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