PURIFICATION OF HEMOGLOBIN. 197 



(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 tubes 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 Kiihiie 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 oxyhaemoglobin 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 oxyhsemoglobin 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 oxyhaemoglobin 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 spectrophotometric constants have changed; in other 

 words, when oxyhaemoglobin 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 

 nitration, 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 oxyhsemoglobin obtained be repeatedly, 

 say five 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 spectrophotometric 

 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 oxyhsemoglobin dried at 11O-115 C. 



Before describing either the physical or chemical properties of the 



1 CentralU.f. d. med. Wissenscli., Berlin, S. 833. 



2 The account of Hu'fner's discovery of this method is contained in a paper by his pupil, 

 F. Otto, "Ueber das Oxyhamoglobin des Schweines," Ztschr.f. physio!. Chem., Strassburg, 

 1882-83, Bd. vii. S. 57. 



