Mass Spectrometry

Ionization Techniques

Mass Spectrometer

All Instruments Have:

1. Sample Inlet

2. Ion Source

3. Mass Analyzer

• Detector

• Data System

http://www.asms.org

Ionization TechniquesGas-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)

Electron Ionization

http://www.noble.org/PlantBio/MS/ion_tech_main.html

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

Electron Ionization-lactam: Effects of ionization energy

Electron Ionization

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

(low picomole)

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

Chemical Ionization

http://www.noble.org/PlantBio/MS/ion_tech_main.html

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)

Chemical Ionization: Methane

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.

Chemical Ionization: Isobutane

H3CCH3

CH3

H

H3CCH3

CH3

-H

-CH3 H3C

CH3

H

m/z 43

m/z 57

Chemical Ionization: Isobutane

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)

EI vs. Methane vs. Isobutane

EI

Methane CI

Isobutane CI

O

O

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

Chemical Ionization

Advantages

• 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

(low picomole)

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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

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

FAB

Advantages

• 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

(nanomole)

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

SIMSGa+ vs. C60

+

J. Phys. Chem. B 2004, 108, 7831-7838 Videos

SIMS

Advantages

• Surface analyses

• Stable Molecular Ion

• Mass Limit ~ 10000 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

28

MALDIMatrix 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

MALDI

30

MALDI

• Choice of matrix based on empirical evidence

• http://polymers.msel.nist.gov/maldirecipes/maldi.html

• Typically singly charged ions observed• Some matrix adducts/cluster ions• Difficult to analyze low MW compounds

due to matrix background• Typically used for MW 500-500,000

31

MALDI

32

MALDI

OOMe

CH3

()

33

UV-MALDI MatricesMatrix Application Structure

α-Cyano-4-hydroxycinnamic acid(CCA)

peptides

OHNC

OHO

3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid)

proteins HO

H3CO

H3CO

O

OH

2,5 Dihydroxybenzoic acid (DHB) peptides, proteins, polymers, sugars

O OH

OH

HO

3-Hydroxypicolinic acid (HPA) oligonucleotidesN

OH

OH

O

Dithranol (anthralin) polymers

OOH OH

MALDI

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

(low femtomole)

35

ESIElectrospray 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.

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.

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

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

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ESI-Multiply Charged Ions

Δm = 1 amuΔ(m/z) ≈ 0.10

Δm = 1 amu ; ∆(m/z) ≈ 0.055; z = 18

z = 10

M=16953

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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)

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ESI-Multiply Charged Ions

z1 =j(m2-mp)

(m2-m1)M = z1(m1-mp)

1303.8 1621.3j=10

z1 =10(1621.3-1.0073)

(1621.3-1303.8)= 51.0 M = 51.0(1303.8-1.0073)

M = 66485

ESI

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

(low femtomole to zeptomole)