196 H. £J/0 GL OB IN. 
obtained may now be purified by being recrystallised. With this object 
the moist crystals are removed by means of a spatula from the filter, 
and placed in a flask or beaker, and about three times their volume 
of distilled water is added. The mixture is heated to 55 1 1, the solu- 
tion filtered : the filtrate is cooled to 0° C, and to every four volumes 
one volume of absolute alcohol, cooled to 0° C, is added" The mixture 
is then cooled to - 5° C. or - 10° C. 
When the oxyhemoglobin separates again, this process of crystallisa- 
tion may be repeated five or even six times, providing the temperature 
at which the various operations are conducted be a very low one. The 
recrystallised hemoglobin obtained by these processes may be employed 
to make standard solutions of the body, or it may lie dried. It is very 
questionable, however, whether the recrystallising of oxyhemoglobin is 
advisable, for reasons to be stated below, it being prol >ably better to ] nirify 
the crystals by repeated washings with ice-cold water. Hoppe-Seyler 
states that oxyhemoglobin can only be dried, without decomposition, in 
vacuo, at a temperature under 0° C. If dried at a higher temperature it 
assumes a dark colour, and ceases to be entirely soluble in distilled water. 
Zinoffsky, 1 who worked with oxyhemoglobin prepared from the 
blood of the horse, found that, when spread out in very thin layers, it 
could be dried in vacuo in eight hours, without undergoing decomposi- 
tion, at a temperature of 10° C. to 20° C. He found that the oxy- 
hemoglobin thus prepared was entirely soluble in distilled water, and 
that the solution was nut precipitated by lead acetate ; proving that no 
metluemoglobin had been formed. 
Haemoglobin which has been dried in cm- no, over sulphuric acid or 
phosphoric anhydride, at a temperature of 0" G, may be heated to 110 c C. 
or 115' C, without undergoing any decomposition. 
Modifications of Hoppe-Seyh r's method. — (a) Among numerous modi- 
fications may be mentioned one employed by Hiifner, 2 and which may 
with advantage be adopted in laboratories provided with centrifugal 
machines. The blood is not treated with salt solution, but the corpuscles 
are separated by the action of the centrifuge alone. Crystals thus 
obtained are treated with ice-cold water, separated by the centrifuge, 
and this process repeated several times. Finally, the crystals are dried 
on porous plates made of cellulose, or solutions are made of the yet 
moist crystals, and the percentage of haemoglobin in them determined. 
(b) The defibrinated blood of the dog is mixed with its own volume 
of distilled water, and the diluted fluid is treated with one-fourth its 
volume of alcohol. The mixture is kept for twenty-four hours, at a 
temperature which must be lower than 0° C. The crystals which 
separate are dissolved in about three times their bulk of distilled water, 
at a temperature of 30° C, and the solution being cooled to 0° C, a 
fourth of its volume of absolute alcohol at 0° C. is added. The fluid 
should be kept in a freezing mixture at a temperature of - 10° C. to 
- 20° C. for twenty-four hours. The whole fluid then becomes con- 
verted into a magma of crystals. The process of recrystallisation may 
be several times repeated. 
1 ••Ueber die Grbsse des Hainoglobin-nioleculs." Zt*chr. f. physiol. Chem., Strassbirrg, 
L886, Bd. x. S. 15-34. See ' ; Darstellung des Hamoglobins," S. 18 24. 
- "Beitragzur Lehre von Blutfarbstotfe, " Beitr. z. Physiol. ft Ludwig z. s. 70 Geburtst. 
etc., Leipzig, 18S7, S. 74-31 ; and "Xeue Versuche, u.s.w.," Arch. f. Physiol., Leipzig, 
1894. S. 134-136. 
