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Define mass spectrometry

MS is a technique used to measure the relative mass of molecules

Requires generation of analyte ions followed by ion detection and mass analysis

Can be applied to both small organic molecules and biomolecules


Applications of mass spectrometry

Mass spectrometry 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

-Provides means for quality control of recombinant proteins (human insulin, interferons, etc)

-Helps in sequencing of proteins, peptides and oligonucleotides

-Can be used in drug discovery, e.g. for identification of expressed proteins


Main principles of mass spectrometry

The molecules of the sample are ionised and then identified according to their mass

Mass spectrum is a plot of intensity (the abundance of each ion) against mass-to-charge ratio (m/z)

Thus, the mass spectrometer is a device for producing and weighing ions


Major stages in mass spectrometry

1) Sample vaporisation
2) Ion generation
3) Ion separation according to mass/charge ratios
4) Ion detection


Where is the sample vaporised?

vacuum chamber


Where is the sample ionised?

electron beam


How are the ions separated? i.e. what by

By a magnetic field that bends the path of the charged particles


How is sample vaporisation achieved?

This can be achieved by
using heat
placing the sample in the vacuum
using fast atom bombardment (e.g. irradiation of sample by beam of Xenon atoms)

Results in even distribution of the individual molecules within the vacuum chamber


How is the sample ionised?

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


Name 6 ionisation techniques

-Electron impact ionisation (EI)
-Matrix Assisted Laser Desorption Ionisation (MALDI)
-Atmospheric pressure chemical ionisation (APCI)
-Fast atom bombardment (FAB)
-Electrospray ionisation (ESI)
-Chemical ionisation (CI)


How does EI (electron impact ionisation) occur?

Sample is vaporised by heat

Ionisation is achieved by bombarding the volatilised molecules with an electron beam

An electron beam has sufficient energy to fragment the molecule (~70 eV)

This results in molecule ionisation (~99% positively charged (cationic) radicals and ~ 1% negatively charged radicals)

The positive fragments produced (cations and radical cations) are accelerated under vacuum through a magnetic field into the deflection chamber

They are analysed on the basis of mass-to-charge ratio


EI mechanism

Rapidly moving electrons knock an electron out of the molecule (in ~99% cases)

This results in formation of cationic radicals:
Electron removal: [M] → [M]+• + 2e-

If the molecule captures an electron (~1%), this produces an anionic radical :

Electron addition: [M] → [M]-•


How does CI (chemical ionisation) occur?

CI uses a stream of electrons to ionise a reagent gas (ammonia or methane)

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


Summarise EI and CI as techniques

EI and CI are NOT gentle techniques – produce lots of fragments

Suitable for small volatile molecules with MW < 1000 Da

Cheap and relatively easy


Describe how fast atom bombardment occurs (FAB)

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

note: FAB can also be used to produce anions


Purpose of the matrix in FAB?
What if it wasn't there?

The matrix helps to protect the analyte from fragmentation

If the matrix were absent, the direct bombardment of the analyte by the fast atoms would lead to extensive fragmentation


Summarise FAB technique

Gentle technique producing very few fragmentations

Does not require sample to be volatile

Allows the analysis of biomolecules

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)


Describe how Electrospray ionisation (ESI) occurs

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


Describe how atmospheric pressure chemical ionisation (APCI) occurs

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


Benefits of atmospheric pressure chemical ionisation (APCI) occurs?
Mass range?

good for less-polar compounds
excellent LC/MS interface
compatible with MS/MS methods

Mass range:
Typically less than 2000 Da


Describe the matrix assisted laser desorption ionisation technique?
molecule size?

MALDI uses a concept similar to that of the FAB technique
Suitable for biomolecules with MW up to ~500000 Da


Main differences between MALDI and FAB?

Main differences between MALDI and FAB:
In MALDI, the energy is transferred to the matrix from a laser beam

In MALDI, the matrix must have a chromophore absorbing energy at wavelength of laser


Degree of fragmentation for different techniques


Molecular ion [M]+• produced by ionisation is often in an excited state

This state corresponds to excess of vibrational energy leading to molecular fragmentation

The fragmentation points correspond to the weakest bonds in the molecule


What are Tandem (or hyphenated) MS Techniques?

Tandem MS techniques are based on an appropriate combination of MS with other analytical methods

Can be used to analyse a complicated mixture of different compounds and provide structural information on each component


Name the most important tandem MS techniques

The most important tandem MS techniques include:
-Gas Chromatography-MS (GC-MS)
-High-Performance Liquid Chromatography-MS (HPLC-MS or LC-MS)


Describe how GC-MS is used
(Gas chromatography mass spectrometry)

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


Describe the LC-MS technique
(Liquid chromatography mass spectrometry)

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


Describe the MS-MS technique

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


How does it occur?

Molecular ion [M]+• produced by ionisation is often in an excited state

This state corresponds to excess of vibrational energy leading to molecular fragmentation

The fragmentation points are associated with the weakest bonds in the molecule

Thus, 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 fragment ions seen depend upon the exact conditions used in the mass spectrometer

Possible fragment ions: B•+ (cation radical), A+ (cation), [A+H]+ (protonated ion) B•+ (cation radical), X•- (anion radical), Y- (anion), [Y-H]- (deprotonated ion)

The process of fragmentation follows simple and predictable chemical pathways

The ions which are formed will reflect the most stable cations and radical cations

The highest molecular weight peak represents the molecular ion (M•+) of the parent molecule


Rules of fragmentation

Simple alkanes tend to undergo fragmentation by the initial loss of a methyl group to form a (m-15) species

This carbocation can 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

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
Other stable carbocations are those with an adjacent heteroatom
The positive charge resulted from the loss of one electron will be normally distributed on the electronegative atom(s)
For molecules containing heteroatoms (O, N, Cl, Br, etc) a very common fragmentation is the cleavage of the a,b-bond


fragmentation of aldehydes and ketones

The predominate cleavage in aldehydes and ketones is loss of one of the side-chains to generate the substituted oxonium ion

This is an extremely favorable cleavage and this ion often represents the base peak in the spectrum

The methyl derivative (CH3C≡O+) is commonly referred to as the "acylium ion"


How does ion separation occur?

Once ionized, the analyte ions are separated by their interaction with an electric or magnetic field in high vacuum (10-9 – 10-12 bar)

High vacuum is applied to minimize the interaction of analyte ions with molecules in the air


How are the ions in a MS detected?

The positive ions and the molecular fragments produced in the ionization chamber are accelerated into an analyzing tube

The path of the charged molecules is bent by an applied magnetic field


Detection of ions
What if the ions have too much momentum or mass?
What if they don't have enough?

High momentum ions (having high mass) will not be deflected enough and will collide with the analyzer wall

Ions having low mass (low momentum) will be deflected most by this field and will also collide with the walls of the analyzer

Ions having the proper mass-to-charge ratio will follow the path of the analyzer 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 analyzed can be continuously varied.


Reassembling the fragments

Once we have a spectrum, the next stage is to determine the chemical structure of the test sample

Fragmentation of the molecular ion allows to help with this

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


When interpreting mass spectra, you need to answer at least two questions. What are these questions?

Whether the molecular ion has the same mass as we would expect?

Whether particular patterns correspond to certain structural elements of the molecule?