Peptide purity and net peptide content

HPLC (high-performance liquid chromatography) separates a sample into its components, and a UV detector records each one as a peak. Purity is the main peak's share of the total. In Bachem's words, the purity determined by HPLC "using UV detection corresponds to the percentage of requested peptide in relation to the total amount of material absorbing at 220 nm."

So purity compares the target peptide with the other things the detector can see, which are mostly related peptides made during synthesis: chains with an amino acid missing, an extra one, or a protecting group left on. It does not see water or most counter-ions, so it says nothing about how much of the powder they make up.

A freeze-dried peptide is a salt. During purification the peptide picks up counter-ions, usually trifluoroacetate (TFA) or acetate, bound to its charged groups, and the powder holds some water. Net peptide content (often abbreviated NPC, and sometimes printed as "assay" or "peptide content") is the share of the weight that is peptide.

Bachem describes the gross weight of a peptide sample as comprising "the weight of the peptide, counter-ions, and adhered water". It determines net peptide content by elemental analysis, which works because counter-ions and water contain no nitrogen, or by amino acid analysis.

One detail matters: net peptide content counts all the peptide in the powder, including related peptide impurities. GenScript defines it as "the combined fractions of the target peptide and peptidic impurities". That is why the two numbers are needed together.

GenScript states the rule plainly: "The amount of the target peptide is calculated by the multiplication of gross weight x net peptide content x HPLC purity."

Bachem's published example uses bradykinin acetate:

• gross weight weighed out: 5 mg
• HPLC purity: 98.7%
• net peptide content: 85.0%
• molecular weight: 1060.22 g/mol
• target peptide: 4.195 mg, which is 3.956 µmol

So a 5 mg weighing holds a little under 4.2 mg of the peptide you want. If you were making a 1 mg/mL solution from that powder by weight alone, it would actually be about 0.84 mg/mL. For accurate work, use the two figures from your own batch certificate.

A complete set of results should account for everything in the vial. GenScript describes the check as:

% Target Peptide + % Peptidic Impurities + % Counter ion + % Water = 100%

If a certificate reports purity, net peptide content, counter-ion and water, you can do this sum yourself. A large gap means something wasn't measured.

Counter-ions bind to the charged groups on a peptide, mainly the free N-terminus and basic side chains. Bachem notes that "A relatively low peptide content can be expected when a peptide contains a large proportion of basic amino acids." A short peptide rich in lysine or arginine can therefore carry a noticeably higher share of counter-ion by weight than a longer, neutral one, without anything being wrong with it.

Water is the other variable. Peptide powders absorb moisture from the air, which lowers the peptide content of what you weigh out. That is one reason to keep vials dry and let them reach room temperature before opening; see peptide storage and handling.

The salt form is printed on a good certificate. Bachem supplies its peptides as trifluoroacetate salts unless stated otherwise, and peptides made in solution as acetate salts or inner salts. The counter-ion affects the molecular weight of the salt you weigh, so it matters for calculations, and some experiments are sensitive to it. If your work needs a particular salt form, say so when you ask for a quote.

There is no single right answer; it depends on what the peptide is for. Custom synthesis companies sell the same sequence at several purity grades for exactly this reason, and higher grades cost more because each extra step of purification loses material.

A few principles help when you specify a peptide:

• The impurities are mostly relatives of the target. A chain missing one amino acid can behave very like the full sequence in some experiments, so purity matters most where the result depends on precise quantities or on the exact molecule.
• Purity and content are specified separately. A high purity says nothing about how much of the powder is peptide, so ask for net peptide content if you need accurate concentrations.
• Specify the salt form if it matters to you. It changes the weight you need and can matter to some assays.
• Ask for the specification before you order, and the certificate with delivery. The specification is the promise; the certificate is the result for your batch.

Very little on its own. A purity figure only means something when it is tied to a batch, a method and a laboratory, and when it comes with an identity result from mass spectrometry. A headline "99%+" with no certificate behind it is a claim, not a result. How we test explains who tests what.

• Bachem, FAQ: analysis of peptides
• GenScript, AccuPep QC testing