Mass Spectrometry

Principle and Applications of Mass Spectrometry

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Mass Spectrometry?

Based on ionization of gas phase molecule followed byanalysis of the masses of the ions produced

The Mass Spectrum : Graph of ion intensity versus mass-to-charge ratio (m/z) (unitsdaltons, Da)

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Mass Spectrum•molecular ion : peak (M+) m/z corresponds to MW of singly

charged molecule

•fragment : peak m/z less than MW of singly-charged molecule

•base : peak most intense m/z

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Instrumentation

•sample introduction system - vaporize sample•ion source - ionizes analyte gas molecules•mass analyzer - separates ions according to m/z•detector - counts ions•vacuum system - reduces collisions between ions and gas molecules

InletSystem

IonSource

MassAnalyzer Detector

SignalProcess

VacuumSystem

Readout

10-5 – 10-8 torr

Sample

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Why Vacuum?

• Increase sensitivity

• Avoid ion-molecule reactions

• Collision free ion trajectories

• Increase filament lifetime

• Avoid electric discharge

• Avoid background interference

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Sample introductionA micromolar or less of sample is introduced into source chamber, which is maintained at a pressure of about 10-5 torr: most commonly the sample is

form of a gas, but liquids and solids can also be accomodated.

Direct Probe/Metal TargetsSample put onto the end of long probe and inserted into the MSSample spotted with matrix onto a metal plate

Gas chromatography: EI, CI, NCISample must be volatile, thermally stable

Liquid chromatography: PB, FAB, ESI & APCIMost widely used separation technique in the pharmaceutical industryLC/MS applicable to thermally labile, high MW compoundsLC/MS suitable for proteins & peptidesOn-line technique

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Sample introductionExternal (Batch) Inlet System :

Sample heated (<400oC) in small external ovenVapor admitted to ionizer through valveGas steam added to entrain analyte

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Sample introductionDirect Probe :

Sample vial inserted through air-lock into ionizer chamberVial heated to vaporize sampleVial can be reduced to capillary or surface plate for small quantities

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Ion Source

Basic Type Name and Acronym Ionizing AgentGas Phase Electron impact (EI)

Chemical ionization (CI)Field ionization (FI)

Energetic electronsReagent gaseous ionsHigh-potential electrode

Desorption Field desorption (FD)Electrospray ionization (ESI)Matrix-assisted desorption ionization (MALDI)Plasma desorption (PD)Fast atom bombarment (FAB)Secondary ion mass spectrometry (SIMS)Thermospray ionization (TS)

High-potential electrodeHigh electric fieldLaser beamFission fragments from 252CfEnergetic atomic beamEnergetic beam of ionsHigh temperature

Table 20-1. Ion sources for molecular mass spectrometry

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Hard or Soft?Hard ion sources leave excess energy in molecule

- Extensive fragmentation

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Hard or Soft?Soft ion sources little excess energy in molecule

- Reduced fragmentation

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Hard or Soft?

Ionization Techniques

ESIMALDI

Fast AtomBombardment(FAB)

ChemicalIonization(CI)

ElectronImpact(EI)

Hard IonizationFragments

Soft IonizationIntact

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Gas Phase Ionization - EI

1eV =1.6x10-19 C x1V (1V = 1 J ×C-1)=1.6x10-19 J = 96.486 kJ/mol

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Gas Phase Ionization - EI

Molecular ionMolecular ion

Fragment ionFragment ion −+−

−+−

++→+

+→+

2eBAeAB

2eABeAB

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Gas Phase Ionization - EI

EI Spectra:• hard source (incident energy 70 eV » than chemical bond)

• molecules electronically, vibrationally and rotationally excited

• extensive fragmentation => fragment ions

• base peak m/z « M+

• complex spectra

- helps identification

- poor for measuring MW of compound

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Gas Phase Ionization - EI

Fragmentation

+

++

+

++

++

++

−+−

+→

+→+→

+→

+→+→

+→+→

+→

+→+

DC

CDCDAB

BA

ABABCD

DBCBCDA

BCDAABCD

2eABCDeABCD

*

*

*

**

*

Rearrangement followed by fragmentation

Collision followed by fragmentation+

+++

+→

+→→

BCAD

ADBCADBCABCD*

***

+++ +→→+ ABCDABCD(ABCD)ABCDABCD **2*

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Gas Phase Ionization - EIFragmentation Patterns

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Gas Phase Ionization - EIWhat about peaks at greater m/z than M+?

Two sources:• Isotope Peaks - same chemical formula but different masses

12C1H235Cl2 m = 84

13C1H235Cl2 m = 85

12C1H235Cl37Cl m = 86

13C1H235Cl37Cl m = 87

12C1H237Cl2 m = 88

heights vary with abundance13C is 1.1 % 12C, 37Cl is 32.5 % 35Cl

• Collision Product Peaks - only common peak is proton transferto give (M+1)+ peak (increases with increasing pressure)

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Gas Phase Ionization - EIAdvantages of EI:

• high ion currents - sensitive

• fragmentation aids identification

Disadvantages of EI:

• weak or absent M+ peak inhibits determination of MW

• molecules must be vaporized (MW < 103 Da)

• molecules must be thermally stable during vaporization

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Gas Phase Ionization - CI

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Gas Phase Ionization - CIAdvantages of Chemical Ionization

1. Large (M+H)+ ion identitfies molecular weight (M)2. Sensitivity is enhanced by

- simple fragmentations (fewer peaks of higher abundance)- direct GC/MS interface

3. CI spectra compliment EI spectra

Disadvantages of Chemical Ionization1. Simple fragmentation gives little structural information2. Ion source contamination3. Control reagent gas flow

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Desorption/Ionization Sources- ESIApplicable to non-volatile (>105 Da) or non-stable analytesEnergy applied to analyte causing desorption and ionizationExact mechanisms still under investigation

(A) Electrospray Ionization (ESI):Explosion of charged droplets containing analytes

- solution analyte pumped through charged (1-5 kV) capillary- small droplets become charged- solvent evaporates, drop shrinks, surface charge density increases- charge density reduced by expulsion of charged analyte molecules

("Coulomb explosion")Soft ionization - little fragmentationEasily adapted to chromatography

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Desorption/Ionization Sources- ESI

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Desorption/Ionization Sources- ESISoft ionization, which generates pseudo molecular ions• MH+, (M+NH4)+, (M+Na)+, (M+K)+ etc.

• (M-H)-, (M+CH3COO)-, (M+Cl)- etc.

Applicable for wide range of compounds with high sensitivity• Up to 150 kDa in case of proteins

• Middle to High polar compounds

• Thermally labile compounds

• Non covalently binding complex

Generates multiply charged ions of biopolymers• (M+nH)n+ / (M-nH)n-

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Desorption/Ionization Sources- ESIUseful mass differences for interpretation of ESI-MS

- Positive ion mode -

(M+H)+

(M+NH4)+

17 (M+Na)+

(M+K)+16

5 (M+MeCN+H)+

2238

41

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Desorption/Ionization Sources- ESIUseful mass differences for interpretation of ESI-MS

- Negative ion mode -

(M+Cl)-

(M-H)-(M+HCOO)-

(M+CH3COO)-36

(M+CF3COO)-

46

11460

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Desorption/Ionization Sources- ESI

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Desorption/Ionization Sources- FAB

(B) Fast Atom Bombardment (FAB):• Hard ionization - fragmentation

• Sample in glycerol solution

• Bombarded by high energy Ar or Xe atoms (few keV)

• Atoms and ions sputtered from surface (ballistic collision)

• Both M+ and M- produced

• Applicable to small or large (>105 Da) unstable molecules

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Desorption/Ionization Sources- MALDI

(C) Matrix-Assisted Laser Desorption/Ionization (MALDI):

Soft ionization•analyte dissolved in solution of UV-absorber and solvent

•solid crystals of analyte + absorber grow (matrix)

•pulsed laser fired at crystals in time-of-flight mass spectrometer (TOF-MS)

•molecular ion desorbed from crystal surface

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Desorption/Ionization Sources- MALDIMALDI spectrum contains :

dimer, trimers...multiply charged molecules no fragmentation

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Mass Analyzer

Mass Analyzer

Ability to separate ions

High accuracy

High sensitivity

High mass range

Structural information

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Mass AnalyzerResolution

- ability to differentiate peaks of similar massResoution = M/∆M = M1/(M1-M2)

- resolution depends on mass!- if R = 1000

separate peaks at m/z = 100 and 100.1or m/z = 1000 and 1001or m/z = 10000 and 10,000

- high resolution necessary for exact MW determination:nominal MW = 28actual MW C2H4

+ = 28.0313CH2N+ = 28.017N2

+ =28.0061R > 2570

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Magnetic Sector Analyzer

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Magnetic Sector Analyzer

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Magnetic Sector Analyzer

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Magnetic Sector Analyzer

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Magnetic Sector Analyzer

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Quadrupole Analyzer

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Time of Flight (TOF) Analyzer

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Time of Flight (TOF) Analyzer

+

+

+

+

Source

V

Drift region (flight tube)

dete

ctor

Small ions reach the detector before large ions

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Applications of Molecular MS

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Applications of Molecular MS

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Summary of Molecular MSOne of most powerful analytical tools:• sensitive (10-6 to <10-13 g)

• range of ion sources for different situations

• elemental composition for small and large MW - biomolecules

• limited structural information

• qualitative and quantitative analysis of mixtures

• composition of solid surfaces

• isotopic information in compounds

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Summary of Molecular MSBut:• complex instrumentation

• expensive high resolution

• structure obtained indirectly

• complex spectra/fragmentation for hard ionization sources

• simple spectra for soft ionization sources