Reverse-phase HPLC (revision 10)
Old revision·11:01, 12 Oct 2024·StubSorterBot
| 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]
An acidic modifier — most often trifluoroacetic acid — is added to suppress ionisation of acidic side chains and to pair with basic ones, sharpening peaks considerably. Its use is why peptides purified by this route emerge as trifluoroacetate salts.[3]
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]
Temperature is the parameter most often omitted from certificates. Elevated column temperature sharpens peaks for large peptides by reducing conformational heterogeneity, and a method developed at 60 °C will not reproduce at ambient.
References
- ^ United States Pharmacopeia, General Chapter <621>, Chromatography.
- ^ a b Mant CT, Hodges RS. "Analysis of peptides by high-performance liquid chromatography." Methods in Enzymology 271:3–50 (1996). PMID 8782429.
- ^ United States Pharmacopeia, General Chapter <1503>, Quality Attributes of Synthetic Peptide Drug Substances.