PREPAR. I TION OF 0XY1I. I.MOGLOBIN. 195 
A. Schmidt' made the same observation in reference to the blood near 
the positive pole, when this liquid is subjected to the action of a 
constant current. 
Without undergoing any other treatment, the blood of asphyxiated 
animals often crystallises. Blood which has been deprived of its gases, 
by boiling in vctcuo, often crystallises, [ndeed, by this method, Preyer 2 
succeeded in the very difficult task of crystallising sheep's blood. 
Methods of preparing considerable quantities of oxyhemo- 
globin. — Certain of the methods already recommended for the prepara- 
tion of haemoglobin^ when small quantities only are needed for the 
purposes of microscopic investigation, might be employed in the pre- 
paration of larger quantities. Other processes, however, are to be 
preferred, and of these sonic which are specially to be recommended 
are given below. Of these processes, the first, or Hiifner's modification 
of it, should, by preference, be employed, especially if a preparation, as 
free as possible from products of decomposition, be desired. 3 
Hoppe-Seylers method. 4 " — Defibrinated blood is mixed with ten times 
its volume of a solution of sodium chloride 5 (made by diluting one 
volume of a saturated solution of XaCl with nine volumes of water), 
and the mixture is poured into shallow basins, which are set aside in a 
cool place, so as to allow the greater part of the blood corpuscles to 
settle. 6 The supernatant liquid is decanted, and the magma of corpuscles, 
mixed with a small quantity of water, is poured into a stoppered 
separating funnel. The contents of this funnel are treated with an 
equal volume of ether. After repeated, but not too violent, agitation, 
the deep red aqueous solution is separated from the supernatant ether, 
and filtered as quickly as possible. The clear red filtrate cooled to 0° C. 
is then mixed with one-fourth of its volume of absolute alcohol, likewise 
cooled to 0° C. The mixture is then maintained for a couple of days 
(and, if crystallisation has not occurred, even longer), at a temperature of 
— 5° C. to - 10° C. In a period varying between twenty-four and 
forty-eight hours crystallisation has usually occurred, and, unless the 
solution of haemoglobin was too dilute, the whole of the liquid has set into 
a mass of crystals. The crystals are now collected on a filter (the process 
of filtration being carried on at as low a temperature as possible, in any 
case below 0° C.) and washed several times with a previously cooled 
mixture, composed of one volume of absolute alcohol and four volumes 
of distilled water. The filter with its contents is now placed between 
sheets of filtering paper, and as much as possible of the adhering mother- 
liquor is removed by gentle pressure. The oxyhemoglobin thus 
1 "Zur Krystallisation ties Blutes," Virchovis Archiv, 1864, Bd. xxix. S. 29. 
2 "Die BlutkiTstalle/' S. 19 and 20. 
3 The blood of the dog, and especially of the horse, are to be preferred for the preparation 
of large quantities of oxyhemoglobin. As the success of the various operations depends 
upon their being conducted at a low temperature, the preparation of haemoglobin lor purposes 
of research should only be attempted in the depth of winter. 
4 " Beitrage zur Kenntniss des Blutes des Menschen und der "Wirbelthiere," Med. 
Chem. Untersuch., Berlin, 1866, S. 170, 180-185; " Haudbuch d. physiologisch. cheni. 
Analyse," Berlin, 1893, Aufl. 6, S. 274. 
5 In the preparation of hemoglobin from the blood of birds, amphibia, and fish, sodium 
sulphate is to be employed in the place of sodium chloride. In the case of mammalian 
blond, it presents no advantages over sodium chloride. 
6 Instead of allowing the corpuscles to separate, as described, it is preferable to employ a 
centrifugal machine. The separation of the corpuscles from the mixture of serum and salt 
solution is not only very much more rapid, but also much more complete, and therefore the 
obtaining of pure oxyhemoglobin is facilitated. 
