What a 1 Da mass tolerance means
Why identity criteria are set at 1 Da, what sits inside that window and what falls outside it, and how to read the numbers on a certificate.
Researchers reasonably ask why 1 Da, whether that is tight or loose, and what a difference of 0.9 Da means when a difference of 1.1 Da fails. This note explains the tolerance, its relationship to instrument accuracy and to average versus monoisotopic mass, and what falls inside and outside it. All Alphex products are for laboratory and in-vitro research only.
What 1 Da is
One dalton is one twelfth of the mass of a carbon-12 atom, close to the mass of a hydrogen atom. Peptide molecular weights are expressed in daltons (equivalently g/mol). A tolerance of 1 Da means the measured mass and the calculated mass agree to within roughly one hydrogen atom, on a molecule that for the Alphex catalogue weighs between 404 and 4,731 Da. As a relative figure that is 0.25% for GHK-Cu and 0.02% for GLP-3 (RT).
Why not tighter?
Modern electrospray instruments routinely achieve accuracy far better than 1 Da; high-resolution instruments reach parts per million. The tolerance is set at 1 Da for a different reason: it must accommodate the difference between average and monoisotopic mass, isotope-peak selection, and small calibration differences between laboratories, while still excluding every chemically meaningful discrepancy. The smallest common modification, deamidation, is +0.98 Da; the smallest common isotope-averaging error on a large peptide is a few daltons if the wrong convention is used. So the criterion is applied against the correctly matched convention (average with average, monoisotopic with monoisotopic), and at that point 1 Da is comfortably tighter than any modification except deamidation, which is caught by HPLC as a separate peak. See molecular weight and molar calculations.
What sits inside the tolerance
- Instrument calibration variation between runs and laboratories.
- Rounding of the theoretical mass.
- For a large peptide, choice of isotope peak within the cluster when reporting monoisotopic mass.
What sits outside it
- Oxidation (+16 Da).
- Sodium and potassium adducts (+22, +38): these are artefacts of the measurement, not of the peptide, and a competent lab reports the deprotonated mass, but they would fail the criterion if reported naively.
- Residual protecting groups (+56, +100 and others).
- Any deletion, truncation or wrong sequence (tens to thousands of daltons).
Deamidation (+0.98) is the borderline case: it can sit just inside 1 Da. It is caught instead by HPLC, where the deamidated form is a separate, slightly earlier peak, and by the fact that a batch would need to be substantially deamidated to shift the observed mass rather than merely show an extra peak. See mass spectrometry identity explained.
Reading the number on a COA
A well-presented result gives theoretical mass, observed mass, the difference, and states the convention. For example: theoretical (average) 1419.5, observed 1419.7, difference +0.2 Da, pass. A COA that gives only "MS: conforms" without numbers has withheld the evidence; ask for the spectrum. A COA whose observed mass differs by exactly zero to four decimal places for several batches is also worth a second look, since real measurements vary slightly.
Frequently asked questions
Would a stricter tolerance be better?
Not meaningfully. Below 1 Da the criterion would start to fail correct material on convention mismatches while catching nothing new that HPLC does not already catch.
Does the tolerance apply to the salt or the free peptide?
The free peptide. Counter-ions dissociate in the electrospray and are not measured.
Can I request the spectrum for a batch?
Yes, with the COA, via the COA request form.
Alphex research peptides are supplied for laboratory and in-vitro research by qualified researchers only. They are not authorised for human or veterinary use.
Research use only. Alphex products are supplied exclusively for laboratory and in-vitro research by qualified researchers. They are not authorised for human or veterinary use, and nothing in this note is advice on any such use.
Published 8 April 2026. Alphex reviews research notes when the testing method, the catalogue or UK guidance changes.