High-performance liquid chromatography: difference between revisions
Diff·revision 5 → 6·06:20, 4 Aug 2024
Difference between revision 5 and revision 6 of High-performance liquid chromatography. 2 lines changed; the page grew by 289 bytes.
| Revision 5 — 11:32, 23 Jul 2024 Chromatokid (talk) add the peptide-content point: area percent alone cannot give a mass 2,620 bytes ±0 | Revision 6 — 06:20, 4 Aug 2024 GradientGus (talk) state that ultraviolet response is not uniform across impurities 2,909 bytes +289 | ||
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| 10 | For peptides the dominant mode is [[Reverse-phase HPLC|reverse phase]], in which the stationary phase is hydrophobic and the mobile phase is a water–acetonitrile mixture whose organic content is increased during the run. Detection is usually by ultraviolet absorbance at 214 nm, where the amide bond absorbs, so response is broadly proportional to the number of peptide bonds rather than to any particular side chain.{{r|usp1503}} | 10 | For peptides the dominant mode is [[Reverse-phase HPLC|reverse phase]], in which the stationary phase is hydrophobic and the mobile phase is a water–acetonitrile mixture whose organic content is increased during the run. Detection is usually by ultraviolet absorbance at 214 nm, where the amide bond absorbs, so response is broadly proportional to the number of peptide bonds rather than to any particular side chain.{{r|usp1503}} |
| 11 | 11 | ||
| + | 12 | Almost every number on a peptide [[Certificate of analysis|certificate of analysis]] originates here. Understanding what the technique measures — and what it cannot measure — is therefore the single most useful piece of analytical background for reading such a document.{{r|usp1503}} | |
| + | 13 | ||
| 12 | == How the separation works == | 14 | == How the separation works == |
| 13 | A sample is injected into a stream of mobile phase and carried onto the column. Each component partitions continuously between the mobile phase, in which it moves, and the stationary phase, in which it does not. The fraction of time spent in each determines how long it takes to traverse the column — its [[Retention time|retention time]].{{r|usp621}} | 15 | A sample is injected into a stream of mobile phase and carried onto the column. Each component partitions continuously between the mobile phase, in which it moves, and the stationary phase, in which it does not. The fraction of time spent in each determines how long it takes to traverse the column — its [[Retention time|retention time]].{{r|usp621}} |