June 13 1923

Professor Serafino Belfanti,
Milano, Italy

Dear Professor Belfanti:-

I presume that you have understood that we were using indicators to determine the hydrogen-ion concentration, in the isoelectric purification. We use a potentiometer for this purpose. I am enclosing a copy of the method of purification worked out by H. H. Dudley of the national institute for Medical Research, Hampstead, England.
Dudley starts with the "crude insulin" obtained with the 80 per cent filtrate in the first method, which was communicated to you, is added to the large volume of the high grade alcohol, that is, the precipitate thrown down by the high alcohol, after drying, is the so-called "crude insulin."
We are still using the rabbit test to determine the potency of insulin. One physiological unit is the amount required to reduce the blood sugar of a normal rabbit (2 Kilograms), which has been starved 24 hours, from its normal level which is approximately .12 per cent to .040 per cent and produces convulsions in three hours.
Since there is a considerable variation in the resistance of rabbits to insulin, as large a number of animals as possible should be used. The convulsions are antidoted with dextrose, and the animals if in good condition can be used repeatedly. We usually allow the rabbit one week to recuperate after the test. The Shaffer-Hartman method is