400 Dr. A. E. Garrod. A Contrilntiuu to the Study [Apr. 2 



viously contained in the extract, the bulk of the dissolved ammonion 

 sulphate being separated with it. 



Stage III. The lower layer having been removed, the alcoholi 

 extract is now evaporated to dryness over a water bath, a few dropi 

 of ammonia being added from time to time so as to maintain ai 

 alkaline reaction. 



This precaution is rendered necessary by the presence in the extrac 

 of a considerable quantity of indoxyl sulphate, which is otherwiw 

 apt to be decomposed during the evaporation, with the formation 

 indigo pigments. 



Such decomposition cannot take place if the liquid be kept alkali 

 for, as Baumann showed, the indoxyl sulphates may be boiled wi 

 caustic alkalies without undergoing change ; and, far from produci 

 any alteration in the yellow pigment, the ammonia tends to prese 

 it from changes to which it is otherwise liable. 



A brown residue remains after the evaporation is complete, w 

 has a treacly consistence, but solidifies on cooling. This resid 

 which emits a powerful urinous odour, and contains some ammoni 

 sulphate, is washed once or twice with acetic ether, which remo 

 the bulk of the indoxyl sulphate, and comparatively little of 

 yellow pigment. It is then transferred to a stoppered bottle a 

 allowed to soak for some hours in absolute alcohol. On filtering 

 beautiful orange-coloured alcoholic solution is obtained, but some 

 the pigment escapes solution and may be in part removed by a 

 soaking in fresh alcohol. 



Water dissolves the undissolved residue readily and complete! 

 and if the aqueous solution so obtained is treated like the origi 

 urine by saturation with ammonium sulphate and extraction wi 

 alcohol, a further supply of absolute alcoholic solution may be o' 

 tained from it, which has the advantage of being free from indo: 

 sulphate. 



It is probable that some of the pigment has undergone a sligl 

 change which renders it very sparingly soluble in alcohol. 



Stage IV. The alcoholic solution, which, when treated in the co 

 with hydrochloric acid and a trace of chloride of lime, still yields 

 indigo reaction, is next concentrated, if necessary, until it has a 

 orange colour. It is then poured into rather more than its own bul 

 of ether, whereupon much of the pigment is precipitated in, 

 amorphous state, and may be collected upon a filter which has 

 first moistened with pure ether to ensure rapid filtration. 



The presence of a very little water prevents the precipitation, 

 few drops of a very concentrated aqueous solution separating out 

 passing through the filter. 



If the various stages of the process have been carefully follow ed, 

 and especially if the second separation of the alcoholic extract frpm 



I 



