Source of Reverse-phase HPLC

{{Infobox method | name = Reverse-phase HPLC | subtitle = Chromatographic mode | image = chromatogram.svg | 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 }} '''Reverse-phase HPLC''' is the mode of [[High-performance liquid chromatography|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 of analysis|certificate]].{{r|usp621}} 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}} 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 counterion|trifluoroacetate salts]].{{r|usp1503}} == Conditions and what they control == | 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]].{{r|mant1996}} 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 == 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.{{r|mant1996}} 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|mass spectrometry]], and the same chemistry scales to [[Preparative HPLC purification|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|semaglutide]] fall into this class. Very hydrophilic peptides may not retain at all and elute in the void, where nothing is separated from anything.{{r|usp1503}} == Reading a reported determination == The fields that make a reverse-phase determination interpretable are the column (chemistry, dimensions, particle size), the mobile phases and modifier, the gradient profile and duration, the flow rate, the column temperature, and the detection wavelength. A certificate stating "purity by RP-HPLC: 99.1%" without them reports a number that cannot be reproduced or compared.{{r|usp621}} Two further fields raise a determination substantially. Naming the retention time of the principal peak allows a subsequent determination to be checked for gross discrepancy. Itemising related substances individually, rather than aggregating them, shows how many species were resolved and at what level — which, as discussed at [[Limit of detection]], says as much about the method as about the material. None of this is a judgement about a supplier. It is a description of which fields make a chromatographic figure checkable, and the criterion applies identically to a supplier certificate and to a [[Third-party testing|third-party]] report.{{r|usp1503,ich_q2}} == References == {{reflist}} <ref name="usp621">United States Pharmacopeia, General Chapter <621>, ''Chromatography''.</ref> <ref name="mant1996">Mant CT, Hodges RS. "Analysis of peptides by high-performance liquid chromatography." ''Methods in Enzymology'' 271:3–50 (1996). PMID 8782429.</ref> <ref name="usp1503">United States Pharmacopeia, General Chapter <1503>, ''Quality Attributes of Synthetic Peptide Drug Substances''.</ref> <ref name="ich_q2">International Council for Harmonisation, ''Q2(R2): Validation of Analytical Procedures'' (2023).</ref> == See also == * [[High-performance liquid chromatography]] * [[Area percent purity]] * [[Retention time]] * [[Trifluoroacetate counterion]] * [[Preparative HPLC purification]] {{DEFAULTSORT:Reverse-phase HPLC}} [[Category:Chromatography]] [[Category:Analytical methods]] [[Category:Analytical science]]