Posts Tagged ‘Electrospray ionization’
- Most biologically interesting chemicals exist as isomers. Isomers have exactly the same mass and cannot normally be differentiated by a mass detector, no matter how expensive it is. Therefore it helps if you can additionally separate the isomers before hand by chromatography.
- When a mixture of chemicals enters the process of ionisation, the chemicals can interact and affect one another’s chances of getting properly ionised. This is called ion suppression. It is usually a problem where you are trying to detect one minor, or poorly ionised chemical in the presence of a large amount of something else, maybe a buffer from the sample. Some pre-purification of the ionisation mixture can get the suppressed away from the suppressors. There are ways to recognise ion suppression.
And why is the chromatography usually reverse-phase?
Why bother with mass spectroscopy?
More sophisticated mass detectors such as triple quadrupole and ion-trap instruments can be set up to carry out more detailed structure-dependant analyses on what is eluting from the HPLC system.
After HPLC separation the sample goes straight into a mass detector. Mass specs detect ions in a vacuum, so the first tasks in the LC-MS are to
- remove the solvent and ionize the metabolites.
- get the ions into a vacuum.
GC-MS came before LC-MS because it is comparatively easy to pump off a small amount of GC carrier gas, but quite difficult to pump off all the vapour that can be created from even a small amount of liquid. One ml water will produce 1.3 litres of vapour at room temperature and pressure…
Written by : Dr. Lionel Hill, John Innes Centre UK
GC-MS and LC-MS typically use totally different mechanisms for ionisation. In GC-MS the sample is usually ionised directly (Electron Impact, EI), or indirectly (Chemical Ionization, CI) by an electron beam. The high-energy electrons cause the formation of free radical ions. These are ions because they’ve lost an electron, so they have the same mass as the parent, but an odd number of electrons.
In fact the electron beam is often energetic enough to cause substantial fragmentation. The fragments are also free radicals, and form the “fingerprint” that is used in confirmation of identity. This fingerprint is compared with library fingerprints; NIST is probably the most widely used library.
An example might be Aspirin, acetyl-salicylate. By EI in GC-MS, this has a tiny peak at 180amu, corresponding to the molecular ion (free radical; the MWt of acetyl salicylate is 180Da). But the major peaks are all fragments: 120, 138, 92, 43amu, and many others.
In contrast, the spectrum below is acetyl-salicylate measured in LC-MS, using electrospray ionisation, ESI (in negative mode):
Notice that the major ion is now 179amu. This is a pseudomolecular ion, formed by loss of H+. This is quite typical in electrospray: the major ion is formed without fragmentation (unlike EI), and it is formed by loss of H+ in negative mode, or by gain of H+, Na+, or some other ion (ammonium and potassium are quite common) in positive mode. Very often in positive mode, one sees a mixture. Peaks 22amu apart nearly always mean the H+ and Na+ ions of the same thing.
Acetyl salicylate is a very easily fragmented molecule, so two fragments are still visible even under the mild conditions of ESI. These are the ions at 137 and 93amu. Unfortunately ESI collision-induced fragmentations are often not very peak-rich, so they are often not such good “fingerprints” as EI spectra. They can also vary with instrument and conditions.
Notice also the peaks at 225 and 381amu. These are adducts. Unfortunately analytes often associate weakly in the spray-chamber in ESI, and then appear as adducts of two things together. 225amu is probably a formate acid adduct of acetyl salicylate (46+179), and 381 is a sodium dimer (179+179+23; notice there is still a single negative charge). Adducts are most problematic at high concentrations.
Download (gift from Dr. Lionel Hill, John Innes Centre, UK) :