Mass Spectrometry 2 Flashcards
The rest of the spectrum: Fragmentation
EI: AB + e- → BA●+ + 2e- most common
-Odd-electron ion = free radical = relatively unstable
- Fragmentation and/or Rearrangement may occur
• Relative energy differences of potential fragments (parent / daughter) crucial
• Kinetics also has a role to play: will ions fragment before they reach the detector?
– Same applies to possible rearrangements
Fragmentation
used in most databases: EI
• Not all 70eV transferred on e- impact
• Ions have distribution of internal energies
• If no collisions (vacuum in mass spectrometer), then uni-molecular processes to revert to lower energy
– Collisions can help when using soft ionisation – Sometimes you can cause collisions to occur on purpose
• Metastable ions: fragmentation after leaving source
– appear at non-integral mass
– Position reveals fragmentation process and hence structural features of the original molecule
• But we mostly rely on stable ions
Electron ionisation (EI)
• Hard ionisation: 70 eV 6700 kJ mol-1
– Extensive fragmentation
– Not all energy passed-on from electrons to molecules
– Not always a molecular ion
Fragmentation (EI)
- No need to apply energy as there is no barrier to overcome. However this depends on the fragment to be made • noEa -> no M+remains • high Ea( >= 4eV =390 kJmol-1) -> little fragmentation -> abundant M+. • Fragm. with loss of stable mol.: -> (substantial) reverse Ea • Fragm. with single scission: -> little or no reverse Ea
Single bond cleavage
Preferred fragmentation path way: Thermodynamics ∆fH(products) - ∆fH(reactant(s))
Fragmentation (EI)
• Energy considerations: predict which decomposition route most favourable
• Statistics -> relative peak intensities
– several pathways can occur at same time
• Single bond cleavage may not be favoured:
– Fragmentation with (multiple) H-rearrangement(s)
– Skeletal reorganisation incl. formation of stable neutrals
• Geometry also important
• Reactions involving bond formation unlikely if NOT to H
– Exception is in case of unsaturation
• Lower energy promotes H-rearrangement over single-bond cleavage
General “rules”
- Formation of very small ions unlikely
- Ease of ion formation (most stable ion):
tertiary > secondary > primary - If ions are stable in solution, same applies to gas
(e.g. delocalised cations, acylium, oxonium, imminium ions) - Radicals more stable with increased substitution
- Vinyl cations: high ∆fH (C=C+ +C-C=C)
- Vinyl radicals unstable
Molecular ions
• If peak below supposed M+/- with
difference 4 - 13 u: – Not M+/- after all
– Mixture
• Peak 14 u below M+/- suggests homologue as ∆fH(:CH2) = very high
– “Same” molecule, but differs in length by –CH2-
• Compounds with C, H, O and # N = 0, 2, … have even mass
EI: Fragmentation of Aromatics
- Ar-X : in general loss of X / part of X
- Tables list ease of fragmentation (e.g. Williams T4.8)
- Competitive losses possible leading to more complex spectrum
- Rearrangements can compete with decomposition, leading to different fragmentation
- Resonance effects may be important
- Ortho-substituted: consider proximity of side groups which may interact
EI: Fragmentation of Aliphatics
- Tables: primary single-bond cleavages for common functional groups
- If primary fragmentation leads to competition then loss of larger radical normally dominant
EI Fragmentation: Example 1 Ketones
• Loss of alkyl group attached to C=O readily predicted
• Acyllium ions formed
C2H5C=O+; C4H9C=O+ • Rearrangement?
see Williams T4.11
• High resolution:
resolve between C4H9+ and C2H5C=O+ at m/z = 57
EI Fragmentation: Example 2 Amines
M+●: Odd molecular weight → odd number of N
• Check tables for ion series (Williams T4.10: 30, 44, 58, 72)
• How? Rearrangement allows for loss of largest radical
Summary of major fragmentation routes
diagram 21
Common Impurities
in lab or in production…
m/z 149, 167, 279
Plasticizers (phtalic acid derived)
129, 185, 259, 329
Plasticizer (tri-n-butyl acetyl citrate)
133, 207, 281, 355, 429
Silicone grease
99, 155, 211
Plasticizer (tributyl phosphate)
EI Fragmentation: using losses
After identification of M+/- look for neutral losses
• Tables for common losses (e.g. Williams T4.13)
• Use your chemistry knowledge
M - 1 -> loss of H
M - 2 -> loss of H2
M - 15 -> loss of CH3
M - 16 -> loss of O
Fingerprinting
- Compare to database of spectra
- Needs to be established first
- Spectra specific for specific conditions
- EI mainly
- Other MS techniques much smaller databases
- Make own dedicated database of limited size
Soft Ionisation
- Only add charge to molecule for easy identification of molecular ion -> molecular mass
- If you know the precise mass very well you can calculate # C, H, O, N, S, …
- But different molecules may have the same molecular formula!
- Different molecules fragment differently
- Control what functionality is ionised
Electron ionisation (EI)
M+ can lose:
- 1 radical only
- Any number of neutrals
- Once radical lost, only neutrals can be lost
CI Fragmentation
• Determine molecular weight = great strength – M+/- – (M+H)+ / (M-H)- – (M-X)+/- / (M+X)+/- • High mass resolution: molecular formula • Tailor ionising agent to – Control level of fragmentation – Detect specific functionalities • Well-suited to MS-MS • Compare to database spectra
Chemical ionisation (CI) table
slide 29
Reagent gas
Reagent ion
Analyte ion
Comment
H2
H3+
(M+H)+
Very energetic
CH4
CH5+
(M+H)+
Energetic
i-C4H10
C4H9+
(M+H)+
Mild, protonates all N-bases
NH3
NH4+
(M+NH4)+
Selective, little fragm.
NH3-CH4
NH4+
(M+H)+
Selective
Biacetyl
CH3CO+
(M+CH3CO)+
Acetylating
Ar
Ar· +
M ·+
Energetic
CS2
CS2·+
M ·+
Mild
CH3ONO-CH4
CH3O-
(M-H)-
Mild
NF3
F-
(M-H)-
Medium
CHCl3-CH4
Cl-
(M+Cl)-
Cl addition
Electrospray Ionisation (ESI)
- “Soft” ionisation: (quasi-)molecular ion
- Polar molecules which have poor vapour pressure
- & -ion spectra
- Very good for large (bio-) molecules
- Multiple charging helps with large biomolecules
- Couples readily with chromatographic separation of mixtures
• Large, polar molecules
• Solvent: mainly MeOH / H2O mixtures
• Additives, e.g. + ions: 0.1% organic acid (CF3COOH) as H+ donor
• +/- droplets
• Desolvation aided by Coulombic repulsion
–»_space; 1 molecule per droplet typical
– Separation of charged molecules aided by repulsion
ESI “Fragmentation”
• (quasi-) Molecular ion intense
• Add ionising agent to solvent
• Induce fragment by collisions: MS-MS
• Sensitivity: down to ~ femto (10-15) moles for proteins
– for a typical 10,000 D (10 kD) protein this means 200 pg!
Step 1/3a: hyphenated techniques
Separate mixtures before MS:
• GC-MS
• LC-MS
Select component during MS: MS-MS
• Select ion
• Fragment
• Identify
(GC or ) LC coupled to MS
• Mixtures
– Natural
– Synthetic organic
• GC or (HP)LC gives some idea of chemical character
– GC if easy to volatilise, mostly LC in pharmaceutical applications
– MS ideal to further analyse minute amounts of eluent
• HPLC:
– Separate complex mixtures
– Especially if comparable polarities
– Remove solvents before MS
– Use solutes with charge (e.g. -NH2; -COOH) or volatile buffer (e.g. NH4OAc; HCO2H)
– Separate ionisation not always necessary
• LC-ESI-MS especially good if only value for [M+H] needed
Tandem MS: MS-MS (MSn)
- n >= 2
- Similar function to other hyphenated techniques
1. Separate interesting ions (molecular or fragments)
2. Study each ion separately - (Mixtures)
- Complex compounds: check “fragmentation pattern”
- Physical coupling of different MS-systems (mass filters), e.g. Q3 : Special type “Ion trap” for MS-MS with single mass filter
-HPLC-MS-MS often used in healthcare applications
• Biomedical, pharmaceutical
• High complexity
• Low concentration of key molecules
Key new developments in MS
• More practical high mass resolutio ninstruments (£££)
• Compact mass spectrometers (resolution, sensitivity)
• Direct Analysis/ Ambient MS
– Ambient conditions
– Desorption ElectroSpray Ionisation (DESI)
– Direct Analysis in Real Time (DART)
– Plasma Assisted Desorption Ionisation (PADI) Very gentle plasma to generate gas-phase ions Under development at Keele!
EI: Fragmentation of Aliphatics - Amine
Simplest ion type?
CH2=N+H2
m/z 30
EI: Fragmentation of Aliphatics -Ether alcohol
Simplest ion type?
CH2=O+H
m/z 31
EI: Fragmentation of Aliphatics - Ketone
Simplest ion type?
CH3C(TRIPLE BOND)O+
m/z 43
EI: Fragmentation of Aliphatics - HC
Simplest ion type?
C2H5+
m/z 29
