Reverse-phase HPLC (revision 30)

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]

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.

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]

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.^[1]

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 report.^[3][4]

• High-performance liquid chromatography
• Area percent purity
• Retention time
• Trifluoroacetate counterion
• Preparative HPLC purification