178 ESSENTIALS OF CHEMICAL PHYSIOLOGY 



so-called ' pathological urobilin ' described by previous writers is ordinary 

 urobilin incompletely separated from various impurities. 



The following are the raethods introduced by Garrod and Hopkins for its 

 separation from the urine : — 



(a) The urine is first saturated with ammonium chloride, and the urate 

 so precipitated is filtered off. The filtrate is then acidified with sulphuric 

 acid and saturated with ammonium sulphate. This causes a precipitate of 

 urobilin, which may be collected and dissolved in water. The aqueous 

 solution is again saturated with ammonium sulphate, and the pigment 

 is thus precipitated in a state of purity. 



(6) The urates are first removed, then the urine is acidified and saturated 

 with ammonium sulphate as before. The urobilin is then extracted from the 

 mixture by shaking it with a mixture of chloroform and ether (1 : 2) in a 

 large separating funnel. The ether-chloroform extract is then rendered 

 faintly alkaline and shaken with distilled water, and the urobilin passes into 

 solution in the water. The aqueous solution is now once more saturated 

 with ammonium sulphate and slightly acidified ; it then once more yields its 

 pigment to ether chloroform. 



By means of either of these methods urobilin is obtained in a pure con- 

 dition ; even normal urine will give some, for the chromogen is partly con- 

 verted into the pigment by the acid employed. 



Urobilin dissolved in alcohol exhibits a green fluorescence, which is 

 greatly increased by the addition of zinc chloride and ammonia. It shows a 

 well-marked absorption band between b and F, slightly overlapping the latter 

 (fig. 63, spectrum 4). 



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

 compounds with bases ; it is liberated from such combinations by the 

 addition of an acid. 



If urobilin is dissolved in caustic potash or soda, and sufficient sulphuric 

 or hydrochloric acid is added to render the liquid faintly acid, a turbidity is 

 produced. This turbid liquid shows an additional band in the region of the 

 E line (fig. 63, spectrum 6), which is probably due to the special light absorp- 

 tion exercised by fine particles of urobilin in suspension. It wholly dis- 

 appears when the precipitate is filtered off, and when it is re-dissolved the 

 ordinary band alone is visible. 



3. Uroerythrin. — This is the colouring matter of pink urate sediments. 

 It may be separated from the sediment as follows : — The deposit is washed 

 with ice-cold water, dried, and placed in absolute alcohol. The alcohol, 

 though a solvent for uroerythrin, does not extract it from the Tirates. The 

 alcohol is poured off, and the deposit dissolved in warm water. From this 

 solution the pigment is easily extracted by amy lie alcohol. 



Uroerythrin has a great affinity for urates, with which it appears to form 

 a loose compoimd. Its solutions are rapidly decolorised by light. Spectro- 

 scopically it shows two rather ill-defined bands (fig. 63, spectrum 7). It 

 gives a green colour with caustic potash, and red or pink with mineral acids. 

 Uroerythrin appears to be a small but constant constituent of urine. Its 

 origin and relationship to other pigments are unknown. 



4. HaBmatoporpliyrin. — This also occurs in small quantities in normal 



