112 PREPARATION AND CRYSTALLOGRAPHY OF HEMOGLOBINS 



(Zeit. f. phys. Chemie, 1878, 11, 149) that the crystals of horse hemoglobin 

 appear to be of two kinds (needles and prisms) which differ in solubility 

 a difference which Hoppe-Seyler thought likely to be due to differences in 

 the amount of water of crystallization. Otto states that there continually 

 appeared, besides little needles which are in relatively greater abundance in 

 recrystallization, long, thick prisms which prevail in the first crystallization. 

 He endeavored, but failed, to separate the needles from the prisms by wash- 

 ing with dilute alcohol, as Hoppe-Seyler states could be done. He also 

 tried to determine differences in the water of crystallization, but he failed 

 to obtain concordant results. 



Crystals from horse's blood were prepared by Hiifner and Biicheler 

 (Zeit. f. phys. Chemie, 1884, viu, 355) by the ordinary alcoholic method 

 and recrystallized three times in a refrigerator. Generally needles were 

 obtained from 2 to 3 mm. long and 0.5 mm. wide. Once they found hex- 

 agonal tablets of reduced hemoglobin, which changed quickly upon coming 

 in contact with the air. Dried at over sulphuric acid and anhydrous 

 phosphoric acid, the crystals retained 3.94 per cent of water, which came 

 off when the crystals were subjected to a stream of hydrogen at 115. They 

 made elementary analyses (page 71), calculated the molecular weight and 

 formula (page 75), and determined the oxygen capacity. 



A new method for preparing hemoglobin crystals was reported by von 

 Stein (Centralblatt f. med. Wissensch., 1884, xxn, 404; Archiv f. path. 

 Anat. u. Physiologic, 1884, xcn, 483), which is applicable to small quan- 

 tities of blood that are readily crystallizable. A drop of defibrinated blood 

 or blood squeezed from a clot was placed on an object-glass and exposed to 

 the air until it began to dry up at the margins. Canada balsam was then 

 applied, first around the drop of blood, in order to prevent any possible 

 escape, and then the remaining space above it was filled. It is to be observed, 

 von Stein states, that the center of the drop of blood is pushed off to the 

 periphery. In this way a clear space is made for crystallization, otherwise 

 the crystals are so small that their outlines can not be made out. Too thick 

 a layer of blood is to be avoided, because the balsam does not penetrate to 

 the deeply lying portions. Von Stein proceeded in another way, by not 

 allowing the blood to evaporate, and by treating it immediately with the 

 reagent and covering the mixture with a cover-glass. Canada balsam is 

 best when it appears yellow and not entirely clear. In liquid balsam the 

 crystals form more quickly, and sometimes have larger dimensions, but 

 they soon become brown (in one or two days), then dull and black, and in a 

 short time are fissured to small pieces. Preparations can be made which 

 retain their form and color for years if the balsam has been exposed to the 

 air for a long time, or is evaporated to such a consistence that it can be 

 drawn out into transparent but not milky threads when lifted with a glass 

 rod. Whichever method is used, it is important that the preparation be left 

 uncovered in the air until the crystallization has been completed, and until 

 the odor of the balsam has completely disappeared, which lasts ordinarily 

 a few days. Then with a knife immersed in ether, turpentine, or oil of cloves 

 (little should be used of either), the upper portion of the balsam is removed, 



