In a large round-bottomed flask are placed 1.4 kg. of dried blood corpuscle paste (Note 1) and 4.5 l. of concentrated hydrochloric acid (sp. gr. 1.18). The flask is warmed on a steam bath until the protein has dissolved, and the mixture is boiled gently under reflux for eighteen to twenty hours. After the hydrolysate has been concentrated under reduced pressure to a thick paste, the distillation is continued with the slow addition of a liter of water, thus eliminating most of the excess hydrochloric acid. The residue is taken up in 8 l. of warm water, cooled, and neutralized to pH 4.4–4.6 (methyl orange or bromcresol green) by the addition of concentrated sodium hydroxide solution (Note 2). After the mixture has stood overnight, the precipitated pigment is removed by filtering through a large Büchner funnel fitted with two layers of filter paper and a 2-mm. layer of infusorial earth. The filtrate, which is dull red in color, is decolorized by warming and stirring for ten minutes with 60 g. of Norite.

The pale yellow filtrate and washings from the Norite are diluted to 25 l. with tap water, and a solution of 600 g. of mercuric chloride in 2 l. of hot 95 per cent alcohol is added, with stirring. A concentrated solution of sodium carbonate (corresponding to about 350 g. of anhydrous sodium carbonate) is added slowly, with stirring, until the mixture reaches pH 7.0–7.5 (phenol red or litmus). After settling for several hours, preferably overnight, the supernatant liquid is siphoned off, and the crock is refilled with water to the original volume (Note 3). The mixture is allowed to settle, the wash liquid siphoned off, and the precipitate washed twice more in the same fashion. After the third washing, the supernatant liquid is siphoned off and the precipitate collected with suction on a large Büchner funnel fitted with two layers of filter paper and a 2-mm. layer of infusorial earth.

The moist histidine-mercury complex is suspended in 5 l. of water and stirred vigorously while a stream of hydrogen sulfide is introduced. When precipitation of mercuric sulfide is complete, the suspension becomes uniformly black and settles sharply on standing. The filtrate and washings from the mercuric sulfide (Note 4) are concentrated under reduced pressure to a volume of about 1 l. and cleared with 5 g. of Norite. The filtrate and washings from the Norite are concentrated further to a volume of about 250 cc. and mixed with three volumes of 95 per cent alcohol. Crystallization is induced by cooling the solution and scratching the walls of the vessel; the histidine monohydrochloride separates in plates. After the material has remained in an ice chest for three or four days, the crystals are separated by suction filtration. The yield of crude histidine monohydrochloride is 85–90 g. The filtrate, on standing for several weeks in an ice chest, usually deposits an additional 4–5 g. of material.

The crude product is dissolved in five times its weight of water, and, after clearing with a little Norite, the solution is diluted with one and one-half volumes of 95 per cent alcohol. The product separates in well-formed, snow-white crystals, and after standing for several days in an ice chest is collected with suction on a Büchner funnel. The yield of purified histidine monohydrochloride is 75–80 g. (Note 5). The compound melts at 251–252°, with decomposition. The amino acid is not racemized by the procedure employed, and it shows the characteristic optical activity, [α]_D26° = +8.0°, in the presence of three moles of hydrochloric acid. The recrystallized product is usually analytically pure and shows the correct Van Slyke amino nitrogen content. Occasionally a second recrystallization is necessary to obtain analytically pure material.