What HPLC purity measures
HPLC separates a sample into its components, and a UV detector draws each one as a peak. Purity is the main peak's share of the total area. Bachem defines it as the percentage of the requested peptide in relation to all the material absorbing at 220 nm.
The other peaks are mostly relatives of the target made during synthesis: chains missing an amino acid, or carrying a leftover protecting group. Water and most counter-ions are invisible to the detector, so purity says nothing about them.
What net peptide content measures
A freeze-dried peptide is a salt. During purification it picks up counter-ions, usually acetate or trifluoroacetate, and the powder holds some water. Net peptide content is the share of the weight that is peptide. On a certificate it may be printed as "peptide content", "assay" or "NPC".
So a batch can be 99% pure and 80% peptide by weight, and both figures are right.
The two side by side
| HPLC purity | Net peptide content | |
|---|---|---|
| Answers | How much of the peptide is the right sequence? | How much of the powder is peptide? |
| Counts | Target peptide against other UV-absorbing material | All peptide against counter-ions and water |
| Method | Reversed-phase HPLC, UV at 220 nm | Nitrogen analysis or amino acid analysis |
| Below 100% because | Synthesis makes close relatives of the target | Counter-ions and water are part of the powder |
How much target peptide is in what you weigh
GenScript gives the rule: gross weight × net peptide content × HPLC purity.
Bachem's published example is bradykinin acetate. Weigh out 5 mg with an HPLC purity of 98.7% and a net peptide content of 85.0%, and you have 4.195 mg of bradykinin. Make that up to 5 mL expecting 1 mg/mL and you actually have about 0.84 mg/mL.
Is 95% purity good? Is 98%?
It depends on the work. There is no single pass mark, which is why synthesis companies sell the same sequence at several purity grades and charge more for the higher ones.
Two things help you decide:
- The impurities are mostly near-copies of the target. Where a result depends on exact quantities or on the exact molecule, they matter more.
- The figure means little without the method and the chromatogram. "99% pure" on a web page is a claim. A certificate for your batch, with the trace, is evidence.
A check that the numbers add up
A full set of results accounts for everything in the vial. GenScript writes it as: target peptide + peptide impurities + counter-ion + water = 100%. If a certificate gives all four, do the sum. A large gap means something was not measured.
Why some peptides have low peptide content
Counter-ions bind to a peptide's charged groups, so a short peptide rich in lysine or arginine carries more counter-ion by weight. Bachem notes that a relatively low peptide content can be expected when a peptide contains a large proportion of basic amino acids. Nothing is wrong with such a batch.
"Third-party tested" and "lab tested"
Every manufacturer tests its batches before release: that is the manufacturer's certificate. Some suppliers also send samples to an independent laboratory. Both are useful. Ask which one you are looking at, which laboratory did the work, and whether the report number can be confirmed with that laboratory.
Read next: how to read a certificate of analysis · how we test · the certificate library