1. Mass Spectrometry
Implication is, we don’t get the sample back; a destructive method
A key Tool for the chemist’s toolbox.
The logic is, we always want the molecular weight.
Second, we can smash out fragments that are intact structurally
These are easier to solve and relate back to the starting structure

2. 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

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

4. How does a mass spectrometer work?
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. Sample Introduction High Vacuum System Ion Source Mass Analyzer Data
Inlet
Detector
HPLC
Flow injection
Sample plate

9. Ion Source High Vacuum System Ion Source Mass Analyzer Data System
Inlet
Detector
MALDI
ESI
FAB
LSIMS EI CI

10. Ion Sources make ions from sample molecules (Ions are easier to detect than neutral molecules.)
Electrospray ionization:
High voltage applied
to metal sheath (~4 kV)
Sample Inlet Nozzle
(Lower Voltage)
Charged droplets +
MH+
MH3+
MH2+
Pressure = 1 atm Inner tube diam. = 100 um
Sample in solution N2
N2 gas
Partial vacuum

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

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

13. 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 exist: for bioanalysis, quadrupole, time-of-flight and ion traps are most used.

14. Mass Spectrometry
A primary tool for chemists from almost every discipline
Molecular Weights are fundamental to almost every structural question. Molecular weight is not ambiguous. A compound has a unique MW.
Our ability to analyze compounds on this basis, depends completely on being able to generate ions from the compound. Specifically molecular ions*, whose weight is equal to the MW of the compound, are critical.
Once produced, our analysis according to MW depends on differential mobility or acceleration of ions proportionate to the MW.
*We can usefully broaden this definition from [M]+ to embrace [M+H]+, [M-H]-, Chemical Ionization adducts, etc.

15. 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
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

16. Molecular Ions give us the molecular mass
Dislodges an electron
Electron Impact M
-e•
2e-•
M+•
Chemical Ionization M H+
[M+H]+
Weighs one more than MW

17. ElectronImpact and ChemicalIonizationEI
Sometimes too energetic for molecular ion to survive
Rich harvest of fragment ions
“fingerprint” nature of fragment patterns lends itself to database library searches CI
Stronger, more reliable molecular ions
Fewer fragments
Can choose different reagent gasses and exploit chemistry, giving different fragmentation. e.g. NH3/ND3
Adduct ions give support to identities
Nitrogen rule works but inverted
Can do negative ion Mass Spec EI CI

18. When would you use CI, EI? EI CI
When “fingerprint” is needed for Identification by comparison screening in databases
Trace analysis
Forensics
Environmental
Total unknowns, e.g natural products
Fragment homology within a series, e.g. of natural products CI
When rapid, reliable identification of molecular ion is needed.
LC-MS
Following a synthetic chemistry route, tentative impurity ID
Biological samples, other fragile or sensitive to decomposition; Drug or other metabolite ID
When reagent gas chemistry is key, e.g. exchange D in for H
Minimize fragmentation, get most intensity in molecular ion.

19. How can I tell which (EI or CI) was run?
Chemical Ionization
Adduct ions higher m/z than MH+, ,[M+C2H5]+ ,[M+C3H5]+ [M+NH4]+
Large molecular ion
Relatively few fragment ions
Electron Impact
No ions higher m/z than M•+
Smaller M•+ intensity
Rich family of fragment ions

20. The “Rule of 13” as an aid to guessing a molecular Formula
Take the Weight of ion, divide by 13
This answer is N, for (CH)N and any numerical remainder is added as H
e.g.; 92
92/13 = 7 with remainder = 1; C7H8 weighs 92. This is our candidate formula
Can evaluate other alternative candidate formulas possessing heteroatoms. For each member of the list below, replace the indicated number of CHs in the above answer
Hetero substitution
CH replacement O
CH4 P
C2H7 N
CH2 S
C2H8
O+N
C2H6
O+S C4 F
CH7 I
C10H7 Si
C2H4
Cl,Br
(use isotopes)

21. Fragmentation or B+ + A· EI [M·]+ A+ + B· (neutral)
Better carbocation wins and predominates (“Stevenson’s Rule”) 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)

22. Reading a Mass Spec from the M+• Down (EI)
Fragment
Due to loss of…
Interpretation
M+• -1
-H•
Aldehydes, tert. Alcohols, cyclic amines
M+• -2
Multiple -H•
Secondary alcohols
M+• -3
Primary alcohols
M+• -4 to -13
(doubtful)
Consider contaminants
M+• -14
CH2• , N• not good losses
M+• -15
CH3•
Available methyl groups, methylesters
M+• -16 O•
Peroxides
M+• -17
OH•
Alcohols, phenols, RCO2H
M+• -18
H2O
alcohols
M+• -19
-F•
M+• -20
-HF
M+• -21 to -25
No peaks expected
M+• -26
HCCH
M+• -27
•HC=CH2 or HCN
HCN from pyridine, anilines
M+• -28
CO or CH2=CH2
Check for McLafferty R&R

23. Nominal Mass
Here for example is a list of the compounds in the Merck Index (9th ed) that weigh nominally, 200
Exact mass measurements can easily distinguish
These instruments (and other) can generate exhaustive lists of possible structure formulas near the exact mass value.

24. Some popular cleavages
Cleave at a branch point. Loss of radical or other neutral to provide a more stable cation C H 3 + .
Obs. in Mass Spec
neutral
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

25. Conclusions
Mass spectrometers can do everything.... including making coffee or
Mass spectrometry can play an important role in almost any biological oriented research...
...if you let it