Mass Spectrometry

Mass Spectrometry

1. Principle: Sorting of ionic species of molecules or compounds based on their mass to charge ratio in a electric field via deflection.

2. Ion Source

   • Function: - To generate ions - To transfer into gas phase
   • Technique choice depends on: - Properties of analyte - Non polar - Strong I - Proteins, thermally unstabe compounds : Soft I (ionizes without degradation)
   • Electron Spray Ionisation (ESI)
     • Principle: Analyte mixed with solvent is passed through a cappillary at high voltage and alllowed to pass through a hole in the centre of a counter electrode
       • Three steps (1) Formation of charged species (2) Shrinking of droplets (3) Formation of gaseous species
       • Solvent : Water or organic solvent like acetonitrile
     • Shrinking of Droplets
       • Charged droplets form at the capillary end with positive charges at the surface of the droplet. Solvent evaporates until the droplet becomes unstable and leads to taylor cone formation
         • Unstability = rayleigh limit ehich means electrostatic forces of repulsion is greater than surface tension
     • Evaporation
       • Charge Residue model (Fission reaction)
       • Ion Evaporation Model (Ion evaporates into charged species)
     • Advantages over MALDI - No sample prep, High reproducibilty, Can be combined with LC to extremely high detection levels, multiple ion formation, high sensitivity
   • Matrix Assisted Laser Desorption Ionisation (MALDI)
     • Principle: To utilize laser to ionize matrix of analyte mixed with a solvent containing light sensistive organic compounds or rich in chromophores . Matrix absorbs energy from the laser and produces plasma that vaporizes the analyte.
       • Commonly used chromophores: * CHCA * SA * DHB ( Matrix molecues have conjugated double bonds or aromatic species that have pi electrons that are easy to excite)
     • Sample Preparation Methods (FEDSPIM)
       • Dried Droplet
         • Droplet of analyte is mixed with matrix and dried = Matrix doped with anayte into MS
       • Fast - Evaporation
         • In two steps (1) Matrix droplet on sample stage and dried (2) Analyte droplet on dried matrix + drying
           • - Improved Resolution - Long lasting Matrices
       • Electrospray
         • Spraying from a high volume capillary (1-5 KV) onto a metal substrate 2 cm away from the capillary
           • - Homogenous or equal distribution of analyte - No sample segregation
       • Particle doped liquid
         • Suspension of particle in solvent. Forms a paste sent into MS.
           • Contamination of sample - Liquid sample promotes Ionization
       • Solid Support
         • Using porous solid support like SiO2
           • - Severe Contamination possible - poor reproducibility
       • Insoluble Samples
         • Mixing with an insoluble compound
           • Thick and long lasting
       • Matrix Precoated Layer
         • Droplet onto a precoated layer
           • Too time consuming
     • LASER
       • UV
         • N2 lasers or Nd: YAG laser 332-337nm
           • - very high energy - always excites samples - ruins bio samples
       • IR
         • TEA CO2 or Er:YAG Laser
           • - Does not destroy the sample - does not always excite (low energy provided)
       • Vis

3. Mass Spectrometer

   • Time of Flight (TOF)
     • Different ionic species have different flight time based on the mass to charge ratio
     • SETUP: (1) Field free drift tube (2) Strong electric field (3) vaccum pump (4) Reflectors (5) detector
     • Type 1 - Linear TOF ( for 350 kDa) Type 2 - Reflector TOF (fpr upto 5000 Da) ( reflector mirrors influence the segregation- ions with higher kinetic energy stay at he mirror longer)
   • Quadrapole
     • Four metallic rods that have voltage passing through - Molecules have different inertia due to weight
     • Only ions with a certain voltage range - matching with the voltage applied will pass to reach the detector. Allows selection of ion based on m/z ratio
   • Triple Quadrapole
     • A quadrapole collision chamber placed in between to fragment the sample and further separation of daughter ions. Sprayed with Ag.
   • Quadrapole Quadrapole TOF
   • MS/MS mode
     • Mode where two mass spec is working in tandem. Also called Multiple rxn engineering. First MS for study of quantification and second MS for study of composition. (1) Ionization (2) MS (3) Fragmentation (4) MS2

4. Detector

5. Vaccum Pump

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