Home / Research Hub / Glossary / HPLC

HPLC

High-performance liquid chromatography — the separation technique behind the purity percentage on a certificate of analysis.

High-performance liquid chromatography pushes a dissolved sample through a densely packed column under high pressure. Components travel at different speeds according to how strongly each interacts with the packing material, so they reach the detector separated in time and each appears as its own peak.

For peptides the usual configuration is reversed-phase: a non-polar stationary phase with a polar mobile phase, where retention tracks hydrophobicity. Purity is then reported as the area of the main peak as a percentage of total peak area.

That figure is method-dependent, which is why a purity number quoted without its method is an incomplete claim. The separation resolves what that particular column, gradient and detector can resolve; a co-eluting impurity or a species the detector does not respond to will not appear. HPLC answers how much of this sample is the main component. It does not, on its own, establish what that component is — that is what mass spectrometry is for.

Topic: Lab Practice

Used in

Guides that cover this

Optimising a Peptide Method by Design, Not One Factor at a Time

Why an axis walk misses the ion-pairing/pH interaction and stalls short of a diagonal optimum, how the screening and response-surface stages divide the work, what replication buys that a lack-of-fit test cannot do without, and the difference between a modelled design space and a range you have actually run.

Counterion quantification by ion chromatography

The counterion can be the largest non-peptide term in a lyophilised solid, and it is not inferable from the sequence. How suppressed-conductivity ion chromatography measures trifluoroacetate, acetate, chloride and the common cations; how to dilute into the linear range; what system suitability to fix for a small anion; and how to read a molar ratio that lands far from expectation.

Charged Aerosol and ELSD Detection for Peptides Without a Chromophore

Nebulisation-based detectors answer a physical question rather than an optical one: how much non-volatile mass reached the detector. What that buys when a sequence has no tryptophan or tyrosine, why the response is a power law, how far mobile-phase composition moves it and what inverse-gradient compensation corrects, and the precise claim a relative mass percent from CAD or ELSD will and will not support.

Kaiser and chloranil: reading a free amine on resin

The ninhydrin test answers a narrower question than the one you are asking: it reports that a reacting primary amine was present, not how much, and it gives no diagnostic blue when the terminus is a secondary amine. Where the detection floor sits, what produces a wrong answer, and which quantitative monitors replace it.

← All terms