Electrospray ionisation (revision 17)

Electrospray ionisation transfers molecules from solution into the gas phase as ions. A solution is sprayed from a capillary held at high potential, producing charged droplets whose solvent evaporates until ions are released.^[1]

It is a soft technique — the molecule survives largely intact — and it produces multiply charged ions from peptides and proteins, so a large molecule appears at a mass-to-charge ratio within the range of ordinary analysers. A single peptide therefore produces a series of peaks at successive charge states, deconvolved by software into one neutral mass.^[2]

Because it works from flowing solution, it couples directly to chromatography, which is what makes LC-MS possible.^[1]

The applied potential draws the liquid into a cone from whose tip a fine spray of charged droplets emerges. Solvent evaporates, the droplets shrink until charge repulsion exceeds surface tension, and they divide. Ions are ultimately released either by field-driven ejection from very small droplets or by complete evaporation of the solvent around them.^[1]

The charge states observed depend on the number of ionisable sites and on solution conditions. Acidic mobile phases favour protonation and positive-mode operation, which is why peptide LC-MS is usually run in positive mode with an acidic modifier.^[2]

Ion suppression is the characteristic difficulty: co-eluting species compete for charge, so a compound's signal depends on what else is eluting at the same time. This is why the technique is not quantitative without calibration.^[3]

Mobile phases must be volatile. Trifluoroacetic acid, the standard modifier for peptide reverse-phase chromatography, suppresses electrospray signal appreciably, so LC-MS methods often substitute formic acid at some cost in peak shape.^[2]

Salts and non-volatile buffers foul the source and suppress signal, so a sample containing substantial inorganic salt requires desalting before analysis.

The multiply charged envelope carries information beyond mass: its distribution shifts with conformation, so a partially unfolded protein produces a different envelope from a folded one. For small peptides this is of little consequence.^[1]

A deconvolved mass on a certificate is the software's estimate of the neutral monoisotopic or average mass from the observed charge envelope. Whether the instrument could resolve small differences depends on the analyser — see Mass spectrometry — and a report that does not name the instrument does not establish what could have been seen.^[2]

Poorly ionising species may be absent from a spectrum while present in the material, so absence from the spectrum is not absence from the sample.^[3]

• Mass spectrometry
• Liquid chromatography-mass spectrometry
• Reverse-phase HPLC
• Trifluoroacetate counterion