In a 500-ml. flask (Note 1), fitted with a mechanical stirrer, are placed 36 ml. of water and 25 g. (0.078 mole) of finely crystalline alloxantin dihydrate (p. 42). The flask and contents are heated on a steam bath to 50°, and 3.6 ml. of fuming nitric acid (sp. gr. 1.62) is added in a fine stream while vigorous stirring is maintained and the temperature is not allowed to rise above 60° (Note 2). After all the fuming nitric acid has been added, the temperature is brought to 55° and the stirrer is stopped. In a few minutes a vigorous reaction begins, and large quantities of oxides of nitrogen are evolved. The stirrer is again started, and, if the reaction becomes too violent, the mixture is cooled somewhat; otherwise, the reaction is allowed to take its course. The reaction is complete when a current of air, introduced into the flask above the mixture, does not produce much color due to formation of nitrogen tetroxide (Note 3). The mixture is then heated to 60–65° for 10–15 minutes, whereupon practically all the solid dissolves. The reaction mixture is poured into a glass (Pyrex) tray and cooled overnight at 0° or below. The large triclinic, colorless crystals of alloxan tetrahydrate are broken up, filtered with suction, washed with ice water, and pressed as dry as possible. The crystals are then added to 25–26 ml. of hot water (Note 4), and the mixture is shaken until solution is complete. The solution is filtered immediately, and the filtrate is cooled overnight in a tray at 0°. The crystals are broken up, filtered, washed with ice water, and pressed as dry as possible. These moist crystals of the tetrahydrate, which weigh 22–24 g., are dried to constant weight in a glass tray over concentrated sulfuric acid. The resulting product is a fine white powder (Note 5) which weighs 16 g.

The mother liquor from the recrystallization is placed in a 250-ml. flask and is concentrated to a volume of 8–10 ml. under reduced pressure at not over 30–40° (Note 6). The concentrate, when cooled overnight at 0°, deposits a solid which is filtered, recrystallized from its own weight of boiling water, and dried over sulfuric acid. This solid weighs about 2 g. The mother liquors from the two crystallizations are combined with the original mother liquor from the oxidation, and the whole is evaporated to dryness under diminished pressure at not above 30–40°. This solid residue is somewhat yellow and possesses a strong odor of nitric acid. It is kept on a tray for several days until the odor of nitric acid disappears, and then it is dissolved in its own weight (2–3 g.) of boiling water, and the solution is cooled for several days below 0° (Note 7). The solid is removed, recrystallized from water, and dried over sulfuric acid (Note 8). This crop weighs about 0.5 g. The total yield of alloxan monohydrate is 18–19 g. (72–76%) (Note 9).

A. Benzalbarbituric acid. A mixture of 128 g. (1 mole) of barbituric acid and 1250 ml. of water in a 2-l. three-necked round-bottomed flask equipped with an efficient stirrer and a reflux condenser is heated on a steam bath to effect solution (Note 1). When the acid has dissolved, 115 g. (110 ml., 1.08 moles) of benzaldehyde is added while heating and stirring are continued. The solution rapidly fills with the insoluble benzalbarbituric acid. The mixture is heated for 1 hour on the steam bath to complete the reaction, and then is filtered by suction (Note 2). The filter cake is washed with several portions of hot water and dried at 100°. The yield is 190–205 g. (88–95%) of product possessing a very pale yellow color. The substance melts at 254–256° and needs no further purification.

B. Alloxan monohydrate. A mixture of 730 ml. of acetic acid, 95 ml. of water, and 162 g. (1.62 moles) of chromium trioxide (Note 3) is placed in a 2-l. three-necked round-bottomed flask fitted with a stirrer and a thermometer; the stirrer is started, and the mixture is warmed to 50°. To the solution 180 g. (0.83 mole) of benzalbarbituric acid is added in small portions during the course of 30 minutes, a cold water bath being used to maintain the temperature at 50–60°. After all the acid has been added, stirring is continued and the temperature maintained at 50–60° by a warm water bath for another 30 minutes to complete the reaction. Alloxan monohydrate generally starts to crystallize from the warm solution. The mixture is cooled to 15° and filtered. The product is washed on the filter with cold glacial acetic acid until the washings are no longer green, and then is dried by washing with ether. The yield is 105–112 g. (79–84%) of yellow crystals which melt at about 254° (Note 4) with decomposition and are sufficiently pure for most purposes.

In order to obtain practically colorless alloxan monohydrate (Note 5), 25 g. of the yellow crystals is dissolved in 37 ml. of hot water, the solution is boiled with Norit, and the hot solution is filtered into a 500-ml. round-bottomed flask. About 15–20 ml. of water is removed by distillation under reduced pressure on a water bath. The colorless crystalline residue is dissolved in the minimal volume of hot water, the solution is cooled somewhat, and to it is added 250 ml. of glacial acetic acid. After the mixture has been kept cold (5–10°) for 4–6 hours, the alloxan monohydrate is filtered. The yield is 20–21 g. (80–84% recovery) (Note 6) of practically colorless crystals which melt at about 254° (Note 7) with decomposition.