UROBILIX. 621 
employed. When a urine rich in urobilin is saturated with ammonium 
sulphate (best after previous removal of tin- urates by preliminary saturation 
with chloride of ammonium), and acidified with sulphuric acid, it will yield 
th^ pigment when shaken with a mixture of ether ami chloroform. From this 
organic solvent distilled water will again remove all the urobilin, and from 
the water it may he 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 urocbrorne, 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 they 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 he 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 the Fraunhofer lines b 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 \ 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 
1 Garrod and Hopkins, Journ. Physiol., Cambridge and London, 1896, vol. xx. p. 120. 
