TFA vs Acetate Peptide Salts: Counter-Ion by Compound and What It Adds to the Mass

TFA and acetate are the two counter-ions research peptides are normally supplied with: a trifluoroacetate (TFA) salt pairs each protonated basic site of the peptide with one CF₃COO⁻ anion (113.02 g/mol), an acetate salt pairs it with one CH₃COO⁻ anion (59.04 g/mol), and the peptide's sequence, molecular formula and monoisotopic mass are the same in both. Each counter-ion adds the mass of one acid molecule to the salt, 114.02 g/mol as trifluoroacetic acid or 60.05 g/mol as acetic acid, so the salt form decides how much of the dry solid is peptide. Calculated from its listed formula, FOXO4-DRI, with 15 basic sites, is 75.8% peptide by mass as the fully salted TFA salt and 85.6% as the acetate salt. This page tabulates that arithmetic for most of the listed peptides.

Why are synthetic peptides supplied as salts?

A synthetic peptide is supplied as a salt because its basic groups are protonated during manufacture and each one keeps an anion when the peptide is freeze-dried. Most synthetic peptides carry basic side chains (lysine, arginine, histidine) and a free N-terminal amine. In the acidic conditions used for cleavage from the resin and for reversed-phase purification, these groups are protonated and pair with an anion from the mobile phase. When the purified fractions are freeze-dried the anion stays behind, so the solid is a peptide salt rather than the neutral free base. The identity of that anion is the counter-ion, and the peptide together with its counter-ion is the salt form. The guide to research peptides covers the lyophilized solid.

What is a trifluoroacetate (TFA) peptide salt?

A trifluoroacetate salt is the form a peptide takes when it is purified by reversed-phase HPLC with trifluoroacetic acid (CF₃COOH) in the mobile phase, which is the standard ion-pairing additive for preparative peptide purification. Peptides purified this way are obtained as trifluoroacetate salts by default. One trifluoroacetate anion (CF₃COO⁻, 113.02 g/mol) can be associated with each protonatable site, so a peptide with four basic sites can carry up to four TFA counter-ions, 456.09 g/mol of non-peptide mass counted as trifluoroacetic acid. Residual free trifluoroacetic acid can also remain in the lyophilized cake, which is why TFA salts are often slightly hygroscopic.

What is a peptide acetate salt?

A peptide acetate salt is the same peptide with acetate (CH₃COO⁻, 59.04 g/mol) in place of trifluoroacetate on each protonated basic site. The manufacturer normally makes it from the trifluoroacetate salt in a separate counter-ion exchange step after purification, and that step changes only the anion; the exchange is described in how research peptides are made. The peptide sequence, molecular formula and monoisotopic mass of the peptide itself are unchanged. Because acetate is lighter than trifluoroacetate, an equal mass of the acetate salt contains a larger mass fraction of peptide.

KPV as an acetate example. The compound record for KPV names a specific acetate form, H-Lys-Pro-Val-OH.AcOH: one acetic acid per tripeptide. Calculated from the listed molecular weight of 342.44 g/mol, the monoacetate comes to 402.49 g/mol and is 85.1% peptide by mass. KPV has two basic sites, the N-terminal amine and the lysine side-chain amine, so with both paired the diacetate comes to 462.54 g/mol (74.0% peptide) and the bis-trifluoroacetate to 570.48 g/mol (60.0% peptide). The same 342.44 g/mol tripeptide is therefore anywhere from 60.0% to 85.1% of the salt, depending only on which counter-ion it carries and how many.

How much of a TFA salt can be counter-ion?

In a fully salted TFA peptide, the counter-ion share of the salt is n × 114.02 / (MW + n × 114.02), where n is the number of basic sites and MW is the molecular weight of the free peptide. The basic sites counted are the free N-terminal amine (absent when the N-terminus is acetylated, capped or a pyroglutamate), each lysine and arginine side chain, and each histidine; a lysine whose side chain is closed into a lactam ring is not counted. The share rises with the number of basic sites and falls with the size of the peptide, which is why a small tripeptide can carry a larger counter-ion share than a 46-residue peptide.

Every value in the table is calculated from the sequence and the listing molecular weight in the Pepta Labs compound records, with 114.02 g/mol per trifluoroacetic acid (C₂HF₃O₂) and 60.05 g/mol per acetic acid (C₂H₄O₂).

FOXO4-DRI is the case with the most counter-ions. Its 46-residue D-retro-inverso sequence, H-LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG-OH, holds ten arginines and four lysines plus the free N-terminal amine: 15 basic sites, more than any other peptide in the table. Fully salted, the TFA salt comes to 7068.48 g/mol, of which 1710.33 g/mol (24.2%) is trifluoroacetic acid and 5358.15 g/mol (75.8%) is peptide; as the acetate salt it comes to 6258.93 g/mol and is 85.6% peptide. Its compound record states the form as "typically supplied as multi-trifluoroacetate or acetate salt".

Small peptides carry the largest share. SS-31, a four-residue peptide amide with three basic sites, is 65.2% peptide as the fully salted TFA salt (the SS-31 buying guide covers its identity record), and KPV is the lowest in the table at 60.0%.

Salt Forms Recorded for Listed Compounds

Where the salt form for a listed compound has been verified, it is recorded below and on the product page.

What is a hydrochloride or free-base peptide?

A hydrochloride peptide carries chloride (35.45 g/mol) as its counter-ion, and a free-base peptide carries no counter-ion at all; both exist but are less common for research peptides than acetate and trifluoroacetate salts. Each chloride adds 36.46 g/mol as hydrogen chloride, so fully salted FOXO4-DRI as the hydrochloride would come to 5905.02 g/mol and be 90.7% peptide (calculated). The glutathione and NAD+ records state the free acid, with no counter-ion. Metal complexes are a different case: in GHK-Cu the copper(II) ion is part of the named compound rather than a counter-ion, the listed formula C₁₄H₂₂CuN₆O₄ (401.91 g/mol) already includes it, and the complex itself is usually an acetate or trifluoroacetate salt.

Registry numbers are often assigned separately for a free peptide and for its specific salts, which is one reason the CAS number on a product page does not always match the first result in a database search. Tesamorelin is an example: its compound record gives CAS 218949-48-5 for the free peptide and CAS 804475-66-9 for the acetate salt.

Does counter-ion mass lower HPLC purity?

Counter-ion mass does not lower HPLC purity, because HPLC purity is the area fraction of the chromatogram that is the target sequence, while the counter-ion's share appears only in net peptide content, the mass fraction of the solid that is peptide; the two numbers answer different questions and are frequently confused.

A solid can therefore be 99% pure by HPLC and have a net peptide content well below 99%, because counter-ion and water are part of its mass. Purity describes the peptide-related material on the chromatogram; peptide content describes the composition of the solid. Multiplying the two gives the mass fraction of the solid that is the target sequence. Lyophilized peptides retain bound water, which is reported only when a water determination was run. The measurement routes are explained in how net peptide content is determined, and the purity side in peptide purity testing explained.

Where does the salt form appear on an analytical report?

The salt form line, if present, states which counter-ion the report applies to (for example "trifluoroacetate salt" or "acetate salt"). The molecular weight used for the mass-spectrometry comparison is that of the peptide calculated from its formula, not the weight of the salt. The HPLC purity is the main-peak area percentage. Peptide content, water content and counter-ion content are separate lines and are only present when those assays were actually run.

The testing Pepta Labs represents is independent third-party HPLC purity analysis and mass-spectrometry identity confirmation, commissioned through the purchasing group on a sampled production run from the source; Pepta Labs does not test in-house, and vials carry no lot number. Because testing is commissioned by the purchasing group, the report's client field names the group or source rather than Pepta Labs where that is the case. No net peptide content or water content value is asserted for any listing; the reported HPLC purity shown on the product page is the purity of the peptide fraction only. Reports are sent by email on request through the report request form.

Does salt form change the mass spectrum or a database comparison?

Salt form does not change the mass a spectrometer reports, because counter-ions dissociate on ionization and the observed ion is that of the peptide. The expected mass for FOXO4-DRI is therefore derived from the 5358.15 g/mol peptide, not from the 7068.48 g/mol fully salted TFA salt. Salt form does not explain a mass discrepancy on a report; sequence errors or chemical modifications do. Expected ions for each listed compound are in the expected mass table.

When comparing the molecular weight on a product page with a value from a database such as PubChem, check whether each refers to the free peptide or to a specific salt. The reference table lists the values recorded for each listed compound.

Frequently asked questions

Why are synthetic peptides supplied as TFA salts?

Synthetic peptides are supplied as TFA salts because trifluoroacetic acid is the standard ion-pairing additive in reversed-phase HPLC purification: the peptide's basic groups are protonated during cleavage and purification, each pairs with a trifluoroacetate anion (113.02 g/mol), and the anions stay with the peptide when the purified fractions are freeze-dried.

Is an acetate salt the same peptide as a TFA salt?

Yes. The acetate salt and the TFA salt of a peptide have the same sequence, molecular formula and monoisotopic mass; only the counter-ion differs, acetate (59.04 g/mol) in place of trifluoroacetate (113.02 g/mol), so the acetate salt holds a larger mass fraction of peptide.

How much of a TFA salt can be counter-ion?

In the fully salted case, with one trifluoroacetate on every basic site and water left out, the counter-ion share among the listed peptides tabulated on this page ranges from 11.7% for thymulin (one basic site) to 40.0% for KPV (two basic sites on a 342.44 g/mol tripeptide); FOXO4-DRI, with the most basic sites (15), is 24.2% trifluoroacetic acid and 75.8% peptide, all calculated from the listed formulas.

Which counter-ion does each Pepta Labs listing state?

Each listing with a verified compound record has a salt form line naming the form usually supplied for that compound: acetate or trifluoroacetate for most listed peptides, acetate alone for TB-500, a named acetate form (H-Lys-Pro-Val-OH.AcOH) or the trifluoroacetate salt for KPV, the free acid for glutathione and NAD+, and the copper(II) complex for GHK-Cu; the line is not a measurement of any production run, and no net peptide content is asserted for any listing.

What is a hydrochloride or free-base peptide?

A hydrochloride peptide carries chloride (35.45 g/mol, or 36.46 g/mol per site counted as hydrogen chloride) as its counter-ion, and a free-base peptide carries no counter-ion; both are less common for research peptides than acetate and trifluoroacetate salts.

Sources

• PubChem compound record CID 167312269 (FOXO4-DRI, C228H388N86O64).
• PubChem compound record CID 125672 (KPV, H-Lys-Pro-Val-OH, C16H30N4O4).
• PubChem compound record CID 16137828 (tesamorelin).
• Acid and anion molar masses, salt masses and peptide shares: computed from the molecular formulas and listing molecular weights.

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