The full chain from gross weight to net peptide
Gross weight is what the vial was filled to. From it, HPLC purity removes non-target species, the counterion correction removes salt, Karl Fischer water content removes moisture, and residual solvent removes whatever the lyophilisation did not drive off.
The waterfall chart makes the sequence visible. Each bar is what remains after one correction, and the red segment above it is what that correction removed, so the relative size of the four losses can be read at a glance rather than inferred from four percentages.
Which correction usually dominates
For most peptides the counterion correction is the largest by a wide margin. A trifluoroacetate salt commonly removes fifteen to thirty percent of the weight, while purity at 99 percent removes one and water at three percent removes three.
This is why the salt form is the field to check first on a certificate, and why a certificate that does not state it leaves the largest term in the calculation unknown.
- •Counterion: typically 5 to 30 percent, depending on salt and peptide size
- •Purity: typically 1 to 5 percent
- •Water: typically 1 to 5 percent
- •Residual solvent: typically under 1 percent
Amino acid analysis, and why it supersedes all of this
Amino acid analysis hydrolyses the peptide and quantifies the released amino acids against standards. It measures the peptide directly, so its answer is already net of counterions, water and solvent.
A certificate reporting peptide content by AAA is reporting the number this calculator is estimating. Where it is present, use it. Where it is absent, this chain of corrections is the reasonable substitute, and it is an estimate rather than a measurement.
What this means for comparing suppliers
Two vials both labelled 10 mg can hold materially different amounts of peptide. One at 99 percent purity as an acetate salt might hold 8.9 mg; another at 95 percent as a TFA salt might hold 7.3 mg. That is a 22 percent difference at the same label.
Cost per milligram calculated on the label figure misses this entirely. Running both through this calculator first and then comparing is the comparison that means something.
How net peptide content is calculated
Four multiplicative corrections in a fixed order, with each intermediate value and each loss reported so the arithmetic can be followed rather than trusted.
after purity = gross x (purity % / 100) after salt = after purity x peptide content fraction after water = after salt x (1 - water % / 100) net peptide = after water x (1 - solvent % / 100) correction factor = net peptide / gross
- Enter the gross weight. The labelled fill weight of the vial, which includes everything in it.
- Apply HPLC purity. Removes the non-target fraction of the eluting material. It cannot remove what the chromatographic method never saw.
- Apply the counterion correction. From the shared counterion table, the same one the purity calculator and the salt form converter use. It is usually the largest of the four corrections.
- Apply water and residual solvent. Two separate fields because they are measured by different methods: Karl Fischer for water, gas chromatography for solvent.
- Chart the losses. Each bar shows the remaining mass and the loss that produced it, so the relative weight of the four corrections is visible rather than having to be inferred.
What this method cannot tell you
- •The salt correction is a typical value for the counterion, not a measurement of the batch. The exact figure depends on the peptide's molecular weight and its number of basic sites.
- •A certificate reporting peptide content by amino acid analysis supersedes this whole chain; applying both double-counts.
- •It cannot detect a certificate that does not describe your batch. Check the lot number.
- •It assumes the corrections are independent, which is a simplification: purity and salt content are both consequences of the same purification.
Net peptide content: frequently asked questions
The fraction of a vial's contents that is actually peptide, after counterions, water, residual solvent and non-target species are excluded.
It is the number that matters for any calculation where the absolute amount of peptide is important.
Purity is a ratio among the species that eluted from a chromatography column. Net peptide content is an absolute fraction of the total weight.
Salt, water and solvent never appear in a chromatogram, so they lower net content without touching purity. A 99 percent pure peptide can be 75 percent peptide by weight.
The counterion, almost always. A TFA salt typically removes fifteen to thirty percent of the weight, against a few percent each for purity, water and solvent.
That is why the salt form is the first field to look for on a certificate, and why its absence is a bigger gap than a missing water content.
A method that hydrolyses the peptide into its constituent amino acids and quantifies each against standards, giving the peptide content directly.
It is the reference method for this question. A certificate reporting AAA peptide content has measured what this calculator estimates.
The AAA figure, whenever it is available. It is a measurement of your batch rather than an estimate from typical values.
Applying the salt and water corrections on top of an AAA figure double-counts the same material.
Organic solvent left over from synthesis and purification, most often acetonitrile, that lyophilisation did not fully remove.
It is usually well under one percent and is measured by gas chromatography. It matters more as a contaminant in sensitive assays than as a weight correction.
Because it shows the relative size of the four losses. Four percentages in a table do not convey that one of them accounts for most of the shortfall.
Substantially. Two vials both labelled 10 mg can differ by more than twenty percent in actual peptide once purity and salt form are accounted for.
Correcting both figures first and then computing cost per milligram is the comparison that reflects what you are buying.
No. Purity is nearly universal, salt form usual, water content common, residual solvent and AAA peptide content much less so.
A certificate missing the salt form leaves the largest correction unquantified, which is worth asking about.
No. Every correction removes material, so the net figure is always below the gross.
A vial can be overfilled relative to its label, which is a separate matter and one only weighing detects.
Not by itself. TFA salt is the normal output of reverse-phase purification, and a TFA salt has lower net content than an acetate one by chemistry rather than by carelessness.
What matters is whether the certificate states the salt form so the correction can be applied. Silence is the problem, not the number.
The concentration is the net peptide divided by the diluent volume, not the labelled mass divided by it.
Enter the corrected mass into the reconstitution calculator to get a concentration that reflects the peptide actually present.
Ask. If the answer is not forthcoming, assume TFA, because it is the default output of reverse-phase purification and it is the least favourable case.
Assuming acetate when the material is TFA overstates the peptide by ten percentage points or more.
Yes, readily. Many are hygroscopic, which is why they are stored sealed and desiccated and why the recommendation is to let a vial reach room temperature before opening it.
Opening a cold vial condenses atmospheric moisture directly onto the powder, which raises water content beyond whatever the certificate measured.
As precise as its weakest input, which is normally the assumed salt correction. Expect a few percent of uncertainty from that term alone.
It is far better than using the label figure uncorrected, which is systematically wrong in a known direction by a known kind of amount.
Yes, if the certificate values differ. Purity, salt form and water content are batch properties, and two lots of the same product routinely differ in all three.
The batch comparison tool puts the certificate values for several lots side by side for exactly this reason.
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