What the instrument does
High-performance liquid chromatography pushes a dissolved sample through a column packed with fine particles under pressure. For peptides the column is almost always reversed-phase: the particles carry hydrophobic chains, and the mobile phase is water with a rising proportion of acetonitrile. Everything in the sample sticks to the column to a degree set by its own hydrophobicity, then lets go as the acetonitrile rises. Hydrophilic species come off first, hydrophobic ones later. A detector at the outlet, usually ultraviolet absorbance at 214 or 220 nm where the peptide backbone absorbs, records how much material is leaving the column at each moment. The plot of that signal against time is the chromatogram.
The axes
- Horizontal: retention time, in minutes. A given peptide on a given column under a given gradient comes off at a reproducible time, so retention time is an identity check in its own right. It is not transferable between laboratories or methods.
- Vertical: detector response, usually milli-absorbance units. The height of a peak depends on how much material is there and how strongly it absorbs; the area under it is what purity calculations use.
What you are looking at
- The main peak. One tall, reasonably symmetric peak is the product. A purity of 99.5% means that peak's area was 99.5% of the total area of every peak the software integrated.
- Small peaks close to the main peak. Usually related impurities: a deletion sequence, an oxidised methionine, a deamidated asparagine, an incompletely deprotected side chain. They elute near the product because they are almost the product. These are what the purity figure is counting against.
- Peaks at the very start. The solvent front and anything that did not interact with the column: salts, very polar small molecules. Good methods exclude this region from the integration; a certificate that counts it will understate purity.
- A rising baseline toward the end. Acetonitrile absorbs slightly at 214 nm, so the baseline climbs as the gradient runs. Laboratories subtract a blank run to flatten it. A baseline that has not been subtracted makes small late peaks hard to judge.
- A shoulder on the main peak. A co-eluting impurity the method could not fully separate, often a diastereomer from racemisation during synthesis. Whether the software splits it off or folds it into the main peak affects the purity figure, which is one reason the same sample can score differently in two laboratories.
Area percent is not mass percent
The purity figure is the fraction of ultraviolet-absorbing material that is the product. Two things follow. Anything that does not absorb at the detector wavelength is invisible: water, trifluoroacetate, acetate, inorganic salts. And impurities that absorb more or less strongly than the product per unit mass are over- or under-counted. HPLC purity therefore says how clean the peptide is relative to other peptides, and nothing about how much of the powder is peptide. That second question is net peptide content, covered on the certificate guide.
Why identity needs a second instrument
A chromatogram shows a peak; it does not say what is in it. A wrong peptide of similar hydrophobicity would produce an equally clean trace. That is why a certificate pairs HPLC with mass spectrometry: the mass of the main peak has to match the theoretical mass of the sequence. The two measurements answer different questions and neither substitutes for the other.
Reading the certificates on this site
The third-party reports linked from our product pages state a purity percentage and a content in milligrams, and the laboratory's verification page can show the underlying data for a report's key. Where the laboratory reports content only, as with NAD+, the product page shows content only. We never fill a gap in a certificate with a figure of our own. Which products currently carry a certificate is listed on the certificate guide.