Water content and Karl Fischer titration, explained
Why lyophilised peptides are not dry, how Karl Fischer measures the water, when the figure matters, and how to work without it.
Lyophilised peptides are not dry. A few percent of their mass is water, bound to the peptide and its counter-ions, and that water is part of the reason a "10 mg" vial does not contain 10 mg of peptide. Some COAs report the figure, measured by Karl Fischer titration; most research peptide COAs, including Alphex's, do not. This note explains what water content is, how Karl Fischer measures it, when the number matters, and how to work without it. All Alphex products are for laboratory and in-vitro research only.
Where the water is
Freeze-drying removes free water by sublimation but leaves a residual fraction bound to polar groups on the peptide and to the counter-ions. Typical residual moisture for a well-dried peptide is 2-8% by mass; hygroscopic peptides and TFA salts sit at the higher end and will absorb more from humid air once opened. This water is chemically important (it is what allows slow hydrolysis and deamidation even in the powder) and gravimetrically important (it is non-peptide mass in the vial). See what lyophilised means and net peptide content versus gross vial weight.
How Karl Fischer titration measures it
Karl Fischer titration is a chemical method specific to water: iodine reacts stoichiometrically with water in the presence of sulfur dioxide and a base, and the amount of iodine consumed (volumetric method) or generated electrochemically (coulometric method, for small samples) gives the water content directly. It is precise, specific, and the reference method for water in pharmaceuticals. It also consumes the sample, needs milligrams of material for a good result on a low-moisture solid, and requires an instrument most research peptide testing does not include by default.
When water content matters
- Absolute concentration. If you need to know how much peptide is in the vial rather than how much solid, water content is one of the two corrections (the other is counter-ion) between gross mass and net peptide.
- Stability studies. Residual moisture predicts how fast a lyophilised peptide degrades in storage.
- Formulation work. Where the peptide is being incorporated into a solid or semi-solid vehicle by mass.
When it does not matter: comparative bench work with a single stock, qualitative assays, and anything where you calibrate against a measured concentration rather than a nominal one.
What Alphex reports, and does not
The Alphex COA reports chromatographic purity (220 nm, reference standard, 99+% threshold), with the trace, batch number and laboratory. It does not report Karl Fischer water content or endotoxin, and we do not claim to run those assays. Net peptide content is therefore not on the standard certificate; researchers who need it should treat the label mass as gross and apply a measured or stated correction. We say this plainly rather than print a moisture figure that was not measured for the batch. See endotoxin testing and research peptides for the parallel case.
Working without the figure
- Assume a documented net peptide content (many laboratories use 75-85% for acetate salts of short peptides) and state it in the methods.
- Measure concentration directly after reconstitution: UV at 280 nm for Trp/Tyr-containing peptides, otherwise amino acid analysis, a peptide assay, or quantitative HPLC against a standard of known concentration.
- Design assays that are internally calibrated, so nominal concentration errors cancel.
- Minimise further water uptake: warm sealed vials before opening, reconstitute promptly, keep the freezer box dry. See long-term storage.
Frequently asked questions
Can I dry the peptide further myself?
Not usefully at vial scale; you risk heat damage and you would still not know the number. Correct by measurement instead.
Does higher purity mean lower water content?
No, they are independent. Purity is a property of the peptide fraction; water is part of the non-peptide fraction.
Can I request Karl Fischer for a batch?
Ask via the COA request form; where the testing laboratory has the data we will send it, and where it does not we will say so.
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 2 June 2026. Alphex reviews research notes when the testing method, the catalogue or UK guidance changes.