Transcript Mass Spectrometry and Organic

Mass Spectrometry
•A key Tool for the chemist’s
toolbox.
•The logic is, we always want the
molecular weight.
•Second, we can smash out
fragments that are intact structurally
•These are easier to solve and relate
back to the starting structure
Implication is, we don’t
get the sample back; a
destructive method
Mass Spectrometry
A primary tool for chemists from almost every discipline
Molecular Weights are fundamental to almost every structural
question. Molecular weight is not ambiguous. A compound has a
unique MW.
Our ability to analyze compounds on this basis, depends
completely on being able to generate ions from the compound.
Specifically molecular ions*, whose weight is equal to the MW
of the compound, are critical.
Once produced, our analysis according to MW depends on
differential mobility or acceleration of ions proportionate to the
MW.
*We can usefully broaden this definition from [M]+ to embrace
[M+H]+, [M-H]-, Chemical Ionization adducts, etc.
What’s in a Mass Spectrum?
Fragment Ions
Derived from
molecular ion
or higher
weight
fragments
[M+H]+(CI)
Or M•+ (EI)
“molecular ion”
Unit mass
spacing
Not usually
scanned below
m/z=32 (Why?)
In CI, adduct ions,
[M+reagent gas]+
High
mass
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
Molecular Ions
give us the molecular mass
Electron Impact
-e•
-e•
2e-•
-e•
H+ H+
-e•
M
-e•
Chemical Ionization
-e•
Dislodges an
electron
M+•
H+
M
H+
H+
[M+H]+
Weighs one more than MW
Identifying Molecular Ions
•Potential question; Is the largest m/z the molecular ion or is it a
prominent fragment from an even heavier molecule?
•Increase sample loading
•In EI, can lower the beam voltage (make the M•+ less energetic,
perhaps more long-lived.)
•Logical interval between significant peaks and suspected M•+ . i.e.
the loss of 3-14 mass units is unusual, as is loss of 19-25 (except F).
Loss of 33, 35, 38 also unusual. However a loss of 15, 18, 31 is
good evidence for a molecular ion.
•Switch to CI, vary reagent gas. Positive, negative probes. Check for
CI adduct ions. e.g. C2H5+ , CH5+, C3H5+
•Find MW by other method Other compounds present may give ions
that deceive us. May be more detectable.
•Prepare derivative
MS intensities are problematic
The “Nitrogen Rule”
•Molecules containing atoms limited to C,H,O,N,S,X,P
of even-numbered molecular weight contain either NO
nitrogen or an even number of N
•This is true as well for radicals as well.
• Not true for pre-charged, e.g. quats, (rule inverts) or
radical cations.
•In the case of Chemical Ionization, where [M+H]+ is
observed, need to subtract 1, then apply nitrogen rule.
•Example, if we know a compound is free of nitrogen and
gives an ion at m/z=201, then that peak cannot be the
molecular ion.
ElectronImpact and ChemicalIonization
EI
CI
Sometimes too energetic for
molecular ion to survive
Stronger, more reliable molecular
ions
Rich harvest of fragment
ions
Fewer fragments
“fingerprint” nature of
fragment patterns lends itself
to database library searches
Can choose different reagent gasses
and exploit chemistry, giving
different fragmentation. e.g.
NH3/ND3
Adduct ions give support to
identities
EI
CI
Nitrogen rule works but inverted
Can do negative ion Mass Spec
When would you use CI, EI?
EI
CI
•When “fingerprint” is needed
for Identification by
comparison screening in
databases
•When rapid, reliable identification
of molecular ion is needed.
•Trace analysis
•Forensics
•Environmental
•Total unknowns, e.g natural
products
•LC-MS
•Following a synthetic chemistry
route, tentative impurity ID
•Biological samples, other fragile or
sensitive to decomposition; Drug
or other metabolite ID
•When reagent gas chemistry is key,
e.g. exchange D in for H
•Fragment homology within a
series, e.g. of natural products •Minimize fragmentation, get most
intensity in molecular ion.
How can I tell which (EI or CI)
was run?
Chemical Ionization
Adduct ions higher m/z than MH+, ,[M+C2H5]+ ,[M+C3H5]+
[M+NH4]+
Large molecular ion
Relatively few fragment ions
Electron Impact
No ions higher m/z than M•+
Smaller M•+ intensity
Rich family of fragment ions
The “Rule of 13” as an aid to guessing
a molecular Formula
Take the Weight of ion, divide by 13
This answer is N, for (CH)N and any numerical remainder is added as H
e.g.; 92
92/13 = 7 with remainder = 1; C7H8 weighs 92. This is our candidate formula
Can evaluate other alternative candidate formulas possessing heteroatoms. For
each member of the list below, replace the indicated number of CHs in the above
answer
Hetero
substitution
CH
replacement
Hetero
substitution
CH
replacement
O
CH4
P
C2H7
N
CH2
S
C2H8
O+N
C2H6
O+S
C4
F
CH7
I
C10H7
Si
C2H4
Cl,Br
(use isotopes)
Analyzing Ion Clusters:
a way to rule candidate structures
in or out
Mass spectrometry “sees” all the isotopomers as distinct ions
An ion with all 12C is one mass unit different from an ion with one
13C and the rest 12C
Since the isotope distribution in nature is known* for all the
elements (13C is 1.1%), the anticipated range and ratios of ions for
a given formula can be predicted and calculated
Follows a binomial expansion: e.g.; for N carbon atoms
(%12C + %13C)N
Clusters of Ions
Spaced by unit mass
The Nominal mass is
m/z of the lowest
member of the
cluster. This is the
isotopomer that has
all the C’s as 12C, all
protons as 1H, all N’s
as 14N, etc.
Each peak is for the same
molecular formula
Different peaks because
there are some molecules
with 13C, 2H etc.
Especially significant for
Cl, Br
m/z
Isotope Patterns in Ion Clusters
Here are two molecular
ions of nearly the same
m/z. One of them is
“carbon-rich”, and has a
larger number of 13C’s
The other, presumably
has proportionately,
more heteroatoms
C24H50
C12H22O11
Why is this Important?
A rule of thumb, made possible by knowing the isotopic
abundance is that the number of C in a formula is given by:
N=
Intensity
M 1 x90.1
M 
C10

All 12C
C100
1 13C
2 13C
1 13C
From this, it is clear that for large or macromolecules, there will
be practically no population having all 12C or even only 113C
Fragmentation
EI
[M·]+
A+
+
B· (neutral)
or
B+
+
A·
Better carbocation wins and predominates (“Stevenson’s Rule”)
CI
[M+H]+
PH+
+
N
(neutral)
The “Even Electron Rule” dictates that even (non-radical) ions will
not fragment to give two radicals (pos• + neutral•) (CI)
Reading a Mass Spec from the M+•
Down (EI)
Fragment
Due to loss of…
Interpretation
M+• -1
-H•
Aldehydes, tert. Alcohols, cyclic amines
M+• -2
Multiple -H•
Secondary alcohols
M+• -3
Multiple -H•
Primary alcohols
M+• -4 to -13
(doubtful)
Consider contaminants
M+• -14
(doubtful)
CH2• , N• not good losses
M+• -15
CH3•
Available methyl groups, methylesters
M+• -16
O•
Peroxides
M+• -17
OH•
Alcohols, phenols, RCO2H
M+• -18
H2O
alcohols
M+• -19
-F•
M+• -20
-HF
M+• -21 to -25
No peaks expected
M+• -26
HCCH
M+• -27
•HC=CH2 or HCN
HCN from pyridine, anilines
M+• -28
CO or CH2=CH2
Check for McLafferty R&R
How Do I go about using Mass
Spec Data for Unknowns?
First, get the molecular weight
Identify prime, smaller mass losses like water, etc.
Now stop. Don’t worry about the fragments till you have
some candidate structures
Based on NMR, IR get some notions of structure candidates
or partial structures, functional groups
Now go back to MS, predict some fragments your structure
will give, calculate the molecular weights and check MS
Back and forth with other data, to corroborate or refute a
possible structure.
Nominal Mass
Here for example is a list of
the compounds in the Merck
Index (9th ed) that weigh
nominally, 200
Exact mass measurements
can easily distinguish
These instruments (and
other) can generate
exhaustive lists of possible
structure formulas near the
exact mass value.
Example, m/z’s for 157
molar mass: 157
Formula
M+1 M+2 MM
e/o dbr
HN2O8
1.05 1.60 156.9732 e 1.5
HN10O
3.75 0.20 157.0337 e 5.5
H3N3O7
1.41 1.40 156.9971 o 1
H3N11
4.12 0.00 157.0576 o 5
H5N4O6
1.78 1.20 157.021 e 0.5
H7N5O5
2.14 1.00 157.0448 o 0
CHO9
1.45 1.80 156.9619 e 1.5
CHN8O2
4.15 0.43 157.0224 e 5.5
CH3NO8
1.81 1.60 156.9858 o 1
CH3N9O
4.51 0.24 157.0463 o 5
CH5N2O7
2.17 1.41 157.0096 e 0.5
CH5N10
4.88 0.04 157.0702 e 4.5
CH7N3O6
2.54 1.21 157.0335 o 0
C2HN6O3
4.55 0.66 157.0111 e 5.5
C2H3N7O2 4.91 0.47 157.0350 o 5
C2H5O8
2.57 1.61 156.9983 e 0.5
C2H5N8O
5.27 0.28 157.0589 e 4.5
C2H7NO7
2.93 1.42 157.0222 o 0
C2H7N9
5.64 0.09 157.0827 o 4
C3HN4O4
4.94 0.89 156.9998 e 5.5
C3H3N5O3 5.31 0.70 157.0237 o 5
C3H5N6O2 5.67 0.51 157.0476 e 4.5
C3H7N7O
6.03 0.33 157.0714 o 4
C3H9N8
6.4 0.14 157.0953 e 3.5
C4HN2O5
5.34 1.11 156.9885 e 5.5
C4H3N3O4 5.70 0.93 157.0124 o 5
C4H5N4O3 6.07 0.74 157.0362 e 4.5
C4H7N5O2 6.43 0.56 157.0601 o 4
C4H9N6O
6.79 0.38 157.0840 e 3.5
C4H11N7
7.16 0.20 157.1078 o 3
C5HO6
5.74 1.32 156.9772 e 5.5
C5H3NO5
6.10 1.15 157.0011 o 5
C5H5N2O4 6.46 0.97 157.025 e 4.5
C5H7N3O3 6.83 0.79 157.0488 o 4
C5H9N4O2 7.19 0.61 157.0727 e 3.5
C5H11N5O 7.55 0.44 157.0965 o 3
C5H13N6
7.92 0.26 157.1204 e 2.5
C6HN6
8.84 0.33 157.0264 e 9.5
C6H5O5
6.86 1.19 157.0136 e 4.5
C6H7NO4
7.22 1.02 157.0375 o 4
C6H9N2O3 7.59 0.84 157.0614 e 3.5
C6H11N3O2 7.95 0.67 157.0852 o 3
C6H13N4O 8.31 0.50 157.1091 e 2.5
C6H15N5
8.68 0.32 157.1329 o 2
C7HN4O
9.23 0.58 157.0151 e 9.5
C7H3N5
9.60 0.41 157.0390 o 9
C7H9O4
7.98 1.07 157.0501 e 3.5
C7H11NO3
8.35 0.90 157.0739 o 3
C7H13N2O2 8.71 0.73 157.0978 e 2.5
C7H15N3O 9.07 0.56 157.1217 o 2
C7H17N4
9.44 0.40 157.1455 e 1.5
C8HN2O2
9.63 0.81 157.0038 e 9.5
C8H3N3O
9.99 0.65 157.0277 o 9
C8H5N4
10.36 0.49 157.0516 e 8.5
C8H13O3
9.11 0.96 157.0865 e 2.5
C8H15NO2 9.47 0.80 157.1104 o 2
C8H17N2O 9.83 0.64 157.1342 e 1.5
C8H19N3 10.20 0.47 157.1581 o 1
C9HO3
10.03 1.05 156.9925 e 9.5
C9H3NO2 10.39 0.89 157.0164 o 9
C9H5N2O 10.75 0.73 157.0403 e 8.5
C9H7N3
11.12 0.57 157.0641 o 8
C9H17O2 10.23 0.87 157.1229 e 1.5
C9H19NO 10.59 0.71 157.1468 o 1
C9H21N2 10.96 0.55 157.1706 e 0.5
C10H5O2 11.15 0.97 157.029 e 8.5
C10H7NO 11.51 0.81 157.0528 o 8
C10H9N2 11.88 0.66 157.0767 e 7.5
C10H21O 11.35 0.79 157.1593 e 0.5
C10H23N 11.72 0.64 157.1832 o 0
C11H9O
12.27 0.90 157.0654 e 7.5
C11H11N
12.64 0.75 157.0892 o 7
C12H13
13.40 0.84 157.1018 e 6.5
C13H
14.32 0.97 157.0078 e 13.5
Clearly, some are
not realistic!
Calculated mass distributions
IMASS for Mac OSX
Version 1.0 (v2A15)
© 2000 - 2002, Urs Roethlisberger,
Isotopic Element Massesand Atomic Weights:Lide,
D.R., Ed., CRC Handbook of Chemistry and
Physics,74th Ed., CRC Press, Boca Raton FL,(1993)
Isotope Distribution:Rockwood, A. L., Van Orden, S.
L.,Smith, R. D.,Anal. Chem. , 67, 2699, (1995)
•iMass is freeware.
•Contact: [email protected]
Fragment Ions
•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
Chemical Ionization
Fragmentation
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 acidbase (proton affinities)
Some popular cleavages
Cleave at a branch point. Loss of
radical or other neutral to provide a
more stable cation
+
H3C
CH3
CH3
H3C
Cleave  to a heteroatom
(capable of supporting
positive charge)
Note the use of “half arrow”
for one-electron movements.
e.g homolytic cleavage
+
CH3
H3C
C
+
Obs. in Mass Spec
RO:
CH3.
neutral
RO +
RO :
Obs. in Mass Spec
Resonance stabilized
+
neutral
Some examples
Primary alcohols, m/z=31
CH2=OH+
Primary amines, m/z=30
CH2=NH2+
Commonly encountered
Electron-Impact fragments
O
+
29
H
CH2 +
43
H
77
H
+
CH2 +
N
92
H
H
+
H
91
+
McLafferty Rearrangements
Radical cations localized on keto-type oxygen give  cleavage
The mechanism limits this to EI fragmentation
Needs a H atom on a  sp3 carbon
Ketones, esters, carboxylic acids all give McLafferty products
H
+
O•
R1
R2
H
+
O
R1
•
Note the use here, of
the “half arrow” to
represent “1-electron
flow”
The new radical
cation is
stabilized by
resonance
••
• OH
+
R2
Loss of
neutral
alkene
Important example of McLafferty
R&R
OH
+
•
OH
m/z = 60
Seen for primary carboxylic acids
Non-Sequential Losses
O
CH3
M+-CH3CO
M+-CH3
MW=152
CH3
Hydrocarbons
Weak [M•]+
Intense CnH2n+1
Good 43 m/z = C3H7 protonated cyclopropane
57 m/z = C4H9+
71 m/z = C5H11
Hydrocarbon chains characterized by successive
losses of m/z=14 (clusters)
Cleavage  to C=O groups
+
:O .
:O +
+
neutral
Prominent for
ketones
:O:
+
Obs. in mass spec.
Acylium ions are
resonance-stabilized
CH3C=O+
m/z=43
Example
O
O
+
O
C
O
O
O
+
+
M+• -45, loss of
ethoxy radical
Example
+
O
O
M+• -43; also
tropylium ion
Cleave  to Heteroatoms like O, N
R
+
R
:O.
•
neutral
: O. :
+
+
Heterolytic cleavage
Observed in Mass Spec
provided that a good stabilized
carbocation can form
Rearrangements and fragmentations
to give good Carbocations
CH 2+
Benzylic
cation
(stabilized
C+
H
including
“tropylium” H+C
ion m/z=91
CH 2
CH 2
CH+
Good cleavage  to
aromatic rings
Example
Br
Tropylium
ion
Bromine
pattern
Carboxylic acids
H present?; can give McLafferty R&R to alkene plus
CH2=C(OH)(OH)•+ at m/z=60
Loss of water, especially in CI
Loss of 44 is loss of CO2
m/z=45 suggests OC–OH+
Amines
N•+
-R•
N
+
R
Cyclic amines will lose adjacent H•,
form iminium ion
In CI, NH+ can eliminate adjacent
alkene, reprotonate
Silyl Ethers
O+
•
Si
Loss of CH3• from Si
Loss of R• in  cleavage
Loss of •CR3 then CH3• to (CH3)2Si=OH+ m/z=75
Total loss of carbinol to (CH3)3Si+ m/z=73
H transfer in heterosubstituted
Anisoles
OR
Loss of CH2O
+
OR
H
OCH3
+•
Extra H
transfer
mediated by
adjacent
heteroatom
H
H
Nitroaromatics
m/z= 93
Loss of
•N=O
+
N
O
+
O
O•
Loss of
CO
Aromatic!
m/z=65
Good test for
aryloxy
CH+
(this can
form
from lots
of
different
origins)
Sulfur Compounds
Fortunately there is an [M+2]+ of 4% for the natural
abundance of 34S. This is diagnostic for S vs 2x16O
Aliphatic thiols can split out H2S, [M-34]
Alpha cleavage at carbon bearing the sulfur in thiols,
thioethers, similar to ethers, etc.
R
-R•
S
•
+
S
+
The “retro Diels-Alder” Cleavage
Cyclohexenes, with favorable 6-membered
transition state. Can include heteroatoms (N,O,
driven by keto-enol like stability.
Observed!
+
•
+
•
+
Typically you see both.
More stable cation will predominate
Also works for hetero-substituted (e.g. make
enol)
Both EI (shown) and in Chemical Ionization.
(protonated molecular ion, cleave, then
reprotonation
+
•
+
Observed!
An Example from Terpenoid
Chemistry
+
•
+
+
•
12-Oleanene
m/z 204
A good example for Retro Diels
Alder fragmentation
HO
EI Mass
Spectrum
HO
4-terpineol
+
mz 68
MW 154
+
+
mz 86
O
Double bonds can isomerize
OH
MW=396
-cleavage
following
double bond
migration
m/z118
136-water
m/z136
C9H12O+
Mass Spectral
“shifts”
Note highly
conserved regions;
series of related
compounds
Losses down to ions
common in series.
Variation can not
influence the
fragmentation or
introduce new
fragmentation, e.g.
internal fission not
possible for homologs
Using the Information in Ion
Clusters--Halogens
The paired
appearance
flags the ions
as to the
number of
halogens
35Cl
37Cl
CH3Cl
CHCl3
One chlorine
Three
chlorines
Fragment ions
with the same
halogen count
preserve the
pattern
79Br
81Br
81Br
81Br
CH3Br
CHBr3
One
bromine
Three
bromines
2
1
Great Websites
http://medlib.med.utah.edu/masspec/elcomp.htm
Calculate potential molecular formulas from m/z
(neutrals only)
http://www.colby.edu/chemistry/PChem/Fragment.html
Wizard calculates both odd, even electron species based
on m/z
The same folks provide a online wizard for calculating
ion clusters (isotope patterns) from a suggested formula.
http://www.colby.edu/chemistry/NMR/IsoClus.html
http://webbook.nist.gov/chemistry/
Free search of name, formulas