Mass Spectrometry Flashcards
What is Mass spectrometry?
MS is a technique used to measure the relative mass of molecules
What does it require the generation of?
Analyte ions followed by ion detection and mass analysis
Why is mass spectrometry useful in pharmacy?
Helps to:
verify the identity of a drug substance
confirm the presence of a particular drug in formulated products
verify the presence of drugs and drug metabolites in clinical samples
identify unknown drug metabolites (to allow you to see how drug acts in body)
Provides means for quality control of recombinant proteins (human insulin, interferons, etc) It does this cheap and with good results.
Helps in sequencing of proteins, peptides and oligonucleotides
Can be used in drug discovery, e.g. for identification of expressed proteins – pathology in humans.
What are the 4 major stages of MS?
- ) Sample needs to be vaporised
- ) Ion generation – need to generate ion
- ) Ion separation according to mass-to-charge ratio
- ) Ion Detection
How can stage 1 (vaporise sample) be achieved?
Using heat
Placing the sample in the vacuum
Using fast atom bombardment (e.g. beam of xenon atoms)
How can stage 2 (sample ionisation) be achieved?
This can be done by bombarding the volatilised molecules with the electrons from the electron gun. This results in molecule ionisation. Charged plates accelerate the ionised molecules into the deflection chamber.
What ionisation techniques are there?
o Electron Impact Ionisation (EI)
o Chemical Ionisation (CI)
o Fast Atom Bombardment (FAB)
o Matrix Assisted Laser Desorption Ionisation (MALDI)
o Electrospray Ionisation (ESI)
o Atmospheric Pressure Chemical Ionisation (APCI)
What is electron impact ionisation?
The lost electron means the analyte becomes positively charged which means they can now be accelerated in the chamber. Achieved by under vacuum or electrical waves.
An electron beam has sufficient energy to fragment the molecule (~70 eV)
What does rapidly moving electrons knocking an electron out of a molecule lead to?
The formation of cationic radicals:
- Electron removal: [M] → [M]+• + 2e-
What happens if the molecule captures an electron?
This produces an anionic radical:
- Electron addition: [M] → [M]-•
What is Chemical ionisation?
CI uses a stream of electrons to ionise a reagent gas (ammonia or methane). The ionisation of the reagent gas results in production of strong acid (e.g. NH4+ or CH5+) Volatilised analyte molecules are ionised by strong acid (CH5+ or NH4+) via protonation.
This results in generation of an [M+H]+ ions.
What sorts of techniques are EI and CI?
NOT gentle techniques - produce lots of fragments.
Suitable for small volatile molecules with mw < 1000
Cheap and easy
Not suitable for large fragments
What is Fast Atom Bombardment?
- ) Bombarding an analyte sample suspended in a viscous matrix with a beam of fast moving Xe atoms.
- ) Energy transfer from Xe atoms to the matrix
- ) Breaking of intermolecular bonds and ionisation
- ) Desorption of analyte ions into the gas phase
What does the matrix in FAB help to do?
The matrix helps to protect the analyte from fragmentation. If the matrix was absent, the direct bombardment of the analyte by the fast atoms would lead to extensive fragmentation. FAB can also be used to generate negatively charged ions.
What sort of technique is FAB?
GENTLE technique producing very few fragments.
Does not require sample to be volatile
Suitable for molecules with MW up to ~6000
The peaks obtained in FAB are denoted as [M+H]+ (resulted from protonation) and [M-H]- (resulted from deprotonation)
What is Electrospray ionisation (ESI)?
ESI is a routine technique for the ‘soft’ ionisation
ESI is normally applied for polar analytes (e.g. biomolecules with MW up to ~100 000 Da)
Analyte is dissolved in (a mixture of) an organic solvent (AN or MeOH)
pH modifier (e.g. formic, acetic acid) is used to produce ions
Ionisation proceeds via protonation or deprotonation mechanism (depending upon the pH modifier used)
[M+H]+ or [M-H]- are detectable ions in the ESI
What is Atmospheric Pressure Chemical Ionisation (APCI)?
Similar interface to that used for ESI
However, the gas-phase ionisation in APCI is more effective than ESI for analyzing less-polar species
Sample introduction is the same as for ESI (e.g. use of organic solvents (AN or MeOH) and pH modifiers (formic or acetic acid ))
ESI and APCI are complementary methods
Mass range – typically less than 2000 Da
What is Matrix Assisted Laser Desorption Ionisation (MALDI)?
MALDI uses a concept similar to that of the FAB technique
Main differences between MALDI and FAB:
The energy is transferred to the matrix from a laser beam
The matrix must have a chromophore absorbing energy at wavelength of laser
Suitable for biomolecules with MW up to ~500000 Da
They are dissolved in matrix and then ionised. They are then draw to detector by electrical field
What is the overall degree of fragmentation for the different techniques?
EI > CI»_space; FAB, ESI, APCI, MALDI
What do the fragmentation points correspond to?
The weakest bonds in the molecule
What are Tandem MS techniques?
Based on combination of MS with other analytical methods:
Gas Chromatography-MS (GC-MS)
High-Performance Liquid Chromatography-MS (HPLC-MS or LC-MS)
MS-MS
What is GC-MS?
GC-MS is used to analyse non-polar, volatile analytes
Interfacing a GC system to an MS instrument allows to
- separate components of the analyte
- obtain mass spectrum for each point on the chromatogram
Unique fragmentation fingerprints can be used for compound identification
Using a library of standard MS spectra it is possible to identify an unknown analyte
What is HPLC-MS?
LC-MS is very much dependent on ionisation and ion vaporisation
Usually ESI or Atmospheric Pressure Chemical Ionisation (APCI) are used as an ionisation method in LC-MS
LC-MS separates out the components of a mixture and provides a MS profile on each fraction
What is MS-MS?
MS-MS is used to produce structural information about a compound
This can be achieved by fragmenting sample ions inside the mass spectrometer and identifying the resulting fragment ions
This information can then be pieced together to generate structural information regarding the intact molecule
A tandem mass spectrometer has more than one analyser
The fragmentation is often carried out within the mass analysis device (instead of MS source)
This gives superior fragmentation results and, thus, better structural information
Complicated method which eventually gives you key structural information which helps in drug development.
What are daughter ions?
the molecular ion [M]+• can generate daughter ions via the loss of either radical or neutral molecule
This produces additional peaks corresponding to the fragment ions with smaller m/z ratio
The ions which are formed will reflect the most stable cations and radical cations
What does the highest molecular weight peak represent?
the molecular ion (M•+) of the parent molecule
What fragmentation do simple alkanes tend to undergo?
fragmentation by the initial loss of a methyl group to form a (m-15) species
What can this carbocation then undergo?
stepwise cleavage down the alkyl chain, expelling neutral fragments of general formula CnH2n+1
Fragmentations that give rise to stable carbocations will be particular favoured
What will branched hydrocarbons form?
Branched hydrocarbons form more stable secondary and tertiary carbocations
These peaks will tend to dominate the mass spectrum
In a branched chain alkanes fragmentation occurs next to the branch site
What is a heteroatom?
O, N, Cl, Br, etc
For molecules containing heteroatoms (O, N, Cl, Br, etc) a very common fragmentation is the cleavage of the gammabeta bond
What are other stable carbocations?
those with an adjacent heteroatom
where will the positive charge resulted from the loss of one electron be?
normally distributed on the electronegative atom(s)
What is the predominate cleavage in aldehydes and ketones?
loss of one of the side-chains to generate the substituted oxonium ion (RC0+)
This is an extremely favorable cleavage and this ion often represents the base peak in the spectrum
What is the methyl derivative (CH3C-=O+) commonly referred to as?
the acylium ion
What is ion separation?
Once ionized, the analyte ions are separated by their interaction with an electric or magnetic field in high vacuum(10-9 to 10-12) High vacuum is applied to minimise the interaction of analyte with molecules in the air.
How are molecular ions then DETECTED?
- ) High momentum ions (having high mass) will not be deflected enough and will collide with the analyser wall – for these could change strength of the magnet to increase magnetic field, which means you would change angle of trajectory.
- ) Ions having low mass (low momentum) will be deflected most by this field and will also collide with the walls of the analyser – for this you would reduce the strength of the magnet, to reduce magnetic field so trajectory would be less so you will be able to reach detector
- ) Ions having the proper mass-to-charge ratio will follow the path of the analyser and collide with the Collector
- ) This generates an electric current, which is then amplified and detected
- ) By varying the strength of the magnetic field, the mass-to-charge ratio which is analysed can be continuously varied.
What is the next stage after having a spectrum?
To determine the chemical structure of the test sample
How can you identify the chemical structure?
By measuring mass of these fragments, we can identify their chemical structure and reconstruct the structure of the test molecule
The analysis of mass spectra involves the re-assembling of fragments, working backwards to generate the original molecule
