Mass SpectroscopyMass SpectroscopyMass Spectrometry is an analytical spectroscopic tool primarily concerned with the separation of molecular (and atomic) species according to their mass.

What information can be determined?

• Molecular weight

• Molecular formula (HRMS)

• Structure (from fragmentation fingerprint)

• Isotopic incorporation / distribution

• Protein sequence (MS-MS)

Pharmaceutical analysisBioavailability studiesDrug metabolism studies, pharmacokineticsCharacterization of potential drugsDrug degradation product analysisScreening of drug candidatesIdentifying drug targets

Biomolecule characterizationProteins and peptidesOligonucleotides

Environmental analysisPesticides on foodsSoil and groundwater contamination

Forensic analysis/clinical

Applications of Mass Spectrometry

Atom or molecule is hit by high-energy electronAtom or molecule is hit by high-energy electron

Principles of Electron-Impact Mass Principles of Electron-Impact Mass SpectrometrySpectrometry

ee––

Atom or molecule is hit by high-energy electronAtom or molecule is hit by high-energy electron

electron is deflected but transfers much of its electron is deflected but transfers much of its energy to the moleculeenergy to the molecule

ee––

This energy-rich species ejects an electron.This energy-rich species ejects an electron.

This energy-rich species ejects an electron.This energy-rich species ejects an electron.

forming a positively charged, odd-electron forming a positively charged, odd-electron species called the species called the molecular ionmolecular ion

ee––++••

Atom or molecule is hit by high-energy Atom or molecule is hit by high-energy electron from an electron beam at 10evelectron from an electron beam at 10ev

ee––

beambeam

forming a positively charged, odd-electron forming a positively charged, odd-electron species called the species called the molecular ionmolecular ion

ee––

++••

Molecular ion passes between poles of a magnet and is deflected by magnetic field

amount of amount of deflection deflection depends depends on mass-to-charge on mass-to-charge

ratioratio

highest m/z highest m/z deflected leastdeflected least

lowest m/z lowest m/z deflected mostdeflected most

++••

If the only ion that is present is the molecular ion, mass spectrometry provides a way to measure the molecular weight of a compound and is often used for this purpose.

However, the molecular ion often fragments to a mixture of species of lower m/z.

The molecular ion dissociates to a cation and a radical.The molecular ion dissociates to a cation and a radical.

++••

The molecular ion dissociates to a cationThe molecular ion dissociates to a cationand a radical.and a radical.

++ ••

Usually several fragmentation pathways Usually several fragmentation pathways are available and a mixture of ions is are available and a mixture of ions is produced.produced.

mixture of ions of mixture of ions of different mass different mass gives separate peak gives separate peak

for each m/zfor each m/z

intensity of peak intensity of peak proportional to proportional to percentage of each percentage of each ion of different ion of different mass in mixturemass in mixture

separation of peaks separation of peaks depends on relative depends on relative massmass

++

++++

++

+

+

mixture of ions of mixture of ions of different mass different mass gives separate peak gives separate peak

for each m/zfor each m/z

intensity of peak intensity of peak proportional to proportional to percentage of each percentage of each atom of different atom of different mass in mixturemass in mixture

separation of peaks separation of peaks depends on relative depends on relative massmass

++ ++ ++ ++

+ +

What’s in a Mass Spectrum?Io

n A

b un d

a nce

(a s

a %

o f B

a se

pea k

)

Mass, as m/z. Z is the charge, and for doubly charged ions (often seen in macromolecules), masses show up at half their proper value

High mass

Not usually scanned below m/z=32 (Why?)

[M+H]+(CI)Or M•+ (EI)“molecular ion”

Unit mass spacing

Fragment Ions Derived from molecular ion or higher weight fragments

In CI, adduct ions, [M+reagent gas]+

• Mass spectrum:Mass spectrum: A plot of the relative abundance of ions versus their mass-to-charge ratio (m/z).

• Base peak:Base peak: The most abundant peak.

– Assigned an arbitrary intensity of 100.

• The relative abundance of all other ions is reported as a % of abundance of the base peak.

Mass SpectrumMass Spectrum

• Molecular ion (M):Molecular ion (M): A radical cation formed by removal of a single electron from a parent molecule in a mass spectrometer = MW.

• For our purposes, it does not matter which electron is lost; radical cation character is delocalized throughout the molecule; therefore, we write the molecular formula of the parent molecule in brackets with:

– A plus sign to show that it is a cation.

– A dot to show that it has an odd number of electrons.

Molecular IonMolecular Ion

M + e- M+ + 2e-

Molecule High EnergyElectron

MolecularIon

(Radical Cation)

1009080706050403020100

Inte

nsi

ty (

% o

f B

ase

Pea

k)

20 30 40 50 60 70 80 90

m / z

1-Pentanol - MW 88

CH3(CH2)3 – CH2OH

CH2OH+M - (H2O and CH2=CH2)

M - (H2O and CH3)

M - H2O

M+ - 1

Molecular Ion Peak

Base Peak

M + e- M+ + 2e-

Molecule High EnergyElectron

MolecularIon

(Radical Cation)

M + e- M+ + 2e-

Molecule High EnergyElectron

MolecularIon

(Radical Cation)

1009080706050403020100

Inte

nsi

ty (

% o

f B

ase

Pea

k)

20 30 40 50 60 70 80 90

m / z

1-Pentanol - MW 88

CH3(CH2)3 – CH2OH

CH2OH+M - (H2O and CH2=CH2)

M - (H2O and CH3)

M - H2O

M+ - 1

Molecular Ion Peak

Base Peak

Mass Spectrum

– A partial MS of dopamine showing all peaks with intensity equal to or greater than 0.5% of base peak.

MS of dopamineMS of dopamine

AN INSTRUMENT THAT GENERATES IONS FROM MOLECULES AND MEASURES THEIR MASSES

THE ESSENTIAL COMPONENTS OF A MASS SPECTROMETER:

SAMPLE INLET

IONSOURCE

ION ACCELERATOR

IONANALYSER

IONDETECTOR

signal

COMPUTERMASS SPECTRUM

DATABASE

0

50

100

0 10 20 30 40 50 60 70 80

2

15

27

41

53

69

84

1-Butene, 3,3-dimethyl-

MASS SPECTROMETERMASS SPECTROMETER

Illustration of the basic components of a mass spectrometry system.

IonizationSource

MassAnalzyer Detector

Inlet all ionsselected

ionsData

System

Diagram of a simple mass spectrometer

Fig. 13.39

2. Atomic & Mass Number

AZ Xatomic numberatomic number

(number of protons)(number of protons)

(number of electrons)(number of electrons)

mass numbermass number

(number of protons plus (number of protons plus neutrons)neutrons)

WAYS TO PRODUCE IONSWAYS TO PRODUCE IONS• Electron impact (EI) - vapor of sample is bombarded with

electrons: M + e 2e + M.+ fragments

• Chemical ionization (CI) - sample M collides with reagent ions present in excess e.g.

CH4 + e CH4

.+ CH5+

M + CH5+ CH4 + MH+

• Fast Atom/Ion Bombardment (FAB)

• Laser Desorption & Matrix-Assisted Laser Desorption (MALDI)- hit the sample with a laser beam

• Electrospray Ionization (ESI) - a stream of solution passes through a strong electric field (106 V/m)

1. Electron Ionization (EI)most common ionization technique, limited to relatively low MW compounds (<600 amu)

2. Chemical Ionization (CI)ionization with very little fragmentation, still for low MW compounds (<800 amu)

3. Desorption Ionization (DI)for higher MW or very labile compounds

4. Spray ionization (SI)for LC-MS, biomolecules, etc.

Ionization MethodsIonization Methods

• vaporized sample is bombarded with high energy electrons (typically 70 eV)

• “hard” ionization method leads to significant fragmentation

• ionization is efficient but non-selective

Electron IonizationElectron Ionization ((EIEI))

Electron IonizationElectron Ionization

Advantages• inexpensive, versatile and reproducible• fragmentation gives structural

information• large databases if EI spectra exist and are

searchable

Disadvantages• fragmentation at expense of molecular

ion• sample must be relatively volatile

Chemical Ionization Chemical Ionization ((CICI))

Vaporized sample reacts with pre-ionized reagent gas via proton transfer, charge exchange, electron capture, adduct formation, etc.

– Common CI reagents:methane, ammonia, isobutane, hydrogen, methanol

• “soft” ionization gives little fragmentation

• selective ionization-only exothermic or thermoneutral ion-molecule reactions will occur

• choice of reagent allows tuning of ionization

CI MS Sources

High Energy electrons

Sample Molecule MH

CH4

CH4 CH4+

CH3+ CH2

+

25243

3544

HHCCHCH

CHCHCHCH

6252

42252

425

HCMMHHC

HCMHMHHC

CHMHMHCH

Molecule Ions

Lets talk about mass!• Atomic mass of Carbon

– 12.000000000000000000000000000 amu

• Atomic mass of Chlorine– 35.4527 amu

• Atomic mass of Hydrogen– 1.00794 amu

1amu = 1 dalton (Da)

Just for clarificationJust for clarification

• Atomic mass• amu, atomic mass units (uma??)• “Da” or Dalton. • kD (kiloDalton for macromolecules) • 1 amu = 1.66056*10-27 kg. • proton, mp = 1.67265*10-27 kg, • neutron, mn = 1.67495*10-27 kg.

• Resolution:Resolution: A measure of how well a mass spectrometer separates ions of different mass.

– low resolution:low resolution: Refers to instruments capable of separating only ions that differ in nominal mass; that is ions that differ by at least 1 or more atomic mass units.

– high resolution:high resolution: Refers to instruments capable of separating ions that differ in mass by as little as 0.0001 atomic mass unit.

ResolutionResolution

Inte

ns

ity

(%

)

0

20

40

60

80

100

Mass [amu]111.95 112.00 112.05 112.10

Inte

ns

ity

(%

)

0

20

40

60

80

100

Mass [amu]111.95 112.00 112.05 112.10

Inte

ns

ity

(%

)

0

20

40

60

80

100

Mass [amu]111.95 112.00 112.05 112.10

RP= 3,000 RP= 5,000 RP= 7,000

All resolving powers are FWHM

C6H5OFC6H5Cl

Resolving Power ExampleResolving Power Example

• High resolution data reports include ppm estimate– ppm = parts per million (1 ppm = 0.0001%)

•5 ppm @ m/z 300 = 300 * (5/106) = ±0.0015 Da•5 ppm @ m/z 3,000 = 3,000 * (5/106) = ±0.015 Da

• A molecule with mass of 44 could be C3H8, C2H4O, CO2, or CN2H4.• If a more exact mass is 44.029, pick the correct structure from

the table:

C3H8 C2H4O CO2 CN2H4

44.06260 44.02620 43.98983 44.03740

High Resolution MSHigh Resolution MS

– C3H6O and C3H8O have nominal masses of 58 and 60, and can be distinguished by low-resolution MS.

– C3H8O and C2H4O2 both have nominal masses of 60.

– Distinguish between them by high-resolution MS.

C2H4O2

C3H8O

60.02112

60.05754

60

60

MolecularFormula

Nominal Mass

PreciseMass

ResolutionResolution

– High resolution MS can replace elemental analysis for chemical formula confirmation

• Atomic number is the number of protons (+) in the nucleus and determines the element identity.

• Isotopes of an element have a different number of neutrons in the nucleus. Electrons (-) form a cloud and most of the volume of the atom.

• Electrons weigh very little. Atomic weight is basically the sum of the number of protons and neutrons.

What about isotopes?What about isotopes?Atomic TheoryAtomic Theory

• Atomic mass of Carbon– 12.000 amu for 12C but 13.3355 for 13C

• Atomic mass of Chlorine– 34.9688 amu for 35Cl and 36.9659 for 37Cl

• Atomic mass of Hydrogen– 1.00794 amu for H and 2.0141 for D!

Get it now?

Most elements have more than one stable isotope.

– For example, most carbon atoms have a mass of 12 Da, but in nature, 1.1% of C atoms have an extra neutron, making their mass 13 Da.

Exact Masses of Some Common Elements and Their Isotopes:Exact Masses of Some Common Elements and Their Isotopes:Element Symbol Exact Mass (u) Rel. Abundance %

Hydrogen 1H 1.007825037 100.0

Deuterium 2H or D 2.014101787 0.015

Carbon 12 12C 12.00000 100.0

Carbon 13 13C 13.003354 1.11223

Nitrogen 14 14N 14.003074 100.0

Nitrogen 15 15N 15.00011 0.36734

Oxygen 16 16O 15.99491464 100.0

Oxygen 17 17O 16.9991306 0.03809

Oxygen 18 18O 17.99915939 0.20048

Fluorine 19F 18.998405 100.0

Sodium 23Na 22.9897697 100.0

Silicon 28 28Si 27.9769284 92.23

Silicon 29 29Si 28.9764964 5.0634

Silicon 30 30Si 29.9737717 3.3612

Phosphorus 31P 30.9737634 100.0

Sulfur 32 32S 31.972074 100.0

Sulfur 33 33S 32.9707 0.78931

Sulfur 34 34S 33.96938 4.43065

Sulfur 36 36S 35.96676 0.02105

Chlorine 35 35Cl 34.968854 100.0

Chlorine 37 37Cl 36.965896 31.97836

Relative Isotope Abundance of Common Elements:

 

Element Isotope Relative Abundance

Isotope Relative Abundance

Isotope Relative Abundance

Carbon 12C 100 13C 1.11    

Hydrogen 1H 100 2H .016    

Nitrogen 14N 100 15N .38    

Oxygen 16O 100 17O .04 18O .20

Sulfur 32S 100 33S .78 34S 4.40

Chlorine 35Cl 100      37Cl 32.5

Bromine 79Br 100     81Br 98.0

• The most common elements giving rise to significant M + 2 peaks are chlorine and bromine.– Chlorine in nature is 75.77% 35Cl and 24.23% 37Cl. – A ratio of M to M + 2 of approximately 3:1 indicates

the presence of a single chlorine in a compound.

M+2 and M+1 PeaksM+2 and M+1 Peaks

– Bromine in nature is 50.7% 79Br and 49.3% 81Br.– A ratio of M to M + 2 of approximately 1:1

indicates the presence of a single bromine in a compound.

M+2 and M+1 PeaksM+2 and M+1 Peaks

• Sulfur is the only other element common to organic compounds that gives a significant M + 2 peak.– 32S = 95.02% and 34S = 4.21%– Also 33S = 0.8%, an M+1 peak.

• Because M + 1 peaks are relatively low in intensity compared to the molecular ion and often difficult to measure with any precision, they are generally not useful for accurate determinations of molecular weight.

M+2 and M+1 PeaksM+2 and M+1 Peaks

Nobel Prizes in Mass Spectrometry

1906- J.J. Thomson- m/z of electron

1911- W. Wien- anode rays have positive charge

1922- F. Aston- isotopes (first MS with velocity focusing)

1989- H. Dehmelt, W. Paul- quadrupole ion trap

1992- R.A. Marcus- RRKM theory of unimolecular dissociation

1996- Curl, Kroto, and Smalley- fullerenes (used MS)

2002- J. Fenn- electrospray ionization of biomolecules K. Tanaka- laser desorption ionization of biomolecules

• Better carbocation wins and predominates ““Stevenson’s RuleStevenson’s Rule””

[M·]+ A+ + B· (neutral)

or

B+ + A·

EI

FragmentationFragmentation

Stevenson’s Rule:Stevenson’s Rule:

– For simple bond cleavage, the fragment with lowest ionization potential takes the charge

(in other words, the most stable ion is formed)

• The Game is, to rationalize these in terms of the structure

• Identify as many as possible, in terms of the parent structure

• Generally, simply derived from the molecular ion

• Or, in a simple fashion from a significant higher mw fragment.

• Simply, here means, ions don’t fly apart, split out neutrals and then recombine.

• Fragments will make chemical sense

• A good approach is the “rule of 13” to write down a molecular formula for an ion of interest.

• Especially in EI, we only identify major fragments

Fragment Ions

The “Even Electron Rule” dictates that even (non-radical) ions will not fragment to give two radicals (pos• + neutral•) (CI)

CI

[M+H]+ PH+ + N (neutral)

– Loss of neutral molecules, small stable, from MH+

– Loss of neutrals from protonated fragments

– Subsequent reprotonation after a loss

– Typically there is no ring cleavage (needs radical) or two bond scissions.

– Depends highly on ion chemistry specifically acid-base (proton affinities)

• Governed by product ion stability• consideration– octet rule– resonance delocalization– polarizability and hyperconjugation– electronegativity

FragmentationFragmentation

General Fragmentation PathwaysGeneral Fragmentation Pathways– One-bond cleavagesOne-bond cleavages -cleavages-cleavages

C OHR- R

C OH C O

Cleave to Heteroatoms like O, N

O

R

: .

+•

R

O:: .

neutral

+

+

HeterolyticHeterolytic cleavagecleavage

Observed in Mass Spec provided that a good stabilized carbocation can form

O

O

O

: .

+

++:

+: :

Obs. in mass spec. Acylium ions are resonance-stabilized

neutral

Prominent for ketones

CH3C=O+ m/z=43

Cleavage to C=O groups

O

O O

M+• -45, loss of ethoxy radical

O+

C+

O

O+

Example

Ethyl 3-oxo-3-phenylpropanoate (Mol. Wt.: 192.21)

O

O+

M+• -43; also tropylium ion

Example

1-Phenylpropan-2-one (Mol. Wt.: 134.18)

-cleavages-cleavages– Cleave to a heteroatom (capable of supporting positive charge)

RO

RO

RO

:

:Obs. in Mass Spec

Resonance stabilized

neutral

+

+

+

Note the use of “half arrow” for one-electron movements. e.g homolytic cleavage

examplesPrimary alcohols, m/z =31 CH2=OH+

Primary amines, m/z =30 CH2=NH2+

m/z 30

++•

-cleavageCH3 CH3

CH3-CH-CH2-CH2-NH2 CH3-CH-CH2 CH2=NH2

Two-bond cleavagesTwo-bond cleavages

– Eliminate H-X

– Retro Diels-Alder

–McLafferty rearrangement

+C

C

O

CCH2

CH2

CR2

H

Y

-R2=CH2

Y = H, R, OH, OR NR2

O

CCH2

H

Y

O

CCH2

H

Y

O

CCH2

H

Y

need -hydrogens

Alkane FragmentationAlkane Fragmentation• Long chains give homologous series of m/z = 14 units

• Long chains rarely lose methyl radical

• Straight chain alkanes give primary carbocation

• Branched alkanes have small or absent M+

• Enhanced fragmentation at branch points

CH3 CH3

CH3CH3

CH3

C+

CH3

CH3.

Obs. in Mass Spec

+

neutral

+