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

A Discussion of Molecular IonsA Discussion of Molecular Ions

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

Making ions: A Practical Making ions: A Practical PrimerPrimer

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

ESI PictureESI Picture

http://newobjective.com/images/electro/spraytip_bw.jpg

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

APCI DiagramAPCI Diagram

http://imaisd.usc.es/riaidt/masas/imagenes/apci1.jpg

APCI ExampleAPCI Example

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

MALDI DiagramMALDI Diagram

Image from http://www.noble.org/Plantbio/MS/iontech.maldi.html

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

MALDI Example I ContinuedMALDI Example I Continued

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 DiagramEI Diagram

Image from http://www.noble.org/Plantbio/MS/iontech.ei.html

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

Timescales for EI-MS

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

H2C

CH2

Br•+

Exact Mass: 135.9882

H3CCH2

H2C

CH2+

Br•

Exact Mass: 57.0699

136

57

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

H3C

H2C

HC

CCH3

O

CH3

Exact Mass: 100.0957

7229

43

100

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

Huygens Probe (on Titan)Huygens Probe (on Titan)

Problem Solving with MSProblem Solving with MS

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)