BILIRUBIN. 407 



alkali bilirubin in water is treated with an excess of ammonia and then 

 with a zinc-chloride solution, the liquid is first colored deep orange and 

 then gradually olive-brown and then green. This solution first gives 

 a darkening of the violet and blue part of the spectrum, and then the bands 

 of alkaline cholecyanin (see below), or at least the bands of this pigment 

 in the red between C and D, close to C. This is a good reaction for 

 bilirubin. The compounds of bilirubin with alkalies are insoluble in 

 chloroform, and bilirubin may be separated from its solution in chloro- 

 form by shaking with dilute caustic alkali (differing from lutein). Solu- 

 tions of alkali bilirubin in water are precipitated by the soluble salts of 

 the alkaline earths and also by metallic salts. 



As EHRLICH first showed, bilirubin forms combinations with diazo 

 compounds, which have been closely studied by PROSCHER, ORNDORFP 

 and TEEPLE. 1 A test suggested by EHRLICH for bilirubin is based upon 

 this behavior with diazobenzenesulphonic acid. 



If an alkaline solution of bilirubin be allowed to stand in contact 

 with the air, it gradually absorbs oxygen, and green biliverdin is formed. 

 This process is accelerated by warming. According to KUSTER, in this 

 case the alkali also has a splitting action upon the pigment, and among 

 the products formed we find haematinic acid. Biliverdin is formed only 

 from bilirubin by oxidation under special conditions (KUSTER). A green 

 coloring-matter similar in appearance is formed by the action of other 

 reagents such as Cl, Br, and I. According to JoLLES, 2 biliverdin is 

 produced by the use of HUBL'S iodine solution, while according to others 

 (THUDICHUM, MALY S ) substitution products of bilirubin are formed. 



GMELIN'S Reaction for Bile-pigments. If one carefully pours nitric acid 

 containing some nitrous acid, under an aqueous solution of alkali biliru- 

 bin. there is obtained a series of colored layers at the juncture of the two 

 liquids in the following order from above downward : Green, blue, violet, 

 red, and reddish yellow. This color reaction, GMELIN'S test, is very delicate, 

 and serves to detect the presence of one part bilirubin in 80,000 parts 

 liquid. The green ring must never be absent; and also the reddish- 

 violet must be present at the same time, otherwise the reaction may be 

 confused with that for lutein, which gives a blue or greenish ring. The 

 nitric acid must not contain too much nitrous acid, for then the reaction 

 takes place too quickly and it does not become typical. Alcohol must 



1 Ehrlich, Zeitschr. f. anal. Chem., 23; Proscher, Zeitschr. f. physiol. Chem., 39; 

 Orndorff and Teeple, 1. c. 



2 Kiister, Ber. d. d. chem. Gesellsch., 35 and 59; Jolles, Journ. f . prakt. Chem. (N. F.), 

 59, and Pfliiger's Arch., 75. 



3 Thudichum, Journ. of Chem. Soc. (2), 13, and Journ. f. prakt. Chem. (N. F.), 

 53; Maly, Wien. Sitzungsber., 72. 



