430 Histidine Preparation 
is removed and the mixture allowed to cool: It is then mixed 
with an equal volume of saturated sodium carbonate, pouring 
the acid mixture slowly into the carbonate solution, with thorough 
mixing. The vessel for this should have a capacity of 40 or 50 
liters to allow for the effervescence of such a viscous mass. Fi- 
nally, after the foam has settled, which requires a few hours, the 
mixture should be made exactly neutral to litmus paper and fil- 
tered. If it has not been carefully neutralized, this filtrate will 
have a reddish purple muddy color instead of a clear yellow. 
The filtrate is now made alkaline with 25 gm. of NaOH and boiled 
-to expel ammonia. After an hour of boiling, it may be necessary 
to add more NaOH to make sure that all the ammonia will be 
driven off. Any NH,Cl left in the solution will interfere in all 
subsequent treatments of the precipitates and filtrates, and will 
eventually have to be separated from the final product. 
After standing for several hours in the cold, the leucine and 
tyrosine will separate out, and should be filtered off. The filtrate 
should then be treated, alternately, with concentrated solutions of 
HgCl, and of NagCOs;, using the NagCQO; to keep the mixture alkaline 
as more HgCl, is added. Continue this until a small filtered por- 
tion, which should be distinctly alkaline to phenolphthalein, 
does not produce the heavy voluminous precipitate of mercuric 
histidine when treated with more of the HgCl, solution. An 
opalescent or milky precipitate will continue to form even after 
all the histidine is precipitated, but should be disregarded.’ Now 
filter off the large volume of precipitate and wash by suspending 
' the latter in a 2 liter quantity of distilled water and again filter- 
ing. This process is repeated a second time to insure the re- 
moval of substances in the original solution, as well as the excess 
of HgCl,. Suspend the twice washed precipitate in five times its 
volume of water and, using concentrated HCl, render the latter 
acid to bromophenol blue (pH = 3). If the dilution is greater 
than five volumes of water, the same volume of concentrated 
HCl would be required, but the pH would be higher; and since 
both the dilution and the pH are more easily measured than the 
strength of the acid and the volume of precipitate, this method is 
suggested. At such a dilution, and pH, the mercuric histidine 
_ will be redissolved, while a large volume of a dirty brown residue 
should remain in suspension. The latter is then removed by 
