Peptide purification by RP-HPLC
Reversed-phase HPLC is the default purification for synthetic peptides because the impurities it has to remove, deletion sequences and incompletely deprotected material, differ from the product mostly in hydrophobicity. A shallow gradient on a C18 column separates them when nothing else will.
Read the crude before you set up the run
Run an analytical injection first, on the same stationary phase you intend to use preparatively. The crude profile tells you what kind of purification problem you have, and the three cases need different responses.
A dominant product peak with well separated minor impurities is a simple loading problem, and you can push the column hard. A product peak with a shoulder means a closely related impurity, usually a single deletion or an epimer, and that needs a shallow gradient and a deliberate sacrifice of yield at the peak edges. A broad, unresolved hump with no dominant peak means the synthesis failed, and no gradient will rescue it. Resynthesizing is faster than trying.
Column and gradient choices
C18 is the starting point and handles most peptides up to about thirty residues. Longer and more hydrophobic peptides tend to smear on C18, and a shorter chain, C8 or C4, gives sharper peaks because the interaction is weaker and the peptide elutes as a band rather than bleeding off.
Pore size matters more than people expect. Use 300 angstrom material for anything above roughly fifteen residues. On 100 angstrom material a larger peptide cannot reach most of the surface area, so the effective capacity collapses and the peak tails.
For the gradient, find the elution point with a fast scouting run, then build a shallow gradient around it. One percent acetonitrile per minute is a reasonable working slope, and half that for a shoulder you are trying to resolve. A gradient that runs from five to ninety-five percent in twenty minutes will separate almost nothing worth separating.
Ion pairing and the counterion you inherit
Trifluoroacetic acid at around 0.1 percent is the standard ion pairing additive because it sharpens peaks and improves resolution. It also leaves the peptide as a TFA salt, and that has consequences downstream.
TFA is cytotoxic in cell assays at concentrations that are easy to reach from residual salt, and it suppresses signal in electrospray mass spectrometry. For material going into cells or into an animal, exchange the counterion, usually to acetate or hydrochloride, by a second short run or by repeated lyophilization from dilute acid. State the counterion on the certificate, because a peptide quoted at ninety-five percent purity can still be twenty percent TFA by mass.
That last point is the one that catches people. Peptide content and peptide purity are different numbers. Purity is the fraction of peptide-related material that is your peptide. Content is the fraction of the powder that is peptide at all, with the balance being counterion and water. Weighing out by dry mass without knowing the content will give you a concentration that is wrong by a quarter, and every downstream potency number inherits the error.
Pooling and recovery
Pool on purity rather than on yield. Collecting the full peak including both edges is how a ninety-eight percent center becomes a ninety-one percent pool, and the material at the edges is exactly the closely eluting impurity you ran the gradient to remove.
Lyophilize from a solution that is mostly water with enough acetonitrile to keep the peptide soluble. Freezing a concentrated organic solution produces a film rather than a cake, and the film redissolves badly and traps solvent.
Confirm identity by mass on the purified material, not on the crude. The crude spectrum will contain the product mass regardless of how little of it is there, and confirming the mass before purification tells you almost nothing about what you are about to ship.