Transcript Chapter 14: Mass Spectrometry

Mass Spectrometry
Mass Spectrometry (MS)
• An analytical technique for measuring the
mass-to-charge ratio (m/z) of ions in the gas
phase.
– Mass spectrometry is our most valuable
analytical tool for determining accurate
molecular masses.
– Also can give information about structure.
– Proteins can now be sequenced by MS.
A Mass Spectrometer
• A mass spectrometer is designed to do three
things:
– Convert neutral atoms or molecules into a
beam of positive (or rarely negative) ions.
– Separate the ions on the basis of their
mass-to-charge (m/z) ratio.
– Measure the relative abundance of each
ion.
Mass Spectrometry (MS)
• Schematic of an electron ionization
mass spectrometer (EI-MS).
A Mass Spectrometer
• Electron Ionization MS
– In the ionization chamber, the sample is
bombarded with a beam of high-energy
electrons.
– Collisions between these electrons and the
sample result in loss of electrons from sample
molecules and formation of positive ions.
H
H C
H
+
H
H + e
H
C
H
H
Molecular ion
(a radical cation)
+2 e
Molecular Ion
• Molecular ion (M): A radical cation formed by
removal of a single electron from a parent molecule
in a mass spectrometer.
• 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
At times, however, we find it useful to depict the
radical cation at a certain position in order to
better understand its reactions.
CH3 CH2 OCH( CH3 ) 2
.
CH3 CH2 OCH( CH3 ) 2
Mass Spectrum
• Mass spectrum: A plot of the relative
abundance of ions versus their mass-tocharge ratio.
• 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.
MS of dopamine
A partial mass spectrum of dopamine showing
all peaks with intensity equal to or greater than
0.5% of the base peak.
Other MS techniques
• What we have described is called electron
ionization mass spectrometry (EI-MS).
• Other mass spectrometry techniques include
– fast atom bombardment (FAB).
– matrix-assisted laser desorption ionization
(MALDI).
– chemical ionization (CI).
– electrospray ionization (ESI).
Resolution
• Resolution: A measure of how well a mass
spectrometer separates ions of different masses.
– Low Resolution MS (LR-MS): 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
(amu).
– High resolution MS (HR-MS): Refers to
instruments capable of separating ions that
differ in mass by as little as 0.0001 amu.
Resolution
– C3H6O and C3H8O have nominal masses of 58
and 60, and can be distinguished by lowresolution MS.
– C3H8O and C2H4O2 both have nominal masses of
60 which occurs due to isotopes of the same
elements.
– distinguish between them by high-resolution MS.
M o l e cu l ar N o m i n al
Form u l a
M as s
P re c i s e
M as s
C3 H8 O
60
60.05754
C2 H4 O 2
60
60.02112
Isotopes
Virtually all
elements
common to
organic
compounds
are mixtures
of isotopes
with different
relative
abundances.
El e m e n t
h y d ro ge n
A to m i c
M as s
w e i gh t I s o to p e (am u )
1.0079
carb o n
12.011
n i tro ge n
14.007
o xyg e n
15.999
s u l fu r
32.066
ch l ori n e
35.453
b rom i n e
79.904
1
R e l ati v e
A b u n d an ce
H
H
1.00783
2.01410
100
0.016
C
C
12.0000
13.0034
100
1.11
N
15
N
16
O
18
O
14.0031
15.0001
15.9949
17.9992
100
0.38
100
0.20
31.9721
33.9679
100
4.40
34.9689
36.9659
100
32.5
78.9183
80.9163
100
98.0
2
12
13
14
32
S
34
S
35
Cl
37
Cl
79
81
Br
Br
Isotopes
– Carbon, for example, in nature is 98.90%
12C and 1.10% 113C.
– There are 1.11 atoms of carbon-13 in
nature for every 100 atoms of carbon-12.
So low intensity M+1 peak is very likely to
appear along with M+ peak.
1.10
13
Cx
100
98.90
12
C
x 100
= 1.11 atoms
13
C per 100 atoms
12
C
M+2 and M+1 Peaks
• 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, as seen in
the MS of chloroethane.
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 atom
in a compound, as seen in the MS of
1-bromopropane.
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%
• 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.
Interpreting a mass spectrum
• The only elements to give significant M + 2 peaks
are Cl and Br.
– If no large M + 2 peak is present, these elements
are absent.
• Is the mass of the molecular ion odd or even?
• Nitrogen Rule: If a compound has
– zero or an even number of nitrogen atoms, its
molecular ion will have an even m/z value.
– an odd number of nitrogen atoms, its molecular
ion will have an odd m/z value.
Fragmentation of the Molecular Ion
• To attain high efficiency of molecular ion
formation and give reproducible mass
spectra, it is common to use electrons with
energies of approximately 70 eV [6750 kJ
(1600 kcal)/mol].
– This energy is sufficient not only to dislodge one or
more electrons from a molecule, but also to cause
extensive fragmentation.
– These fragments may be unstable as well and, in
turn, break apart to even smaller fragments.
Fragmentation of M
• Fragmentation of a molecular ion,
produces a radical and a cation.
– Only the cation is detected by MS.
A •
+
A -B
+
R ad i cal
(a rad i cal c atio n )
A
+
C ati on
B+
Cati o n
•
M o l e cu l ar i on
M,
+
• B
R ad i cal
Fragmentation of M
• A great deal of the chemistry of ion fragmentation
can be understood in terms of the formation and
relative stabilities of carbocations in solution.
– Where fragmentation occurs to form new cations,
the mode that gives the most stable cation is
favored.
– The probability of fragmentation to form new
carbocations increases in the order.
CH 3
+
2°
3°
< 1° < 1° al l yl i c < 2° al l yl i c <
3° al l yl i c
3° b e n z y l i c
1° b e n z y l i c
2° b e n z y l i c
Interpreting a mass spectrum
• The only elements to give significant M + 2 peaks
are Cl and Br.
– If no large M + 2 peak is present, these elements
are absent.
• Is the mass of the molecular ion odd or even?
• Nitrogen Rule: If a compound has
– zero or an even number of nitrogen atoms, its
molecular ion will have an even m/z value.
– an odd number of nitrogen atoms, its molecular
ion will have an odd m/z value.
Alkanes
• Fragmentation tends to occur in the middle of
unbranched chains rather than at the ends.
• The difference in energy among allylic, benzylic,
3°, 2°, 1°, and methyl cations is much greater
than the difference among comparable radicals.
– Where alternative modes of fragmentation are
possible, the more stable carbocation tends to
form in preference to the more stable radical.
Alkanes
– Mass spectrum of octane.
Alkanes
– Mass spectrum of 2,2,4-trimethylpentane.
Alkanes
– Mass spectrum of methylcyclopentane.
Alkenes
• Alkenes characteristically
– show a strong molecular ion peak.
– cleave to form resonance-stabilized allylic cations.
+
[ CH 2 = CH CH 2 CH 2 CH 3 ] •
CH 2 = CHCH 2
+
+
•
CH 2 CH 3
Cyclohexenes
– Cyclohexenes give a 1,3-diene and an alkene, a
process that is the reverse of a Diels-Alder
reaction.
CH 3
+
•
CH 3
H 3C
C
H3 C
C
+
•
+
CH 2
CH 2
Li m on e n e
(m / z 1 36 )
A n e u tra l d i e n e
(m / z 6 8)
A ra d i c a l ca ti o n
(m / z 6 8)
Alkynes
• Alkynes typically
– show a strong molecular ion peak.
– cleave readily to form the resonance-stabilized
propargyl cation or substituted propargyl cations.
3-P ro p y n y l cati o n
HC
(P ro p argy l cati o n )
C-CH 2
+
+
HC
C= CH 2
Alcohols
• One of the most common fragmentation patterns of
alcohols is loss of H2O to give a peak which
corresponds to M-18.
• Another common pattern is loss of an alkyl group
from the carbon bearing the OH to give a
resonance-stabilized oxonium ion and an alkyl
radical.
R'
•
R C
+
O H
••
+
R•
R"
Molecular ion
(a radical cation)
+
R' - C = O - H
+
R' - C ••O H
R"
A radical
••
A res onance-s tabilized
oxonium ion
R"
Alcohols
– Mass spectrum of 1-butanol.
Aldehydes and Ketones
• Characteristic fragmentation patterns are
- -cleavage
– McLafferty rearrangement
O
+
+•
O
+
•
m/z 43
 - cleavage
O
m/z 128
CH3 •
+
+
m/z 113
H
+•
O
Molecular ion
m/z 114
McLafferty
rearrangement
H
+
O
m/z 58
+•
Aldehydes and Ketones
– Mass spectrum of 2-octanone.
Carboxylic Acids
• Characteristic fragmentation patterns are
-cleavage to give the ion [CO2H]+ with m/z 45.
– McLafferty rearrangement.
O
 - cleavage
•
+
OH
O = C-O - H
m/z 45
Molecular ion
m/z 88
H
+•
O
McLafferty
rearrangement
H
+
O
OH
Molecular ion
m/z 88
+•
OH
m/z 60
+
Carboxylic Acids
– Mass spectrum of butanoic acid.
Esters
 -cleavage and McLafferty rearrangement
O
+
•
O
+
 - cleavage
+
•
OCH3
m/z 71
OCH3
O
Molecular ion
+
+
m/z 102
OCH3
m/z 59
H
+
•
O
OCH3
Molecular ion
m/z 102
McLafferty
rearrang ement
H
+
•
O
+
OCH3
m/z 74
Esters
– Mass spectrum of methyl butanoate.
Aromatic Hydrocarbons
– Most show an intense molecular ion peak.
– Most alkylbenzenes show a fragment ion of m/z 91.
H
+
•
CH3
T oluene radical
cation
- H•
H
H
+
H
H
H
H
Tropylium cation
(m/z 91)
Amines
• The most characteristic fragmentation pattern of
1°, 2°, and 3° aliphatic amines is -cleavage.
CH3
C H 3 - C H - CH 2 - C H 2 - N H 2
 - cleavage
CH3
C H 3 - C H - CH 2 •
+
+ CH2 = N H2
m/z 30
Mass spectrometry by chemical ionization
• In chemical ionization (C.I.) mass spectrometry the
electron beam is used to ionize a simple molecule
(reagent gas) such as methane which in turn ionizes our
molecule by collision and transfer of a proton.
Under electron bombardment, methane loses a bonding electron
to give CH4+• which reacts with an unionized methane molecule
to give CH3• and CH5+.
CH5+ does have a carbon atom with five bonds. But it has only
eight electrons! These are distributed between five bonds (hence
the + charge) and the structure is thought to be trigonal
bipyramidal.
This structure has not been determined as it is too unstable. It is
merely proposed from theoretical calculations.
Chemical Ionization (CI)
• This unstable compound is a powerful acid, and can
protonate just about any other molecule.
When it protonates our sample, a proton has been added rather
than an electron removed, so the resulting particles are simple
cations, not radical cations, and are generally more stable than the
radical cations produced by direct electron impact.
So the molecular ion has a better chance of lasting the necessary
20 ms to reach the detector.
Note :
We observe [M + H]+ (i.e. one more than the molecular mass)
rather than M+ by this method.
Fast Atom Bombardment (FAB)
• Polar molecules, such as peptides, with molecular
weights up to 10,000 Da can be analyzed by a "soft"
ionization technique called fast atom bombardment
(FAB).
• The bombarding beam consists of xenon (or argon)
atoms of high translational energy (Xe). This beam is
produced by first ionizing xenon atoms with electrons to
give xenon radical cations:
• The radical cations are accelerated to 6-10 keV to give radical
cations of high translational energy (Xe)∙+, which are then passed
through xenon. During this passage, the charged high-energy
xenon obtains electrons from the xenon atoms to become highenergy atoms (Xe) or fast atoms, and the (Xe)∙+ ions are removed
by an electric field.
• The compound of interest is dissolved in a high-boiling viscous
solvent such as glycerol; a drop is placed on a thin metal sheet,
and the compound is ionized by the high-energy beam of xenon
atoms (Xe). Ionization by translational energy minimizes the
amount of vibrational excitation, and this results in less
destruction of the molecule.
Matrix Assisted Laser Desorption Ionization (MALDI)
Laser
Sample plate
hn
1. Sample (A) is mixed with
excess matrix (M) and dried
on a MALDI plate.
AH+
2. Laser flash ionizes matrix
molecules.
3. Sample molecules are ionized
by proton transfer from matrix:
MH+ + A  M + AH+.
+20 kV
Variable Ground
Grid
Grid
Matrix Assisted Laser Desorption lionization (MALDI)
• In the MALDI procedure - used mainly for large biomolecules - the
sample in a matrix is dispersed on a surface, and is desorbed and
ionized by the energy of a laser beam.
• The MALDI procedure has been used recently in several variations to
determine the molecular weight of large protein molecules - up to
several hundred kDa.
• Peptide sequencing is another application.
• Matrix selection is critical and depends on the wavelength of the laser
beam and on the nature of the sample.
• ESI (Electrospray ionization) and MALDI are the preferred
procedures for large biopolymers.