Reversed-phase HPLC for peptides

Separating a peptide from its related substances and quantifying the result as area percent.

Purpose

Separating a peptide from its related substances and quantifying the result as area percent.

This page exists so that a discussion about a number can start from a shared understanding of what produced it. It is written for a reader who is technically literate but not a practising analyst.

How it works

Reversed-phase liquid chromatography separates on hydrophobicity. A peptide is retained on a non-polar stationary phase and eluted by increasing the proportion of organic solvent in the mobile phase. For peptides the mobile phase almost always contains an ion-pairing acid, typically 0.1% trifluoroacetic acid or formic acid, which suppresses secondary interactions and sharpens peaks.

The parameters that determine the result, in rough order of influence: gradient slope, stationary phase chemistry and particle size, column temperature, detection wavelength, and injection mass. A shallower gradient over a longer run resolves more; a steep gradient produces a tidier-looking chromatogram with fewer visible impurities. Both are legitimate methods and they will not produce the same number.

What determines the result

Detection is usually by ultraviolet absorbance at 214 nm, which detects the peptide bond and is therefore relatively insensitive to composition, or at 280 nm, which detects aromatic residues and is strongly composition-dependent. Area percent at one wavelength is not area percent at the other.

Area percent is not mass percent. It is a proportion of absorbance, weighted by each species' molar extinction coefficient at the detection wavelength. For closely related substances the approximation is usually good. For a structurally dissimilar impurity it can be poor.

Typical criteria

These are typical values, not universal standards. The criteria that apply to a specific result are the ones in the validated method that produced it, which is why a report without a method identifier is harder to interpret than one with.

What it cannot tell you

Every analytical method answers one question. The most common error in this community's discussions is treating an answer to one question as an answer to another: a mass match read as a purity result, a purity figure read as a content determination, a sterility test read as a guarantee of safety.

When you read a report, write down which question was asked. The rest follows.

Related pages

Other method notes in this commons cover the neighbouring techniques, and the certificate interpretation page covers how these results get reported. The hplc subcategory is where the discussion happens.