Every number we print,
and how we got it.

RP-HPLC area purity

Agilent 1260 Infinity II, DAD @ 214 nm

Reports

Main peak area %, largest single impurity, baseline drift

Method

Roughly 20 µg of the finished powder is dissolved in 0.1% TFA and injected onto a C18 core-shell column, 2.7 µm, 150 × 4.6 mm, held at 30 °C and run at 1.0 mL/min. The gradient goes from 5% to 65% acetonitrile over 30 minutes — shallow enough that a deletion sequence one residue short pulls away from the main peak instead of riding on its shoulder. Detection is at 214 nm, the amide bond, so the response is roughly proportional to peptide length rather than to whatever aromatic residues happen to be present. Purity is reported as the main peak area over total integrated area, with the injection solvent front excluded. System suitability runs first: five replicate injections of a reference peptide must return under 2.0% RSD on area and a tailing factor below 1.5, or the sequence does not start.

Identity by ESI-MS

Bruker amaZon SL ion trap

Reports

Observed vs. theoretical monoisotopic mass, ±0.5 Da

Method

A diluted aliquot is infused directly, or taken from the HPLC eluent, and ionised by electrospray in positive mode. Peptides of this size come off multiply charged, so the raw spectrum is a series of [M+nH]ⁿ⁺ envelopes rather than a single line; those are deconvoluted to a neutral monoisotopic mass and compared against the mass calculated from the sequence. The acceptance window is ±0.5 Da. A result that lands 18 Da low means a dehydration somewhere, 16 Da high means an oxidised methionine, and either sends the lot back rather than onto a certificate.

Net peptide content

Elemental nitrogen determination

Reports

Peptide as % of gross powder mass

Method

The number buyers most often overlook. A weighed sample is combusted and its total nitrogen measured, then back-calculated against the nitrogen content the sequence predicts, which gives the fraction of the powder that is peptide at all. The balance is trifluoroacetate counterion and bound water — typically 15–20% of the mass in the vial. A lot can be 99% pure by HPLC and still be 82% peptide by weight, and both numbers are true: purity describes the peptide present, net content describes the powder. We print both, because a supplier quoting only the first is letting you weigh out about a fifth less material than you think you are.

Water by Karl Fischer

Metrohm 899 coulometer

Reports

Water as % w/w

Method

Coulometric titration on a sample transferred under dry nitrogen, because a lyophilised cake will pull moisture out of room air fast enough to move the result while you are weighing it. Iodine generated at the anode reacts stoichiometrically with the water present, and the charge consumed gives the mass of water directly — no calibration curve, no drying oven. The specification is ≤ 6.0% and results usually land between 2 and 4%. High moisture means the drying cycle was cut short or the stopper seated badly, and either way it shortens the shelf life of the material rather than merely inflating its weight.

Residual TFA

Ion chromatography, suppressed conductivity

Reports

TFA as % w/w against a 5-point curve

Method

Trifluoroacetate arrives with the purification buffer and stays with the peptide as its counterion. The sample is dissolved in ultrapure water and run on an anion-exchange column against a hydroxide eluent, with suppressed conductivity detection and quantitation against a five-point trifluoroacetate calibration curve. We specify ≤ 1.0% and typically report under 0.3%. It matters because TFA is cytotoxic at concentrations that are easy to reach in cell work, and because it is a large part of the mass that is not peptide — a figure worth having in front of you before you weigh anything out.

Bacterial endotoxin

Kinetic chromogenic LAL

Reports

EU/mg, with positive product control recovery

Method

A reconstituted sample is incubated with limulus amoebocyte lysate and a chromogenic substrate at 37 °C, and the time taken to reach a fixed absorbance change is read against a standard curve of control standard endotoxin. Every sample is run alongside a positive product control spiked with a known endotoxin load; if that spike does not recover between 50 and 200%, the sample is inhibiting or enhancing the reaction and the result is discarded rather than reported. Specification is ≤ 1.0 EU/mg. Glassware is depyrogenated and the water is endotoxin-free, so a failure here almost always points at handling rather than at the peptide.