Services and techniques

Our instrumentation and expertise allows the determination of the molecular mass of a compound to typically better than 5 ppm accuracy.

Offered both as staffed and walk-up services, the latter including the ability to analyse compounds by HPLC-MS or directly from a TLC plate. Useful for reaction monitoring and analysing reaction intermediates.

Used in combination with other analytical techniques, in particular NMR spectroscopy, analysis of fragmentation patterns in mass spectra can help to confirm chemical structure.

Complex mixtures can be analysed using either liquid chromatography (HPLC) or gas chromatography (GC) followed by mass spectrometry analysis of the components.

Ion mobility is a structure identification technique that can be used to distinguish between two ions of identical mass but different structures. Ions are distinguished by the way in which their shape affects their passage though the time-of-flight (TOF) tube and can also be differentiated according to their charge state.

Users are advised to consult a member of the Facility team to discuss their individual analyses and requirements.

Sample	ESI	APCI	ASAP	APPI	EI	CI	MALDI
Small amount / low concentration	X
Mr < 400 Da	X				X	X
Mr ~ 150 Da - > 70 kDa	X
Mr > 2 kDa	X						X
Biological molecules	X						X
Polar compounds	X
Non polar compounds			X	X
Compounds requiring high temperature to vaporise			X
Good UV excitation				X
Difficult to ionise		X
Significant fragmentation desirable					X
Significant fragmentation not desirable	X					X	X
Volatile or soluble in a volatile solvent					X	X

Applications

ESI is an extremely versatile ionisation technique.  ESI is a sensitive technique, enabling molecular-weight information to be obtained on very small amounts of sample. Samples can be ionised in either positive or negative ionisation modes, allowing for the detection of a positively charged ion or negatively charged ion.

ESI can be used to analyse low (Mr >50 Da) to high (Mr <100,000 kDa) mass, and is suitable for both polar, ionic and organometallic compounds.  It is equally useful for the analysis of biological molecules including peptides, proteins, saccharides and oligonucleotides.  The sample must dissolve in water, methanol or acetonitrile.

ESI is a soft ionisation technique and usually only molecular ions are produced, usually as their [M + H]⁺, [M + NH₄]⁺ or [M + Na]⁺ ions ion positive ionisation mode or [M-H]^‑ or [M + Cl]^‑ in negative ionisation mode, as examples.  Ions which are able to hold more than one charge will form ions of the type [M + nH]^{n +} and will appear on the spectrum at [M+nH]/n (mass spectrometry reports an m/z value where m is the ion and z is the number of charges).  If greater fragmentation is required (e.g. for structure elucidation purposes), a technique called tandem mass spectrometry (MS/MS) or another called MS^e can be applied post-ionisation.

The ESI technique

In ESI, a pure sample is injected into a stream of solvent, which is sprayed through a needle.  A voltage potential of 1 to 4 kV is applied to the needle which forms a spray containing charged droplets.  As the solvent evaporates, the charge density upon the surface of the solvent-sample droplet increases until a charged sample ion is expelled. The ions then enter the mass analyser whilst neutral molecules are lost.  With a Time-of-Flight (TOF) analyser, the time taken for the ion to travel to the detector can be recorded very precisely, and the mass-to-charge ratio (m/z) can be measured to better than 5 ppm accuracy.

Applications

APCI is an alternative to ESI and particularly useful for compounds which are less polar, or do not dissolve easily in water, methanol or acetonitrile. 

The APCI Technique:  The dissolved sample passes into the APCI source and undergoes heating.  The sample and solvent are vaporised.  The solvent molecules are subjected to a corona discharge (~4KV) and form stable reactant ions.  Proton transfer occurs between these reactant ions and the sample molecules, the sample can gain or loss protons to form [M + H]⁺ or [M – H]^- ions respectively.  The ionisation can be modified with the use of chemical additive within the solvent stream, such as ammonium ions to form species such as [M + NH₄]⁺.

Prior to the application of both of ESI or APCI, a mixture can be separated using High Pressure Liquid Chromatography (HPLC). The compound mixture is first dissolved in a suitable solvent, which passes (elutes) through a column containing a suitable separation medium (stationary phase). The components of the mixture travel at different rates through the medium allowing their separation.  Upon elution from the column, each component of the mixture can be ionised ready for mass spectrometry analysis.

Applications

This ionisation technique is an alternative to ESI and APCI for samples that do not dissolve easily in solvent suitable for ESI or APCI and the sample can be analysed as a solid.  The sample needs to be volatile under 500C.  Ions such as [M + H]⁺ (or [M + NH₄]⁺ with the addition of a source of ammonium) are formed.

Applications

APPI is another alternative to ESI and useful for ionising relatively non-polar compounds with good ultra-violet (UV) excitation.

The APPI technique

The solvent, which must have good UV absorbance properties, is ionised through absorption of UV and then produces secondary ionisation in the sample.

Applications

EI is most frequently used for low molecular weight compounds (Mr < 1000 Da using a direct insertion probe).  EI is an energetic ionisation technique and produces fragment ions, which are smaller parts of the original molecule and are useful for structure confirmation.  It is possible that a molecular ion will not be seen if a fragment ion is more stable.

The EI technique

The sample (analyte) is introduced into the ion source in the gaseous state.  A beam of electrons produced by a heated filament of Tungsten collides with the sample gas molecules, removing an electron and producing a positively charged ion corresponding to the relative molecular mass of the sample being analysed.

The ions are accelerated out of the source and pass through a series of slits to produce a focused beam. This beam of ions then enter a 'time-of-flight' (TOF) analyser which allows their mass to be determined.

Applications

CI is softer ionisation technique compared to EI.  It is therefore useful for analysing compounds for which fragment too easily under EI.  CI is suitable for volatile samples <1500 Da using a probe.  Chemical ionisation can be performed in both positive and negative modes.

The CI technique

A gas is introduced into the source of the mass spectrometer, this is usually either methane, iso-butane or ammonia.  The gas is ionised by stream of electrons from a Rhenium filament.  These ionised gas molecules are then able to undergo secondary ionisation passing their charge on to the sample.  Ions formed are of the type [M + H]⁺, [M + CH₃]⁺, [M + NH₄]⁺; the ions formed depend upon the gas used.

Prior to the application of either EI or CI, the components of a mixture can be separated by Gas Chromatography (GC).  The sample, dissolved in a suitable solvent is injected into a heated inlet.  The mixture is vaporised prior to passing through the column (stationery phase). The components must therefore volatile (under 350C), making this separation technique especially well-suited for analysing low molecular weight species (EI: Mr <650 Da; CI Mr <800 Da). Ionic and salt samples are not suitable for analysis by GCMS.

Applications

MALDI is used for ionising molecules of medium to very large size (Mr > 1,000 Da), commonly peptides, proteins, polymers and organometallics.  MALDI provides molecular species information, often forming ions of the type [M]⁺ or [M + H]⁺ with a single charge.  If, however, more extensive fragmentation is required MS/MS can be used post source ionisation.

The MALDI technique

In MALDI, a sample-matrix spot is dried upon a sample holder which is shot with a UV laser.  There are several methods to prepare samples, one common methodology is that a suitable matrix is applied to a stainless steel plate at a concentration of 10mg/mL and allowed to dry.  A sample-matrix solution is then applied on top of this matrix spot and allowed to dry to a thin film.  The optimal matrix and sample deposition method will vary from one sample to another; the matrix is chosen for its ability to desorb the laser energy and pass its charge to the sample molecules.

The sample plate is placed in the instrument and a laser beam fired at the matrix-sample spot, resulting in ionisation of the sample. The ions are extracted into the Time-of-Flight analyser and separate according to their mass.  By calibrating the instrument with a compound of known molecular mass, an accurate spectrum can be obtained.