Reverse-phase HPLC (revision 15)
Old revision·09:05, 9 Jan 2025·SecondarySrc_Seb
| Reverse-phase HPLCChromatographic mode | |
|---|---|
| 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.
Why it suits peptides
[edit]Peptides are amphipathic and their hydrophobicity varies with sequence, so retention differences between closely related species are often large enough to separate them. A single-residue deletion changes hydrophobicity measurably in most cases, which is why the technique can resolve synthesis impurities that differ very little in mass.[2]
The technique is also tolerant. Samples can be injected in aqueous solution, the mobile phases are simple and volatile enough to be compatible with mass spectrometry, and the same chemistry scales to preparative loadings.
Its limitations follow from the same properties. Very hydrophobic peptides retain strongly and may need a shorter alkyl chain or a higher organic proportion; acylated analogues such as semaglutide fall into this class. Very hydrophilic peptides may not retain at all and elute in the void, where nothing is separated from anything.[3]
See also
References
- ^ United States Pharmacopeia, General Chapter <621>, Chromatography.
- ^ a b c Mant CT, Hodges RS. "Analysis of peptides by high-performance liquid chromatography." Methods in Enzymology 271:3–50 (1996). PMID 8782429.
- ^ a b United States Pharmacopeia, General Chapter <1503>, Quality Attributes of Synthetic Peptide Drug Substances.