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Molecular mass Molecular mass spectrometryspectrometry
Chapter 20Chapter 20
The study of “molecular The study of “molecular ions”ions”
M + eM + e-- M M..++ + 2e + 2e--
Ionization energy?
EI Ionization??
MS TermsMS Terms FragmentationFragmentation Molecular ionsMolecular ions Daughter ionsDaughter ions Base peakBase peak Isotopic peaksIsotopic peaks Collision product peaks (MHCollision product peaks (MH++……)……) Hard and soft ionization sourcesHard and soft ionization sources Gas-phase sourcesGas-phase sources Desorption sourcesDesorption sources
Mass Spectrum of Ethyl BenzeneMass Spectrum of Ethyl Benzene
Ion Sources for Mass Ion Sources for Mass SpectrometersSpectrometers
Unless you “create” an ion you won’t see it!
Energy driven process HARD IONIZATION, SOFT IONIZATION
MS with “Hard” and “Soft” MS with “Hard” and “Soft” SourcesSources
Electron Impact SourceElectron Impact Source
Electron Ionization (EI) M + eM + e-- M M.+.+ + 2e + 2e--
Why vacuum?Why vacuum?
Ensure filament does not burn Ensure filament does not burn outout
Help to vaporize samplesHelp to vaporize samples Reduce collision between formed Reduce collision between formed
ions and atmospheric gasesions and atmospheric gases Remove sample from instrument Remove sample from instrument
after analysisafter analysis
Typical Reactions during Typical Reactions during Electron ImpactElectron Impact
Energy = 70eV 6700 kJ/mol
Typical bond energies 200 to 600 kJ/mol EXTENSIVE FRAGMENTATION
2(ABCD).+
Electron Impact SpectraElectron Impact Spectra
•Different molecules behave differently
•Good molecular ion little fragmentation
•No molecular ion extensive fragmentation
•Isotopes are extremely important!
•Molecular ion isotopic cluster
CI REAGENT GASCI REAGENT GAS
Chemical Ionization MS Chemical Ionization MS SourcesSources
High Energy electrons
Sample Molecule MH
CH4
CH4 CH4+
CH3+ CH2
+
25243
3544
HHCCHCH
CHCHCHCH
6252
42252
425
HCMMHHC
HCMHMHHC
CHMHMHCH
Molecule Ions
EI vs. CI
Why EI and CI not Why EI and CI not enough?enough?
Sample must be in gas phaseSample must be in gas phase Not for nonvolatile or thermally Not for nonvolatile or thermally
unstable compoundsunstable compounds
Electrospray Ionization Electrospray Ionization SourceSource
Several KV
ELCTROSPRAY IONIZATION DETAILSELCTROSPRAY IONIZATION DETAILS
Iribarne-Thomson Iribarne-Thomson ModelModel
Charge density increasesCharge density increases Rayleigh limit (Coulomb repulsion Rayleigh limit (Coulomb repulsion
= surface tension)= surface tension) Coulomb explosion (daughter Coulomb explosion (daughter
driblets)driblets) Evaporation of daughter dropletsEvaporation of daughter droplets Desorption (desolvation) of ions Desorption (desolvation) of ions
from the droplets into the ambient from the droplets into the ambient gas (gas (IONS FORMEDIONS FORMED))
Electrospray Ionization MS of Electrospray Ionization MS of Proteins and PeptidesProteins and Peptides
Special Features of ESISpecial Features of ESI
Little fragmentLittle fragment Multiple chargesMultiple charges Linear relationship between AVG Linear relationship between AVG
charge and molecular weight charge and molecular weight Coupling to HPLC directly!Coupling to HPLC directly!
An exampleAn exampleApplications:Applications:Determination of MW and charges for each Determination of MW and charges for each
peak (Smith et al. Anal. Chem., 1990, 62, peak (Smith et al. Anal. Chem., 1990, 62, 882-899):882-899):
AssumptionsAssumptions The adjacent peaks of a series differ by The adjacent peaks of a series differ by
only one chargeonly one charge For proteins, the charging is due to For proteins, the charging is due to
proton attachment to the molecular ion. proton attachment to the molecular ion. This has been an excellent (but not This has been an excellent (but not
crucial) assumption of nearly all proteins crucial) assumption of nearly all proteins studied to data where alkali attachment studied to data where alkali attachment contributions are small.contributions are small.
Ionization of only the intact molecule.Ionization of only the intact molecule.
Advanced Analytical Chemistry – CHM 6157Advanced Analytical Chemistry – CHM 6157 ® Y. CAI® Y. CAI Florida Florida International UniversityInternational UniversityUpdated on 10/9/2006Updated on 10/9/2006 Chapter 7Chapter 7Chromatogr./Mass Spec. Coupling Chromatogr./Mass Spec. Coupling
Given these Given these assumptions, eq 1 assumptions, eq 1 describes the describes the relationship between relationship between a multiply charged a multiply charged ion at m/z Pion at m/z P11 with with charge zcharge z11 and and molecular weight M.molecular weight M.
Z1
Z2
M/ZP1 P2
P1Z1 = M + MaZ1 = M + 1.0079Z1 [1]Assume that the charge carrying species (Ma) is a proton. The molecular weight of a second multiply protonated ion at m/z P2 (where P2 > P1) that is j peaks away from P1 (e.g. j = 1 for two adjacent peaks) is given by
P2(Z1-j) = M + 1.0079(Z1-j) [2]
Equations 1 and 2 can be solved for the charge of P1.
Z1 = j(P2-1.0079)/(P2-P1) [3]The molecular weight is obtained by taking Z1 as the nearest integer valve.
FAB Ionization: Fast Atom Bombardment
+
SECONDARY ION MASS SPECTROMETRY SIMS
FAB Ionization
Xe+
Xe0
Primary Ion Beam
Atom Gun
Secondary ion Beam to MS
MS Instrument MS Instrument ComponentsComponents
Gas/Liquid Inlet SystemGas/Liquid Inlet System
Solid/Matrix Inlet SystemsSolid/Matrix Inlet Systems
Sample
More MS AnalyzersMore MS Analyzers
Resolution?Resolution?
The capability of a MS to differentiate The capability of a MS to differentiate between massesbetween masses
R = R = m/∆mm/∆m
∆∆m: mass different between two adjacent m: mass different between two adjacent peaksthat are just resolved (height of the peaksthat are just resolved (height of the valley < 10% of the peak).valley < 10% of the peak).
M: the norminal mass of the first or the M: the norminal mass of the first or the mean of the two peaks mean of the two peaks
ResolutionResolution
Estimate the accuracy of the Estimate the accuracy of the measurement if resolution is knownmeasurement if resolution is known
If R = 5000, at mass 500 rangeIf R = 5000, at mass 500 range
5000 = 500/ ∆m5000 = 500/ ∆m
∆∆m = 0.1m = 0.1
Determine R requiredDetermine R required
CC22HH44++ and CH and CH22NN++ have masses of have masses of
28.0313 and 28.0313 and 28.018728.0187
R = 28.025/0.0126 = 2220R = 28.025/0.0126 = 2220
Magnetic Sector AnalyzerMagnetic Sector Analyzer
2
1 zeVKE
Magnetic Sector PhysicsMagnetic Sector Physics
curvature of radius2
v
vv
F F
vF ForcelCentripeta
vF Force Magnetic
v2
1VKE
22
2
c M
2
c
M
2
rV
erB
z
m
m
Bzerr
mBze
r
m
Bze
mze
Magnetic Sector PhysicsMagnetic Sector Physics
curvature of radius2
v
vv
F F
vF ForcelCentripeta
vF Force Magnetic
v2
1VKE
22
2
c M
2
c
M
2
rV
erB
z
m
m
Bzerr
mBze
r
m
Bze
mze
Single Focusing Magnetic Single Focusing Magnetic SectorSector
Ions at source with same mass-to-Ions at source with same mass-to-charge ratiocharge ratio
Ions with diverging directional Ions with diverging directional distribution will be acted upon in distribution will be acted upon in the same waythe same way
Brings ions with different Brings ions with different directional orientations to focusdirectional orientations to focus
Limits the resolutionLimits the resolution
DoubleDoubleFocusing Mass Focusing Mass SpectrometersSpectrometers
Passes ions through an electrostatic Passes ions through an electrostatic analyzer (ESA) which limits the range analyzer (ESA) which limits the range of the kinetic energy of ions reaching of the kinetic energy of ions reaching the magnetic sectorthe magnetic sector
Only ions with the same average Only ions with the same average kinetic energy pass through the ESA kinetic energy pass through the ESA slits into the magnetic sectorslits into the magnetic sector
Two focal planes at the ion collectorTwo focal planes at the ion collector Energy focal planeEnergy focal plane Directional focal planeDirectional focal plane
Increases resolutionIncreases resolution
DoubleDoubleFocusing Mass SpectrometerFocusing Mass Spectrometer
MS-MS
Daughter ion scanDaughter ion scan
The parent ion mass is fixed and the masses of The parent ion mass is fixed and the masses of all daughter ion formed from the parent ion are all daughter ion formed from the parent ion are measured. measured.
The first stage of analysis selects ions of The first stage of analysis selects ions of particular m/z. This parent ion is passed onto the particular m/z. This parent ion is passed onto the reaction region. The daughter ions formed in the reaction region. The daughter ions formed in the reaction region are then analyzed by mass. These reaction region are then analyzed by mass. These daughter ions are generally characteristic of the daughter ions are generally characteristic of the structure of the parent ions and thus provide a structure of the parent ions and thus provide a means of determining that structure.means of determining that structure.
Instrument designInstrument design
Daughter ion scanDaughter ion scan
The parent ion mass is fixed and the The parent ion mass is fixed and the masses of all daughter ion formed from the masses of all daughter ion formed from the parent ion are measured. parent ion are measured.
The first stage of analysis selects ions of The first stage of analysis selects ions of particular m/z. This parent ion is passed particular m/z. This parent ion is passed onto the reaction region. The daughter onto the reaction region. The daughter ions formed in the reaction region are then ions formed in the reaction region are then analyzed by mass. These daughter ions are analyzed by mass. These daughter ions are generally characteristic of the structure of generally characteristic of the structure of the parent ions and thus provide a means the parent ions and thus provide a means of determining that structure.of determining that structure.
Instrument designInstrument design
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