Counter-Ions in Synthetic Peptides: TFA Salts and Acetate Salts

Why a peptide is a salt

Amino acid side chains can be basic, acidic or neutral. Lysine, arginine and histidine carry positive charge under acidic conditions, as does the free amino terminus. Those positive charges have to be balanced by negative ions, called counter-ions. The overview literature on peptide counter-ions describes exactly this: peptides frequently occur as salts with different counter-ions, and the choice influences structure, physical properties and formulation.^[1]

Where trifluoroacetate comes from

Trifluoroacetic acid (TFA) appears twice in the making of a synthetic peptide. It is the strong acid that removes the finished peptide from its solid support and strips protecting groups; see solid-phase peptide synthesis. And it is the standard ion-pairing additive in reversed-phase HPLC purification, because it improves peak shape and separation.^[2] A peptide purified this way is, as one study puts it, often in the form of a trifluoroacetate salt.^[3] A material that arrives as a TFA salt is the default outcome of that route, not an anomaly.

Making acetate or hydrochloride forms

Other salt forms are made by exchanging the counter-ion after purification. Typical routes are using acetic acid in place of TFA in the chromatography, passing the peptide through an anion-exchange resin loaded with acetate, or dissolving the peptide in dilute acid and freeze-drying it, sometimes more than once. A comparison of procedures found that freeze-drying from hydrochloric acid, and also from acetic acid, lowered trifluoroacetate to satisfactory levels.^[4] In another study, replacing TFA with hydrochloric acid in the purification steps of a bacterial peptide gave similar yields and removing the hydrochloride afterwards was easier than removing TFA.^[5]

Exchange is rarely complete and some material is usually lost, so an acetate salt or a hydrochloride salt still contains whatever TFA was not removed. The amount can be measured; see below.

Why the counter-ion matters in the laboratory

• It adds mass. Counter-ions are part of the weighed powder. Each trifluoroacetate or acetate is a real contribution to the mass, which lowers the share of the powder that is peptide; see the worked example below.
• It sets the pH of a solution. TFA is a much stronger acid than acetic acid, so a solution of a TFA salt in unbuffered water is usually more acidic than the same amount of an acetate salt.
• It can interfere with assays. One widely cited study found that trifluoroacetate on its own reduced cell numbers in cultures of several cell types at very low concentrations, and that TFA and hydrochloride forms of the same peptides gave different results in the same cell assay. Its authors recommended converting peptides to a hydrochloride or comparable salt before cell-based work.^[3] A separate report on a bacterial peptide found TFA interfered with infrared analysis of its structure and changed its folding by circular dichroism.^[5]
• It changes physical form. Different salts differ in hygroscopicity, solubility, crystallinity and handling, which is part of why counter-ions are chosen deliberately for formulation.^[1]

A worked example of the mass effect

Take a made-up peptide of molecular weight 1000 g/mol with three basic sites, each paired with one counter-ion. Trifluoroacetic acid has a molar mass of about 114.02 g/mol; acetic acid is about 60.05 g/mol.

As a TFA salt the formula mass is 1000 + 3 × 114.02 = 1342.07 g/mol, so the peptide itself is 1000 ÷ 1342.07 = 74.5 % of the salt by mass. As an acetate salt it is 1000 + 3 × 60.05 = 1180.16 g/mol, and the peptide is 84.7 % of it. These are the best cases, with no water or anything else in the powder. A real powder also holds moisture, so its peptide share is lower again. See purity versus net peptide content, and enter your own values into the purity and content calculator.

Measuring counter-ions

The counter-ion content of a peptide is measured, not guessed. Ion chromatography, capillary electrophoresis and capillary isotachophoresis have all been compared for acetate, trifluoroacetate and chloride in synthetic peptides; ion chromatography gave the best results in that comparison.^[4] A report that lists a net peptide content will usually have accounted for counter-ions and water together.

Counter-ions in analytical methods

The same acid that makes peaks sharp in ultraviolet HPLC is less welcome in electrospray mass spectrometry, where strong ion-pairing anions are known to reduce the signal of positively charged peptide ions. The review of counter-ions also covers the use of salts and acids as mobile-phase additives in analysis.^[1] For that reason, methods built to measure purity by UV and methods built to measure mass by LC-MS often use different additives, and the same sample can look somewhat different in the two. Neither picture is wrong; they are different experiments. See reading an HPLC chromatogram and mass spectrometry for peptide identity.

Questions to ask about any material

1. What is the stated salt form? If a specification does not state one, treat it as unknown.
2. Has the counter-ion content been measured, or only assumed from the route?
3. Does your assay tolerate the counter-ion present? If it does not, plan an exchange or a control.
4. Does your weighing take the counter-ion into account? If the answer is no, the concentration you calculate will be higher than the true peptide concentration.

Questions about the salt form or composition of a material listed on this site can be sent to support.