Reverse-phase HPLC (revision 4)
Old revision·11:30, 20 Jul 2024·ForestPlotFinn
| Reverse-phase HPLC | |
|---|---|
| Abbreviation | RP-HPLC |
| Stationary phase | Alkyl chains bonded to silica, usually C18 |
| Mobile phase | Water and acetonitrile with an acidic modifier |
| Detection | UV at 214 nm; sometimes 280 nm |
| Analytical method infobox · conventions | |
Reverse-phase HPLC is the mode of liquid chromatography in which the stationary phase is non-polar and the mobile phase is polar — the reverse of the normal-phase arrangement from which the name derives. It is the dominant analytical technique for peptides and the source of almost every purity figure on a peptide certificate.[1]
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.[2]
Conditions and what they control
[edit]| Parameter | Typical value | What it changes |
|---|---|---|
| Column chemistry | C18, sometimes C8 or C4 | Retention strength; C4 for large or very hydrophobic peptides |
| Particle size | 1.7–5 μm | Efficiency and back-pressure |
| Modifier | 0.1% trifluoroacetic acid | Peak shape; ion pairing |
| Gradient | 5–60% acetonitrile | Elution window and resolution |
| Gradient duration | 15–60 min | Resolution of close-eluting species |
| Temperature | 25–60 °C | Selectivity, peak shape, viscosity |
| Detection | 214 nm | Sensitivity to the peptide bond |
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.[2]