Liquid chromatography-mass spectrometry (revision 11)
Old revision·16:13, 24 Feb 2025·CiteBot
| Liquid chromatography-mass spectrometryHyphenated technique | |
|---|---|
| Abbreviation | LC-MS |
| Combines | Chromatographic separation and mass detection |
| Interface | Usually electrospray |
| Adds | Identity to a retention-based separation |
| Analytical method infobox · conventions | |
Liquid chromatography-mass spectrometry couples a chromatographic separation to a mass spectrometer, so that each eluting peak is characterised by both its retention time and its mass. It converts a weak identification into a strong one.[1]
The interface is almost always electrospray, which works directly from flowing solution. This compatibility is what made the technique routine, and it constrains the chromatography: mobile phases must be volatile and free of non-volatile buffers.[2]
For peptide work its principal use is identity confirmation — establishing that the peak dominating a chromatogram has the expected mass — and characterisation of impurities, since each minor peak carries a mass that often identifies what it is.[3]
What it adds over either alone
[edit]Chromatography alone establishes that one species dominates; mass spectrometry alone establishes that a species of the expected mass is present. Together they establish that the dominant species has the expected mass, which is a materially stronger statement than either.[1]
Impurity identification is where the combination is most valuable. A deletion sequence appears as a peak of lower mass by one residue; an oxidised species by 16 Da more; an incompletely deprotected species by the mass of the retained group. See Resin cleavage and Solid-phase peptide synthesis.[3]
Tandem operation — selecting a precursor, fragmenting it, and measuring the fragments — adds sequence information and can locate a modification to a residue rather than merely detecting it.[1]
Quantification
[edit]LC-MS is not inherently quantitative: ionisation efficiency varies by orders of magnitude between species and with what else is eluting. Quantification requires calibration against a standard of the same substance, ideally with an isotopically labelled internal standard.[1]
This is why a purity figure is normally reported from ultraviolet detection rather than from mass detection: ultraviolet response at 214 nm is roughly proportional to the number of peptide bonds and is therefore a reasonable proxy for mass across related peptides, whereas mass-spectrometric response is not.[3]
See also
References
- ^ a b c d Aebersold R, Mann M. "Mass spectrometry-based proteomics." Nature 422(6928):198–207 (2003). PMID 12634793.
- ^ Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. "Electrospray ionization for mass spectrometry of large biomolecules." Science 246(4926):64–71 (1989). PMID 2675315.
- ^ a b c United States Pharmacopeia, General Chapter <1503>, Quality Attributes of Synthetic Peptide Drug Substances.