mass spectrometry - caucrf.cau.ac.kr/ms.pdf · 2009-07-01 · secondary ion mass spectrometry...
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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