The treatment with lead acetate removes all tannin, phosphates, etc, and the hopresin and lupulin are also completely precipitated, while all or nearly all hop substitutes remain in solution.

The next object is to separate the sugar, dextrin, and mineral constituents of the beer as perfectly as possible from the active principles of the various hop substitutes. Instead of precipitating the carbohydrates, etc, by excess of strong alcohol, I prefer to remove the bitters from the aqueous liquid itself by agitation with suitable immiscible solvents.

The object being to extract as many active principles as possible in the simplest possible way, leaving their mutual separation and recognition for farther consideration, I employ chloroform as having the most general solvent action. It separates with tolerable ease from the aqueous liquid, and should be employed as long as it leaves a notably bitter residue on evaporation. In many of my experiments I relied on the traces of lactic and other acids naturally present in beer to produce the requisite degree Of acidity, but I am now of opinion that the addition of a little dilute sulphuric acid is advantageous, if not actually necessary, in some cases. The extraction with chloroform being complete, ether should next be used, the treatment being repeated as long as any notably bitter principle is extracted. Finally, the aqueous liquid is rendered alkaline with ammonia, and agitated with chloroform or ether-chloroform, to extract any alkaloids.

The following arrangement shows the behavior of the more important bitter principles when the aqueous liquid is agitated in succession with chloroform, ether, and ammonia, and ether-chloroform. I have personally verified the behavior of the substances to the names of which asterisks are attached.

(1) Extracted by chloroform from acid solutions:

Absinthin (wormwood). Anthemin (chamomiles). Colchicine (colchicum), imperfectly.

* Colocynthin (colocynth, or bitter apple), imperfectly.

* Calumbin, and probably some berberine (calumba), bright yellow and highly fluorescent.

* Gentipicrin (gentian), very imperfectly.

* Picric acid (artificial), yellow, imperfectly. Picrotoxin (cocculus indicus), with difficulty.

* Quassiin (quassia wood).

(2) Subsequently extracted by ether from acid solutions :

* Chiratin (chiretta).

* Colocynthin (colocynth, or bitter apple).

" Gentipicrin (gentian). Picric acid, yellow. Picrotoxin (cocculus indicus).

(3) Subsequently extracted by ether-chloroform from alkaline solutions:

* Berberine (calumba root). Colchicine (colchicum.)

By evaporating off the solvent, warming the residue with a little alcohol, and then adding water, solutions are obtained which will be bitter If any of the above substances be present. A very small quantity of the substance is required for this test; indeed, the use of too large an amount must be carefully avoided or the sense of taste will be found to be wholly paralysed for the remainder of the day at any rate.

II will be seen that chloroform or ether extracts from acidulated aqueous liquids almost the whole of the above bitters. The subsequent treatment with ether-chloroform in alkaline solution is usually unnecessary, as the principles of calumba and colchicum are in part extracted by acid chloroform. Seeing that the bitter principles of hops are entirely precipitated by neutral acetate of lead, the presence of some hop substitute is absolutely certain if the chloroform or ether residue has a marked bitter taste. This can be ascertained in the course of a few hours by the simple method above indicated, and half a pint of the beer is amply sufficient for the purpose.

The presence of a hop substitute being proved by the marked bitter taste of the chloroform or ether extract it will of course be very desirable to ascertain its nature, and in some important cases this can fortunately be effected very satisfactorily. In others we may expect future investigation to afford the necessary assistance.

I have made special endeavors to ascertain the possibility of definitely recognizing quassia, which is one of the most important of the hop substitutes actually employed.

For this purpose I prepared quassiin in a moderately pure state by exhausting quassia wood with hot water and treating the decoction with acetate of lead and chloroform, in the manner recommended for beer. The quassiin was obtained with some difficulty in a distinctly crystalline state and otherwise it presented a close general resemblance to the description of it given by other observers. The following characters and tests were specially verified :

Quassiin is intensely and persistently bitter, sparingly soluble in cold water, more readily in hot, and easily soluble in alcohol. Its best solvent is chloroform, which extracts it readily from acidulated solutions.

An aqueous solution of quassiin does not reduce Fehling's solution, or ammonio-nitrate of silver. The solid substance gives no coloration (or merely yellow) when treated with strong sulphuric acid or with nitric acid of 1. 25 specific gravity, nor is any color produced on warming. These four negative reactions are important, for picrotoxin reduces Fehling's solution and gives an orange-red color with sulphuric acid; gentipicrin and menyanthin reduce ammonio-nitrate of silver, and the former gives a red color and the latter a yellowish brown, changing to violet red when warmed with sulphuric acid, and other bitters mostly give more or less characteristic reactions.

A solution of quassiin gives a white precipitate with tannin. The reaction is used by Christensen, Oliveri, and others to isolate quassiin from its solutions, and by En-ders to separate it from picrotoxin. In my hands the reaction has not proved satisfactory. The liquid is very difficult to filter and the filtrate still retains an intensely bitter taste, showing that the precipitation is very incomplete. As an analytical method the reaction is useless, but it is of some value as a qualitative test. The test must be made in a cold solution.