Karl Fischer titration: measuring water in a peptide powder

Residual moisture is defined in What lyophilized means; this article covers how it is measured. In the Karl Fischer reaction, iodine oxidizes sulfur dioxide, and the oxidation needs water. In a solution that also holds a base and an alcohol (classically methanol), the sulfur dioxide sits as an alkyl sulfite, and iodine converts it to an alkyl sulfate only by using up water. In the accepted mechanism, one mole of iodine reacts with one mole of water.

The endpoint is found electrochemically with a double platinum electrode. A small constant current is held across the pair, and the first excess of iodine depolarizes it, so the measured voltage drops abruptly (bivoltammetric indication).

Volumetric and coulometric Karl Fischer titration

In volumetric Karl Fischer titration, an iodine reagent is added from a burette until the endpoint, and water is calculated from the volume used and the titer (mg of water per mL). The titer is set against a water standard such as sodium tartrate dihydrate (15.66% water by mass), because a reagent's water equivalent is not fixed: it varies by batch and falls as the reagent takes up moisture. This form suits larger amounts of water.

In coulometric Karl Fischer titration, the iodine is made in the cell: iodide is oxidized at an anode and the instrument integrates the charge. By Faraday's law, at two electrons per iodine and one iodine per water, 1 mg of water takes about 10.7 coulombs. The charge is an absolute measure, so no titrant needs standardizing (performance is still checked with a water standard), and the form suits microgram to low-milligram amounts of water. A 10 mg cake at 2% w/w water, for illustration, holds only 0.2 mg. Coulometric results have been compared with gravimetric and thermogravimetric ones for freeze-dried biological products in their final containers.

The oven technique

In the oven technique, the weighed sample is heated in a sealed vial and a stream of dry carrier gas sweeps the released water into the titration cell. The solid never enters the reagent, so the technique serves samples that do not dissolve cleanly or that would react with it (free thiols, for example, which consume iodine without any water).

The oven temperature is part of the method: too low and water stays behind, too high and the sample may decompose and release water that was not free moisture. A study of five water methods on six substances judged direct plus oven Karl Fischer the best combination for substances of unknown chemical and thermal behaviour.

Why loss on drying is a different number

Loss on drying weighs a sample, heats it under stated conditions and weighs it again. The loss is everything volatile under them: water, but also residual solvent and volatile acids such as acetic acid. Karl Fischer responds to water, apart from known interferences such as thiols, which also consume iodine. The two figures can differ in either direction: non-water volatiles raise loss on drying, and tightly bound water may stay behind at the drying temperature. Each is reported with its method.

Handling a hygroscopic cake

A lyophilized cake is dry, porous and hygroscopic, and takes up water vapour quickly. Samples are therefore weighed in low humidity, in a glove box or dry bag, from a vial opened only for the test.

Where Solara's documentation stands