HPLC

High-Performance Liquid Chromatography, almost always abbreviated HPLC, is the workhorse analytical technique used to separate, identify, and quantify the components of a liquid mixture. In a peptide research context, HPLC is the primary method by which a laboratory answers the question "what is actually in this vial, and how much of each thing is there?" A pump pushes a pressurized solvent (the mobile phase) through a packed column (the stationary phase); different molecules interact with the column packing to different degrees and therefore exit the column at different times. A detector records each molecule as it elutes, producing a chromatogram whose peaks represent the compounds present and whose peak areas are proportional to how much of each compound is present.

How HPLC is used on synthetic peptides

Peptides are typically assessed by reversed-phase HPLC (RP-HPLC), in which a nonpolar stationary phase (most commonly a C18-bonded silica) is paired with a polar aqueous mobile phase containing an ion-pairing additive such as trifluoroacetic acid. The main peak in the chromatogram corresponds to the intended peptide sequence; smaller peaks correspond to truncated sequences, deletion sequences, diastereomers, oxidation products, and residual synthesis impurities. The ratio of the main peak area to the total area under the chromatogram is reported as "HPLC purity" and is the single number most commonly cited on a peptide Certificate of Analysis. The United States Pharmacopeia uses RP-HPLC as a compendial method for peptide-related substances testing (see USP General Chapter <621> Chromatography and the USP monographs for peptide drug substances). FDA chemistry, manufacturing, and controls guidance for synthetic peptide substances likewise centers on chromatographic purity as the core release test (FDA ANDAs for Certain Highly Purified Synthetic Peptide Drug Substances, 2021).

What a purity number does and does not tell you

A reported purity value is only as meaningful as the method that produced it. A 99% figure collected with a short gradient on a low-resolution column can easily hide co-eluting impurities that a longer, orthogonal method would resolve. For that reason, rigorous peptide characterization pairs HPLC with a complementary separation mode, typically a second HPLC method run at a different pH, or hyphenated detection by mass spectrometry (LC-MS). Peer-reviewed methodology on orthogonal RP-HPLC for peptide-related impurities is summarized in D'Hondt et al., J. Pharm. Biomed. Anal., 2014 (NLM 24661963). HPLC also does not, on its own, establish identity: two peptides of similar hydrophobicity can elute at the same retention time. Identity confirmation requires an orthogonal technique such as mass spectrometry or amino-acid analysis.

How Open Assay uses HPLC data

Every vendor COA surfaced in the Open Assay database is parsed for HPLC purity, method conditions where disclosed (column chemistry, gradient, detection wavelength), and whether an orthogonal method was run. Listings that report a single purity number with no method detail are tagged as lower-evidence than listings whose COAs disclose the gradient and include a second orthogonal method or LC-MS confirmation. The goal is not to rank vendors on a single number but to make the underlying evidence legible to a researcher comparing reference materials for in vitro work.