated from natural sources, oligonucleotides, drug candidates, peptides, synthetic organic chemicals, polymers etc

3. Applications of Mass Spectrometry
Pharmaceutical analysis
Bioavailability studies
Drug metabolism studies, pharmacokinetics
Characterization of potential drugs
Drug degradation product analysis
Screening of drug candidates
Identifying drug targets
Bio-molecule characterization
Proteins and peptides
Oligonucleotides
Environmental analysis
Pesticides on foods
Soil and groundwater contamination
Forensic analysis/clinical

4. Theory: Ionization
A gas phase molecule subjected to energy* greater than the ionization energy
electron can be removed which results in the formation of a molecular ion:

5. Working of mass spectrometer
Sample
Ion source: makes ions
Mass analyzer: separates ions
Mass spectrum: presents information

6. Mass Spectrometer Block Diagram
High Vacuum System
Ion
source
Mass
Analyzer
Data
System
Inlet
Detector

7. Mass Spectrometer Block Diagram
Turbo molecular pumps
High Vacuum System
Ion
source
Mass
Analyzer
Data
System
Inlet
Detector

8. Types of Vacuums Rough High Ultra High Mechanical Pumps
oil diffusion/ turbomolecular pump in series with a mechanical pump
Ultra High
cyropumps or ion pumps in series with mechanical pumps

9. Sample Introduction High Vacuum System Ion Source Mass Analyzer Data
Inlet
Detector
HPLC
Flow injection
Sample plate

10. Ion Source High Vacuum System Ion Source Mass Analyzer Data System
Inlet
Detector
MALDI
ESI
LSI (Liq. Sec. Ion)
FAB EI CI

11. Ionization Techniques
Gas-Phase Methods
Electron Impact (EI)
Chemical Ionization (CI)
Desorption Methods
Matrix-Assisted Laser Desorption Ionization (MALDI)
Fast Atom Bombardment (FAB)
Spray Methods
Electrospray (ESI)

12. Electron Impact Advantages Well-Established Fragmentation Libraries
No Supression
Insoluble Samples
Interface to GC
Non-Polar Samples
Disadvantages
Parent Identification
Need Volatile Sample
Need Thermal Stability
No Interface to LC
Low Mass Compounds (<1000 amu)
Solids Probe Requires Skilled Operator

13. Electron Impact

14. Chemical Ionization Disadvantages Advantages No Fragment Library
Parent Ion
Interface to GC
Insoluble Samples
Disadvantages
No Fragment Library
Need Volatile Sample
Need Thermal Stability
Quantitation Difficult
Low Mass Compounds (<1000 amu)
Solids Probe Requires Skilled Operator

15. MALDI: Matrix Assisted Laser Desorption Ionization
Sample plate
Laser hn
Sample is mixed with matrix (X) and dried on plate.
Laser flash ionizes matrix molecules.
Sample molecules (M) are ionized by proton transfer: XH+ + M  MH+ + X.
MH+
Grid (0 V)
+/- 20 kV

16. FAB: Fast Atom Bombardment
Advantages
Parent Ion
High Mass Compounds (10,000 amu)
Thermally Labile Compounds (R.T.)
Disadvantages
No Fragment Library
Solubility in Matrix (MNBA, Glycerol)
Quantitation Difficult
Needs Highly Skilled Operator
Relatively Low Sensitivity

17. Electrospray ionization
Advantages
Parent Ion
High Mass Compounds (>100,000 amu)
Thermally Labile Compounds (<0º C)
Easy to Operate
Interface to HPLC
Zeptomole sensitivity with nanospray
Disadvantages
No Fragmentation
Need Polar Sample
Need Solubility in Polar Solvent (MeOH, ACN, H2O, Acetone are best)
Sensitive to Salts
Supression

18. Mass Analyzer High Vacuum System Ion source Mass Analyzer Data System
Inlet
Detector
Double Focusing
Time of flight (TOF)
Quadrupole
Ion Trap
Magnetic Sector
FTMS

19. Mass analyzers separate ions based on their mass-to-charge ratio (m/z)
Operate under high vacuum (keeps ions from bumping into gas molecules)
Actually measure mass-to-charge ratio of ions (m/z)
Key specifications are resolution, mass measurement accuracy, and sensitivity.
Several kinds like, quadrupole, time-of-flight and ion traps etc. are mostly used.

20. Quadrupole Mass Analyzer
Uses a combination of RF and DC voltages to operate as a mass filter.
Has four parallel metal rods.
Lets one mass pass through at a time.
Can scan through all masses or sit at one fixed mass.

21. Quadrupoles have variable ion transmission modes
mass scanning mode m1 m3 m4 m2
single mass transmission mode m2 m3 m1 m4

22. Time-of-flight (TOF) Mass Analyzer
Source
Drift region (flight tube) + +
detector + + V
Ions are formed in pulses.
The drift region is field free.
Measures the time for ions to reach the detector.
Small ions reach the detector before large ones.

23. Quadrupole Ion Trap Electric/magnetic fields trap, store, eject ions
Varying RF frequency will vary the m/z ratios that are trapped
Ions may be stored either destructively or non-destructively
Additional fragmentation can be performed on ions stored in the ion trap
Cut away side view

24. Detector High Vacuum System Ion source Mass Analyzer Data System Inlet
Microchannel Plate
Electron Multiplier
Hybrid with photomultiplier
Faraday Cup

25. Electron Multiplier

26. Microchannel plate + e -
primary ion L D
1000V
100V
L >> D

27. Data System High Vacuum System Ion source Mass Analyzer Data System
Inlet
Detector

28. The mass spectrum shows the results as

29. Mass spectrometers get their names based upon Types of ion sources:
Electrospray (ESI)
Matrix Assisted Laser Desorption Ionization (MALDI)
Types of mass analyzers:
Quadrupole (Quad, Q)
Ion Trap
Time-of-Flight (TOF)
- Either source type can work with either analyzer type: “MALDI-TOF,” “ESI-Quad.”
Analyzers can be combined to create “hybrid” instruments. ESI-QQQ, MALDI QQ TOF, Q Trap etc.

30. Summary: acquiring a mass spectrum
Ionization
Mass Sorting (filtering)
Detection
Ion Source
Ion
Detector
Mass Analyzer
Form ions
(charged molecules)
Sort Ions by Mass (m/z)
Detect ions
100 75
Inlet
• Solid
• Liquid
• Vapor 50 25
1330
1340
1350
Mass Spectrum

31. Important Performance Factors
Mass accuracy: How accurate is the mass measurement?
Resolution: How well separated are the peaks from each other?
Sensitivity: How small an amount can be analyzed?

32. Gas Chromatography/Mass Spectrometry
MS is the most dependable technology for forensic chemistry
analysis of drugs and toxicological analysis
The use of combined gas chromatography-mass spectrometry (GS/MS) is applied in forensic chemistry
Gas chromatography helps provide the identification for the presence of a compound of interest
Mass spectrometer provides a fragmentation pattern, which can be compared with the referenced sample

33. MS/MS or Tandem Mass Spectrometry
MS/MS means using two mass analyzers (combined in one instrument) to select an analyte (ion) from a mixture, then generate fragments from it to give structural information.
Mixture of ions
Single ion
Fragments
Ion source
MS-1
MS-2

34. Thank you!