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 SpectrometryA 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?Io

n A

b un d

a nce

(a s

a %

o f B

a se

pea k

)

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

High mass

Not usually scanned below m/z=32 (Why?)

[M+H]+(CI)Or M•+ (EI)“molecular ion”

Unit mass spacing

Fragment Ions Derived from molecular ion or higher weight fragments

In CI, adduct ions, [M+reagent gas]+

Molecular Ionsgive us the molecular mass

Chemical Ionization

MH+

H+

H+

H+

H+

[M+H]+

Weighs one more than MW

Dislodges an electron

Electron Impact

M-e• -e•

-e•

-e•-e•

-e•

2e-•

M+•

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

•Prepare derivative

Other compounds present may give ions that deceive us. May be more detectable. 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

Sometimes too energetic for molecular ion to survive

Rich harvest of fragment ions

“fingerprint” nature of fragment patterns lends itself to database library searches

CI

Stronger, more reliable molecular ions

Fewer fragments

Can choose different reagent gasses and exploit chemistry, giving different fragmentation. e.g. NH3/ND3

Adduct ions give support to identities

Nitrogen rule works but inverted

Can do negative ion Mass Spec

EI

CI

When would you use CI, EI?EI

•When “fingerprint” is needed for Identification by comparison screening in databases

•Trace analysis

•Forensics

•Environmental

•Total unknowns, e.g natural products

•Fragment homology within a series, e.g. of natural products

CI

•When rapid, reliable identification of molecular ion is needed.

•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

•Minimize fragmentation, get most intensity in molecular ion.

How can I tell which (EI or CI) was run?

Adduct ions higher m/z than MH+, ,[M+C2H5]+ ,[M+C3H5]+ [M+NH4]+

Large molecular ion

Relatively few fragment ions

Chemical Ionization

No ions higher m/z than M•+

Smaller M•+ intensity

Rich family of fragment ions

Electron Impact

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 outMass 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

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

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.

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?

All 12C

1 13C

C10 C100

1 13C

2 13C

From this, it is clear that for large or macromolecules, there will be practically no population having all 12C or even only 113C

A rule of thumb, made possible by knowing the isotopic abundance is that the number of C in a formula is given by:

N=

€

IntensityM +1[ ]

M[ ]x90.1

Fragmentation

The “Even Electron Rule” dictates that even (non-radical) ions will not fragment to give two radicals (pos• + neutral•) (CI)

Better carbocation wins and predominates (“Stevenson’s Rule”)

[M·]+ A+ + B· (neutral)

or

B+ + A·

EI

CI

[M+H]+ PH+ + N (neutral)

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 HCCH

M+• -27 •HC=CH2 or HCN HCN from pyridine, anilines

M+• -28 CO 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.

molar mass: 157Formula M+1 M+2 MM e/o dbrHN2O8 1.05 1.60 156.9732 e 1.5HN10O 3.75 0.20 157.0337 e 5.5H3N3O7 1.41 1.40 156.9971 o 1H3N11 4.12 0.00 157.0576 o 5H5N4O6 1.78 1.20 157.021 e 0.5H7N5O5 2.14 1.00 157.0448 o 0CHO9 1.45 1.80 156.9619 e 1.5CHN8O2 4.15 0.43 157.0224 e 5.5CH3NO8 1.81 1.60 156.9858 o 1CH3N9O 4.51 0.24 157.0463 o 5CH5N2O7 2.17 1.41 157.0096 e 0.5CH5N10 4.88 0.04 157.0702 e 4.5CH7N3O6 2.54 1.21 157.0335 o 0C2HN6O3 4.55 0.66 157.0111 e 5.5C2H3N7O2 4.91 0.47 157.0350 o 5C2H5O8 2.57 1.61 156.9983 e 0.5C2H5N8O 5.27 0.28 157.0589 e 4.5C2H7NO7 2.93 1.42 157.0222 o 0C2H7N9 5.64 0.09 157.0827 o 4C3HN4O4 4.94 0.89 156.9998 e 5.5C3H3N5O3 5.31 0.70 157.0237 o 5C3H5N6O2 5.67 0.51 157.0476 e 4.5C3H7N7O 6.03 0.33 157.0714 o 4C3H9N8 6.4 0.14 157.0953 e 3.5C4HN2O5 5.34 1.11 156.9885 e 5.5C4H3N3O4 5.70 0.93 157.0124 o 5C4H5N4O3 6.07 0.74 157.0362 e 4.5C4H7N5O2 6.43 0.56 157.0601 o 4C4H9N6O 6.79 0.38 157.0840 e 3.5

C4H11N7 7.16 0.20 157.1078 o 3C5HO6 5.74 1.32 156.9772 e 5.5C5H3NO5 6.10 1.15 157.0011 o 5C5H5N2O4 6.46 0.97 157.025 e 4.5C5H7N3O3 6.83 0.79 157.0488 o 4C5H9N4O2 7.19 0.61 157.0727 e 3.5C5H11N5O 7.55 0.44 157.0965 o 3C5H13N6 7.92 0.26 157.1204 e 2.5C6HN6 8.84 0.33 157.0264 e 9.5C6H5O5 6.86 1.19 157.0136 e 4.5C6H7NO4 7.22 1.02 157.0375 o 4C6H9N2O3 7.59 0.84 157.0614 e 3.5C6H11N3O2 7.95 0.67 157.0852 o 3C6H13N4O 8.31 0.50 157.1091 e 2.5C6H15N5 8.68 0.32 157.1329 o 2C7HN4O 9.23 0.58 157.0151 e 9.5C7H3N5 9.60 0.41 157.0390 o 9C7H9O4 7.98 1.07 157.0501 e 3.5C7H11NO3 8.35 0.90 157.0739 o 3C7H13N2O2 8.71 0.73 157.0978 e 2.5C7H15N3O 9.07 0.56 157.1217 o 2C7H17N4 9.44 0.40 157.1455 e 1.5C8HN2O2 9.63 0.81 157.0038 e 9.5C8H3N3O 9.99 0.65 157.0277 o 9C8H5N4 10.36 0.49 157.0516 e 8.5C8H13O3 9.11 0.96 157.0865 e 2.5C8H15NO2 9.47 0.80 157.1104 o 2C8H17N2O 9.83 0.64 157.1342 e 1.5C8H19N3 10.20 0.47 157.1581 o 1C9HO3 10.03 1.05 156.9925 e 9.5C9H3NO2 10.39 0.89 157.0164 o 9

C9H5N2O 10.75 0.73 157.0403 e 8.5C9H7N3 11.12 0.57 157.0641 o 8C9H17O2 10.23 0.87 157.1229 e 1.5C9H19NO 10.59 0.71 157.1468 o 1C9H21N2 10.96 0.55 157.1706 e 0.5C10H5O2 11.15 0.97 157.029 e 8.5C10H7NO 11.51 0.81 157.0528 o 8C10H9N2 11.88 0.66 157.0767 e 7.5C10H21O 11.35 0.79 157.1593 e 0.5C10H23N 11.72 0.64 157.1832 o 0C11H9O 12.27 0.90 157.0654 e 7.5C11H11N 12.64 0.75 157.0892 o 7C12H13 13.40 0.84 157.1018 e 6.5C13H 14.32 0.97 157.0078 e 13.5

Example, m/z’s for 157

Clearly, some are not realistic!

Calculated mass distributions

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: ursr@mac.com

IMASS for Mac OSXVersion 1.0 (v2A15)© 2000 - 2002, Urs Roethlisberger,

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 acid-base (proton affinities)

Some popular cleavages

Cleave at a branch point. Loss of radical or other neutral to provide a more stable cation

Cleave to a heteroatom (capable of supporting positive charge)

Note the use of “half arrow” for one-electron movements. e.g homolytic cleavage

CH3 CH3

CH3CH3

CH3

C+

CH3

CH3.

Obs. in Mass Spec

+

neutral

+

RO

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

N

CH2+

92+ +

91

H

H

H

H

H

+

77

CH2+

43

H

O

+29

McLafferty RearrangementsRadical cations localized on keto-type oxygen give cleavageThe mechanism limits this to EI fragmentationNeeds a H atom on a sp3 carbonKetones, esters, carboxylic acids all give McLafferty products

O+

HR2

R1

• O+

H

•

OH

R1 R2

•••

+ Loss of neutral alkene

The new radical cation is stabilized by resonance

Note the use here, of the “half arrow” to represent “1-electron flow”

Important example of McLafferty R&R

OH

OH

m/z = 60

+•

Seen for primary carboxylic acids

Non-Sequential Losses

CH3

O

CH3

MW=152

M+-CH3CO

M+-CH3

HydrocarbonsWeak [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

O

: .

+

++:

+: :

Obs. in mass spec. Acylium ions are resonance-stabilized

neutral

Prominent for ketones

CH3C=O+ m/z=43

Example

O

O O

M+• -45, loss of ethoxy radical

O+

C+

O

O+

Example

OO

+

M+• -43; also tropylium ion

Cleave to Heteroatoms like O, N

O

R

: .

+•

Heterolytic cleavage

R

O:: .

neutral

+

Observed in Mass Spec provided that a good stabilized carbocation can form

+

Rearrangements and fragmentations to give good Carbocations

CH2+

CH

+ CH2

H C

CH2

CH+

+

Benzylic cation (stabilized including “tropylium” ion m/z=91

Good cleavage to aromatic rings

Example

Bromine pattern

Tropylium ion

Br

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 OC–OH+

Amines

N•+ N+

-R•

R

Cyclic amines will lose adjacent H•, form iminium ion

In CI, NH+ can eliminate adjacent alkene, reprotonate

Silyl Ethers

SiO+

•

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

OCH3

ORLoss of CH2O

Extra H transfer mediated by adjacent heteroatom

H

O+

R

H

H

+•

Nitroaromatics

N+

O•

OO

+

Loss of •N=O

Loss of CO

m/z= 93

(this can form from lots of different origins)

CH+Aromatic!

m/z=65

Good test for aryloxy

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.

S+

S+R

•

-R•

The “retro Diels-Alder” Cleavage

+

+•

+•

+

+•

Observed!

Observed!

Typically you see both.More stable cation will predominateAlso works for hetero-substituted (e.g. make enol)Both EI (shown) and in Chemical Ionization. (protonated molecular ion, cleave, then reprotonation

Cyclohexenes, with favorable 6-membered transition state. Can include heteroatoms (N,O, driven by keto-enol like stability.

An Example from Terpenoid Chemistry

+

•

+

•

+

12-Oleanene m/z 204

A good example for Retro Diels Alder fragmentation

4-terpineol

MW 154

OH

OHO

mz 86+

+

mz 68

EI Mass Spectrum

+

Double bonds can isomerize

OH

MW=396

m/z136C9H12O+

m/z118136-water

-cleavage following double bond migration

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

CH3Cl

One chlorine

CHCl3

Three chlorines

35Cl

37Cl

CH3Br

One bromine

CHBr3

Three bromines

79Br

81Br

81Br2

81Br1

The paired appearance flags the ions as to the number of halogens

Fragment ions with the same halogen count preserve the pattern

Great Websiteshttp://medlib.med.utah.edu/masspec/elcomp.htmCalculate potential molecular formulas from m/z(neutrals only)

http://www.colby.edu/chemistry/PChem/Fragment.htmlWizard 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