Two grammes of the fatty acids are now dissolved in neutral alcohol saturated with some solvent, preferably a light fraction of benzoline, a quantity of the solvent added to take up the unsaponifiable matter, and the whole boiled under a reflux condenser. After cooling, the liquid is titrated with N/2 aqueous KOH solution, using phenol-phthalein as indicator, this figure giving the amount of the total fatty acids present. The whole is then poured into a separating funnel, when separation immediately takes place. The alcoholic layer is withdrawn, the benzoline washed with warm water (about 32 deg. C.) followed by neutral alcohol (previously saturated with the solvent), and transferred to a tared flask, which is attached to a condenser, and the benzoline distilled off. The last traces of solvent remaining in the flask are removed by gently warming in the water-oven, and the flask cooled and weighed, thus giving the amount of unsaponifiable matter. _Constitution of the Unsaponifiable Matter._--Unsaponifiable matter may consist of cholesterol, phytosterol, solid alcohols (cetyl and ceryl alcohols), or hydrocarbons (mineral oil). Cholesterol is frequently found in animal fats, and phytosterol is a very similar substance present in vegetable fats. Solid alcohols occur naturally in sperm oil, but hydrocarbons, which may be generally recognised by the fluorescence or bloom they give to the oil, are not natural constituents of animal or vegetable oils and fats. The presence of cholesterol and phytosterol may be detected by dissolving a small portion of the unsaponifiable matter in acetic anhydride, and adding a drop of the solution to one drop of 50 per cent. sulphuric acid on a spot plate, when a characteristic blood red to violet coloration is produced. It has been proposed to differentiate between cholesterol and phytosterol by their melting points, but it is more reliable to compare the crystalline forms, the former crystallising in laminae, while the latter forms groups of needle-shaped tufts. Another method is to convert the substance into acetate, and take its melting point, cholesterol acetate melting at 114.3-114.8 deg. C., and phytosterol acetate at 125.6 deg.-137 deg. C.