fragmentation Flashcards
fragmentation
generation of smaller parts of your analyte during the ionisation process
fragmentation is structurally dependent and so knowledge of the way your analyte fragments can provide valuable insight into structure/ based on ionisation
fragmentation basic principles
where the charge is located will determine which fragmentation route(s) are followed. often multiple locations are possible so multiple fragmentation routes are possible
ionisation sites - all imitate fragmentation: non-bonded electrons (lp), pi-electrins, aromatic electrons and hydrocarbons
fragmentation rules
- ionisation:e removed from site with lowest ionisation potential i.e. non-bonding > pi bond > sigma bond
- sigma bond dissociation: charge retained by fragment with lowest ionisation potential
- even-electron ions cannot cleave to pair of odd-electron fragments
- odd-electron ions can fragment to give odd or even-electron fragments
- loss of mass 14 from M+ is extremely rare (seen in hydrocarbons but not from M+)
- order of radical/carbocation loss: tertiary > secondary > primary> methyl > hydrogen
- probability of bond cleavage : bond strength and fragment stability
- fragmentations can occur sequentially if leads to increased stability
- loss of a stable neutral molecule will happen whenever it is possible
- best to remember a few key diagnostic fragment ions/neutral losses
- small differences in spectra can be used to distinguish between structural isomers or some stereoisomers
- not all rules are binding
fragmentation by EI
EI = hard ionisation technique so produces considerable fragmentation
ions either have odd or even number of electrons depending on how they are produced
significant influence on any secondary fragmentation routes
analyte+’ –> analy’ + te+
even electrons, lower energy, less fragmentation
analyte+’ –> ana + lyte’+
odd electrons, higher energy, more fragmentation
fragmentation by EI - branched alkanes
the loss of an alkyl group can lead to four possible carbocations
alkyl groups are lost preferentially by size - larger radical neutrals are more stable
loss of largest alkyl radical (forming a secondary cation)is totally dominant over tertiary carbocation formation (through loss of H)
fragmentation by EI - linear alkanes
molecular ion observed at good intensity
loss of methyl radical ‘CH3 at low intensity
C-C bond cleavage produces mix of alkyl radicals and carbocations
homologous series of ions spaced by 14 (CH2)
alkene carbocations observed due to loss of H2
charge retained by smallest fragment - higher intensity
propyl and butyl usually dominant
fragmentation by EI - aldehydes and ketones
Homolytic fission
Homolytic fission: cleavage of bond alpha to carbonyl group - forms a radical alkyl group expulsion and more stable even electron cation of high intensity
e.g for pentanal
2 possible alpha-cleavages resulting in m/z 85 and 29
and 2 for 43 and 71
fragmentation by EI - aldehydes and ketones
McLafferty rearrangement
cleavage of bond beta to carbonyl group
forms unstable odd electron fragment (low intensity) and neutral alkene that is lost
e.g. for pentanal
2 possible beta-cleavages resulting in m/z 44 and 43
elimination of Co gives m/z 58 as does a beta-cleavage
fragmentation of aromatic compounds
controlled by carbocation resonance stabilisation through McLafferty rearrangements. strong molecular ions due. to loss of electron from pi-system, fragmentation of alkyl side chains lead to benzyl and tropylium which is aromatically stabilised
fragmentation by chemical ionisation (CI)
CI is a relatively soft ionisation technique. the ions have less residual energy after ionisation and so there is less fragmentation than in EI. fragmentation is dominated by 2-electron processes which ‘mimic’ that seen in the condensed phase. the site of protonation is not always known and multiple routes can occur from each site
fragmentation by CI - charge directed neutral elimination
the heteroatom is protonated ( good lg) which is eliminated forming a carbocation which can then undergo further fragmentation
fragmentation by CI - charge remote neutral elimination
an example of a 1,3 proton shift leading to an elimination of HX
there is charge somewhere on the molecule but not on the heteroatom
fragmentation by CI - charge remote beta-cleavage(C-C cleavage)
an example of a McLafferty rearrangement, leading to C-C cleavage. which of the 2 products is observed depends on the site of protonation
hybrid mass spectrometer
contains two or more types of mass analyser
- to try to get benefits of both
- enables tandem mass spectrometry experiments
tandem mass spectrometry (MS/MS)
makes use of 2 separate mass analysis steps with a fragmentation step in-between
most often used to generate fragment ions in ESI MS, but can be used with EI and CI when you want to see specific fragmentation
EI and CI produce non-specific fragmentation
MS/MS methods used with triple quadrupole mass spectrometer
product ion scanning
precursor ion scanning
constant neutral loss scanning
precursor ion
fragmented during MS/MS
product ion
generated during MS/MS
product ion scanning
the product ions of a specific precursor ion can be studied independently of any other ions present in the spectrum
precursor ion scanning
all precursor ions of a mixture scanned for those that produce a specific product ion
constant neutral loss scanning
all precursor ions of a mixture scanned for those that produce a specific neutral loss
sequential mass spectrometry (MS^n)
makes use of an ion trap instrument to perform repeated fragmentation of product ions
sequential mass spectrometry overview (steps)
- mass spectrum recorded MS1 spectrum
- required precursor ion isolated
- precursor ion then fragments with collision gas
- product spectrum recorded - MS^2 spectrum
MS^2 product ion(s) isolated as MS^3 precursor ions - precursor ion(s) then fragment with collision gas
- product ion spectra recorded - MS^3
- repeated for MS^4 et.c
collision induced dissociation (CID)
most common for fragmentation analysis
- collision cell filled with an inert collision or CID gas (N2, CO2 or Ar)
- CID gas energised
- precursor ions collide with the CID gas and energy is transferred
- the precursor ions undergo repeated collins and get increasingly energised until the fragmentation threshold is reached
- at that point they will fragment into smaller product ions
- small neutrals lost + controlled structural fragmentations
- some fragment ions are unstable and continue to fragment
the power of product ion scanning (steps to confirm peak is due to a particular compound)
- perform an isolation experiment in quadrupole 1 (results in only the precursor ion passing through to the collision cell)
- precursor ion is fragment in quadrupole 2 - the collision cell and the product ion spectrum is measured in quadrupole 3
- compare the product ion scan from your extract to that of the pure compound (database)
fragmentation in CID-MS/MS
common functional groups often proceeds by protonation of the heteroatom leading to neutral loss and formation of a carbocation - leaving group lost during CID induced fragmentation to produce the carbocation product ion
fragmentation in CID-MS/MS:
acids and esters
tend to fragment in 2 step process initiated by protonation of the hydroxyl or ether oxygen leading to loss of water or the corresponding alcohol, followed by loss of CO to form carbocation
fragmentation in CID-MS/MS: amino acids
complex, two possible protonation sites - N or OH
at N lose -HNH2 then water
at OH can lose -HOH then CO
major route is protonation of O and loss of -HCOOH through N lone pairs
fragmentation in CID-MS/MS: charge remote
alpha carbon must have a proton to transfer to the heteroatom in a concerted manner
alcohols
ethers
amines
nitriles
all form alkenes
acids undergo decarboxylation to generate CO2
fragmentation in CID-MS/MS: C-C bond cleavage
McLafferty rearrangement
Retro Diels-Alder reaction
Bond cleavage and elimination (X= heteroatom)
Grab fragmentation
1,3 hydride shifts
Ring contractions supported by neighbouring aromatic ring
fragmentation in CID-MS/MS: charge directed amides and esters
elimination to lose either the alcohol or amine
1 step
