MASS SPECTROSCOPY.ppt

Mass Spectroscopy
Mass 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
• Structure (from fragmentation fingerprint)
• Isotopic incorporation / distribution
• Protein sequence

Pharmaceutical analysis
Bioavailability studies
Drug metabolism studies, pharmacokinetics
Characterization of potential drugs
Drug degradation product analysis
Screening of drug candidates
Identifying drug targets
Biomolecule characterization
Proteins and peptides
Oligonucleotides
Environmental analysis
Pesticides on foods
Soil and groundwater contamination
Forensic analysis/clinical
Applications of Mass Spectrometry

Atom or molecule is hit by high-energy electron
Principles of Electron-Impact Mass Spectrometry
e–

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

This energy-rich species ejects an electron.

This energy-rich species ejects an electron.
forming a positively charged, odd-electron species
called the molecular ion
e–
+
•

from an electron beam at 10ev
e–
beam
forming a positively charged, odd-electron
species called the molecular ion
e–
+
•

Molecular ion passes between poles of a
magnet and is deflected by magnetic field
amount of
deflection depends
on mass-to-charge
ratio
highest m/z
deflected least
lowest m/z
deflected 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.
+ •
Usually several fragmentation pathways are
available and a mixture of ions is produced.

mixture of ions of
different mass
gives separate peak
for each m/z
intensity of peak
proportional to
percentage of each
ion of different
mass in mixture
separation of peaks
depends on relative
mass
+
+
+
+
+
+

mixture of ions of
different mass
gives separate peak
for each m/z
intensity of peak
proportional to
percentage of each
atom of different
mass in mixture
separation of peaks
depends on relative
mass
+ + + +
+ +

What’s in a Mass Spectrum?
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
[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: A plot of the relative abundance
of ions versus their mass-to-charge ratio (m/z).
• 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 Spectrum

• 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 Ion

M + e-  M+ + 2e-
Molecule High Energy
Electron
Molecular
Ion
(Radical Cation)
100
90
80
70
60
50
40
30
20
10
0
Intensity
(%
of
Base
Peak)
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-
Electron
Molecular
Ion
(Radical Cation)
M + e-  M+ + 2e-
Electron
Molecular
Ion
(Radical Cation)
100
90
80
70
60
50
40
30
20
10
0
Intensity
(%
of
Base
Peak)
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 dopamine

AN INSTRUMENT THAT GENERATES IONS FROM MOLECULES
AND MEASURES THEIR MASSES
THE ESSENTIAL COMPONENTS OF A MASS SPECTROMETER:
SAMPLE
INLET
ION
SOURCE
ION
ACCELERATOR
ION
ANALYSER
ION
DETECTOR
signal
COMPUTER
MASS 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 SPECTROMETER

Illustration of the basic components of a mass spectrometry system.
Ionization
Source
Mass
Analzyer
Detector
Inlet all ions
selected
ions
Data
System
Diagram of a simple mass spectrometer

2. Atomic & Mass Number
A
Z X
atomic number
(number of protons)
(number of electrons)
mass number
(number of protons plus neutrons)

WAYS 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 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 Ionization (EI)

 Electron 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 (CI)
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
+
2
5
2
4
3
3
5
4
4
H
H
C
CH
CH
CH
CH
CH
CH










6
2
5
2
4
2
2
5
2
4
2
5
H
C
M
MH
H
C
H
C
MH
MH
H
C
CH
MH
MH
CH















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)

• Resolution: A measure of how well a mass
spectrometer separates ions of different
mass.
– 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: Refers to instruments
capable of separating ions that differ in
mass by as little as 0.0001 atomic mass unit.
Resolution

In
ten
sity
(%)
0
20
40
60
80
100
Mass [amu]
111.95 112.00 112.05 112.10
In
ten
sity
(%)
0
20
40
60
80
100
Mass [amu]
111.95 112.00 112.05 112.10
In
ten
sity
(%)
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
C6H5OF
C6H5Cl
Resolving 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 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.
C2 H4 O2
C3 H8 O
60.02112
60.05754
60
60
Molecular
Formula
Nominal
Mass
Precise
Mass
Resolution
– 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?
Atomic 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:
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 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 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 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 Rule”
[M·]+ A+ + B· (neutral)
or
B+ + A·
EI
Fragmentation
 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
Fragmentation

 General Fragmentation Pathways
– One-bond cleavages
a-cleavages
C OH
R
- R C OH C O
Cleave a to Heteroatoms like O, N
O
R
: .
+
•
R
O:
: .
neutral
+
+
Heterolytic cleavage
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 a 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)

b-cleavages
– Cleave b 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
examples
Primary alcohols, m/z =31 CH2=OH+
Primary amines, m/z =30 CH2=NH2
+
m/z 30
+
+
•
b-cleavage
CH3 CH3
CH3 - CH- CH2 -CH2 -NH2 CH3 - CH- CH2 CH2 = NH2

 Two-bond cleavages
– Eliminate H-X
– Retro Diels-Alder
–McLafferty rearrangement
+
C
C
O
C
C
H2
CH2
CR2
H
Y
-R2=CH2
Y = H, R, OH, OR
NR2
O
C
CH2
H
Y
O
C
CH2
H
Y
O
C
CH2
H
Y
need g-hydrogens

 Alkane 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
C
H3 CH3
CH3
C
H3
C
H3
C
+
CH3
CH3.
Obs. in Mass Spec
+
neutral
+

MASS SPECTROSCOPY.ppt

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MASS SPECTROSCOPY.ppt