Reverse-phase HPLC: difference between revisions
Diff·revision 2 → 3·01:17, 12 Jul 2024
Difference between revision 2 and revision 3 of Reverse-phase HPLC. 13 lines changed; the page grew by 958 bytes.
| Revision 2 — 06:25, 10 Jul 2024 DiluentDiya (talk) add the note on co-elution and what it hides 1,347 bytes ±0 | Revision 3 — 01:17, 12 Jul 2024 ResolutionRoo (talk) add the ionisation mode to the mass-spectrometry description 2,305 bytes +958 | ||
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| 11 | Separation follows hydrophobicity. A peptide adsorbs to the alkyl stationary phase from a largely aqueous mobile phase and desorbs as the organic proportion rises; the organic content at which it desorbs is characteristic and reproducible under fixed conditions.{{r|mant1996}} | 11 | Separation follows hydrophobicity. A peptide adsorbs to the alkyl stationary phase from a largely aqueous mobile phase and desorbs as the organic proportion rises; the organic content at which it desorbs is characteristic and reproducible under fixed conditions.{{r|mant1996}} |
| 12 | 12 | ||
| + | 13 | == Conditions and what they control == | |
| + | 14 | | Parameter | Typical value | What it changes | | |
| + | 15 | |---|---|---| | |
| + | 16 | | Column chemistry | C18, sometimes C8 or C4 | Retention strength; C4 for large or very hydrophobic peptides | | |
| + | 17 | | Particle size | 1.7–5 μm | Efficiency and back-pressure | | |
| + | 18 | | Modifier | 0.1% trifluoroacetic acid | Peak shape; ion pairing | | |
| + | 19 | | Gradient | 5–60% acetonitrile | Elution window and resolution | | |
| + | 20 | | Gradient duration | 15–60 min | Resolution of close-eluting species | | |
| + | 21 | | Temperature | 25–60 °C | Selectivity, peak shape, viscosity | | |
| + | 22 | | Detection | 214 nm | Sensitivity to the peptide bond | | |
| + | 23 | ||
| + | 24 | Gradient duration is the parameter with the largest effect on a reported purity figure. A shallow gradient run over an hour resolves species a fifteen-minute gradient merges, and the merged determination returns a higher number on identical material. Neither is wrong; they are different measurements. See [[Purity claim inflation]].{{r|mant1996}} | |
| + | 25 | ||
| 13 | == References == | 26 | == References == |
| 14 | {{reflist}} | 27 | {{reflist}} |