196 HEMOGLOBIN. 



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 fiask or beaker, and about three times their volume 

 of distilled water is added. The mixture is heated to 55 C., the solu- 

 tion filtered ; the filtrate is cooled to C., and to every four volumes 

 one volume of absolute alcohol, cooled to C., is added. The mixture 

 is then cooled to - 5 C. or - 10 C. 



When the oxyhsemoglobin 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 haemoglobin obtained by these processes may be employed 

 to make standard solutions of the body, or it may be dried. It is very 

 questionable, however, whether the refcrystallising of oxyhaemoglobin is 

 advisable, for reasons to be stated below, it being probably better to purify 

 the crystals by repeated washings with ice-cold water. Hoppe-Seyler 

 states that oxyhsemoglobin can only be dried, without decomposition, in 

 vacuo, at a temperature under 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 oxyhaemoglobin 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- 

 hsemoglobin thus prepared was entirely soluble in distilled water, and 

 that the solution was not precipitated by lead acetate ; proving that no 

 methaemoglobin had been formed. 



Haemoglobin which has been dried in vacuo, over sulphuric acid or 

 phosphoric anhydride, at a temperature of C., may be heated to 110 C. 

 or 115 C., without undergoing any decomposition. 



Modifications of Hoppe-Seylers method. (a) Among numerous modi- 

 fications may be mentioned one employed by Hlifner, 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. 



(&) 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 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 C., a 

 fourth of its volume of absolute alcohol at 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 Grosse des Hamoglobin-moleciils," Ztschr. /. physiol. Chem., Strassburg, 

 1886, Bd. x. S. 15-34. See "Darstellung des Hamoglobins, ' S. 18-24. 



2 " Beitrag zur Lehre von Blutfarbstoffe, " Beitr. z. Physiol. C. Ludioig s. s. 70 Gelurtst. 

 etc., Leipzig, 1887, S. 74-81; and "Neue Versuche, u.s.w.," Arch. f. Physiol., Leipzig, 

 1894, S. 134-136. 



