Why peptides are supplied as salts
Reverse-phase purification runs in a mobile phase containing trifluoroacetic acid, which ion-pairs with the peptide's basic residues and sharpens the peaks. When the fractions are lyophilised, the TFA comes with them, so a peptide straight out of purification is a TFA salt whether or not anyone chose that.
Salt exchange to acetate is a deliberate extra step, done by ion exchange or by repeated lyophilisation from dilute acetic acid. It costs material and money, which is why it is usually offered as an option rather than performed by default.
How much weight the counterion adds
Each counterion adds its own mass per basic site. Trifluoroacetic acid is 114.02 daltons, acetic acid 60.05, hydrochloric acid 36.46. A peptide with three basic residues carrying three TFA counterions is 342 daltons heavier than the free base.
The proportional effect depends entirely on the peptide's own weight. Those same three TFAs on a 700 dalton peptide take peptide content down to about 67 percent; on a 4,500 dalton peptide they take it to 93 percent. This is why a single fixed percentage per salt form is a poor approximation, and why this tool computes it from molecular weight.
How many counterions does a peptide carry?
Roughly one per basic site: each lysine, arginine and histidine, plus the free N-terminus. It is an approximation rather than a rule, because the actual stoichiometry depends on the purification conditions and the drying.
For a rough count, the amino acid composition tool gives the number of basic residues. The exact figure, where it matters, comes from elemental analysis or ion chromatography on the certificate.
Why TFA is worth avoiding in cell work
Trifluoroacetate is cytotoxic at concentrations a TFA-salt peptide can readily produce in culture medium, roughly above 0.1 percent. The peptide may be fine and the experiment may still fail because of the counterion.
The symptom is a dose-dependent effect that scales with peptide concentration and is reproduced by a vehicle control containing TFA and no peptide. That control is the diagnostic, and it is worth running whenever a TFA-salt peptide gives an unexpected cytotoxic result.
The two ways this tool answers the question
The equivalence route asks how much of the target salt carries the same amount of actual peptide as a given mass of the source salt. That is the practical question when substituting one form for another in an existing protocol.
The molecular weight route reaches the same answer from the total weights of the two salts. Because both now derive from the same molecular weight arithmetic, they agree. They used to disagree, because the peptide content fractions were fixed guesses while the weights were computed.
How the salt conversion is calculated
Peptide content is derived from molecular weight rather than assumed per salt form, so the answer scales correctly with the size of the peptide.
salt MW = free base MW + counterion mass x number of sites content = free base MW / salt MW peptide in a labelled mass = mass x content(source) equivalent target mass = peptide / content(target) counterion masses, free acid convention: TFA 114.02 acetate 60.05 HCl 36.46
- Take the free base molecular weight. The peptide alone, without counterions. The molecular weight calculator gives it from the sequence, or it can be read from the certificate.
- Add the counterion mass per site. The free acid mass of the counterion, multiplied by the number of basic sites carrying one. All counterions use the free acid convention, so the table is internally consistent.
- Derive peptide content. Free base weight over total salt weight. This scales correctly with peptide size, which a fixed per-salt percentage does not.
- Convert the labelled mass to peptide. Labelled mass times the source form's content fraction gives the actual peptide present.
- Convert peptide back to the target salt. Divided by the target form's content fraction. The molecular weight ratio route gives the same number by construction.
What this method cannot tell you
- •The number of counterions is an assumption. One per basic site is the usual approximation; the real stoichiometry depends on purification and drying conditions.
- •It assumes complete salt exchange. A partially exchanged batch carries a mixture of counterions.
- •The typical content ranges shown alongside are reference figures for orientation, not measurements of your batch.
- •It does not model water content, which is a separate correction that the purity and net peptide content calculators apply.
Salt form converter: frequently asked questions
Because reverse-phase purification runs in a trifluoroacetic acid mobile phase, and the TFA lyophilises along with the peptide. A peptide straight from purification is a TFA salt by default.
Exchanging to acetate is a deliberate extra step that costs material, which is why it is usually an option rather than the default.
114.02 daltons per counterion. A peptide with three basic sites carrying three TFAs is 342 daltons heavier than the free base.
The proportional effect depends on peptide size: on a 700 dalton peptide that is a third of the weight, and on a 4,500 dalton one it is seven percent.
Roughly one per basic site: each lysine, arginine and histidine plus the free N-terminus.
It is an approximation. The exact stoichiometry depends on purification conditions, and where it matters it comes from elemental analysis or ion chromatography.
It is cytotoxic above roughly 0.1 percent in the medium, a concentration that a TFA-salt peptide can reach at ordinary working concentrations.
The diagnostic is a vehicle control containing TFA at the same concentration and no peptide. If it reproduces the effect, the counterion is responsible.
Chemically, by ion exchange chromatography or by repeated lyophilisation from dilute acetic acid. Both require equipment and both lose material.
Buying the acetate form is usually cheaper than converting it, and it is worth specifying when ordering for cell-based work.
Work out the peptide in the TFA salt, then divide by the acetate form's content fraction.
For a 1,419.5 dalton peptide with one counterion, 5 mg of TFA salt holds 4.63 mg of peptide, which corresponds to 4.83 mg of the acetate salt.
Because they mixed two conventions. HCl and succinate used the acid mass while acetate, sulfate, citrate and phosphate used the bare anion mass, so the table was not internally consistent.
Every counterion now uses the free acid convention, which is how a certificate states a salt: free base plus acid.
Because a fixed percentage per salt form is only right at one peptide size. TFA was fixed at 80 percent, which is roughly right for a 500 dalton peptide and badly wrong for a 4,000 dalton one.
Computing it from molecular weight gives the correct figure at any size, which is what the stoichiometry actually says.
Not generally. Free base peptides are less common, often less stable, and frequently less soluble, since the salt form is part of what keeps a peptide dissolved.
The free base is the right reference point for calculations and not necessarily the right form to buy.
Yes. Salt forms are generally more soluble than the free base, and different counterions differ from each other.
It also affects the pH of the reconstituted solution: a TFA salt dissolved in water gives a mildly acidic solution, since TFA is a strong acid.
Acetate for cell-based work, to avoid TFA cytotoxicity. TFA is acceptable where the counterion cannot interfere, and it is usually cheaper.
Hydrochloride is occasionally offered and has the highest peptide content of the common forms, because chloride is the lightest counterion.
The peptide is chemically identical in either form. What changes is how much peptide a given weight of powder contains, and what else goes into the solution with it.
An apparent potency difference between salt forms is usually a concentration error from not correcting for peptide content.
Yes. Incomplete salt exchange leaves a mixture, which is one reason a measured peptide content can differ from a calculated one.
A certificate that reports the counterion by ion chromatography is reporting a measurement rather than an assumption.
It changes what a milligram of powder is worth. Two vials at the same price and the same label weight hold different amounts of peptide if their salt forms differ.
Correcting both to peptide mass before comparing prices is what makes the comparison meaningful.
Usually 85 to 95 percent, depending on peptide size and the number of counterions.
Acetate's 60 dalton counterion is roughly half TFA's, so acetate salts carry noticeably more peptide per milligram of powder.
Yes, or you will overstate the concentration. Weighing out a salt and using the free base molecular weight counts counterion mass as peptide.
Either use the salt's molecular weight against the weighed mass, or correct the mass to peptide and use the free base weight. Mixing the two is the error.
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