Mass spectrometry theory and applications Flashcards
What are types of clinical mass spectrometry?
- GC-MS
- ToF (time of flight)
- Orbitrap
- LC-MS/MS
What is an overview of LC-MS/MS?
- Inlet
- Ionise
- Mass Analyse
- Fragment
- Mass Analyse
- Detection
How does the source work in LC-MS?
- Eluate from LC enters the “source”
- This is at atmospheric pressure Involves a method of removing solvent and creating ions
- Uses heating element and heated nitrogen gas for desolvation
- Charge applied to stainless steel capillary to aid ion formation (ESI)
- Can create both negative and positive ions
How does APCI work?
- A sample solution flows through a heated tube where it is volatilized and sprayed into a corona discharge with the aid of nitrogen nebulization
- Ions are produced in the discharge and extracted into the mass spectrometer
- APCI is best suited to relatively polar, semi-volatile samples. An APCI mass spectrum usually contains the quasi-molecular ion, [M+H]+
What are the steps in the ionisation process of APCI?
- EI-ionisation of gas molecules: N2+, O2+
- Gas molecules ionize solvent molecules: H3O+, CH3OH2+
- CH3OH2+ and/or H3O+ transfer proton to analyte molecule: [M+H]+
What ionisation modes?
- ESI
- APCI
What are advantages and disadvantages of ESI?
Advantages
- Most versatile mode for LC/MS
- Ionises small polar and mid-polarity molecules to large proteins
- “Soft” ionisation, very little fragmentation
Disadvantages
- Can be dependent on mobile phase used
- Matrix effects
- Analytes must be in solution
What are advantages and disadvantages of APCI?
Advantages
- Better for less polar compounds
- Compatible with higher flow rates
- Good sensitivity
- Fewer matrix effects than ESI
Disadvanatges
- Thermal degradation can occur
- Not good for high or low masses
- Manufacturer dependent
What is ion focussing?
After ions have entered the mass spectrometer through the sample cone(s), they enter a region of ion focussing
What is mass separation?
- Quadrupole mass filters
- Four rods arranged precisely with DC and RF alternating voltages applied to pairs
- Ions enter quadrupole region
- Because of RF voltage and DC offset the polarity of each pair of rods continually changes
- Ions oscillate within quadrupoles
- Amplitude of oscillation unique for a particular m/z
- Length of quadrupole determines the mass scale of instrument
What are collision cells?
- Can be quadrupoles, hexapoles or multi-plate wave guide
- Filled with inert gas such as argon (or nitrogen)
- Collision energy is applied to inert gas to give it kinetic energy
- Collision with molecules of interest causes fragmentation
- Collision energy and collision gas flow can be optimised
- Can be straight (e.g. Waters) or curved (e.g. Thermo, ABI)
What are features of second quadrupoles?
Have the same functionality as the first quadrupoles
What are detectors?
Electron multiplier Photomultiplier
How do mass spectrometers operate?
Mass spectrometers operate on the basis of the mass-to-charge ratio (m/z).
- Singly charge m/z = (M + H+)/1z m/z = (M – H-)/1z
- Doubly charged m/z = (M + 2H+)/2z
Multiple charging extends the mass range of the mass spectrometer
What is Fragmentation?
- Same mechanism as selected reaction monitoring (SRM)
- MS/MS will do one SRM then switch to do another
- SRM for a different m/z
- The time spent counting for any particular m/z is the dwell time
- The MS/MS can switch between m/z in milliseconds
- Allows simultaneous determination of multiple analytes and their internal standards
- m/z 300>100 aka “transition”
