SINCE PREYER'S INVESTIGATIONS. 113 



and the whole covered with a cover-glass and sealed with asphalt or balsam. 

 Crystals from human, horse, guinea-pig, and rat blood were obtained by 

 the above methods. 



Von Stein's methods were extended by Smreker and Zoth (Sitzungsber. 

 d. Wiener Acad., 1886, xcni, Abth. in; Maly's Jahr. ii. d. Fort. d. Thier- 

 chemie, 1886, xvi, 102), who used Canada balsam, turpentine, Peru and other 

 balsams; solutions of colophony, damar, and mastic dissolved in xylol; 

 fixed oils; xylol solutions of rosin; fatty acids, etc. 



The doubt as to whether or not hemoglobin is a chemical individual, 

 together with the fact of the discrepancies in the centesimal analyses of 

 hemoglobin, led Zinoffsky (Zeit. f. physiol. Chemie, 1886, x, 16) to prepare 

 crystals of hemoglobin in several ways and to make careful determinations 

 of the iron and sulphur contents. In preliminary experiments he found 

 that the washing of the corpuscles by common salt solution, according to 

 the directions of Hoppe-Seyler, is not only superfluous, but also undesir- 

 able, because the washing introduces the danger of decomposition, owing to 

 the fact that from 3 to 5 days are required in the process, and because it 

 is not of importance in removing the small quantity of protein in solution. 

 In experiments in relation to the separation of the hemoglobin from the 

 stromata he found that, when the corpuscle pulp is heated to 35 with 3 

 volumes of distilled water, the hemoglobin dissolves and crystallizes and that 

 the stromata remain undissolved and cling so tenaciously to the hemoglobin 

 crystals that they can not be removed by nitration. They must, therefore, 

 be dissolved before the crystallization of the hemoglobin, either (1) by the 

 addition of very little ammonia to the fluid heated to 35, which must 

 then be carefully neutralized with dilute hydrochloric acid (according to 

 the direction of Schmidt), or (2) by the addition of ether (30 c.c. of ether 

 being sufficient for 9 liters of blood). To crystallize the hemoglobin the 

 solution was cooled to 0, mixed by titration with one-fourth its volume 

 of absolute alcohol, and left standing for 72 hours. The crystals were 

 washed by decantation with a mixture of 1 part of alcohol to 4 parts of 

 water cooled to 0. To obtain pure crystals, the crystals were dissolved 

 in 3 volumes of distilled water at 35, the solution was filtered, and the 

 filtrate was mixed with dilute alcohol as before. Tests showed that two 

 recrystallizations of the first product sufficed to obtain pure crystals. 



Zinoffsky also makes note of the fact that the drying of the crystals in 

 vacua at 0, according to the directions of Hoppe-Seyler, is an exceptionally 

 lengthy process, and that the crystals can be dried in about 8 hours at 18 

 to 20 without being placed in a vacuum. After these preliminary investi- 

 gations he prepared crystals from horse blood by three methods, viz : 



First method: 20 liters of horse's blood were defibrinated; the blood- 

 corpuscle pulp, which after 3 hours' standing in the cold had been deposited, 

 was separated from the serum and mixed with 8 volumes of a 2 per cent 

 solution of common salt. After 3 days the corpuscles were collected and 

 placed in 3 volumes of distilled water at 35, to which were then added 

 16 c.c. of one-tenth normal ammonia solution. After 5 minutes the ammonia 

 was neutralized by titration with a very dilute hydrochloric acid. The 



