PURTFICA TION OF HjEMO GL OB IN. 1 9 7 
(c) Defibrinated blood is treated with about one-sixteenth its 
volume of ether (say 31 c.c. of ether to 500 c.c. blood), and the mixture 
shaken for some minutes. It is then set aside in a cool place. After a 
period, varying from twenty-four hours to three days, the liquid has 
been converted into a thick magma of crystals. These may be separated 
by placing in tidies and using the centrifugal apparatus. The cakes of 
crystals are treated with a mixture of one part of absolute alcohol and 
four parts of distilled water, and again centrifugalised. By repeating 
this process the crystals are ultimately obtained free from serum 
albumin. The crystals may be dissolved in water and recrystallised, as 
described in Hoppe-Seyler's method. 
In addition to the methods described, many others have been 
suggested, and to these only a passing reference need be made. 
Thus Kiihne devised a method based upon the fact that the stroma 
of the coloured corpuscles is dissolved by the addition of a watery 
solution of crystallised- bile (a mixture of sodium glycocholate and 
taurocholate). 1 Hiifner 2 and his pupil Otto employed a 1 per cent, 
alcoholic solution of chinoline, or a watery solution of the hydrochlorate 
of the same base, to prepare oxyhemoglobin from pig's blood, though 
Otto afterwards found 2 that, by taking special precautions, Hoppe-Seyler's 
method is available, even in the case of pig's blood, and indeed preferable 
to all others. 
Remarks on the purification of haemoglobin. — It has, until lately, 
been assumed that in the preparation of pure oxyhemoglobin the body should 
be recrystallised as frequently as possible, with the object of getting rid of all 
traces of adherent albuminous and saline impurities derived from the plasma 
or serum. Since spectrophotometry has supplied us with a method of deter- 
mining, with an accuracy previously unattainable, the purity of a colouring 
matter, it has been found that although oxyhemoglobin which has been 
recrystallised, when examined in the ordinary manner, exhibits a spectrum 
which appears identical with that of the colouring matter which has been only 
once crystallised, its spectrophotometry constants have changed ; in other 
words, when oxyhemoglobin is recrystallised it undergoes a change, possibly 
only affecting its physical, but more probably affecting its chemical constitution 
also. The knowledge of these facts has caused Hiifner in his recent researches 
to employ haemoglobin which has not been recrystallised. 
If precautions are taken in the first instance to separate (by the most perfect 
filtration, followed by prolonged centrifugalising) all formed elements and acci- 
dental solid impurities from the solution of blood corpuscles which is to be 
crystallised, and if the crystalline mass of oxyhemoglobin obtained be repeatedly, 
say live or six times, treated with ice-cold water, the resulting solution being 
each time separated from the undissolved crystals by very rapid and very 
prolonged centrifugalising, the portion of the original crystals still left undis- 
solved will be found, on chemical, microscopical, and spectro photometric 
investigation, to furnish evidence of being a pure substance. 
The new method is more easily and much more expeditiously carried out 
than the old. 
Elementary composition of oxyhemoglobin dried at 110°-115° C. 
— Before describing either the physical or chemical properties of the 
1 C'entralbl. f. d. vied. Wissmsch., Berlin, S. 833. 
2 The account of Hiifher's discovery of this method is contained in a paper by his pupil, 
F. Otto, "Ueberdas Oxyhemoglobin des Schweines," Ztschr. f. physiol. Chem., Strassburg, 
1882-83, Bd. vii. S. 57. 
