1. Ionization Techniques
Mass Spectrometry
Ionization Techniques

2. Mass Spectrometer All Instruments Have: Sample Inlet Ion Source
Mass Analyzer
Detector
Data System

3. Ionization Techniques
Gas-Phase Methods
Electron Ionization (EI)
Chemical Ionization (CI)
Desorption Methods
Matrix-Assisted Laser Desorption Ionization (MALDI)
Fast Atom Bombardment (FAB)
Secondary Ion MS (SIMS)
Spray Methods
Electrospray (ESI)
Atmospheric Pressure Chemical Ionization (APCI)

4. Electron Ionization

5. Electron Ionization Samples must be vaporized in the ion source
Typically 1 of 1000 molecules entering the source is ionized
10-20 eV of energy is imparted to the molecule
~10eV is enough to ionize most molecules
Up to 230 kcal/mol is left to cause fragmentation
Using lower energies can reduce fragmentation, but since efficiency falls off, less molecular ion is generated under these conditions

6. Electron Ionization b-lactam: Effects of ionization energy
Beta Lactam gives lower overall efficiency at 15 eV negating the effects of reduced fragmentation.

7. Electron Ionization (low picomole) Advantages Disadvantages
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

8. Chemical Ionization
Reagent gas is introduced into the source at ~0.5 torr
Reagent gas is preferentially ionized. Ions react mostly with neutral reagent gas
Reactions occurring depend on the nature of the reagent gas
Ions in the reagent gas plasma react with the analyte

9. Chemical Ionization

10. Chemical Ionization: Methane
Methane primarily forms CH4+• with CH2+• and CH3+
CH4+• + CH4 → CH5+ + CH3 (m/z 17)
CH2+• + CH4 → C2H3+ + H2 + H•
C2H3+ + CH4 → C3H5+ + H2 (m/z 41)
CH3+ + CH4 → C2H5+ + H2 (m/z 29)

11. Chemical Ionization: Methane

12. Chemical Ionization: Methane
Ions other than saturated hydrocarbons react via proton transfer
CH5+ + M → MH+ + CH4 (or via C2H5+ or C3H5+)
For saturated hydrocarbons, hydride abstractions is common
CH5+ + RH → R+ + CH4 + H2
For polar molecules, adducts can form
CH3+ + M → (M+CH3)+
MH+, R+, and adducts are pseudomolecular ions.

13. Chemical Ionization: Isobutane

14. Chemical Ionization: Isobutane

15. Chemical Ionization: Isobutane
Reacts through Proton Transfer
C4H9+ + M → MH+ + C4H8
For saturated hydrocarbons, no reaction
For polar molecules, adducts can form
C4H9+ + M → (M+C4H9)+
Lack of reaction with hydrocarbons can be used for selective detection of compounds in mixtures containing hydrocarbons
Less fragmentation is observed with isobutane. (molecular species is more reliably formed)

16. EI vs. Methane vs. Isobutane
Methane CI
Isobutane CI

17. Chemical Ionization: Negative Ions
Low energy electrons are present in the CI plasma
These can attach to molecules with high electron affinities
There are two principal pathways: N2O/CH4 reagent gas
AB + e- → AB-• (associative resonance capture)
AB + e- → A• + B- (dissociative resonance capture)
Deprotonation can also occur if a basic ion is formed in the reagent gas plasma

18. Chemical Ionization (low picomole) Advantages Disadvantages
Molecular 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

19. Ionization Sources - II
EI and CI have limitations
Both require a volatile sample
Samples must be thermally stable
Neither lends itself to LC/MS analysis
Other techniques have been developed
FAB (Fast Atom Bombardment)
SIMS (Secondary Ion MS)
MALDI (Matrix Assisted Laser Desorption)
ESI (Electrospray)

20. FAB Sample is dissolved in a non-volatile liquid matrix
Glycerol and m-Nitrobenzyl alcohol are common matrices
A high energy (5kV) beam of neutral atoms (typically Ar or Xe) is focused onto the sample droplet
Dissolved Ions and Molecules are ejected into the gas phase for analysis

21. FAB

22. FAB For Organic Molecules M+H and M+Na ions are typically observed
M+H ions typically fragment more than M+Na ions
Salts such as NaI can be added to the matrix to induce M+Na formation

23. FAB (nanomole) Advantages Disadvantages No Fragment Library
Stable Molecular 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
Not amenable to automation
Relatively Low Sensitivity
Can be used with HPLC (Continuous flow FAB) Difficult to implement
Has been largely replaced by ESI/APCI/MALDI

24. SIMS Analysis of surfaces in situ
A high energy (15-40 keV) beam of primary ions (In+, Ga+) or clusters (SF6, Au3, C60) is focused onto the sample droplet
Surface and slightly sub-surface atoms or molecules are ejected and ionized
Clusters increase secondary ion yield dramatically

25. SIMS
Courtesy of Mike Kurczy, Winograd Group, Penn State University

26. SIMS Ga+ vs. C60+
J. Phys. Chem. B 2004, 108,
Videos

27. SIMS Advantages Disadvantages No Fragment Library Surface analyses
Stable Molecular Ion
Mass Limit ~ amu
Small primary ion beam spots and beam rastering make imaging possible
Disadvantages
No Fragment Library
Quantitation Difficult
Needs Highly Skilled Operator
Not amenable to automation
Relatively Low Sensitivity
Can be used with HPLC (Continuous flow FAB) Difficult to implement
Has been largely replaced by ESI/APCI/MALDI

28. MALDI Matrix Assisted Laser Desorption
Sample dissolved in a solid matrix
Typically mixed in solution
Small droplet applied to target and dried
A wide variety of matrices exist
Choose based on hydrophobic/hydrophilic character of sample
Also based on laser absorbance (usually UV)
An ionization agent is often added
Agent must bind to the sample
TFA and its Na+ Ag+ salts are common
Acid for basic analytes such as peptides/proteins
Na for good lewis bases. Ethers/esters
Ag for pi systems (polystyrene, polyisoprene, polybutadiene

29. MALDI

30. MALDI Choice of matrix based on empirical evidence
Typically singly charged ions observed
Some matrix adducts/cluster ions
Difficult to analyze low MW compounds due to matrix background
Typically used for MW ,000

31. MALDI
MALDI of a large protein (monoclonal antibody)

32. MALDI
MALDI of polymethylmethacrylate

33. UV-MALDI Matrices Matrix Application Structure
α-Cyano-4-hydroxycinnamic acid
(CCA)
peptides
3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid)
proteins
2,5 Dihydroxybenzoic acid (DHB)
peptides, proteins, polymers, sugars
3-Hydroxypicolinic acid (HPA)
oligonucleotides
Dithranol (anthralin)
polymers

34. MALDI (low femtomole) Advantages Parent Ion
High Mass Compounds (>100,000 amu)
Thermally Labile Compounds (R.T.)
Easy to Operate
Easily Automated
Disadvantages
No Fragment Library
Wide variety of matrices
Quantitation Difficult
Matrix Background

35. ESI Electrospray Ionization
Sample dissolved in a polar solvent
Solution flows into a strong electric field (3-6 kV potential)
Electric field induces a spray of highly charged droplets (charges at surface)
As droplets shrink, repulsion increases until they break into smaller droplets
In small enough droplets, surface charges can be desorbed into the gas phase.
Solvents: MeOH, H2O, ACN,

36. ESI

37. ESI Ions formed via charge-residue or ion- evaporation
Molecules form M+H+ or M-H- ions
Large molecules: 1 charge / 1000 amu
Small molecules: Usually singly charged
Molecules with no acid/base groups
Can form adduct ions with Na+ K+ NH4+ Cl- OAc-, etc.
Salts may be added or already present in sample.
At higher concentrations, non-covalent dimers, trimers, etc can be observed.

38. ESI
ESI ions formed at high pressure must be transferred into high vacuum
Differential pumping is needed to move ions through small openings while maintaining low pressures
Ions become super-cooled by expansion. Solvent can recondense
Two methods to reduce cluster formation
High temperature transfer tube
Heated counter-current flow of N2

39. ESI

40. ESI-Multiply Charged Ions
Large Molecules produce an envelope of charge states
Deconvolution must be done to determine the charge states if isotopic resolution is not possible
Typically, MS data systems use software to deconvolute automatically

41. ESI-Multiply Charged Ions
Δm = 1 amu ; ∆(m/z) ≈ 0.055; z = 18
Δm = 1 amu
; Δ(m/z) ≈ 0.10
z = 10

42. ESI-Multiply Charged Ions
Consider (M+zH)z+
z1m1 = M + z1mp (m1 = measured m/z)
Consider a peak of m/z=m2 which is (j- 1) charge states away from peak m1
m2(z1-j) = M + (z1-j)mp
z1 =
j(m2-mp)
(m2-m1)
M = z1(m1-mp)

43. ESI-Multiply Charged Ions
j=10
1303.8
1621.3
z1 =
j(m2-mp)
(m2-m1)
M = z1(m1-mp)
z1 =
10( )
( )
= 51.0
M = 51.0( )
M = 66485

44. ESI (low femtomole to zeptomole) Advantages Disadvantages 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