Identity vs purity: the two questions a COA must answer

Identity asks: is the molecule in this vial the one named on the label?

Purity asks: of everything detectable in this vial, what proportion is that molecule?

They are answered by different techniques. Identity comes from mass spectrometry, which measures mass-to-charge ratio and compares the observed molecular weight against the value calculated from the stated sequence. Purity comes from HPLC, which separates components and reports the main peak as a percentage of total detected area.

Neither substitutes for the other. An instrument that tells you what something is does not tell you how much of it there is, and vice versa.

Because they are independent measurements, all four outcomes occur:

• Correct identity, high purity. The intended result: the right molecule, and little else detectable.
• Correct identity, low purity. The right peptide is present but accompanied by significant related species — deletion sequences, truncations, oxidised forms. Common in poorly purified synthesis.
• Wrong identity, high purity. The most dangerous combination, and the reason purity alone is insufficient. A single, clean, well-resolved peak of the wrong compound reports as high purity. The chromatogram looks excellent. Only MS catches it.
• Wrong identity, low purity. A failed synthesis. Usually obvious.

The third row is why a COA quoting only a purity percentage — however impressive — leaves the central question unanswered.

MS measures molecular weight, and molecular weight is a strong but not conclusive identity check. It confirms that the observed mass matches the mass calculated from the sequence, within instrument tolerance.

What it does not immediately distinguish are isomers and sequence permutations. Two peptides containing the same amino acids in a different order have identical molecular formulas and therefore identical masses. Leucine and isoleucine are structural isomers and are indistinguishable by mass alone.

Resolving sequence order requires tandem mass spectrometry (MS/MS), which fragments the molecule and reads the resulting ion series, or classical Edman degradation. Neither appears on a typical research-market COA. What mass spectrometry tells you goes further into this.

Used together the two measurements are considerably stronger than the sum of their parts:

• MS establishes that the target molecule is present.
• HPLC establishes that it is the dominant detectable species.
• Where the MS is taken on the collected main HPLC peak — LC-MS, rather than two separate experiments — the two are explicitly linked: the peak being integrated is confirmed to be the target.

That last point is worth asking about. A COA showing an HPLC trace and a separate MS spectrum leaves an inferential gap: nothing formally proves the mass measured came from the peak that was integrated. LC-MS closes it.

A point worth restating because it survives both measurements: identity and purity together still do not establish net peptide content.

Confirmed identity, 99% purity, and a vial that is 25% water and trifluoroacetate by mass are entirely compatible. The water and counterion are invisible at 214 nm and irrelevant to the mass spectrum of the peptide. Only amino acid analysis or nitrogen determination closes that gap, and it is reported far less often than it should be.

See molar mass and solution concentration for how this propagates into every calculation made from a vial label.