Mass Spectrometry Intro Flashcards
Why Mass Spectrometry?
• Very high sensitivity / small samples (pg)
‒ Quantify (e.g. drugs/ metabolites in blood)
• To determine:
– Elements present (Cl, Br, S, Si, …) NMR, IR not useful
– Molecular weight
• Soft ionisation
– Molecular formula
• High resolution
– Structure (biomolecules, sequence peptides & proteins, …)
• Fragmentation patterns (MS/MS)
– High spatial resolution label-free imaging
• Formulations
• Tissue sections / Single cells
Limitations of MS
• (relatively) Complex equipment – Portable / handheld versions • Not al lmolecules equally easily... – ... into vapour phase (required) – ... form ions (also required) • Not inherently quantitative – Internal/external standards (beyond scope here) • Mixture analysis can be challenging – GC-MS / (HP)LC-MS
Basic Steps in MS
- ( Generate molecule in gas phase )
- Ionisation
- Mass separation
- Ion detection
- Data interpretation
Step 1: Gas phase
• Some molecules already in the gas phase
– Breath analysis (drink-driving, acetone on breath linked to diabetes, other non-invasively accessible disease markers)
– “Smelly” chemicals – drug enforcement dog
• Easiest for others: Heat until evaporation occurs
– “non-polar”: 1 kD (1 Dalton is 1 mass unit; so Mw of CH4 is ~16g, a single CH4 molecule weighs ~16 D
– medium-polar: 300 D
– Problem: a lot of interesting molecules will react or break down before they evaporate
E.g. sugars will caramelise; starchy foods → acrylamide
– Traditional small molecule drugs → new larger (bio)molecules
Step 2: Ionisation
• Most common: electron ionisation (EI)
Commonly 70 eV = 6700 kJ mol-1
Compare: C-C ~350 kJ mol-1
EI (EI-MS)
• Only for molecules that can readily be in gas phase without decomposing
– Size limited
– Polarity limited
• Electron beam interacting with evaporated molecules
• Accelerate molecules into mass filter and then onto detector (count charged particles)
EI principles
- Mostly forms cations – which we will mostly look at
- Counter-intuitive: electron to ionise molecule
- High energy electron fired at molecule in gas phase
- This knocks out an electron , generating a cation: M + e- => M+. + 2e- forming radical cation (molecular ion)
- Weight of electron negligible compared to atoms, so mass of ion = mass of original molecule
- E.g. CH4 + e- → CH4+. (resonance!) + 2e-
Odd electron molecular ions
• Location of missing electron:
– Bonding electron: CH4
– Lone pair preferred if available: ether with charge (positive!) and radical located on O
– Alkene: pi orbital is HOMO orbital and hence loses e-: R-CH-C+H-.CH2 with resonance structures switching position of + and radical
• If there is fragmentation of the molecular ion: – one part has even nr of e- and positive charge
– other part has odd nr e- and no charge
– So charge and electron separate on fragmentation
Step 3: Mass separation
• Accelerate ions so they move in a straight line into a “mass filter” region (in vacuum so no collisions)
• 2 basic methods used most:
– Deflection of ions in electromagnetic field
Ions deflect more if charge (z) higher or mass (m) lower (magnetic sector), quadrupole
– Time-of-flight
Ions fly faster if charge (z) higher or mass (m) lower
In common: m/z rather than m
(especially important for “soft” ionisation techniques)
Electromagnetic field mass filter
– Oldest type, conceptually easiest
– Not used that often anymore
– Detector in fixed position which ions can only reach if they are deflected into it
– Vary magnetic field
Only specific m/z deflected precisely into detector
– Maths: convert strength magnetic field → m/z
– Graph m/z on x-axis and number of detected ions (Intensity) on y-axis: the mass spectrum
Diagram and equation
m/ z = B2 r2/ 2V
1. r is fixed
2. Select combination of B and V
3. If ions are detected for this setting you know
what m/z value these ions have
4. Repeat for other combinations of B and V to cover the part spectrum you are interested in
Quadrupole filter
- Similar principle as previous
- Now detector is at the end of the filter which looks like 4 rods alongside each other: see next slide
- Only detect ions that carry straight on
- Alternating current on rods – vary so only specific m/z can fly on to reach detector
- Detect 1 m/z value at a time
- Unit mass resolution
Quadrupole Analyser
- Mass filter: only 1 mass at a time reaches the detector
- Low mass resolution
- Cheap, robust
- < 1000 m/z, but ion-trap version:
- m/z ~ 70,000
- Complex (biological) mixtures
- very sensitive (~2.5 10-18 mol peptide)
- Couple to chromatographic separation (GC, LC)
A different method: ToF
• All ions reach the detector!
• But separated by time
• Give all ions same kinetic energy (Ekin) at the same time and place
• Basic physics: Ekin = mv2
• So if m(ass) is different, but Ekin the same, v must be
different.
• v is velocity of the moving ions
• So heavier ions take longer to reach the detector
– Depends on charge, so again linked to m/z rather than m
• Convert time to m/z, counted ions at each time on y-axis
• Higher mass resolution possible
Time-of-Flight analyser
E kinetic = 1/2 m v2
