1. Mass Spectroscopy What information can be determined?
Mass Spectrometry is an analytical spectroscopic tool primarily concerned with the separation of molecular (and atomic) species according to their mass.
What information can be determined?
Molecular weight
Molecular formula (HRMS)
Structure (from fragmentation fingerprint)
Isotopic incorporation / distribution
Protein sequence (MS-MS)

2. 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

3. Principles of Electron-Impact Mass Spectrometry
Atom or molecule is hit by high-energy electron e– 2

4. Atom or molecule is hit by high-energy electron
electron is deflected but transfers much of its energy to the molecule 2

5. This energy-rich species ejects an electron.2

6. This energy-rich species ejects an electron.
+ •
forming a positively charged, odd-electron species called the molecular ion 2

7. Atom or molecule is hit by high-energy electron from an electron beam at 10ev
+ •
forming a positively charged, odd-electron species called the molecular ion 2

8. Molecular ion passes between poles of a magnet and is deflected by magnetic field
amount of deflection depends on mass-to-charge ratio
highest m/z deflected least
lowest m/z deflected most
+ • 5

9. If the only ion that is present is the molecular ion, mass spectrometry provides a way to measure the molecular weight of a compound and is often used for this purpose.
However, the molecular ion often fragments to a mixture of species of lower m/z. 6

10. The molecular ion dissociates to a cation and a radical.
+ • 2

11. The molecular ion dissociates to a cation and a radical.+ •
Usually several fragmentation pathways are available and a mixture of ions is produced. 2

12. mixture of ions of different mass gives separate peak for each m/z
intensity of peak proportional to percentage of each ion of different mass in mixture
separation of peaks depends on relative mass + + + + + + 6

13. mixture of ions of different mass gives separate peak for each m/z
intensity of peak proportional to percentage of each atom of different mass in mixture
separation of peaks depends on relative mass + + + + + + 6

14. What’s in a Mass Spectrum?
Derived from molecular ion or higher weight fragments
Fragment Ions
[M+H]+(CI)
Or M•+ (EI)
“molecular ion”
Ion Abundance (as a %of Base peak)
Unit mass spacing
In CI, adduct ions, [M+reagent gas]+
Not usually scanned below m/z=32 (Why?)
High mass
Mass, as m/z. Z is the charge, and for doubly charged ions (often seen in macromolecules), masses show up at half their proper value

15. Mass Spectrum
Mass spectrum: A plot of the relative abundance of ions versus their mass-to-charge ratio (m/z).
Base peak: The most abundant peak.
Assigned an arbitrary intensity of 100.
The relative abundance of all other ions is reported as a % of abundance of the base peak.

16. Molecular Ion
Molecular ion (M): A radical cation formed by removal of a single electron from a parent molecule in a mass spectrometer = MW.
For our purposes, it does not matter which electron is lost; radical cation character is delocalized throughout the molecule; therefore, we write the molecular formula of the parent molecule in brackets with:
A plus sign to show that it is a cation.
A dot to show that it has an odd number of electrons.

17. Mass Spectrum

18. MS of dopamine
A partial MS of dopamine showing all peaks with intensity equal to or greater than 0.5% of base peak.

19. MASS SPECTROMETER
AN INSTRUMENT THAT GENERATES IONS FROM MOLECULES AND MEASURES THEIR MASSES
THE ESSENTIAL COMPONENTS OF A MASS SPECTROMETER:
SAMPLE
INLET
ION
SOURCE
ION
ACCELERATOR
ION
ANALYSER
signal
ION
DETECTOR
MASS SPECTRUM
COMPUTER
DATABASE

20. Diagram of a simple mass spectrometer
Illustration of the basic components of a mass spectrometry system.
Ionization
Source
Mass
Analzyer
Detector
selected
ions
Data
System
Inlet
all ions

21. Fig

22. (number of protons plus neutrons)
2. Atomic & Mass Number
mass number
(number of protons plus neutrons)
atomic number
(number of protons)
(number of electrons)

23. WAYS TO PRODUCE IONS
Electron impact (EI) - vapor of sample is bombarded with electrons: M + e 2e + M fragments
Chemical ionization (CI) - sample M collides with reagent ions present in excess e.g.
CH4 + e CH4.+ CH5+
M + CH5+ CH4 + MH+
Fast Atom/Ion Bombardment (FAB)
Laser Desorption & Matrix-Assisted Laser Desorption (MALDI)
- hit the sample with a laser beam
Electrospray Ionization (ESI) - a stream of solution passes through a strong electric field (106 V/m)

24. 1. Electron Ionization (EI)
Ionization Methods
1. Electron Ionization (EI)
most common ionization technique, limited to relatively low MW compounds (<600 amu)
2. Chemical Ionization (CI)
ionization with very little fragmentation, still for low MW compounds (<800 amu)
3. Desorption Ionization (DI)
for higher MW or very labile compounds
4. Spray ionization (SI)
for LC-MS, biomolecules, etc.

25. “hard” ionization method leads to significant fragmentation
Electron Ionization (EI)
vaporized sample is bombarded with high energy electrons (typically 70 eV)
“hard” ionization method leads to significant fragmentation
ionization is efficient but non-selective

26. Electron Ionization Advantages inexpensive, versatile and reproducible
fragmentation gives structural information
large databases if EI spectra exist and are searchable
Disadvantages
fragmentation at expense of molecular ion
sample must be relatively volatile

27. Chemical Ionization (CI)
Vaporized sample reacts with pre-ionized reagent gas via proton transfer, charge exchange, electron capture, adduct formation, etc.
Common CI reagents:
methane, ammonia, isobutane, hydrogen, methanol
“soft” ionization gives little fragmentation
selective ionization-only exothermic or thermoneutral ion-molecule reactions will occur
choice of reagent allows tuning of ionization

28. CI MS Sources CH4 CH4 CH4+ CH3+ CH2+ High Energy electrons 
Sample Molecule MH 
Molecule Ions 

29. Lets talk about mass! Atomic mass of Carbon Atomic mass of Chlorine
amu
Atomic mass of Chlorine
amu
Atomic mass of Hydrogen
amu
1amu = 1 dalton (Da)

30. Just for clarification
Atomic mass
amu, atomic mass units (uma??)
“Da” or Dalton.
kD (kiloDalton for macromolecules)
1 amu = *10-27 kg.
proton, mp = *10-27 kg,
neutron, mn = *10-27 kg.

31. Resolution
Resolution: A measure of how well a mass spectrometer separates ions of different mass.
low resolution: Refers to instruments capable of separating only ions that differ in nominal mass; that is ions that differ by at least 1 or more atomic mass units.
high resolution: Refers to instruments capable of separating ions that differ in mass by as little as atomic mass unit.

32. Resolving Power Example
RP= 3,000
RP= 5,000
RP= 7,000
C6H5Cl
C6H5OF
All resolving powers are FWHM

33. High Resolution MS High resolution data reports include ppm estimate
ppm = parts per million (1 ppm = %)
5 m/z 300 = 300 * (5/106) = ± Da
5 m/z 3,000 = 3,000 * (5/106) = ±0.015 Da
A molecule with mass of 44 could be C3H8, C2H4O, CO2, or CN2H4.
If a more exact mass is , pick the correct structure from the table:
C3H8 C2H4O CO2 CN2H4

34. Resolution
C3H6O and C3H8O have nominal masses of 58 and 60, and can be distinguished by low-resolution MS.
C3H8O and C2H4O2 both have nominal masses of 60.
Distinguish between them by high-resolution MS.
High resolution MS can replace elemental analysis for chemical formula confirmation

35. What about isotopes? Atomic Theory
Atomic number is the number of protons (+) in the nucleus and determines the element identity.
Isotopes of an element have a different number of neutrons in the nucleus. Electrons (-) form a cloud and most of the volume of the atom.
Electrons weigh very little. Atomic weight is basically the sum of the number of protons and neutrons.

36. Atomic mass of Chlorine Atomic mass of Hydrogen
Most elements have more than one stable isotope.
For example, most carbon atoms have a mass of 12 Da, but in nature, 1.1% of C atoms have an extra neutron, making their mass 13 Da.
Atomic mass of Carbon
amu for 12C but for 13C
Atomic mass of Chlorine
amu for 35Cl and for 37Cl
Atomic mass of Hydrogen
amu for H and for D!
Get it now?

37. Exact Masses of Some Common Elements and Their Isotopes:
Symbol
Exact Mass (u)
Rel. Abundance %
Hydrogen 1H
100.0
Deuterium
2H or D
0.015
Carbon 12
12C
Carbon 13
13C
Nitrogen 14
14N
Nitrogen 15
15N
Oxygen 16
16O
Oxygen 17
17O
Oxygen 18
18O
Fluorine
19F
Sodium
23Na
Silicon 28
28Si
92.23
Silicon 29
29Si
5.0634
Silicon 30
30Si
3.3612
Phosphorus
31P
Sulfur 32
32S
Sulfur 33
33S
Sulfur 34
34S
Sulfur 36
36S
Chlorine 35
35Cl
Chlorine 37
37Cl

38. Relative Isotope Abundance of Common Elements:
Relative Abundance
Carbon
12C
100
13C
1.11
Hydrogen 1H 2H
.016
Nitrogen
14N
15N
.38
Oxygen
16O
17O
.04
18O
.20
Sulfur
32S
33S
.78
34S
4.40
Chlorine
35Cl
 37Cl
32.5
Bromine
79Br
81Br
98.0

39. M+2 and M+1 Peaks
The most common elements giving rise to significant M + 2 peaks are chlorine and bromine.
Chlorine in nature is 75.77% 35Cl and 24.23% 37Cl.
A ratio of M to M + 2 of approximately 3:1 indicates the presence of a single chlorine in a compound.

40. M+2 and M+1 Peaks Bromine in nature is 50.7% 79Br and 49.3% 81Br.
A ratio of M to M + 2 of approximately 1:1 indicates the presence of a single bromine in a compound.

41. M+2 and M+1 Peaks
Sulfur is the only other element common to organic compounds that gives a significant M + 2 peak.
32S = 95.02% and 34S = 4.21%
Also 33S = 0.8%, an M+1 peak.
Because M + 1 peaks are relatively low in intensity compared to the molecular ion and often difficult to measure with any precision, they are generally not useful for accurate determinations of molecular weight.

42. Nobel Prizes in Mass Spectrometry
1906- J.J. Thomson- m/z of electron
1911- W. Wien- anode rays have positive charge
1922- F. Aston- isotopes (first MS with velocity focusing)
1989- H. Dehmelt, W. Paul- quadrupole ion trap
1992- R.A. Marcus- RRKM theory of unimolecular dissociation
1996- Curl, Kroto, and Smalley- fullerenes (used MS)
2002- J. Fenn- electrospray ionization of biomolecules
K. Tanaka- laser desorption ionization of biomolecules

43. Fragmentation or B+ + A· EI [M·]+ A+ + B· (neutral)
Better carbocation wins and predominates “Stevenson’s Rule”
Stevenson’s Rule:
For simple bond cleavage, the fragment with lowest ionization potential takes the charge
(in other words, the most stable ion is formed)

44. Fragment Ions
The Game is, to rationalize these in terms of the structure
Identify as many as possible, in terms of the parent structure
Generally, simply derived from the molecular ion
Or, in a simple fashion from a significant higher mw fragment.
Simply, here means, ions don’t fly apart, split out neutrals and then recombine.
Fragments will make chemical sense
A good approach is the “rule of 13” to write down a molecular formula for an ion of interest.
Especially in EI, we only identify major fragments

45. CI
[M+H]+ PH+ + N (neutral)
The “Even Electron Rule” dictates that even (non-radical) ions will not fragment to give two radicals (pos• + neutral•) (CI)
Loss of neutral molecules, small stable, from MH+
Loss of neutrals from protonated fragments
Subsequent reprotonation after a loss
Typically there is no ring cleavage (needs radical) or two bond scissions.
Depends highly on ion chemistry specifically acid-base (proton affinities)

46. Fragmentation
Governed by product ion stability
consideration
octet rule
resonance delocalization
polarizability and hyperconjugation
electronegativity

47. General Fragmentation Pathways
One-bond cleavages
a-cleavages
Cleave  to Heteroatoms like O, N
Observed in Mass Spec provided that a good stabilized carbocation can form
Heterolytic cleavage

48. Cleavage  to C=O groups+ : + : . O + O
neutral +
Prominent for ketones :
CH3C=O+ m/z=43 O :
Obs. in mass spec. Acylium ions are resonance-stabilized

49. Example Ethyl 3-oxo-3-phenylpropanoate (Mol. Wt.: 192.21)
M+• -45, loss of ethoxy radical

50. Example 1-Phenylpropan-2-one (Mol. Wt.: 134.18)
M+• -43; also tropylium ion

51. b-cleavages
Cleave  to a heteroatom (capable of supporting positive charge) R O :
Obs. in Mass Spec
Resonance stabilized
neutral + +
Note the use of “half arrow” for one-electron movements. e.g homolytic cleavage
examples
Primary alcohols, m/z =31 CH2=OH+
Primary amines, m/z =30 CH2=NH2+

52. Two-bond cleavages Eliminate H-X Retro Diels-Alder
McLafferty rearrangement
need g-hydrogens

53. Alkane Fragmentation
Long chains give homologous series of m/z = 14 units
Long chains rarely lose methyl radical
Straight chain alkanes give primary carbocation
Branched alkanes have small or absent M+
Enhanced fragmentation at branch points C H 3 + .
Obs. in Mass Spec
neutral