Water content by Karl Fischer titration

Karl Fischer titration quantifies water by a stoichiometric reaction of water with iodine, sulfur dioxide and an alcohol in a pyridine-free base medium. In the coulometric variant the iodine is generated electrochemically and the water content is calculated from the charge passed: 10.71 coulombs per milligram of water. It is the reference technique for residual moisture in lyophilised material and it is sensitive to about 10 µg of water, which is what makes it usable on a 5 mg sample.

Procedure as applied in the index

1. Condition the coulometric cell until the drift is below 15 µg/min. This is the step most often skipped and it invalidates everything downstream.
2. Determine the vial's gross and tare mass on a five-place balance under nitrogen. Record ambient relative humidity.
3. Introduce the whole vial contents, or a weighed aliquot of 3–10 mg, through a septum using a dry syringe. Do not open the vial to room air.
4. Titrate to a stable end-point. Record the sample mass, the water mass, and the blank.
5. Run in duplicate. The index requires duplicate agreement within 0.4 pp absolute; a third determination is required if they disagree.

Worked calculation

Sample mass                       6.412 mg
Water found (coulometric)       360.4 µg
Blank (matched solvent)           4.1 µg

water content = (360.4 − 4.1) / 6 412 × 100
              = 356.3 / 6 412 × 100
              = 5.557 %  → 5.56 %

Duplicate: 5.71 %.  |Δ| = 0.15 pp  ✓ within the 0.4 pp requirement.
Reported: mean of duplicates, 5.64 %.

Reading the number

Residual moisture in a well-lyophilised peptide runs 1–4 %. Between 4 and 7 % the cycle was probably cut short at secondary drying, or the product was above its collapse temperature. Above 7 %, either the cycle failed or the material has taken up water since — and the distinction is answerable from the cake appearance, because a cake that has absorbed atmospheric water shrinks away from the vial wall and loses its structure.

The proline-rich short peptides in the index are the worst offenders: selank, KPV and the bioregulator tetrapeptides routinely return 8–13 % and the cakes are visibly hygroscopic. NAD+ is worse again, at 8–14 %. In those cases a high water content is a property of the compound rather than a criticism of the supplier, and the index's class specifications are set accordingly — 12 % for the bioregulator and neuro classes against 8 % for the incretins.