Karl Fischer titration (revision 7)

Karl Fischer titration is a titrimetric method for determining the water content of a sample, based on the stoichiometric oxidation of sulfur dioxide by iodine in the presence of water. It is the reference technique for moisture determination in lyophilised pharmaceutical solids and is specified in pharmacopoeial moisture chapters worldwide — USP <921> and Ph. Eur. 2.5.12.^[1]

The method matters to readers of a certificate of analysis for a specific and frequently misunderstood reason: a chromatographic purity figure is a statement about the relative composition of the material that dissolved, not about how much peptide the vial contains. Water, counterions and residual salts occupy mass. A vial of material reported at 99% purity by HPLC may nevertheless be 6% water and 8% trifluoroacetate by mass, in which case its peptide content is closer to 85%.^[2]

The underlying reaction is the oxidation of sulfur dioxide by iodine, which consumes water stoichiometrically:

H₂O + I₂ + SO₂ + CH₃OH + 2\,RN → 2\,RNH⁺I^- + RNH⁺CH₃SO₄^-

Methanol participates directly, forming a methyl sulfite intermediate that is the species actually oxidised; the base (historically pyridine, now typically imidazole) buffers the liberated acid and keeps the reaction in the pH window where the stoichiometry holds at 1 mol iodine per mol water.^[3]