MASS SPECTROMETRY: BASIC Pavia 8.1-8.5
EXPERIMENT
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Molecule
-eMolecule
+.
Types of Ionization
Electron Ionization, EI
Chemical Ionization, CI
Desorption Ionization Techniques
Secondary Ion MS, SIMS
Fast Atom Bombardment, FAB
Matrix Assisted Laser Desorption Ionizaton, MALDI
Electrospray Ionization, ESI
Electron Ionization
High vacuum
en.wikipedia.org
M + e-
M+ . + 2e-
MW=142
Effect of electron energy on appearance of E.I
spectra
M + e–
M +• + 2e–
70 eV
E.I.
20 eV
E.I.
14 eV
E.I.
11 eV
E.I.
http://science.widener.edu/svb/massspec/ei.ht
ml
1-propanol (C3H8O, M = 60)
Chemical Ionization
Introduction of
Ionizing reagent
CH4, NH3,
etc
CH5+
CH5+
CH4
CH4 CH
M4
CH5+
CH
4
CH
4
vacuum
CH4 + e-
.
CH4+ + 2e-
CH4+. + CH4
CH5+ + .CH3
M + CH5+
(M+H)+ + CH4
MW=142
EI
CH4
(CH3)2CHCH3
Desorption Ionization Techniques:
SIMS: secondary ion bombardment
Xe: Fast AtomBombardment
h: Matrix Assisted Laser Desorption Ionization
The matrix can be many compounds (see list Fiqs 8.6 & 8.7).
Very often the ion with the highest MW is M+matrix.
Electrospray Ionization (ESI)
Used for very high molecular weight biological molecules
because
multiple charged species are formed.
ESI mass spectrum of horse heart myoglobin (mass 16955 Da)
Mass Analyzers
Magnetic sector
scienceaid.co.uk
Kinetic energy = 1/2mv2 = zV
r = mv /zB
m/z = B2r2/2V
Double focusing Mass Spectrometer
home.postech.ac.kr
epa.gov
Quadrapole Mass Spectrometer
Ion Trap
Low MW < 1000 and unit mass resolution.
Very compact and relatively cheap.
Time of Flight (ToF)
Detects all ions so this is very sensitive.
Can be used for very high molecular weight.
High resolution.
When coupled to MALDI, ToF is excellent for biological samples
Pavia 8.8
IONIZATION
Where does the electron come from?
M + e-
M+ . + 2e-
Pavia 8.8
FRAGMENTATION
Radical cations formed upon electron impact are unstable!
Remember: You will only see positively charged species (cations and
radical cations), not neutral radicals.
Fragmentation of Radical Cations
Radical cations are very reactive species
Radical cations are very unstable. Fragmentations are unimolecular
chemical reactions. The relative stability of cations and radical cations
can often be understood in terms of substitution patterns and
resonance.
Remember: Ease of formation of carbocations
less stable
more stable
c.f. ease of SN1 reactions of R-Hal, or E1 dehydration of ROH
Alkyl groups are inductive electron donating substituents
Allylic and benzylic cations are resonance stabilized
Pavia 8.8 I
Alkanes
57
M–29
E.I.
43
M–43
29
M–57
71
M–15
base peak
57
86
M+
Spectral Database for Organic Compounds (SDBS)
hexane (C6H14; M = 86)
small
M–15
Linear Alkanes - Molecular ions fragment by cleavage of C-C
bonds to give one cation and one radical
29
M–57
43
M–43
57
M–29
71
M–15
86
M+
Peaks appear in clusters separated by 14 mass units ….
…with other fragments corresponding to 1 and 2 mass units
fewer.
Alkyl radical cations can lose a hydrogen atom (radical) to give a
new cation radical, e.g.:
2-methylpentane (C6H14; M = 86)
71
M–15
57
M–29
29
M–57
base peak
43
86
M+
Spectral Database for Organic Compounds
(SDBS)
E.I.
43
M–43
smaller
M+
43
M–43
57
M–29
3-methylpentane (C6H14; M = 86)
29
M–57
71
M–15
86
M+
57
M–29
E.I.
43
M–43
71
M–15
base peak
57
86
M+
Spectral Database for Organic Compounds (SDBS)
29
M–57
even smaller
M+
29
M–57
43
M–43
57
M–29
71
M–15
86
M+
M–43 ?! (Radical) Carbocations rearrange:
43
M–43
57
M–29
71
M–15
E.I.
29
M–57
86
M+
Spectral Database for Organic Compounds (SDBS)
2,2-dimethylbutane
(C6H14; M = 86)
No
M+ !!
29
M–57
43
M–43
57
M–29
71
M–15
86
M+
Branched alkanes give weaker M•+ ions, and stronger M – 15
peaks.
Which is which?
(all C6H14)
hexane
2-methylpentane
2,2-dimethylbutane
70
43
Which is which?
(all C10H22; M = 142)
57
113
142
3,4-diethylhexane
3,3-dimethyloctane
5-methylnonane
71
43
57
113
43
85
57
Summary: Alkanes
For straight chain alkanes
- Molecular ion peak is usually present but weak
- Clusters of fragments appear spaced by m/z of 14,
corresponding to CH2
- The largest peak in each cluster corresponds to an alkyl radical
cation, CnH2n+1
- A peak for M – CH3 is often weak or absent
- The intensity of lower m/z fragments is greater, with the
intensities decreasing smoothly up to M – C2H5.
Branched alkanes
- Smaller molecular ion peak
- More fragmentation at highly branched positions
Pavia 8.8 J
Cycloalkanes
56
M–28
cyclohexane (C6H12; M = 84)
E.I.
Spectral Database for Organic Compounds
(SDBS)
84
M+
41
M–43
69
M–15
base peak
has even m/z
M–28
methylcyclopentane (C6H12; M = 84)
strong
M+ !!
56
M–28
41
M–43
84
M+
Spectral Database for Organic Compounds
(SDBS)
E.I.
Summary: Cycloalkanes
1. Relatively large molecular ion peak
2. Significant peak at M – 28 (often the base peak)
3. M–15: from rearrangement
Pavia 8.8 K
Alkenes
1-hexene (C6H12; M = 84)
56
M–28
E.I.
84
M+
strong
M–43
base peak
has even m/z
M–28
Spectral Database for Organic Compounds
(SDBS)
41
M–43
Summary: Alkenes
1. Relatively strong M+ ion
2. Strong peak arises from fragmentation to form a resonance
stabilized allylic cation
3. Difficult to identify position of alkene since the double bond
migrates easily.
cyclohexene (C6H10; M = 82)
82
M+
base peak
has even m/z
M–28
?!
Spectral Database for Organic Compounds (SDBS)
E.I.
54
M–28
Retro-Diels-Alder Reaction
E.I.
base peak
has even m/z
M–68
www.chem.sc.edu/analytical/chem621/lab/spme.html
limonene (C10H16; M = 136)
69
41
Which is which?
56
(all C8H16; M = 112)
83
97
55
112
propylcyclopentane
2-ethyl-1-hexene
2,5-dimethyl-1-hexene
70
41
83
112
56
41
69
112
Pavia 8.8 L
Alkynes
1-hexyne (C6H10; M = 82)
39
81
M–1
82
M+
M–1 !!
Spectral Database for Organic Compounds
(SDBS)
E.I.
Summary: Alkynes
M–1 peak observed
strong peak arises from fragmentation to form a resonance
stabilized propargyl cation
Pavia 8.8 M
Aromatic Hydrocarbons
91
M–1
E.I.
92
M+
Spectral Database for Organic Compounds
(SDBS)
toluene (C7H8; M = 92)
strong
m/z = 91
E.I.
106
M+
Spectral Database for Organic Compounds (SDBS)
91
M–15
ethylbenzene (C8H10; M = 106)
strong
m/z = 91
91
M–43
E.I.
134
M+
strong
m/z = 91
Spectral Database for Organic Compounds (SDBS)
1-butylbenzene (C10H14; M = 134)
-bromotoluene (C7H7Br, M = 170[79Br])
E.I.
Spectral Database for Organic Compounds (SDBS)
91
M–79
170 172
M+ M+2
strong
m/z = 91
What are all those m/z = 91 for PhCH2X?
1,4-diethylbenzene, M =134
Pavia 8.8 N,O,T
Alcohols, Ethers and Amines
56
M–46
E.I.
43
M–59
31
M–71
69
M–33
84
M–18
m/z = 31
Base peak
m/z = even !!
M–18
??
102
M+
Spectral Database for Organic Compounds
(SDBS)
1-hexanol (C6H14O, M = 102)
Summary: Alcohols
1. Weak M+ peak
2. -cleavage of an alkyl radical
3. Dehydration
4. Dehydration with loss of CH2=CH2
45
M–57
E.I.
69
M–33
87 102
84
M–18 M–15 M+
Spectral Database for Organic Compounds (SDBS)
2-hexanol (C6H14O, M = 102)
70
56
Which is which?
43
(all C7H16O; M = 116)
98
83
59
41
1-heptanol
2,2-dimethyl-3pentanol
3-ethyl-3-pentanol
87
69
101
87
45
69
98
45
M–57
E.I.
87
M–15
102
M+
Spectral Database for Organic Compounds (SDBS)
diisopropyl ether
(C6H14O, M = 102)
Summary: Ethers
1. -cleavage of alkyl radical
2. C-O bond cleavage
86
M–43
E.I.
Spectral Database for Organic Compounds (SDBS)
dibutyl amine (C8H19N, M = 129)
The Nitrogen Rule:
C,H,N,O,S molecules with
an odd molecular mass
must contain an odd
number of nitrogen atoms
129
M+
129
M+
58
M–71
Spectral Database for Organic Compounds (SDBS)
44
M– 85
129
M+
Spectral Database for Organic Compounds (SDBS)
1-methylheptylamine (C8H19N, M = 129)
E.I.
Which amine?
E.I.
43
Identify each
compound
31
59
45
73
102
There is one alcohol,
one ether and one
amine
57
69
87
102
86
30
58
101
dipropylether
3,3-dimethyl-2-butanol
triethylamine
C12H27N (MW = 185)
E.I.
C.I.
(CH4)
science.widener.edu/svb/massspec/ci.html
E.I and C.I of tri-n-butylamine
Pavia 8.8 P-Q
Aldehydes and ketones
propanal (C3H6O, M = 58)
E.I.
Spectral Database for Organic Compounds
(SDBS)
58
M+
57
M–1
2-butanone (C4H8O, M = 72)
43
M–29
E.I.
57
M–15
72
M+
Spectral Database for Organic Compounds (SDBS)
29
M–29
44
M–56
100
M+
2-hexanone
(C6H12O, M = 100)
43
M–57
E.I.
58
M–42
29
M–81
Spectral Database for Organic Compounds (SDBS)
E.I.
56
M–44
85
M–15
100
M+
Spectral Database for Organic Compounds (SDBS)
hexanal (C6H12O, M = 100)
106
M+
105
M–1
105
M–15
acetophenone (C8H8O, M = 120)
E.I.
Spectral Database for Organic Compounds (SDBS)
77
M–29
E.I.
77
M–43
120
M+
Spectral Database for Organic Compounds (SDBS)
benzaldehyde (C7H6O, M = 106)
Summary: Aldehydes and Ketones
1. -cleavage: C–C(=O) and C(=O)-H
2. McLafferty rearrangement for CH-C-C-C(=O)
Which is which?
Which ketone?
105
M–43
77
M–60
148
M+
Spectral Database for Organic Compounds (SDBS)
E.I.
Pavia 8.8 R,S
Carboxylic acids and derivatives
propanoic acid (C3H6O2, M = 74)
29
M–45
45
M–29
73
M–1
57
M–17
Spectral Database for Organic Compounds
(SDBS)
E.I.
74
M+
60
M–28
45
M–43
88
M+
71
M–17
105
M–17
benzoic acid (C7H6O2, M = 122)
77
M–45
Spectral Database for Organic Compounds (SDBS)
43
M–45
E.I.
E.I.
122
M+
Spectral Database for Organic Compounds (SDBS)
butanoic acid (C4H8O2, M = 88)
Summary: Carboxylic acids
1. M-1
2. Cleavage of OH
3. Cleavage of R-C(O) → [CO2H]+, M = 45
→ R+
4. McLafferty rearrangement for CH-C-C-C(=O)OH
methyl acetate (C3H6O2, M = 74)
74
M+
59
M–15
Spectral Database for Organic Compounds (SDBS)
E.I.
43
M–31
Summary: Esters
1. Cleavage of C(=O)–OR
2. -cleavage of R-C(O)
3. McLafferty
4. Elimination to give RCO2–H + alkene derives from R’
Which ester?
E.I.
71
89
56
172
M+
Spectral Database for Organic Compounds (SDBS)
43
43
Identify each
ester
57
101
74
43
57
85
C6H12O2 esters, MW = 116
102
tert-butyl acetate
CH3C(=O)OC(CH3)3
Methyl pentanoate
CH3CH2CH2CH2COOCH3
87
Isopropyl propionate
CH3CH2COOCH(CH3)2
57
43
75
101
101
Pavia 8.8 V
Halogen Containing Compounds
2-chloropropane (C3H7Cl, M = 78 [35Cl])
63
M–15
65
Spectral Database for Organic Compounds
(SDBS)
E.I.
43
M–35
78 80
M+ M+2
1-chloropropane (C3H7Cl, M = 78 [35Cl])
78
M+
Spectral Database for Organic Compounds (SDBS)
E.I.
42
M–36
2-chloroheptane (C7H15Cl, M = 134 [35Cl])
98
M+–36
134 136
M+ M+2
Spectral Database for Organic Compounds (SDBS)
E.I.
56
M+–78
2-bromopropane (C3H7Br, M =122 [79Br])
E.I.
Spectral Database for Organic Compounds (SDBS)
43
M–Br
122 124
M+ M+2
Summary: Alkyl halides
C-X bond cleavage
-cleavage
Dehydrohalogenation (-HX)
Dehydrohalogenation with loss of an alkene
SUMMARY: SOME COMMON
FRAGMENT PEAKS Pavia Appendix 12
Peak
Fragment
lost
Interpretation
M–1
M–2
M–3
M–15
M–17
M–18
M–26
M–27
M–28
H•
Multiple H •
Multiple H •
CH3 •
HO •
H2O
HCCH
• HC=CH
2
CH2=CH2
aldehydes, 3° alcohols, cyclic amines
2° alcohols
1° alcohols
methyl groups
alcohols, phenols, carboxylic acids
alcohols
cyclic alkanes, alkenes
CH3CH2CH2C(=O)X [McLafferty rearr.]
•
M–19,35/37,79/81
halogen
•
M–15,29,43,57,71,… alkyl
M–31,45,59,73,87… • alkoxy esters, ethers
Peak
39
Fragment
observed
+
H2C–CCH
Interpretation
Pavia Appendix 11
from alkyne
propargyl cation
41
+
H2C–CH=CH2
from terminal alkene
allyl cation
+
42
H2C=C=NH2
43
H2C=C=OH
from 1° alcohol
77
C6H5+
from substituted phenyl
91
C7H7+
from C-substituted phenyl
+
tropylium cation
from 1° amine (R–CH2NH2)
Compound X revisited, yet again
57
M-31
E.I.
88
M+
Spectral Database for Organic Compounds (SDBS)
Analysis of fragmentation  STRUCTURAL information
Fragmentation:
Structure contains:
Georgia Tech Mass Spec Facility
http://www.chemistry.gatech.edu/research/facilities/spectrometry.php
VG Instruments 70SE
Two sector MS; high resolution and accurate mass measurement (< 5 ppm);
equipped with a GC; ionization by EI or CI for analysis of low molecular mass
(< 700 Da). FAB for much larger (< 5 kDa) polar, non-volatile molecules.
Applied Biosystems 4000 QTrap
Hybrid quadrupole / linear ion trap tandem mass spectrometer; HPLC
interfaced.
Applied Biosystems 4700 Proteomics Analyzer
Time-of-flight (TOF/TOF) MALDI. Molecular mass species up to 10 kDa with
low resolution, and high resolution and accurate mass measurement <10
kDa.
Micromass Quattro LC
Low resolution, moderate mass accuracy triple quadrupole (QQQ) tandem
MS; ionization via ESI and atmospheric pressure chemical ionization (APCI).
Thermo Orbitrap
Very high-resolution, very sensitive ESI instrument.
Bruker ToF/ToF
For imaging and proteomics, with a quadrupole ion trap. Interfaced with LC
NEVER ASK A MASS
SPECTROMETRIST
FOR JUST A MASS SPEC!
1. Always define the technique
that you want.
2. Even Better, discuss the
problem you are working on!
Reporting MS Spectra in Papers and Progress Reports
A
molecular ion
MS (EI, 70 kV):
m/z 91 (tropilium, 100%), 205 (M-29, 64%), 234(M+, 35%), …
B
C
base peak
… 235 (M+1, 4.6%).
A
B
C
D
D
Fragments that help in
structure determination
M+1 peak (or others)
that indicate something
about molecular
structure based on
isotope distribution
Ionization technique/method (e.g., EI, CI, FAB, MALDI).
Mass
Assignment of the peak to a particular ion
Height of the peak relative to the base peak (100%)
Reporting HRMS Spectra in Papers and Progress Reports
A
HRMS (EI, 70 kV):
m/z 100.0639, 39%; Calc. for C4H8N2O,  = 2 ppm.
B
A
B
C
D
E
C
D
E
Ionization technique/method (e.g., EI, CI, FAB, MALDI).
Mass
Height of the peak relative to the base peak (100%)
Suggested formula
The difference between the experimental value of molecular weight
and the value calculated for the putative molecular formula, in ppm:
 = | Mexp – Mcalc |  106/Mexp
WORK AS MANY PROBLEMS AS
POSSIBLE!
Pavia, chap 8 questions:
7 (a)-(x), 8, 9, 10, (a)-(f), 11, (a)-(g), 12 (a)-(c), 13,
14, 15 (a)-(b)
Problem sets based on M.S. alone
http://science.widener.edu/~svanbram/chem465/htmldocs/ms_unk.html
Intensities of peaks are provided, you need to download free
spectral visualization software to view actual spectra
Determine the structures of the compounds for
which the mass spectra and IR spectra are
provided on the following slides. In some cases
you might be able to identify a single compound.
For others, a 1H NMR would really help!!
Compound A
IR → functional group
fragmentation → structure!
Exact mass = 114.1043
C7H14O
Compound B
IR → functional group
MS probably lets you
identify the exact isomer
C.I.  Exact mass = 116.1203
C7H16O
Compound C
Quickly hone in on
formula and 4 possible
structures
Exact mass = 169.9735
C7H7Br
Compound D
Formula, IR → a few
possibilities
MS probably might let you
identify the exact isomer
Exact mass = 150.0041
C5H11Br
Compound E
IR → functional group
McLafferty → small
number of possible
structures!
Exact mass = 100.0893
C6H12O
Compound F
Small molecule!
IR → functional group
→ structure!
Exact mass = 74.0363
C3H6O2
Compound G
Only a few possibilities
here
Exact mass = 73.0896
C4H11N
Compound H
Formula, IR → structure!
C.I.  Exact mass = 56.0261
C3H4O
Compound I
Which functional
groups contains N and
O and have a
distinctive set of IR
peaks?
C.I.  Exact mass = 89.0474
C3H7NO2
Compound J
NMR would help here,
but what can you tell
about the structure
based on IR and MS?
Exact mass = 138.0687
C8H10O2
Compound K
What can you tell
about the structre
based on IR and MS?
When do we get the
NMR?
Exact mass = 122.0733
C8H10O
Compound L
functional group?
m/z = 74
m/z = 71
→structure!
Exact mass = 102.0678
C3H10O2
Compound P
What N-containing functional
group?
Sites of unsaturation?
3 possible strucutres?
Exact mass = 156.9934
C6H4ClNO2
Compound Q
You will need an NMR to
solve this, but what do you
know from the IR and MS?
C.I.  Exact mass = 208.0094
C7H13BrO2
Compound R
You will need an NMR to
solve this, but what do you
know from the IR and MS?
Exact mass = 98.0740
C6H10O
Compound S
You might only get a
limited amiount of
information from this
data
Exact mass = 126.1041
C8H14O
Compound T
McLafferty
says…?
C.I.  Exact mass = 116.0842
C6H12O2
Compound U
Data → a few
possible
compounds
Exact mass = 161.9637
C6H4Cl2O
Compound V
Data → a few
possible
compounds
Exact mass = 122.0733
C8H10O
Compound W
C=O (low freq?); M-28;
C6H8O
How many possibilities?
Exact mass = 96.0572
C6H8O