Introduction to Organic Mass Spectrometry
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Transcript of Introduction to Organic Mass Spectrometry
Introduction to Walk-Up Mass Introduction to Walk-Up Mass SpectrometrySpectrometry
Jonathan A. Karty, Ph.D.Jonathan A. Karty, Ph.D.September 27 & 29, 2010September 27 & 29, 2010
Topics CoveredTopics CoveredMolecular Weight and Isotope Molecular Weight and Isotope
DistributionsDistributionsAccuracy and ResolutionAccuracy and ResolutionEI, ESI, and APCI ionizationEI, ESI, and APCI ionizationEI FragmentationEI FragmentationA Handful of MS ApplicationsA Handful of MS Applications
Why Mass SpectrometryWhy Mass Spectrometry Information is composition-specificInformation is composition-specific
Very selective analytical techniqueVery selective analytical techniqueMost other spectroscopies can describe Most other spectroscopies can describe
functionalities, but not chemical formulaefunctionalities, but not chemical formulaeMS is VERY sensitiveMS is VERY sensitive
MSF personnel dilute NMR samples 1:500MSF personnel dilute NMR samples 1:500Picomole sensitivity is common in the MSFPicomole sensitivity is common in the MSF
Mass spectrometers have become Mass spectrometers have become MUCH easier to use in the last 15 yearsMUCH easier to use in the last 15 years
Three QuestionsThree Questions Did I make my compound?Did I make my compound?
Molecular weight is an intrinsic property of a Molecular weight is an intrinsic property of a substancesubstance
Did I make anything else?Did I make anything else? Mass spectrometry is readily coupled to Mass spectrometry is readily coupled to
chromatographic techniqueschromatographic techniques
How much of it did I make?How much of it did I make? Response in the mass spectrometer is proportional to Response in the mass spectrometer is proportional to
analyte concentration (R = analyte concentration (R = α[M])[M])Each compound has a unique response factor, Each compound has a unique response factor, α
Common MS ApplicationsCommon MS ApplicationsReaction monitoringReaction monitoring
Crude reaction mixture MSCrude reaction mixture MSStable isotope labelingStable isotope labelingStability studiesStability studiesQuick product identification (TLC spot)Quick product identification (TLC spot)
Confirmation of elemental compositionConfirmation of elemental compositionMuch more precise then EAMuch more precise then EA
Selective detector for GC/HPLCSelective detector for GC/HPLCMS provides molecular weight information MS provides molecular weight information
about each chromatographic peakabout each chromatographic peak
Important Concepts to RememberImportant Concepts to Remember Mass spectrometers analyze gas-phase ions, not Mass spectrometers analyze gas-phase ions, not
neutral moleculesneutral molecules Neutrals don’t respond to electric and magnetic fieldsNeutrals don’t respond to electric and magnetic fields If a molecule cannot ionize, MS cannot helpIf a molecule cannot ionize, MS cannot help
MS is not a “magic bullet” techniqueMS is not a “magic bullet” technique MS can describe atomic composition of an ionMS can describe atomic composition of an ion Connectivity of the atoms is much more challengingConnectivity of the atoms is much more challenging
Although MS requires a vacuum, it cannot be Although MS requires a vacuum, it cannot be performed in a vacuum of informationperformed in a vacuum of information Deriving useful information from MS data often requires Deriving useful information from MS data often requires
some knowledge of the system under investigationsome knowledge of the system under investigation
Resolution is the ability to separate ions of Resolution is the ability to separate ions of nearly equal mass/chargenearly equal mass/charge e.g. Ce.g. C66HH55Cl and CCl and C66HH55OF @ 112 m/zOF @ 112 m/z
CC66HH55Cl = 112.00798 amu (all Cl = 112.00798 amu (all 1212C, C, 3535Cl, Cl, 11H)H) CC66HH55OF = 112.03244 amu (all OF = 112.03244 amu (all 1212C, C, 1616O, O, 11H, H, 1919F)F) Resolving power >4700 required to resolve these twoResolving power >4700 required to resolve these two
Two definitions Two definitions Resolution = Resolution = ΔΔm/m (0.024/112.03 = 0.00022 or 2.2*10m/m (0.024/112.03 = 0.00022 or 2.2*10-4-4)) Resolving power = m/Δm (112.03/0.024 = 4668)
What is Resolution?What is Resolution?
Resolving Power ExampleResolving Power Example
Inte
nsity
(%)
0
20
40
60
80
100
Mass [amu]111.95 112.00 112.05 112.10
Inte
nsity
(%)
0
20
40
60
80
100
Mass [amu]111.95 112.00 112.05 112.10
Inte
nsity
(%)
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
C6H5OFC6H5Cl
Mass AccuracyMass Accuracy MSF reports mass accuracy as a relative valueMSF reports mass accuracy as a relative value
ppm = parts per million (1 ppm = 0.0001%)ppm = parts per million (1 ppm = 0.0001%) 5 ppm @ mass 300 = 300 * (5/105 ppm @ mass 300 = 300 * (5/1066) = ) = ±0.0015 Da±0.0015 Da
High resolving power facilitates precise mass High resolving power facilitates precise mass measurementsmeasurements Accurate mass spectrometry is used to confirm a Accurate mass spectrometry is used to confirm a
molecular formulamolecular formula
Walk-up instruments in the MSF should be Walk-up instruments in the MSF should be treated as “nominal mass” accuracytreated as “nominal mass” accuracy +/- 0.15 Da mass accuracy+/- 0.15 Da mass accuracy
Molecular Weight CalculationsMolecular Weight Calculations Calculate molecular weights of expected
components PRIOR to performing MS
The molecular weight of a compound is computed The molecular weight of a compound is computed by summing the masses of all atoms that by summing the masses of all atoms that comprise the compound.comprise the compound.Morphine: CMorphine: C1717HH1919NONO33 = 12.011(17) = 12.011(17)
+1.008(19)+ 14.007 + 15.999(3) = 285.34 Da+1.008(19)+ 14.007 + 15.999(3) = 285.34 Da Yet 285.136 is observed by EI-MSYet 285.136 is observed by EI-MS
Molecular weight is calculated assuming a natural Molecular weight is calculated assuming a natural distribution of isotopesdistribution of isotopes Molecular weights calculated with average masses for Molecular weights calculated with average masses for
Br, Cl, and many metals will differ greatly from MS dataBr, Cl, and many metals will differ greatly from MS data
Monoisotopic vs. Average MassMonoisotopic vs. Average Mass Most elements have a variety of isotopesMost elements have a variety of isotopes
C C 1212C is 98.9% abundant, C is 98.9% abundant, 1313C is 1.1% abundantC is 1.1% abundant For CFor C2020, 80% chance , 80% chance 1313CC00, 18% chance , 18% chance 1313CC11, 2% chance , 2% chance 1313CC22
Sn has 7 naturally occurring isotopes @ >5% ab.Sn has 7 naturally occurring isotopes @ >5% ab. F, P, Na, Al, Co, I, Au have only 1 natural isotopeF, P, Na, Al, Co, I, Au have only 1 natural isotope Mass spectrometers can resolve isotopic distributionsMass spectrometers can resolve isotopic distributions
Monoisotopic masses must be considered Monoisotopic masses must be considered Monoisotopic masses are computed using the most Monoisotopic masses are computed using the most
abundant isotope of each element (abundant isotope of each element (1212C, C, 3535Cl, Cl, 7979Br, Br, 5858Ni, Ni, 1111B, etc.)B, etc.)
For morphine, monoisotopic mass = 285.1365For morphine, monoisotopic mass = 285.1365 (12.0000 * 17) + (1.0078 * 19) + 14.0031 + (15.9949 * 3)(12.0000 * 17) + (1.0078 * 19) + 14.0031 + (15.9949 * 3)
Isotopic EnvelopesIsotopic EnvelopesMass spectrometers measure ion populationsMass spectrometers measure ion populations
101022 – 10 – 1066 ions in MS peaks ions in MS peaksAny single ion only has 1 isotopic compositionAny single ion only has 1 isotopic composition
The observed mass spectrum represents the sum The observed mass spectrum represents the sum of all those different compositionsof all those different compositions
Inte
nsity
(%
)
0
20
40
60
80
100
Mass [amu]285 286 287 288 289 290
“M+ peak”
“M+1 peak”
“M+2 peak”
CC1717HH1919NONO33 Mass Spectrum Mass SpectrumIn
tens
ity (
%)
0
20
40
60
80
100
Mass [amu]285 286 287 288 289 290
13C0, 15N0
13C1
or 15N1
13C2 or13C1+15N1
285.36avg. mass
Isotopic Envelope ApplicationsIsotopic Envelope Applications Isotopic envelopes can be used to preclude Isotopic envelopes can be used to preclude
some elements from ionic compositionssome elements from ionic compositionsLack of intense M+2 peak precludes Cl or BrLack of intense M+2 peak precludes Cl or BrMany metals have unique isotopic signaturesMany metals have unique isotopic signatures
M+1/M+ ratio can be used to count carbonsM+1/M+ ratio can be used to count carbons [(M+1)/M+]/0.011 [(M+1)/M+]/0.011 ≈ # carbon atoms≈ # carbon atomsFor morphine: (0.1901/1)/0.011 = 17.28 For morphine: (0.1901/1)/0.011 = 17.28 17 17
Isotope table can be found on NIST websiteIsotope table can be found on NIST websiteLink from MSF “Useful Information” pageLink from MSF “Useful Information” page
Inte
nsity
(%)
0
20
40
60
80
100
Mass [amu]1759 1760 1761 1762 1763 1764 1765
Inte
nsity
(%)
0
20
40
60
80
100
Mass [amu]362 364 366 368 370 372 374 376 378
Inte
nsity
(%)
0
20
40
60
80
100
Mass [amu]131 132 133 134 135 136 137 138 139
A few isotope patternsA few isotope patternsC2H3Cl3
trichloroethane
C12H27SnBrtributyltin bromide
C83H122N24O19
A 14-mer peptide
Last Comments on Molecular IonsLast Comments on Molecular Ions Be aware of ionization mechanismBe aware of ionization mechanism EI, LDI, and CI generate radical cationsEI, LDI, and CI generate radical cations
MM++•• is an odd electron ionis an odd electron ion Nitrogen rule is normalNitrogen rule is normal
Odd molecular ion mass implies odd # of N atomsOdd molecular ion mass implies odd # of N atoms MM++•• for morphine by EI is 285.136, odd # N (1) for morphine by EI is 285.136, odd # N (1)
ESI, APCI, MALDI, and CI make cation adductsESI, APCI, MALDI, and CI make cation adducts M+H and M+Na are even electron ionsM+H and M+Na are even electron ions Nitrogen rule is inverted for these ionsNitrogen rule is inverted for these ions
Even molecular ion mass implies odd # of N atomsEven molecular ion mass implies odd # of N atoms M+Na for morphine by ESI is 308.126, odd # N (1)M+Na for morphine by ESI is 308.126, odd # N (1)
Metal atoms and pre-existing ions or radicals Metal atoms and pre-existing ions or radicals can override these rulescan override these rules
Some useful software toolsSome useful software tools The “exact mass” feature in ChemDraw will give The “exact mass” feature in ChemDraw will give
you a monoisotopic massyou a monoisotopic mass Not always correct for complex isotope patternsNot always correct for complex isotope patterns
Two freeware apps are available from MSF Two freeware apps are available from MSF website “Links” pagewebsite “Links” page These can be used to predict the entire isotopic pattern as an These can be used to predict the entire isotopic pattern as an
exportable imageexportable image
MS-Search program on GC-MS computer can be MS-Search program on GC-MS computer can be used to retrieve mass spectra from NIST’02 used to retrieve mass spectra from NIST’02 librarylibrary
Mass Spectrometer ComponentsMass Spectrometer Components
InletInlet Get samples into the instrumentGet samples into the instrument
Source Ionize the molecules in a useful way
Mass Analyzer Separates the ions by mass to charge (m/z) ratio
DetectorDetector Converts ions into an electronic signal or photonsConverts ions into an electronic signal or photons
Data systemData system From photographic plates to computer clustersFrom photographic plates to computer clusters
Electrospray Ionization (ESI)Electrospray Ionization (ESI)Dilute solution of analyte (<1 mg/L) infused Dilute solution of analyte (<1 mg/L) infused
through a fine needle in a high electric fieldthrough a fine needle in a high electric fieldVery small, highly charged droplets are Very small, highly charged droplets are
createdcreatedSolvent evaporates, droplets split and/or ions Solvent evaporates, droplets split and/or ions
ejected to lower charge/area ratio ejected to lower charge/area ratio Warm nebulizing gas accelerates dryingWarm nebulizing gas accelerates drying
Free ions are directed into the vacuum Free ions are directed into the vacuum chamberchamber
Ion source voltage depends on solventIon source voltage depends on solventUsually Usually ±±2500 – 2500 – ±±4500 V4500 V
Advantages of ESIAdvantages of ESIGentle ionization processGentle ionization process
High chance of observing molecular ionHigh chance of observing molecular ionVery labile analytes can be ionizedVery labile analytes can be ionized
Molecule need not be volatileMolecule need not be volatileProteins/peptides easily analyzed by ESIProteins/peptides easily analyzed by ESISalts can be analyzed by ESISalts can be analyzed by ESI
Easily coupled with HPLCEasily coupled with HPLCBoth positive and negative ions can be Both positive and negative ions can be
generated by the same sourcegenerated by the same source
Characteristics of ESI IonsCharacteristics of ESI Ions ESI is a thermal process (1 atm in source)ESI is a thermal process (1 atm in source)
Little fragmentation due to ionization (cf EI) Little fragmentation due to ionization (cf EI) Solution-phase ions are often preservedSolution-phase ions are often preserved
e.g. organometallic saltse.g. organometallic salts ESI ions are generated by ion transferESI ions are generated by ion transfer
(M+H)(M+H)++, (M+Na), (M+Na)++, or (M-H), or (M-H)--, rarely M, rarely M++•• or M or M--••
ESI often generates multiply charged ionsESI often generates multiply charged ions (M+2H)(M+2H)2+2+ or (M+10H) or (M+10H)10+10+
Most ions are 500-1500 m/zMost ions are 500-1500 m/z ESI spectrum x-axis must be mass/charge (m/z or Th, ESI spectrum x-axis must be mass/charge (m/z or Th,
not amu or Da)not amu or Da)
ESI DisadvantagesESI Disadvantages Analyte must have an acidic or basic siteAnalyte must have an acidic or basic site
Hydrocarbons and steroids not readily ionized by ESIHydrocarbons and steroids not readily ionized by ESI Analyte must be soluble in polar, volatile solventAnalyte must be soluble in polar, volatile solvent ESI is less efficient than other sourcesESI is less efficient than other sources
Most ions don’t make it into the vacuum systemMost ions don’t make it into the vacuum system ESI is very sensitive to contaminantsESI is very sensitive to contaminants
Solvent clusters can dominate spectraSolvent clusters can dominate spectra Distribution of multiple charge states can make Distribution of multiple charge states can make
spectra of mixtures hard to interpretspectra of mixtures hard to interpret e.g. polymer mass spectrae.g. polymer mass spectra
ESI Example IESI Example ILCT KC366js-29-1
m/z200 300 400 500 600 700 800 900 1000 1100 1200 1300
%
0
100
js-29-1 54 (1.086) Cm (54:60) 1: TOF MS ES+ 6.40e3395.1219
304.0758
396.1333
397.1367
C26H18O4
(M+H)+
O
O
O
OOTBDPS
C
O
O
Me
OTIPS
OMOM
Me
MeOTIPS
OTBS
Me
Na+
O
O
O
HOOTBDPS
C
O
O
Me
OTIPS
OMOM
Me
MeOTIPS
OTBS
Me
Na+
OH
Chemical Formula: C77H136NaO13Si4+
Exact Mass: 1403.9m/z: 1404.9 (100.0%), 1403.9 (94.7%), 1405.9 (67.1%), 1406.9 (33.1%), 1407.9 (13.1%),
1408.9 (4.3%), 1409.9 (1.2%)
Chemical Formula: C77H134NaO12Si4+
Exact Mass: 1385.9m/z: 1386.9 (100.0%), 1385.9 (94.7%), 1387.9 (66.9%), 1388.9 (32.8%), 1389.9 (12.9%), 1390.9 (4.2%), 1391.9 (1.2%)
H2O
ESI Example IIESI Example II
O
O
O
HOOTBDPS
C
O
O
Me
OTIPS
OMOM
Me
MeOTIPS
OTBS
Me
Na+
OH
O
O
O
OOTBDPS
C
O
O
Me
OTIPS
OMOM
Me
MeOTIPS
OTBS
Me
Na+
78%22%
Atmospheric Pressure Chemical Atmospheric Pressure Chemical Ionization (APCI)Ionization (APCI)
APCI uses a corona discharge to generate acidic APCI uses a corona discharge to generate acidic solvent cations from a vaporsolvent cations from a vapor
These solvent cations can protonate hydrophobic These solvent cations can protonate hydrophobic species not amenable to ESIspecies not amenable to ESI APCI can be done from hexane or THFAPCI can be done from hexane or THF Often used to study lipids and steroidsOften used to study lipids and steroids In MSF, completely protected macrocycles are routinely In MSF, completely protected macrocycles are routinely
studied by APCIstudied by APCI APCI is harsher than ESIAPCI is harsher than ESI Large # of variables in APCI make it less reproducible Large # of variables in APCI make it less reproducible
than ESIthan ESI
Agilent 6130 Multi-mode SourceAgilent 6130 Multi-mode Source
http://www.chem.agilent.com/Library/Images1/MMS_schematic_300dpi_039393.jpg
Matrix-Assisted Laser Desorption/Ionization Matrix-Assisted Laser Desorption/Ionization (MALDI)(MALDI)
Analyte is mixed with UV-absorbing matrixAnalyte is mixed with UV-absorbing matrix ~10,000:1 matrix:analyte ratio~10,000:1 matrix:analyte ratio Analyte does not need to absorb laserAnalyte does not need to absorb laser
A drop of this liquid is dried on a targetA drop of this liquid is dried on a target Analyte incorporated into matrix crystalsAnalyte incorporated into matrix crystals
Spot is irradiated by a laser pulseSpot is irradiated by a laser pulse Irradiated region sublimes, taking analyte with itIrradiated region sublimes, taking analyte with it Matrix is often promoted to the excited stateMatrix is often promoted to the excited state Charges exchange between matrix and analyte in the Charges exchange between matrix and analyte in the
plume (very fast <100 nsec)plume (very fast <100 nsec) Ions are accelerated toward the detectorIons are accelerated toward the detector
Some Common MALDI MatricesSome Common MALDI MatricesC
N
CO
OH
HO O
O
HO
H3C
CH3
CO
OH
C
O
OH
OH
HO
NH
C
O
OHC
N
C
N
C
N
C
N
-cyano-4-hydroxycinnamic acidCCA C10H7NO3
peptides and small molecules
sinapinic acidSA C11H12O5
proteins
2,5 dihydroxybenzoic acidDHB C7H6O4
oligosaccharides
N
3-indoleacrylic acidIAA C10H9NO2
organic polymers
7,7,8,8 tetracyanoquinodimethaneTCNQ C12H4N4
polyaromatic hydrocarbons
CO
OH
OH
3-hydroxypicolinic acidHPA C6H5NO3oligonucleotides
MALDI AdvantagesMALDI AdvantagesRelatively gentle ionization techniqueRelatively gentle ionization techniqueVery high MW species can be ionizedVery high MW species can be ionizedMolecule need not be volatileMolecule need not be volatileVery easy to get sub-picomole sensitivityVery easy to get sub-picomole sensitivitySpectra are easy to interpretSpectra are easy to interpretPositive or negative ions from same spotPositive or negative ions from same spotWide array of matrices availableWide array of matrices available
MALDI DisadvantagesMALDI DisadvantagesMALDI matrix cluster ions obscure low m/z MALDI matrix cluster ions obscure low m/z
(<600) range(<600) rangeAnalyte must have very low vapor pressureAnalyte must have very low vapor pressurePulsed nature of source limits compatibility Pulsed nature of source limits compatibility
with many mass analyzerswith many mass analyzersCoupling MALDI with chromatography can Coupling MALDI with chromatography can
be difficultbe difficultAnalytes that absorb the laser can be Analytes that absorb the laser can be
problematicproblematicFluorescein-labeled peptidesFluorescein-labeled peptides
MALDI ExampleMALDI Example
(Ubiq+H)+(Ins+H)+(U
biq+
2H)2+
(ACTH 7-38+H)+(A
CTH
18-
37+H
)+
O
N
N
O
HN
HN
O
O
C12H25O
C12H25O
C12H25O
C12H25O
OC12H25
OC12H25
O O OO
N
N
O
NH
NH
O
O
OC12H25
OC12H25
OC12H25
OC12H25
C12H25O
C12H25O
Chemical Formula: C230H354N8O23Exact Mass: 3596.68
Electron Ionization (EI)Electron Ionization (EI) Gas phase molecules are irradiated by Gas phase molecules are irradiated by
beam of energetic electronsbeam of energetic electrons
Interaction between molecule and beam Interaction between molecule and beam results in electron ejectionresults in electron ejectionM + eM + e-- M M++•• + 2e + 2e--
Radical species are generated initiallyRadical species are generated initially
EI is a very energetic processEI is a very energetic processMolecules often fragment right after ionizationMolecules often fragment right after ionization
EI Mass SpectrumEI Mass Spectrum
Figure from Mass Spectrometry Principles and ApplicationsE. De Hoffmann, J. Charette, V. Strooband, eds., ©1996
More EI Mass SpectraMore EI Mass Spectra
(m a in lib ) C o c a ine10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310
0
50
100
15 27
4251
59 68
77
82
94105
122140 152 166
182
198 272303
N
H O
O
H OO
Cocaine
(m a in lib ) 3,4-Pyrid in e d im e th a n o l, 5-h yd ro xy-6-m e th yl-10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
0
50
100
27 3139 53 67
81
94106
122136
151
169
HO
O H
O H
NVitamin B6
(m a in lib ) A n d ro st-4-e n e -3,17-d io n e10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
0
50
100
18 2941 55 67
79
86
91 97109
124
131
148
162173 187
201
216 229
244
258 271
286
O
O Androstenedione
Basic Rules• Electron is first removed from site with lowest ionization
potential– non-bonding electrons > pi bond electrons > sigma bond electrons– NB > π > σ (think No Pizza from Sigma)
• Stevenson’s Rule: During a sigma bond dissociation, the charge will likely be retained on the fragment with the lowest ionization potential
• Odd electron species can fragment to give odd or even electron products
• Even electron species can only fragment to yield even electron products
• Only CHARGED species are detected
Four Basic Mechanisms to Learn
• Sigma Cleavage• Alpha Cleavage• Inductive Cleavage• McLafferty Rearrangement
Sigma Bond Cleavage• Removal of an electron from a sigma bond weakens it• As bond breaks, one fragment gets the remaining electron, and
is neutral (R•)• The other fragment is a charged, even electron species (R+)• Highly substituted carbocations are more stable (Stevenson’s
Rule)– Cleavage of the C1-C2 bond in long n-alkanes is not favored– Lower IE fragments are favored
• Long n-alkane chains tend to make many fragments spaced by 14 from m/z 20-90
Sigma Cleavage Example: Hexane
H3C
CH2
H2C
CH2
H2C
CH3
H3C
CH2
H2C
CH2+
•H2C
CH3
8.0 eV
8.4 eV
8.2eV
5743
29
86
H3C
CH2
H2C
CH2
H2C
CH3
H3C
CH2
H2C
CH2+
•H2C
CH3
Exact Mass: 86.11 Exact Mass: 57.07H3C
CH2
H2C
CH2
H2C
CH3
H3C
CH2
H2C
CH2•
+H2C
CH3
Exact Mass: 86.11
Exact Mass: 29.04
H3C
CH2
H2C
CH2
H2C
CH3
H3C
CH2
+H2C
CH2•
H2C
CH3
Exact Mass: 43.05
71
43 CH3
C
CH2
H2C
CH3
H3CCH3
•
+HC
H2C
CH2
CH3
CH3
Exact Mass: 71.09
CH3
CH
CH2
H2C
CH3
H3C
Exact Mass: 86.11 CH3+HC
CH2•
CH2
H3C
CH3
CH3
CH
CH2
H2C
CH3
H3C
Exact Mass: 43.0542
Homolytic cleavage – Radical Site Driven• Cleavage is caused when an electron from a bond to an atom
adjacent to the charge site pairs up with the radical– Weakened α-sigma bond breaks– This mechanism is also called α-cleavage
• The charge does not move in this reaction• Charged product is an even electron species• α-cleavage directing atoms: N > S, O, π, R• > Cl, Br > H
– Loss of longer alkyl chains is often favored– Energetics of both products (charged and neutral) are important
CH3
CH2
CH3C
O
43
57
72
57
43
H3CC
CH2
CH3
O•+
Exact Mass: 72.06
H3CC
CH2
CH3
O•+
CH3•
CCH2
CH3
O+
Exact Mass: 57.03
1
H3CC
CH2
CH3
O•+
H3CC
•H2CCH3
O+
2
Exact Mass: 43.02
ΔHf = +117 kJ/mol
ΔHf = +145 kJ/mol
H3C
H2C
CH2
C
+•HO CH3
CH2
CH3
Exact Mass: 116.12
101
87
73
H3C
H2C
CH2
C
+•HO CH3
CH2
CH3
H3C
H2C
CH2
C
+HO CH3
•H2CCH3
Exact Mass: 116.12 Exact Mass: 87.08H3C
H2C
CH2
C
+•HO CH3
CH2
CH3
H3C
H2C
CH2
C
+HO CH3•
CH2
CH3
Exact Mass: 116.12
Exact Mass: 101.10
H3C
H2C
CH2
C
+•HO CH3
CH2
CH3
H3C
H2C
CH2•
C
+HO CH3
CH2
CH3
Exact Mass: 116.12
Exact Mass: 73.06
H3C
H2C
CH2
C
+•HO CH3
CH2
CH3
H3C
H2C
CH2
C
+HO CH3
•H2CCH3
H3C
H2C
CH2
C
+HO CH3•
CH2
CH3
H3C
H2C
CH2•
C
+HO CH3
CH2
CH3
Exact Mass: 116.12 Exact Mass: 87.08
Exact Mass: 101.10
Exact Mass: 73.06
Heterolytic Cleavage: Charge Driven
• Charged site induces a pair of electrons to migrate from an adjacent bond or atom– This breaks a sigma bond
• Also called inductive cleavage• The charge migrates to the electron pair donor
– The electron pair neutralizes the original charge
• Even electron fragments can further dissociate by this mechanism
• Inductive cleavage directing atoms: Halogens > O, S, >> N, C
H3CCH2
CCH2
CH3
O•+
Exact Mass: 86.07
H3CCH2
CCH2
CH3
O•+
H3CCH2
•C
CH2
CH3
O+
Exact Mass: 57.03
H3CCH2
CCH2
CH3
O•+
H3CCH2
C+
CH2
CH3
O•
H3CCH2
+
•CCH2
CH3
O
i
Exact Mass: 29.04
H3CCH2
C+
CH2
CH3
O•
29
57
86
CHC
FF
FBr•+
Cl
Exact Mass: 195.8897
196
127
117
69
CHC
FF
FBr•+
Cl
Exact Mass: 195.89
iC
CH+
FF
F
Br•
ClExact Mass: 116.97
C•
HC
FF
FBr+
ClExact Mass: 126.89
CHC
FF
FBr•+
Cl
C+•HC
FF
F
Br
Cl
CHC
FF
FBr•+
ClExact Mass: 68.99
i
Benzylic Bond Cleavage• The charge stabilizing ability of the aromatic group can
dominate EI spectra • Alkylbenzenes will often form intense ions at m/z 91
– Tropylium ion– 7-membered ring favored by >11 kJ/mol
• Tropylium ion can fragment by successive losses of acetylene– 91 65 39– Phenyl ions (C6H5)+• decompose the same way
• (77 51)
120
91
6539
CH
HC
HC
HCCH+
C•
CH2
H2C
CH3
Exact Mass: 120.0934
CH
HC
HC
HCCH+
CCH2
•H2CCH3
Exact Mass: 91.0542CH+
CH
HC
HC
HCCH
CCH2
+
120
+•H2N CH C
CH2
OH
O
Exact Mass: 165.08
91
74
165
+•H2N CH C
CH2
OH
O
+H2N CH C
CH2•
OH
O
Exact Mass: 74.0237
H2N CH C
CH2
OH
O
C•+HC
H2NH•C C
CH2
OH
O
C+HC
Exact Mass: 91.0542
H2N CH C
CH2
OH
O•+
i
H2N CH+ •C
CH2
OH
O
Exact Mass: 120.08
3-methyl-2-pentanone ions
Exact Mass: 100.09
H3C
H2C
HC
CCH3
O•+
CH3
H3C
H2C
CH•C
CH3
O+
CH3Exact Mass: 43.02
2
H3C
H2C
HC
C+
O•
CH3
H3C
H2C
CH+•C
O
CH3
Exact Mass: 57.07
i2
CH3 CH3
H3C
H2C
HC
C+
O•
CH3
CH3
i3
H3CCH2
+
•HCC
O
CH3
CH3
Exact Mass: 29.04
What about m/z 72?
McLafferty Rearrangement• 72 Th fragment requires elimination of ethene
• A hydrogen on a carbon 4 atoms away from the carbonyl oxygen is transferred– The “1,5 shift” in carbonyl-containing ions is called the McLafferty
rearrangement– Creates a distonic radical cation (charge and radical separate)– 6-membered intermediate is sterically favorable– Such rearrangements are common
• Once the rearrangement is complete, molecule can fragment by any previously described mechanism
CH2•
H2CHC
C
O+
CH3
CH3
H
CH2
CH2•HC
C
O+
CH3
CH3
H
Exact Mass: 72.06
H2C
H2CHC
C
O•+
CH3
CH3
H
rH CH2•
H2CHC
C
O+
CH3
CH3
H
EI AdvantagesEI Advantages Simplest source design of allSimplest source design of all
EI mass spectrometers even go to other planets!EI mass spectrometers even go to other planets!
Robust ionization mechanismRobust ionization mechanism Even noble gases are ionized by EIEven noble gases are ionized by EI
Fragmentation patterns can be used to identify Fragmentation patterns can be used to identify moleculesmolecules NIST ’08 library has over 220,000 spectraNIST ’08 library has over 220,000 spectra Structures of novel compounds can be deducedStructures of novel compounds can be deduced
EI DisadvantagesEI Disadvantages Fragmentation makes intact molecular ion Fragmentation makes intact molecular ion
difficult to observedifficult to observe
Samples must be in the gas phaseSamples must be in the gas phase
Databases are very limitedDatabases are very limitedNIST’08 only has 190,000 unique compoundsNIST’08 only has 190,000 unique compounds
Interpreting EI spectra is an artInterpreting EI spectra is an art
Problem Solving ExamplesProblem Solving ExamplesFormula matching with accurate mass Formula matching with accurate mass
ESI-TOF dataESI-TOF dataDiscovery of a novel steroid (UCLA)Discovery of a novel steroid (UCLA)Diagnosing a reaction with LC-MS and Diagnosing a reaction with LC-MS and
accurate mass LC-MSaccurate mass LC-MS
Formula Matching BasicsFormula Matching Basics Atomic weights are not integers (except Atomic weights are not integers (except 1212C)C)
1414N = 14.0031 Da; N = 14.0031 Da; 1111B = 11.0093 Da; B = 11.0093 Da; 11H = 1.0078 DaH = 1.0078 Da 1616O = 15.9949 Da; O = 15.9949 Da; 1919F = 18.9984 Da; F = 18.9984 Da; 127127I = 126.9045 DaI = 126.9045 Da
Difference from integer mass is called “mass Difference from integer mass is called “mass defect” or “fractional mass”defect” or “fractional mass” Related to nuclear binding energyRelated to nuclear binding energy
Sum of the mass defects depends on Sum of the mass defects depends on compositioncomposition H, N increase mass defectH, N increase mass defect
Hydrogen-rich molecules have high mass defects Hydrogen-rich molecules have high mass defects Eicosane (CEicosane (C2020HH4242)= 282.3286)= 282.3286
O, Cl, F, Na decrease itO, Cl, F, Na decrease it Hydrogen deficient species have low mass defectsHydrogen deficient species have low mass defects Morphine, (CMorphine, (C1717HH1919NONO33) = 285.1365) = 285.1365
More Formula MatchingMore Formula Matching Accurate mass measurements narrow down Accurate mass measurements narrow down
possible formulas for a given molecular weightpossible formulas for a given molecular weight 534534 entries in NIST’08 library @ mass 285 entries in NIST’08 library @ mass 285 Only Only 33 formulas within 5 ppm of 285.1365 formulas within 5 ppm of 285.1365 4646 compounds with formula C compounds with formula C1717HH1919NONO33
Mass spectrum and user info complete the Mass spectrum and user info complete the picturepicture Isotope distributions indicate/eliminate elements Isotope distributions indicate/eliminate elements User-supplied info eliminates others (e.g. no F)User-supplied info eliminates others (e.g. no F) Suggested formula has to make chemical senseSuggested formula has to make chemical sense
Formula Matching ExampleFormula Matching Example
Only 9 ways to combine up to 40 C, 50 H, 5 N, 5 O, and 2 Cl to get a mass within 20 ppm (0.0061 u) of 306.0820, only 3 have 2 Cl
Elemental Composition ReportTolerance = 20.0 PPM / DBE: min = -1.5, max = 50.0Selected filters: NoneMonoisotopic Mass, Even Electron Ions370 formulas evaluated with 9 results within limitsElements Used:C: 0-40 H: 0-50 N: 0-5 O: 0-5 Cl: 0-2 Error 20 ppm
Mass intensity Calc. Mass mDa PPM i-FIT
306.082 100 306.0816 0.4 1.3 39.7 C17 H18 N Cl2 306.0776 4.4 14.4 376 C12 H18 N3 O2 Cl2 306.0875 -5.5 -18 701.7 C10 H22 N O5 Cl2 306.0798 2.2 7.2 1945.8 C18 H13 N3 Cl 306.0857 -3.7 -12.1 2205.2 C11 H17 N3 O5 Cl 306.0766 5.4 17.6 9102.8 C18 H12 N O4 306.078 4 13.1 9195.6 C19 H8 N5 306.0879 -5.9 -19.3 9289.5 C17 H12 N3 O3 306.0838 -1.8 -5.9 9543.2 C12 H12 N5 O5
error in:Formula
Zoloft C17H18Cl2N
Discovery of a Novel SteroidDiscovery of a Novel Steroid A researcher at UCLA was given an athlete’s used A researcher at UCLA was given an athlete’s used
syringe that contained a suspected steroidsyringe that contained a suspected steroid GC-MS revealed a mass spectrum that matched no GC-MS revealed a mass spectrum that matched no
known steroidknown steroid Compound was NOT detected by normal steroid screenCompound was NOT detected by normal steroid screen The mass spectrum was similar to two other steroidsThe mass spectrum was similar to two other steroids Accurate mass spectrometry indicated a molecular Accurate mass spectrometry indicated a molecular
formula of Cformula of C2121HH2828OO22 (312.2080 Da) (312.2080 Da)
Rapid Communications in Mass Spectrometry vol. 18, page 1245 (2004)
Unknown mass spectrum
Trenbalone mass spectrumTrenbalone is used to aid growth in
US beef cattle
Gestrinone mass spectrumUsed in Europe to treat endometriosis
New molecule dubbed THG or tetrahydrogestrinone is active ingredient in “The Clear”
Staudinger Rxn Gone Wrong?Staudinger Rxn Gone Wrong?
NH
O OO N3
O
O
OH
OH
O NH
S
NH
O OO NH2
O
O
OH
OH
O NH
S
PPh3,
THF:H2O (2:1),r.t.
NH
O OO N
O
O
OH
OH
O NH
SPPh3
observed by MSisolation not attempted
Chemical Formula: C47H44N3O8PSExact Mass: 841.2587
Chemical Formula: C29H29N5O8SExact Mass: 607.1737 Chemical Formula: C29H31N3O8S
Exact Mass: 581.1832
Base
M+H for product is 582.19M+Na is 604.18
LC-MS ChromatogramLC-MS Chromatogramsdc2-271-001
Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
%
0
100
sdc2-271-001 1: TOF MS ES+ BPI
4.25e310.20
3.23
5.82
5.62
4.08
9.97
8.15
15.32
18.73
Mass Spectra from PeaksMass Spectra from Peakssdc2-271-001
m/z100 200 300 400 500 600 700 800 900 1000
%
0
100
%
0
100
%
0
100
sdc2-271-001 489 (8.153) Cm (481:499) 1: TOF MS ES+ 1.91e4583.144
142.119539.151
335.097237.565 348.092
584.177
585.166
sdc2-271-001 350 (5.835) Cm (350:359) 1: TOF MS ES+ 1.06e4278.094
142.119 229.140279.110
453.347
sdc2-271-001 194 (3.234) Cm (188:205) 1: TOF MS ES+ 3.36e4278.094
279.110
8.15 min peak1 Th off from product (M+H)+
5.82 minute peakunknown contaminant
3.2 minute peakunknown, non-retained compound
Mass Spectra from Peaks 2Mass Spectra from Peaks 2sdc2-271-001
m/z100 200 300 400 500 600 700 800 900 1000
%
0
100
%
0
100
%
0
100
sdc2-271-001 922 (15.373) Cm (916:926) 1: TOF MS ES+ 9.21e3295.061
185.168 229.147296.075
420.977651.071475.337 697.035
sdc2-271-001 612 (10.204) Cm (610:618) 1: TOF MS ES+ 3.59e4279.056
142.125
280.089
342.100 579.145557.179343.115 580.174
sdc2-271-001 596 (9.937) Cm (595:600) 1: TOF MS ES+ 1.03e4608.141
142.125397.338
609.184
610.194
15.32 minute peakunknown hydrophobic compound
10.20 minute peaktriphenyl phosphine oxide
(M+H)+, (2M+H)+, (2M+Na)+
9.97 minute peakstarting material M+H
Proposed Side ReactionProposed Side Reaction
NH
O OO NH
O
O
OH
OH
O NH
S
NH
O OO N
O
O
OH
OH
O NH
SPPh3
observed by MSisolation not attempted
Chemical Formula: C47H44N3O8PSExact Mass: 841.2587
NH
O OO
O
O
OH
OH
O NH
S
Chemical Formula: C29H30DN3O8SExact Mass: 582.1895
OH
Chemical Formula: C29H30N2O9SExact Mass: 582.1672
D
OR
Accurate Mass DataAccurate Mass Datasdc2-271-003
m/z475 500 525 550 575 600 625 650 675 700
%
0
100
%
0
100
sdc2-271-003 (0.035) Is (1.00,1.00) C29H30N2O9SH1: TOF MS ES+ 6.67e12583.1750
584.1782
585.1772
sdc2-271-003 417 (8.707) AM (Cen,8, 80.00, Ar,6000.0,622.57,0.70,LS 4); Sm (Mn, 2x4.00)1.40e3583.1733
539.1458475.3204
584.1797
585.1721623.4202 667.4045
Theoretical M+H forC29H30N2O9S
Acc. Mass LC-MS data
M+H for deuterated amine is 583.1967 (-40 ppm)