Peptide purity: what "99 %" HPLC on a certificate means — and what it does not

HPLC — high-performance liquid chromatography — separates the components of a sample by pushing a solution of it through a packed column under pressure. In reverse-phase HPLC, the standard for peptides, the column packing is non-polar (typically C18 chains bonded to silica) and the mobile phase is water with an increasing proportion of acetonitrile, usually with 0.1 % trifluoroacetic acid. Components leave the column at different times depending on how strongly they cling to the packing: more hydrophobic, later.

At the outlet, a UV detector records absorbance against time. Peptides absorb at 214–220 nm because of their peptide bonds, so that is the wavelength used. The recording is the chromatogram: a baseline with peaks, each peak one component, its area proportional to how much of that component passed the detector.

The purity figure is a ratio of areas:

purity (%) = area of the main peak ÷ sum of the areas of all peaks × 100

If the main peak accounts for 99.2 % of the total integrated area, the certificate reads 99.2 %. Everything else in the trace — the 0.8 % — is impurity peaks: other molecules that absorb at the same wavelength and eluted at other times.

Three details determine whether that number is worth anything: the laboratory reported the method (column, gradient, wavelength), the chromatogram is shown with its axes, and the integration is visible — which peaks were counted. A percentage printed on its own, with no trace, is a claim, not a measurement. The general USP chapter on chromatography describes what a reportable method includes; a serious certificate follows it. How to read the rest of the document is in how to read a peptide COA.

It says: of the UV-absorbing material that came off the column, 99 % eluted as a single peak. The sample is chromatographically clean.

It does not say:

• That the vial contains 99 mg of peptide per 100 mg of powder. Lyophilized peptide is never pure peptide by mass. It carries residual water (a few percent), counter-ions left from purification — acetate or trifluoroacetate bound to the basic residues — and sometimes salts. None of these absorb at 220 nm, so none appear in the chromatogram. The mass fraction of actual peptide is a separate measurement called net peptide content, obtained by amino-acid analysis or elemental (nitrogen) analysis, and it commonly sits between 70 and 90 % for lyophilized peptides. A 99 % HPLC figure and an 80 % net peptide content describe the same vial without contradiction.
• That the main peak is the compound on the label. A wrong sequence, purified well, also gives one clean peak. Identity is a different question, answered by mass spectrometry.
• Anything about the lot you hold, unless the certificate carries that lot's number.

Mass spectrometry weighs the molecule. The sample is ionized (electrospray, ESI, or MALDI), and the instrument reports the mass-to-charge ratio of the ions; from it the laboratory calculates the observed molecular mass and compares it with the theoretical mass of the compound. For GHK-Cu the expected mass is about 404 Da; for BPC-157, about 1,419 Da. Agreement within a fraction of a dalton confirms that the main HPLC peak is the peptide named on the label.

HPLC without MS gives purity of an unidentified substance; MS without HPLC gives identity without a purity figure. A certificate of analysis carries both lines, from the same sample, on the same report.

The small peaks around the main one are not mysterious. Solid-phase peptide synthesis and the handling that follows produce a predictable set of by-products, and each has a chemical name:

• Truncated sequences. A residue failed to couple during synthesis, so a fraction of chains are missing one amino acid (deletion sequences). They elute close to the main peak and are the most common impurity.
• Incompletely deprotected chains. A protecting group from synthesis (t-butyl, trityl) stayed attached. Slightly more hydrophobic: elutes later.
• Deamidation. An asparagine or glutamine side-chain amide converted to a carboxylic acid. One dalton heavier; often visible as a shoulder on the main peak.
• Methionine oxidation. The sulfur of a methionine residue picked up an oxygen (methionine sulfoxide, +16 Da). Elutes earlier. Semax contains methionine, which is why it ships sealed and cold.
• Aspartimide formation and racemization. Rearrangements at aspartate residues during synthesis; minor, but characteristic.
• Dimers and aggregates. Two chains linked, usually through cysteine; much later elution, and a doubled mass on MS.

A laboratory that reports these peaks by name, or at least by retention time and area, is showing its integration. One that shows a single number is not.

The phrase appears on thousands of listings. Read literally it is a specification — "we intend at least 98 %" — not a measurement of any lot. Without a trace, a method and a lot number, it cannot be checked, and a figure that cannot be checked has the same information content as no figure.

The same goes for percentages given to no decimal place on every product of a supplier. Measured values come out as 98.7 % or 99.31 %; a catalog where every item reads "99 %" was typed, not tested. Supplier checks that follow from this are in where to buy research peptides in the US.

A worked example of a certificate for a single compound — sizes, lot, what the report shows — is in the BPC-157 buying guide.