UROBILIN. 621 



employed. When a urine rich in urobilin is saturated with ammonium 

 sulphate (best after previous removal of the urates by preliminary saturation 

 with chloride of ammonium), and acidified with sulphuric acid, it will yield 

 the pigment when shaken with a mixture of ether and chloroform. From this 

 organic solvent distilled water will again remove all the urobilin, and from 

 the water it may be precipitated by the further use of ammonium sulphate. 

 A method of separation may be based upon these facts which will yield a 

 very pure product in comparatively large amount. 1 



Properties, Urobilin is an extremely soluble substance, dissolving 

 freely in all ordinary solvents. It is, however, proportionately less 

 soluble in water than is urochrome, though much more readily soluble 

 than the latter in alcohol and other organic liquids. Its solutions, when 

 concentrated, have a brown colour ; when more dilute tbey are yellow ; 

 on great dilution they exhibit a highly characteristic change to a dull 

 pink colour. 



An alcoholic solution of the pure pigment free from extraneous acid 

 or alkali exhibits a green fluorescence quite apart from the addition of 

 reagents. When, however, zinc chloride and ammonia are added, a 

 greatly increased fluorescence is produced. This striking reaction is of 

 much value in the identification of urobilin ; it may be obtained after 

 great dilution. 



Solutions of urobilin exhibit very definite spectroscopic phenomena. 

 In clear acid solutions of moderate strength, a single absorption-band is 

 seen between tbe Fraunhofer lines I and F, slightly overlapping the 

 latter ; situate, therefore, at the junction of the green and blue of the 

 spectrum (Fig. 57, Spectrum 4). In highly concentrated solution this band 

 is lost in a general absorption of the more refrangible rays. On diluting 

 such a concentrated solution a broad band first appears with a region of 

 complete blackness towards red, and a dark shading towards violet. As 

 dilution proceeds the shading first disappears, and then the dark portion 

 of the band shrinks till its limits extend from about X 508 to X 486. 

 After this the width of the band is constant, until with very large 

 dilution it grows faint and ultimately disappears (Fig. 57, Spectrum 5). 

 The activity of the pigment in absorbing light in this region is enormous, 

 and a solution so dilute as to have a very faint colour indeed, will show 

 a well-marked band. An absorption-band of an intensity such as is 

 occasionally seen in normal urine, would correspond to that of an 

 almost colourless solution of the pure substance. 



Urobilin, like most animal pigments, shows acidic tendencies, and 

 forms compounds with bases, being liberated from these combinations 

 on the addition of an acid. 



If ammonia be added to a solution of the free pigment, the colour 

 changes to a canary-yellow, and unless the solution be very strong the 

 absorption-band disappears. The sodium and potassium compounds 

 have a colour in solution more like that of the free pigment, and show 

 an analogous band, which is situate, however, somewhat nearer the red. 

 The zinc compound in ammoniacal solution fluoresces, as we have 

 already stated, and shows with the spectroscope a band almost identical 

 with that of the potassium and sodium compounds. The calcium com- 

 pound is yellow in solution and shows no band. Mercury forms a pink 

 compound, with a band nearer to the red than any of those previously 

 referred to. A solution of mercuric chloride will develop a pink colour 

 ] Garrod and Hopkins, Journ. Physiol., Cambridge and London, 1896, vol. xx. p. 120. 



