Why 99% HPLC purity is not 99% peptide in the vial

The certificate for a batch says 99%. We put the powder on the balance and want to know something else: of the milligram we have weighed, how much is peptide? The answer is not 99%, and not because anyone got the analysis wrong. The two numbers measure different things, and between them sit two components the chromatogram does not count, counter-ions and water.

What the 99% actually counts

HPLC purity is a ratio of areas. The area of the main peak is divided by the sum of the areas of all integrated peaks on the chromatogram, with ultraviolet detection at 214–220 nm, where the peptide bond absorbs. So the denominator holds the target peptide and the impurities related to it: shorter chains, chains missing an amino acid, oxidised forms.

That is all. Anything that does not show up as an integrated peak stays outside the ratio. A sample can be 99% pure and, at the same time, a good share of its mass can be something other than peptide. There is no contradiction here, only two different questions put to the same powder.

Where the counter-ions come from

A peptide has basic groups: the amine at the N-terminus and the side chains of lysine, arginine and histidine. Under acidic conditions they are protonated and carry a positive charge. A positive charge does not sit alone in a solid. Next to each one settles a negative ion, and that is the counter-ion. Chemically speaking, a lyophilised peptide is a salt.

Trifluoroacetate

Trifluoroacetic acid (TFA) turns up twice in the making of a synthetic peptide. It is the usual reagent for cleaving the chain from the resin at the end of solid-phase synthesis, and it is also the standard additive in the water/acetonitrile mobile phase used for purification on a C18 column. During lyophilisation most of the free acid leaves, being volatile. The trifluoroacetate ions bound to the basic groups stay. That is why the “default” form of many research peptides is the TFA salt.

Acetate

Through a separate ion-exchange step, trifluoroacetate can be replaced with acetate. Some laboratories ask for this form for cell culture work, where residual TFA can influence the outcome of certain assays. In terms of mass the difference is simple: acetic acid has a molar mass of about 60 g/mol, trifluoroacetic acid about 114 g/mol. The same number of counter-ions weighs almost twice as much when they are trifluoroacetate.

A calculation on an invented peptide

Take a hypothetical peptide of 1000 g/mol with three basic groups. The figures are chosen to be easy to follow and do not describe a real batch.

If each basic group carries one trifluoroacetate, a mole of peptide gains 3 × 114, that is 342 g of counter-ion. The salt weighs 1342 g, of which the peptide is 1000. About a quarter of the mass is TFA. With acetate, the same peptide would carry 3 × 60, that is 180 g in 1180, around 15%.

The calculation assumes all three groups are occupied, which in practice is not always the case, so we take it as an upper limit. It still shows why the sequence matters. A peptide with many basic residues carries more counter-ion than one with few. Selank, for instance, has one lysine and one arginine in its chain of seven amino acids, plus the terminal amine, so three places where a counter-ion can sit. The peptide is also listed in the PEPTIDÁE catalogue, on the Selank product page (in Romanian, like the rest of their site). How much counter-ion a particular batch contains cannot, however, be deduced from the sequence. It is measured, usually by ion chromatography.

The water that stays after lyophilisation

Lyophilisation removes water by sublimation, but not all of it. Some remains bound to the peptide. On top of that, lyophilised powder is hygroscopic: it draws moisture from the air every time the vial stands open. A batch weighed on a humid day can look different from the same batch weighed straight out of the desiccator.

Water content is determined by Karl Fischer titration and appears on some certificates as a separate line. The way the vial is stored has a direct influence on this figure.

Purity and net content, side by side

Net peptide content is the fraction of the powder's mass that is peptide material, the rest being counter-ions and water. It is determined by amino acid analysis after hydrolysis or by elemental nitrogen analysis. It usually comes out somewhere between 60 and 90%, depending on the sequence and the salt form.

“Peptide material” includes the peptide impurities too. To get to the target peptide, we multiply the two numbers. An example, again a constructed one: net content 80%, HPLC purity 99%. Of the weighed mass, the target peptide accounts for 0.80 × 0.99, that is 79.2%. Not 99.

For anyone keeping batch records, the consequence is a practical one. Two batches with the same purity can have different net content, and then the same mass on the balance means different amounts of peptide. A result expressed per mass of powder is, in fact, expressed per peptide plus salt plus water. When we compare data series obtained on different batches, this is where we look first for the explanation of a small, constant difference.

There is also the question of the quantity on the label. Sometimes it means the gross mass of powder, sometimes the net mass of peptide. The certificate should make this clear, and if it does not, we ask the supplier. For the other checks related to purity, the same site's blog has an article on the criteria for verifying purity. In the laboratory notebook, next to the batch number, we record three values when we have them: purity, net content and salt form.