Mass Spectrometry

• Precursor Ion:

  Precursor Ion ---> Transition (Q1) ---> MS Detector
• Selected Ion Monitoring:

  Precursor Ion ---> Filter for Precursor Ion (Q1) ---> Transition (Q2) --- MS Detector
• Multiple Reaction Monitoring:

  Precursor Ion ---> First Filter for Precursor Ion (Q1) ---> Fragmentation in Collision Cell (Using Argon or Helium)(Q2) ---> Second Filter for Product Ion (Q3) ---> MS Detector
• Parallel Reaction Monitoring:

  Precursor Ion ---> Filter for Precursor Ion ---> Fragmentation in the Collision Cell (Using Argon or Helium) (Q2) ---> Orbitrap (MS Detector)
• Characteristics:

  Ion Transmission - for sensitivity

  Scan Speed - Number of MS experiments that can be done

  Mass Accuracy - difference between the theoretical and measured mass

  Mass Resolution - separation of narrow mass spectral peaks

  Resolving Power - degree to which separation of narrow peaks can occur

  Mass Range Limit - Low Resolution can do up to 2000 m/z but High Resolution can do up to 200,000
• Resolving Power (Full Width at Half Maximum) Ranking

  At the top:

  FT-ICR-MS - 1,000,000

  FT-Orbitrap

  High-Res-TOF - 6000

  TOF

  Quadrupole and Ion Trap (ultrazoom mode)

  Quadrupole /Ion Trap - 1000
• Ion Transmission:

  How better the transfer of ions from the source to the detector through the mass analyser.

  • ion optics
  • ion guides
  • hexapoles or octupoles
  • t-wave
  • step-wave
• Quadrupole Ion Trap 3D:

  • doughnut-shaped to confine space
  • does a pre-scan to estimate number of ions
  • only space for so many
  • ions will repel
• Quadrupole Ion Trap Linear:

  • Box-shaped
  • better for quantitation than 3D
  • less chancing of gating
• Things to Consider when selecting Operation Mode:

  • sensitivity
  • Reduction of noise
  • Selectivity
  • Speed
• Selected Ion Monitoring:

  • ions of interest monitored
  • less discriminating
  • high resolution
  • better S:N (3:1)
• Multiple Reaction Monitoring:

  • ions of interest
  • longer focus on precursor and fragment ions (good sensitivity and selectivity)
• Duty Cycle - proportion of time ions are accepted from an ion source and transferred through to the detection (time from ion source to detector)

  Scan Speed - time taken for RF (AC and/or DC) potentials to be applied

  Dwell Time - time taken for two quadrupoles (Q1 and Q2 or Q1 and Q3) to scan through the transition
• Tandem in Time:

  • Quadrupole Ion Trap
  • Linear Ion Trap
  • Ion Cyclotron Resonance
  • Orbitrap

  Tandem in Space:

  • Triple Quadrupole
  • High Resolution Magnetic Sector
  • TOF/TOF
• Types of Ion Dissociation:

  Metastable Ions - spontaneously dissociate en route from the ion source to the detector

  Collisionally Activated Dissociation - ions activated by collision with neutral gases (neutral losses and product ion)

  Electron Capture Dissociation - dissociates the ions using neutral gases and then recombines them by capturing low energy electrons using multiply charged ions (used for peptides and proteins)

  Electron - Transfer Dissociation - multiply charged gaseous molecules transfer electrons. ion-ion reaction and from a CI source
• QIT

  Disdvantages:

  • product ion scan only
  • Low mass cut off
  • spectra more complex

  Advantages:

  • Simple to operate
  • MSn is possible
  • full scan sensitivity
  • higher m/z than QQQ
• QQQ

  Advantages:

  • Simple to operate
  • sensitive to SIM and SRM
  • No loss mass cut off

  
  

  Disadvantages:

  • MS/MS only
  • Low resolving power
  • Acquisition is 1 spectrum/second
  • Maximum m/z range is 4000
• Gas Phase:

  • Electron Impact Ionisation - Energetic Electrons
  • Chemical Ionisation - Reagent Gaseous Ions (NH3, CH4 or C4H10)
  • Photo-ionisation
• Liquid Ionisation:

  • Atmospheric Pressure Ionisation
  • Electrospray Ionisation
  • Atmospheric Pressure Chemical Ionisation
  • Atmospheric Pressure Photo-ionisation

  Ionisation occurs outside the vacuum region

  All soft ionisation techniques

  For polar, low volatility (high molecular mass) and thermolabile (changes based on heat) compounds

  Mechanism of ionisation has the same principles for all of them which is desolvation and ion transmission
• Soft Ionisation - leads to fewer fragments, lower energy and gives the high molecular ion

  Hard Ionisation - will have higher energy, will give more fragments, and will give a low molecular ion, produces predictable ions
• TOF

  Sample (ionised by MALDI source) ---> Grid ---> Flight Tube ---> Grid ---> Detector

  
  

  The space between the sample and the grid is the acceleration region the flight tube is the L drift path.

  
  

  At the grid, the weird ions are neutralised - similar to ion optics
• Linear TOF Analyser:

  • Upper m/z range has not limits
  • Has higher transmission efficiencies therefore -
  • Very sensitive
  • Fast (covers a wide range in microseconds)
  • gives 1000 of spectra

  However

  • if an ion packet energy is normally distributed then there will be less kinetic energy and therefore issues with resolution resulting in broader peaks
• Mass Resolution of TOF:

  TOF has poor mass resolution ---> due to distribution of flight times among the ions with the same m/z ---> space it takes up, the time it takes and the speed it then goes at leads to peak broadening.
• TOF Extraction of Ions:

  • Continuous Extraction - ions have the same m/z but different velocities (still give broad peaks). Acceleration region starts at 20kV
  • Delayed Pulsed Extraction - acceleration region starts at 20kV then half way between the sample and the grid there is another 20kV region acting in a pulse way - helps push the smaller ions further
  • Ion Extraction - acceleration region starts at 26kV then halfway before the grid there is another 20kV, time delay between the two voltages - helps peaks get wider and narrower
• Reflectron TOF:

  • cheaper but only works to a certain concentration
  • The flight tube has a bend to it, and where it bends there are ring electrodes that have increased voltages that causes the larger ions to repel
  • the increased voltage will allow the smaller ions to stay on the flight tube but it also increases the flight path length as it creates a broader bend
• Orthogonal Acceleration TOF

  • beam goes down the flight tube and hits reflectron lenses that reflect the beam back to hit the detector near where the beam enters the flight tube.
• Tandem TOF

  • can be done with quadrupole
  • used for Post Source Decay and generates metastable ions
• Electron Ionisation:

  • Hard ionisation technique
  • Vaporisation of the sample before ionisation
  • Only for volatile and thermostable compounds
  • enhance volatility and stability for polar analytes by derivatisation
  • used in GC-MS more than LC
• Chemical Ionisation:

  • Soft ionisation technique
  • CI source is a reagent gas inside a modified EI source
  • no extensive fragmentation
  • high sensitivity and selectivity
  • only for volatile and thermostable compounds
• ESI Flow Rate:

  Depending on the flow rate three types of ESI can be done

  • Conventional ESI - high flow rate, for larger molecules that have a difference of 20 mm compared to the smallest molecules
  • Micro ESI - flow rate less than 1 ul/min for molecules that have a difference of 2 mm
  • Nan ESI - flow rate less than 0.5 ul./min and a molecule size difference of 1-2 mm

  This is down to the source and the sensitivity
• ESI:

  Advantages:

  • soft ionisation
  • generates ions from liquid phase
  • efficient ion production
  • suitable for a wide range of analytes
  • forms multiply charged ions for the bigger molecules

  
  

  Disadvantages:

  • sensitive to matrix effects
  • Adduct formation which decreases signal and complicates results
• APCI:

  Advantages:

  • soft ionisation
  • single charged and less adduct ions
  • performs well at high flow rate
  • less susceptible to matrix components

  Disadvantages

  • not suitable for thermolabile compounds
  • suitable for small molecules but not peptides
  • trial and error
• Which source to choose?

  Low mass number - APPI and APCI

  High mass number - ESI

  Low Polarity - APPI and APCI

  Mid-range Polarity - APPI, APCI and ESI

  High Polarity - ESI

  High mass number - ESI
• Ambient Ionisation:

  Solid Phase

  • Matrix-assisted laser desorption ionisation (MALDI)
  • Desorption electrospray ionisation (DESI)
  • Laser diode thermal desorption ionisation (LDTD)
  • Atmospheric solids analysis probe (ASAP)
• MALDI:

  • non-volatile and high molecular mass analytes
  • dissolved in matrix absorbing light from short pulse of laser
  • ionise sample and then extract
  • mostly used with TOF
  • peptides and proteins
  • reproducibility issues
• Polar - there is room for more bonds as there are unpaired electrons

  Non-polar - no unpaired electrons which means the molecule is stable
• Quadrupole

  Advantages:

  • Energy and spatial distribution of ions not critical
  • Simple Scanning Method
  • Low Cost
  • Easy coupling to other quadrupoles or other MS analysers
  • polar switching
  • Disadvantages:
  • Low Resolution (1000 m/z)
  • Low mass accuracy (100 - 1000 ppm)
  • Limited mass range (approximately 2000 m/z)
  • Application:
  • General low resolution instrument
  • Molecular weight determination
• 3-D Ion Trap

  Advantages:

  • Energy and spatial distribution of ions not critical
  • Low Cost
  • Small Device
  • Inherent MS/MS and MSn capabilities
  • Disadvantages:
  • Low resolution (1000 - 2000 m/z). Unless special methods like ZoomScan are used
  • Low mass accuracy (100 - 1000 ppm)
  • Application
  • General low resolution MS instrument
  • Molecular weight determination
  • Molecular structure elucidation
• Linear Ion Trap

  Advantages:

  • High resolution achievable at low acquisition rate (30000 in Ultra-zoom scan mode)
  • High sensitivity
  • High scan speed (>15000 amu/s)
  • MSn capabilities up to n=15
  • Disadvantages:
  • Limited mass range (approximately m/z 2000)
  • Higher price than 3-D ion traps
  • Applications:
  • High sensitivity measurements
  • Molecular structure refined elucidation
• Time of Flight

  Advantages:

  • High scan rate (up to 20000 scans per second)
  • High resolution (up to 20000) when reflectrons and high speed electronics are used
  • Virtually no limit on mass range (actual limit 106 amu)
  • High sensitivity (femtomoles)
  • Good mass accuracy (10 - 100 ppm)
  • Disadvantages:
  • Strict demands on initial energy and spatial distribution of ions
  • High performance electronics needed
  • Applications:
  • High resolution measurement using reflectrons
  • High sensitivity measurements