1. OUTLINE Introduction to Mass Spectrometry Ionization Methods
Mass Analyzer
Fragmentation and MS Interpretation

2. Chemical Identification
Comparison of
Physical Properties
Boiling Point
Melting Point
Density
Optical rotation
Appearance
Odor
Elemental Analysis
Burn the compound and measure the amounts of CO2, H2O and other components that are produced to determine the empirical formula

3. Spectroscopic Methods for Structure Determination
Ultraviolet-Visible (UV/Vis) spectroscopy:
determination of solutions of transition metal ions and highly conjugated organic compounds
Infrared (IR) spectroscopy:
Functional groups
Mass spectrometry (MS):
Molecular mass and formula and structure information
Nuclear magnetic resonance (NMR) spectroscopy:
Map of carbon-hydrogen framework

4. Definition of Mass Spectrometry
Mass spectrometry (MS) :
An analytical technique by using mass spectrometry for the determination of the composition of a sample or molecule and elucidation of the chemical structures of molecules, such as peptides and other chemical compounds.
Mass spectrometry has been described as the smallest scale in the world, not because of the mass spectrometer’s size but because of the size of what it weighs -- molecules.

5. What information can be determined?
Molecular weight
Molecular formula (HRMS)
Structure (from fragmentation fingerprint)
Isotopic incorporation / distribution
Protein sequence (MS-MS)

6. MS Principles Find a way to “charge” an atom or molecule (ionization)
Place charged atom or molecule in a magnetic field or subject it to an electric field and measure its speed or radius of curvature relative to its mass-to-charge ratio (mass analyzer)
Detect ions using microchannel plate or photomultiplier tube

7. General principles on mass spectrometry
Sample introduction
Ionization of sample in the ion source
Separation of ions by mass analyzer
Detector
Computer system
Resulting chromatogram

8. Analysis by GC-MS GC MS Mixture Separation Identification B A C*
07/16/96
Analysis by GC-MS GC MS
Mixture
Separation
Identification B
m/z A
m/z C
m/z
A + B + C B A C 8 * ##

9. Components of a mass spectrometry
Instrumentation
Components of a mass spectrometry
1) Inlet system : introduce a very small amount of sample ( micromole or less) into the mass spectrometer, where its components are converted to gaseous ions.
2) Ion source : converts the components of a sample into ions by bombardment with electrons, ions, molecules, or photon.
3) Mass analyzer : separates the analyte ions according to their m/z ratios.
4) Detector : converts the beam of ions into an electrical signal(currents) ; the detector output can be displayed or stored, to yield the mass spectrum.
5) Electronics of power supply and control of the systems.
6) Vacuum systems : maintain low pressures ( 10–5 to 10–8 torr); rotary vacuum oil pump, diffusion pump, turbomolecular pump.

10. Three Components of an MS
A typical mass spectrometer contains
Ionizer
Mass analyzer
Detector
Ion source charges the to-be-measured molecules.
Charge can be negative but often positive.
Two common types: MALDI and ESI.
John B. Fenn & Koichi Tanaka 2002 Nobel Prize in Chemistry for Electrospray and MALDI
Mass analyzer separates ions according to the mass to charge ratio (m/z) of the ions.
Iontrap, TOF, Quadrupole, FTICR.
Detector detects the ions.

11. Ionization Methods Electron bomb Ionization EI Chemical Ionization
Matrix Assisted Laser Desorption Ionization
Fast atom bombardment
Electro Spray Ionization

12. Sample introduction / ionization method:
Typical Analytes
Sample Introduction
Mass Range
Method Highlights
Electron  Impact (EI)
Relatively  small  volatile
GC or  liquid/solid  probe
to  1,000  Daltons
Hard method  versatile  provides  structure info
Chemical Ionization (CI)
Soft method  molecular ion  peak [M+H]+
Electrospray (ESI)
Peptides  Proteins  nonvolatile
Liquid  Chromatography  or syringe
to  200,000  Daltons
Soft method  ions often  multiply  charged
Fast Atom Bombardment (FAB)
Carbohydrates  Organometallics  Peptides  nonvolatile
Sample mixed  in viscous  matrix
to  6,000  Daltons
Soft method  but harder  than ESI or  MALDI
Matrix Assisted Laser Desorption  (MALDI)
Peptides  Proteins  Nucleotides
Sample mixed  in solid  matrix
to  500,000  Daltons
Soft method  very high  mass

13. Electron Ionization ( EI )
Sample is heated and energized by a beam of electrons, usually gives a molecular ion (M+) and a lot of fragments。 M+
(M-R2)+
(M-R3)+
Mass Spectrum
(M-R1)+

14. Electron Impact Ionization (EI)
Most widely used method
Analytes are bombared with high-energy electrons (usually 70eV)
As a result of collision, an electron is removed from the analytes (M), generating a molecular ion M+ (radical cation)
M + e M+ + 2e-

15. Electron Impact Ionization (EI)
Due to excess internal energy, fragmentation of the molecular ion will occur.
The fragmentation is reproducible and characteristic of the compund.
It is also possible to predict the fragmentation on the basis of chemical structures which is why MS is good tool for structure elucidation of unknown compounds

16. Chemical Ionization (CI)
Softer ionization technique Less fragmentation  Easier to find molecular ions.
Two different modes: Negative chemical ionization (NCI) and Positive Chemical Ionization (PCI).
NCI is used for analytes that are able to form stable negative ions, for example samples containing acidic groups or halogens. NCI is often used to analyze pesticides (contains Cl or Br).
PCI is used for samples that can form positive ions (most compounds).

17. Chemical Ionization (CI)
The principle for NCI and PCI is similar:
Reagent gas (usually methane, isobutane or ammonia) is introduced into the source where it is ionized:
PCI (simplified): CH4 + e- CH4+ +2e-
CH4 + CH4+ CH3 + C H5+
NCI: CH4 + e- CH4-
The ionized gas collide with the sample molecules generating a [M+H]+ or [M+H]- ion that is detected:
PCI: CH5+ + M  [M+H]+ + C H4
NCI: CH4- + M  [M+H]- + C H4

18. Field ionization (FI)
Field ionization (FI) is a method that uses very strong electric fields to produce ions from gas-phase molecules. 阳极 + 阴极
d<1mm

19. Field ionization (FI)

20. Matrix Assisted Laser Desorption Ionization (MALDI)
sample is co-crystallized with a matrix and then irradiated
with laser.
MALDI is achieved in two steps. In the first step, the compound to be analyzed is dissolved in a solvent containing in solution small organic molecules, called the matrix. The second step occurs under vacuum conditions inside the source of the mass spectrometer.

21. Properties of MALDI Good solubility
Vapour pressure must be sufficiently low to maintain vacuum conditions
Viscosity must allow diffusion of the analyte from the bulk to the surface
Polar : to solvate and separate preformed ion
Less Sensitive to Salts
Lower PRACTICAL detection limits
Easier to interpret spectra (less multiple charges)
Quick and easy
Higher mass detection
Higher Throughput (>1000 samples per hour)

22. Principle of MALDI
MALDI mass spectrometry has become a powerful analytical
tool for both synthetic polymers and biopolymers.

23. ElectroSpray Ionization (ESI)
Electrospray is abbreviated to ESI ，ample is sprayed out of
a narrow nozzle in a high potential field. Generates positive
(M+nH)n+ and negative (M - nH)n- ions and almost no
fragmentation. Generates multiple charged ions.

24. Electrospray (Detail)

25. Electrospray Ionization
Can be modified to “nanospray” system with flow < 1 mL/min
Very sensitive technique, requires less than a picomole of material
Strongly affected by salts & detergents
Positive ion mode measures (M + H)+ (add formic acid to solvent)
Negative ion mode measures (M - H)- (add ammonia to solvent)

26. Fast atom bombardment ( FAB)
Softer than EI and CI. Ions are produced by bombardment with heavy atoms. Gives (M+H)+ ions and litle fragmentation. Good for more polar compounds.
Ar + e Ar acceleration (5-15 KeV)
Ar Ar Ar + Ar+
fast slow KeV
fast slow

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

28. Mass analyzers
After analytes have been ionized they are separated according to their mass-to-charge ratio (m/z) in a mass analyzer (mass filter).
Quadrupoles and ion traps are common mass filters in GC-MS systems.
Time of flight (TOF) mass filter is very much used nowadays in LC-MS systems.
Sample introduction
Ionization of sample
Separation of ions by mass analyzer
Detector
Computer system

29. Transport of ions to the mass filter
The ionization takes place in the ion source.
Ions are then transported to the mass filter by focusing lenses. These have a voltage running through them and by either attracting or repelling the ions they guide them into the mass filter.

30. Different Mass Analyzers
Magnetic Sector Analyzer (MSA)
High resolution, exact mass, original MA
Quadrupole Analyzer (Q)
Low (1 amu) resolution, fast, cheap
Time-of-Flight Analyzer (TOF)
No upper m/z limit, high throughput
Ion Trap Mass Analyzer (QSTAR)
Good resolution, all-in-one mass analyzer
Ion Cyclotron Resonance (FT-ICR)
Highest resolution, exact mass, costly

31. Magnetic Sector Analyzer
Magnetic sector analyzer – Uses electric and/or magnetic fields to separate ions

32. Advantages
Double focusing magnetic sector mass analyzers are the "classical" model against which other mass analyzers are compared.
Classical mass spectra
Very high reproducibility
Best quantitative performance of all MS analyzers
High resolution
High sensitivity
10,000 Mass Range
Linked scan MS/MS does not require another analyzer

33. Disadvantages Applications Requires Skilled Operator
Usually larger and higher cost than other mass analyzers
Difficult to interface to ESI
Low resolution MS/MS without multiple analyzers
Applications
All organic MS analysis methods
Accurate mass measurements
Quantitation
Isotope ratio measurements

34. Time of Flight Analyzer
TOF analyzer – ions are accelerated through a flight tube and the time of light to the detector is measured

35. Ions are accelerated and their time of flight to the detector is measured.

36. Principle of TOF Analyzer
Uses a pulse of ion mixtures, not steady stream
Ions accelerated into drift tube by a pulsed electric
field called the ion-extraction field
Drift Tube is usually 1-2 m long, under vacuum
Ions traverse the drift tube at different speeds
( L / t ) = v = ( 2zV / m )½

37. Advantages of TOF Analyzer
Good for kinetic studies of fast reactions and for
use with gas chromatography to analyze peaks
from chromatograph
High ion transmission
Can register molecular ions that decompose in the
flight tube
Extremely high mass range (>1MDa)
Fastest scanning

38. Disadvantages
Requires pulsed ionization method or ion beam switching (duty cycle is a factor)
Low resolution (4000)
Limited precursor-ion selectivity for most MS/MS experiments
Applications
Almost all MALDI systems
Very fast GC/MS systems

39. Quadrupole Analyzers
Quadrupole analyzers – ions are filtered or trapped in a
device consisting of several metal rods using specifically tailored electromagnetic fields

40. Quadrupole Analyzers Electric/magnetic fields trap, store, eject ions
Requires an in-line quadrupole to act as
mass pre-filter
Contains a single ring electrode and a top
and bottom cap electrode
Varying RF frequency will vary the m/z ratios
that are trapped
Additional fragmentation can be performed
on ions stored in the ion trap

41. Easy to use ,simple construction,fast
Advantages
Easy to use ,simple construction,fast
Good reproducibility
Relatively small and low-cost systems
Quadrupoles are now capable of routinely
analyzing up to a m/q ratio of 3000,which is
useful in electrospary ionization of biomolecules,
which commonly produces a charge distribution
below m/z 3000

42. MS detectors Many different types available
Electron multipliers (EM) are often used
Continuous –Dynode Version mainly in GC-MS
Sample introduction
Ionization of sample
Separation of ions by mass analyzer
Detector
Computer system

43. Continious dynode EM
The EM multiplies incident charges, thereby amplifying the signal. The current is measured that is proportional to the amount of analyte in the sample.
EMs have limited lifetime which is dependent on the number of ions that hits the device, i.e., the amount of samples introduced and number of samples analyzed.
+Fast response
+ High sensitivity
Watson, Introduction to Mass Spectrometry, 4th ed

44. Ions are detected with a microchannel plate+ e -
primary ion L D
1000V
100V
L >> D

45. What does a mass spectrometer do?
1. It measures mass better than any other technique.
2. It can give information about chemical structures.
What are mass measurements good for?
To identify, verify, and quantitate: metabolites, recombinant proteins, proteins isolated from natural sources, oligonucleotides, drug candidates, peptides, synthetic organic chemicals, polymers

46. 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
Biomolecule characterization
Proteins and peptides
Oligonucleotides
Environmental analysis
Pesticides on foods
Soil and groundwater contamination
Forensic analysis/clinical

47. The mass spectrum shows the results
MALDI TOF spectrum of IgG
MH+
10000
20000
30000
40000
Relative Abundance
(M+2H)2+
(M+3H)3+
50000
100000
150000
200000
Mass (m/z)

48. ESI Spectrum of Trypsinogen (MW 23983)
M + 15 H+
1599.8
M + 16 H+
M + 14 H+
1499.9
1714.1
M + 13 H+
1845.9
1411.9
1999.6
2181.6
m/z
Mass-to-charge ratio

49. Protein Identification
1. Peptide Mass Finger Printing (PMF)
from MS data
2. Database search using fragment ion masses
from MS/MS data
3. Sequence Tags

50. What protein was isolated?
PROBLEM
Bank President
Who robbed the bank?
Biologist
What protein was isolated?

51. GATHER EVIDENCE Mass Spectrometrist
Police Officer
1. Interview witnesses
2. Dust for fingerprints
Mass Spectrometrist
1. Interview biologist who isolated the protein
2. Cleave protein to obtain peptide mixture
3. Analyze peptide mixture by MS to obtain peptide molecular masses!
enzyme

52. Approx. molecular weight: 30,000
DATABASE SEARCH
Mass Spectrometrist
Approx. molecular weight: 30,000
Origin: bovine liver
Peptide mass list from MS analysis: , , , ,
Police Officer
Height: 5’7”
Weight: 160 lbs
Gender: male
Age:
Fingerprints
search
search
DATABASE OF
KNOWN FELONS
PEPTIDE MASS
DATABASE
OF KNOWN
PROTEINS

53. DATABASE SEARCH RESULTS
Police Officer
Identifies the robber
Anthony J. Felon
Mass Spectrometrist
Identifies the protein
bovine carbonic anhydrase