Mass Spectrometry
General Principles
Mass Spectrometer:
• Converts molecules to ions
• Separates ions (usually positively charged) on the basis of their mass/charge (m/z) ratio
• Quantifies how many units of each ion are formed over a given period
Provides:
• Molecular Weight
• Fragmentation information
propane
ionization
Mass Spectrum
100
29
H H H H H H H H H
80
relative intensity
H C C C H H C C C H H C C C H 28
H H H H H H H H H 60
m/z = 15 m/z = 44 m/z = 29 40
44
(molecular ion) 43
20
H H H H H 15
H C C C H C C C 0
0 10 20 30 40 50
H H H H H H
m/z
m/z = 43 m/z = 28
Mass Spectrometry
Basic Mass Spectrometer
acceleration
• Many different methods for ionization and detection are known
- useful for different types of molecules
- vary in amount of energy delivered à impacts ionization / fragmentation
Mass Spectrometry
Spectrum Terminology
base peak
parent
ion
parent ion: molecular ion ( [M]•+ ) or quasimolecular ion ( [M+H]+, [M+Na]+, etc.)
base peak: the tallest peak in the spectrum
Mass Spectrometry
Ionization Methods
Ionization Method Typical Analytes Sample Mass Ionization
Introduction Range [mass]
GC or
relatively small, up to 1000 hard, [M]•+ if
Electron Impact (EI) liquid/solid
volatile Daltons observed
probe
GC or
relatively small, hard to soft;
liquid/solid up to 1000
Chemical Ionization (CI) volatile varies with carrier
probe Daltons
[M+H]+
carbohydrates, sample mixed up to 6000 soft, but harder
Fast Atom Bombardment
organometallics, in viscous Daltons than MALDI, ESI
(FAB)
peptides, nonvolatile matrix [M+Na]+, [M+H]+
up to
soft
Matrix Assisted Laser peptides, proteins, sample mixed 500,000
[M+H]+
Desporption (MALDI) nucleotides in solid matrix Daltons
up to 6000
peptides. proteins, HPLC or soft
Electrospray (ESI) Daltons
nonvolatile syringe [M+Na]+, [M+H]+
Mass Spectrometry
Ionization Methods
Hard Ionization
e
*
e M M FRAGMENTS
Soft Ionization
H+ (etc) + M MH
Mass Spectrometry
Ionization Methods
Electron Impact (EI)
• Hard ionization method
sample in
• Used for low MW, nonpolar compounds (MW <750)
à sample must be volatile (& thermally stable)
• Method that is most commonly used for GC-MS
• Sample bombarded with high energy electrons
à typically 70 eV
• Ejects one electron from sample à radical cation (molecular ion)
à ionization potenial (IP) of an organic molecule is 8-15 eV
• Molecular ion is often small or not observed à fragmentation
• Fragmentation pattern is highly reproducible; unique to each compound
à provides structural information
Mass Spectrometry
Ionization Methods
Effect of electron energy on Impact of EI Spectrum
Mass Spectrometry
Ionization Methods
Chemical Ionization (CI)
• Soft ionization method (more controlled)
à some fragmentation observed
• Indirect ionization of sample
• Collisions between sample & gas ions cause proton transfers
à produces [M+H]+ ions, not M+ ions (so parent is M+1)
• More controlled than EI à reduced fragmentation à greater sensititivty
à typically ~5 eV energy transfer
• Good for molecular weight determination
• Provides less information about structure
Mass Spectrometry
Ionization Methods
Chemical Ionization (CI)
• Ionization methods include proton transfer and adduct formation
• Results in formation of "quasimolecular" ion
M + H3+ [M+H]+ + H2
M + CH5+ [M+H]+ + CH4
M + C2H5+ [M+C2H5]+
M + (CH3)3C+ [M+H]+ + (CH3)2=CH2
M + (CH3)3C+ [M+C(CH3)3]+
M + NH4+ [M+H]+ + NH3+
Mass Spectrometry
Ionization Methods O
CH3O
CH3
EI vs. CI
CH3O
MW = 180
EI
CI (methane)
CI (methane)
Mass Spectrometry
Ionization Methods
EI vs. CI
EI
MW = 142
CI (methane)
[M+H]+
Mass Spectrometry
Ionization Methods
Chemical Ionization (CI)
reagent gas reagent ion sample ion
hard H2 H3+ [M+H]+
CH4 CH5+, C2H5+, C3H5+ [M+H]+, [M+C2H5]+
i-C4H10 C4H9+ [M+H]+, [M+C4H9]+
soft NH3 NH4+ [M+H]+
reagent gas proton affinity notes
H2 101 kcal/mol produces significant fragmentation
CH4 132 kcal/mol less fragmentation than H2; can form adducts
(CH3)3CH 196 kcal/mol mild, selective protonation; little fragmentation; some
adduct formation
NH3 204 kcal/mol selective ionization; little fragmentation; some
adduct formation
Mass Spectrometry
Ionization Methods
O
Chemical Ionization (CI)
O
carrier gas
MW = 196
CH4
CH3
H
H3C CH3
NH3
Mass Spectrometry
Ionization Methods
Fast Atom Bombardment (FAB)
• Soft ionization method
• Sample mixed with a condensed phase matrix (glycerol)
protects sample from excess energy
• Mixture ionized by bombarding with beam of high energy atoms à Xe or Ar (6-10 keV)
• Ionization from protonation ([M+H]+) or cation attachment ([M+Na]+)
• High resolution, exact mass determination is possible
fast (6-10 KeV) beam
of Xe, Ar or Cs ions
M
M
M M
M
M H or M Na
M
secondary ionization and
desorption from matrix
Mass Spectrometry
Ionization Methods
Matrix Assisted Laser Desorption Ionization (MALDI)
• Soft ionization method
• Sample is mixed with a condensed phase matrix that contains a chromophore
• Mixture is ionized with a laser à proton tranfer from matrix to sample
• Charged molecules are ejected from matrix
• Little excess energy → little fragmentation
• Good for large molecules (proteins, polymers, carbohydrates)
Mass Spectrometry
Ionization Methods
Electrospray Ionization (ESI)
• Soft Ionization method
• Does not require vacuum pressures
• Sample solution pumped through a narrow, stainless steel capilliary
à aerosol of charged droplets
• Solvent evaporates until electrostatic repulsion within droplets too great
à Coulombic explosion
• Sample ions released into vapor phase [M+H]+, [M+Na]+
• Good for large molecules (proteins, polymers, carbohydrates)
Mass Spectrometry
Ionization Methods
Electrospray Ionization (ESI)
OH
O CO2Et
Mass Spectrometry
Mass Analysis
Magnetic Sector Mass Analyzer
• Magnetic field is used to deflect ions around a curved path
• Radius of curvature of an ion depends on m/z ratio and strength of magnetic field
• Ions with correct m/z pass through the detector
• Ions too heavy or too light do not make it through
• Can vary strength of magnetic field so that all ions can be detected
• Can increase resolution further by subjecting ions to an additional electric field
double focusing mass analyzer
double focusing mass analyzer
Mass Spectrometry
Mass Analysis
Quadripole Mass Analyzer
• Ions fly through a tunnel of four charged rods
• Voltage of the rods is changed in order to focus ions
• Ions with correct m/z are able to fly through to detector
• less sensitive à low resolution mass spec
Mass Spectrometry
Determination of Molecular Weight
Identification of the Molecular Ion
e + M M + 2e
• Don’t forget about isotopes
100
12CH MW = 16 monoisotopic
4 mass
polyisotopic
mass CH4
13CH MW = 17
4
m/z 16 17
Mass Spectrometry
Determination of Molecular Weight
Effects of Isoptope Differences
Relative Isotopic Abundances of Common Elements
Relative Relative Relative
Element Isotope Abundance Isotope Abundance Isotope Abundance
12C 100 13C 1.11
Carbon
1H 100 2H 0.016
Hydrogen
14N 100 15N 0.38
Nitrogen
16O 17O 18O
Oxygen 100 0.04 0.2
19F 100
Fluorine
28Si 29Si 30Si
Silicon 100 5.1 3.35
31P 100
Phosphorous
32S 33S 34S
Sulfur 100 0.78 4.4
35Cl 37Cl
Chlorine 100 32.5
79Br 81Br
Bromine 100 98
127I 100
Iodine
see also: Pavia Table 3.5
Mass Spectrometry
Determination of Molecular Weight
Effects of Isoptope Differences
Exact Masses of Isotopes
Atomic
Element Weight Nuclide Mass
1.00794 1H 1.00783
Hydrogen
D (2H) 2.01410
12.01115 12C 12.00000 (std)
Carbon
13C 13.00336
14.0067 14N 14.0031
Nitrogen
15N 15.0001
15.9994 16O 15.9949
Oxygen
17O 16.9991
18O 17.9992
18.9984 19F 18.9984
Fluorine
28.0855 28Si 27.9769
Silicon
29Si 28.9765
30Si 29.9738
30.9738 31P 30.9738
Phosphorous
32.0660 32S 31.9721
Sulfur
33S 32.9715
34S 33.9679
35.4527 35Cl 34.9689
Chlorine
37Cl 36.9659
79.9094 79Br 78.9183
Bromine
81Br 80.9163
126.9045 127I 126.9045
Iodine
see also:
Pavia Table 3.4
atom exact mass unit mass
12C: 12.00000 12
Mass Spectrometry
!1H: 1.00783 1
14N:
Determination of Molecular Weight 14.0031 14
!16O: 15.9994 16
Effects of Isoptope Differences
C7H7NO
MW = 121 121
isotope relative abundance
13C: 1.11
Mass Spectrometry
!2H: 0.016
15N: 0.38
Determination of Molecular Weight
!17O: 0.04
C7H7NO
MW = 121 121
[M+1]
[M+1] (1.1 x # of C atoms) + (0.016 x # of H atoms)!+ (0.38 x # of N atoms) …etc.
% [M+1] = x 100 = x 100
M M
O isotope relative abundance
13C: 1.11
Mass Spectrometry
!2H: 0.016
N
15N: 0.38
Determination of Molecular Weight
C7H7NO !17O: 0.04
Relative abundance of [M+1] MW = 121
[M+1] (1.1 x # of C atoms) + (0.016 x # of H atoms)!+ (0.38 x # of N atoms) …etc.
% [M+1] = x 100 = x 100
M M
[M+1] (1.1 x 7 C atoms) + (0.016 x 7 H atoms)!+ (0.38 x 1 N atoms) + (0.4 x 1 O atom)
% [M+1] = x 100 = x 100
M 121
121
[M+1] 8.232
% [M+1] = x 100 = x 100
M 121 relative abundance
100%
[M+1] 8
% [M+1] = x 100 = x 100 = 6.6%
M 121
[M+1] 6.6% (calc)
13C
7.7% (est)
BUT: most abundant of isotopes
for every C, 1.1% are 13C
can estimate: 7C x 1.1% = 7.7%
Mass Spectrometry
Determination of Molecular Weight
Effects of Isoptope Differences
M+3
M+
M+1
M+2
M+3 M+
A. peptide with 96 B. insulin (257 carbon atoms)
carbon atoms
# carbons?
Mass Spectrometry 1. calculate relative % [M+1]
2. divide by abundance 13C
Determination of Molecular Weight
relative
m/z intensity
122.0 4.0
121.0 47.9
107.0 6.9
106.0 100.0
79.0 15.9
78.0 92.7
65.0 2.0
63.0 2.0
53.0 2.8
52.0 11.0
51.0 37.4 M+
50.0 13.4
49.0 2.5
43.0 30.1
42.0 2.8
39.0 7.2
38.0 3.5 [M+1]
37.0 2.4
28.0 3.3
27.0 3.1
26.0 2.4
15.0 6.5
1. calculate relative % [M+1] 2. divide by natural abundance of 13C
[M+1] 4.0 8.0
% [M+1] = x 100 = x 100 = 8% = 7
M 47.9 1.1
Mass Spectrometry
Determination of Molecular Weight
Effects of Isoptope Differences
Relative Isotopic Abundances of Common Elements
Relative Relative Relative
Element Isotope Abundance Isotope Abundance Isotope Abundance
12C 100 13C 1.11
Carbon
1H 100 2H 0.016
Hydrogen
14N 100 15N 0.38
Nitrogen
16O 17O 18O
Oxygen 100 0.04 0.2
19F 100
Fluorine
28Si 29Si 30Si
Silicon 100 5.1 3.35
31P 100
Phosphorous
32S 33S 34S
Sulfur 100 0.78 4.4
35Cl 37Cl
Chlorine 100 32.5
79Br 81Br
Bromine 100 98
127I 100
Iodine
see also: Pavia Table 3.5
Mass Spectrometry
Determination of Molecular Weight
Calculation of Relative Intensities
• Isotopic peak distribution using ChemDraw
O Chemical Formula: C7H7NO
Exact Mass: 121.05
Molecular Weight: 121.14
m/z: 121.05 (100.0%), 122.06 (7.7%)
N
SiMe3 Chemical Formula: C8H18OSi
O
Exact Mass: 158.11
Molecular Weight: 158.31
m/z: 158.11 (100.0%), 159.12 (8.9%), 159.11 (5.1%), 160.11 (3.3%), 160.12 (1.0%)
[M+1] [M+2] [M+3] [M+4]
Mass Spectrometry
Determination of Molecular Weight
Effects of Isoptope Differences
Relative Isotopic Abundances of Common Elements
Relative Relative Relative
Element Isotope Abundance Isotope Abundance Isotope Abundance
12C 100 13C 1.11
Carbon
1H 100 2H 0.016
Hydrogen
14N 100 15N 0.38
Nitrogen
16O 17O 18O
Oxygen 100 0.04 0.2
19F 100
Fluorine
28Si 29Si 30Si
Silicon 100 5.1 3.35
31P 100
Phosphorous
32S 33S 34S
Sulfur 100 0.78 4.4
35Cl 37Cl
Chlorine 100 32.5
79Br 81Br
Bromine 100 98
127I 100
Iodine
see also: Pavia Table 3.5
Mass Spectrometry natural abundance
35Cl !100%!!
Determination of Molecular Weight 37Cl 32.5% ca. 3:1 ratio
Effects of Isoptope Differences
Cl
MW = 78
[M]•+ [M+2]
9.0% 3.1%
Mass Spectrometry natural abundance
78
Br !100%!!
80
Determination of Molecular Weight Br 98% ca. 1:1 ratio
Effects of Isoptope Differences
Br
MW = 122
[M]•+ [M+2]
8.6% 8.3%
Mass Spectrometry
Determination of Molecular Weight
Effect of Isoptope Differences
Mass Spectrometry
Identification of the Molecular Ion
Ease of Fragmentation
less fragmentation aromatics higher relative
abundance of M+
alkenes
unbranched hydrocarbons
ketones
amines
esters
ethers
carboxylic acids
branched hydrocarbons lower relative
more fragmentation alcohols abundance of M+
Mass Spectrometry
Identification of the Molecular Ion
Ease of Fragmentation
Stability plays a factor in whether or not a molecular ion is observed
Decreasing ability to give prominent M+:
aromatics > conjugated alkenes > cyclic compounds >
organic sulfides > alkanes > mercaptans
Decreasing ability to give recognizable M+:
ketones > amines > esters > ethers >
carboxylic acids ~ aldehydes ~ amides ~ halides
M+ is frequently not detectable from:
aliphatic alcohols, nitrites, nitrates, nitro compounds,
nitriles, highly branched compounds
Mass Spectrometry
Identification of the Molecular Ion
Molecular Ion Requirements
1. Peak must correspond to ion of highest mass in the spectrum
- excluding those from isotopic compounds (usually lower intensity)
2. Decreasing energy in the ionizing electron stream (EI) should result in an increase in
relative intensity of the molecular ion
3. The ion must have an odd number of electrons.
4. The “Nitrogen Rule” must be obeyed
- a molecule with an odd number of N atoms has an odd atomic mass
- a molecule with an even number of N will have an even atomic mass
5. Observed fragmentation must be reasonable
- ion must be capable of forming the fragment ions seen by loss of neutral ions.
Unlikely losses Losses that identify M+
[M-4] – [M-14] [M-15] – CH3
[M-21] – [M-25] [M-18] – H2O
[M-33], [M-37], [M-38] [M-31] – CH3O
[M-16], [M-18] likely only if O present
Mass Spectrometry
Determination of the Molecular Formula
Molecular Ion Requirements
