Peptide purity: reading an HPLC figure and a certificate of analysis

Every vial carries a number: 98%, 99%, "not less than 98% by HPLC". It looks like a grade and is often read as one, though it describes something narrower than most people assume. It is worth knowing exactly what.

What HPLC measures

High-performance liquid chromatography separates a mixture into its components and measures absorbance as they leave the column. The purity percentage is the area of the main peak as a share of the total area of all peaks — usually at 214 nm, where the peptide bond absorbs.

The first limitation follows from that: the method sees only what absorbs at that wavelength and what came off the column at all. Salts, bound water and leftover synthesis solvents produce no peak and are absent from the figure. Two preparations both labelled "98% HPLC" can contain different amounts of actual peptide.

Purity versus net peptide content

Net peptide content is a separate parameter: it states how much of the mass in the vial is peptide, and how much is counter-ion salt, water and residue. For material made by Fmoc synthesis, 70–90% is typical, the remainder usually acetate or trifluoroacetate and hygroscopic water.

Concentrations are calculated from that parameter, not from the chromatographic purity. A vial labelled 5 mg at 80% net content holds 4 mg of peptide — a difference that stops being a detail the moment the work is quantitative.

What else the certificate says

Besides the one number, a certificate of analysis usually carries the molecular mass confirmed by mass spectrometry, the molecular formula, the sequence, appearance, batch number and date of analysis. The mass spectrometry matters more than it looks: it confirms identity, while HPLC speaks only to homogeneity. Material can be homogeneous and not be what the label claims — two questions, two different methods.

The batch number ties the certificate to the vial in front of you. A certificate with no batch number, or with an analysis date far from the production date, describes some other material.

When the difference matters

For a qualitative observation, the gap between 95% and 99% rarely changes the conclusion. For quantitative work, where concentration is the point, it starts to — and less through purity itself than through net content and whether it was converted correctly.

The practical conclusion is dull and effective: read the whole certificate, keep it with the batch, and calculate concentrations from net content rather than from the nominal mass.

Disclaimer

The materials described in this shop are supplied for research use only and are not for human or veterinary use. The text above concerns the reading of analytical documentation.