Mass Spectrometry Flashcards
What is an overview of the mass spec system?
Inlet - could be a HPLC system
Ionisation - performed at atmospheric pressure
~under vacuum~
Mass analysis
Fragmentation - in a collision cell
Mass analysis
DetectionWhat are the features of the sample inlet and source and how do they allow ESI?
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)
Nebuliser spray mobile phase comes out of the end of the probe
Can create both negative and positive ions
Spray is forces into close proximity to a sampling cone, this has a hole in and contains opposing charges so that ions are drawn towards it at a right angle. A vacuum also encourages this. All neutral species flow past.
What is Atmospheric Pressure Chemical Ionisation (APCI)?
A sample solution flows through a heated tube where it is volatilized and sprayed into a corona discharge with the aid of nitrogen nebulisation. Corona discharge is very hot, so is better at ionisation - but perhaps too intense and can breakdown species
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]+
Three step ionisation process:
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 are the advantages and disadvantages of ESI and APCI?
ESI Advantages:
- Most versatile mode for LC/MS
- Ionises small polar and mid-polarity molecules to large proteins
- “Soft” ionisation, very little fragmentation
ESI Disadvantages:
- Can be dependent on mobile phase used
- Matrix effects (manufacturer dependent)
- Analytes must be in solution
APCI Advantages:
- Better for less polar compounds
- Compatible with higher flow rates
- Good sensitivity
- Fewer matrix effects than ESI
APCI Disadvantages:
- Thermal degradation can occur
- Not good for high or low masses
- Manufacturer dependent
How is ion focussing performed?
The ion cloud under atmospheric pressure is forced under vacuum causing a dispersed cloud of ions.
Individually controlled concentric disks propel sample along. This process of ion focussing forces them into a narrow beam.
Some neutrals that have passsed through are filtered out.
Only ions can drop down to the unaligned (lower) second region.
What does the process of mass separation involve?
Filters out everything so you only see the m/z charges you want.
Uses 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 - most work from 50-200 m/z
What are collision cells?
Can be quadrupoles, hexapoles or multi-plate wave guides
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 is the purpose the second quadrupoles?
The second quadrupoles are placed slightly out of alignment with the first. However, they have the same functionality as the first quadrupoles
Ions are propelled along to the second quadrupole to separate out to the desired fragment ion.
What is the nature of the detectors?
Electron multiplier - incident ion beam is the select fragment ion. Give amplification of signal
Photomultiplier - has 3 stages that allow amplification of the signal at each step:
- Conversion dynode = ions -> electrons
- Phosphor screen = electrons -> photons
- Photomultiplier = photons -> electrons
What is ion formation important?
Mass spectrometers operate on the basis of the mass-to-charge ratio (m/z), where bigger analytes become more highly charged.
Singly charged 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
How does adduct formation occur?
M = mass of interest.
Rather than binding with proton, M collides with anything of the correct opposing charge.
NH4+ + M -> (M+NH4)+ (M + 18)
Na+ + M -> (MNa)+ (M + 23)
K+ + M -> (MK)+ (M + 39)
Cl- + M -> (MCl)- (M + 35)
Ammonium is used on purpose for immunosuppressant assays as they do not fragment. Instead force this then cleave.
What is selected-reaction monitoring in fragmentation?
A species moves into the first quadrupoles and oscillates at the correct frequency allowing it to pass through into the collision cell.
The collision cell, filled with Ar for example- the fragments the species.
These pass further into the second quadrupoles, similar to the first, only the fragments here that oscillate at the correct frequency will hit the detector.
This way only the fragments of interest are measured.
What is multi-reaction monitoring in fragmentation?
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”
What are the clinical applications of MS in biochemistry?
Immunosuppressant drug monitoring post-transplant
Steroid analysis - immunoassays cant differentitate between some steroids eg cortisol and progneselone
Metabolic diseases
What are the clinical applications of MS in toxicology?
Drugs of abuse
Legal highs
Post-mortem work
