6 



Scientific Proceedings (30). 



This reaction is caused by the fact that thymol dissolves in 

 urine when the latter is treated with it even in solid form, and it is 

 noteworthy that more thymol is dissolved if the urine is neutral 

 or alkaline, than when acid. Accordingly, an alkaline thymolized 

 urine will give Heller's test more pronouncedly than a strongly 

 acid one. But while the thick and heavy ring in the first case will 

 have an appearance somewhat different from the protein ring and 

 thus will hardly mislead one, the delicate thymol ring in the acid 

 urine closely resembles a protein ring and is therefore more apt to 

 cause confusion. 



In urine containing both albumin and thymol in various amounts 

 each ring may be discerned. The albumin ring is somewhat the 

 wider of the two (about 2-3 mm.), and is whitish ; whereas the 

 thymol ring forms directly underneath and is grayer and thinner. 

 The albumin ring may be completely covered by the thick thy- 

 mol ring, so that the detection of protein may be seriously inter- 

 fered with. 



Further investigation proved that this ring is first formed by 

 the precipitation of thymol by the concentrated acid. At this 

 stage it closely resembles the albumin ring. Nitration of the thy- 

 mol soon occurs, resulting in the formation of nitroso- and pos- 

 sibly nitro-thymol. This accounts for the gradual color-change 

 from white to yellowish white, which the precipitate undergoes. 

 A partially successful attempt has been made to isolate the nitro- 

 substance or substances produced. With the aid of chloroform 

 as the solvent, crystals of a yellowish brown color were obtained 

 that gave Liebermann's nitroso reaction. These crystals melt at a 

 temperature slightly above 50° C, whereas nitroso-thymol melts at 

 160° C. Thymol melts at 50°. It is quite probable, therefore, 

 that very incomplete nitration occurred and that the brown crys- 

 talline product referred to was a mixture of thymol and a small 

 amount of nitroso-thymol. Besides, some chloroform may have 

 been occluded in the crystals. Work in this direction is proceeding. 



To guard against this source of error, Heller's test cannot be 

 directly applied to urine preserved with thymol. The latter must 

 first be removed by extraction. Petroleum ether is very suitable 

 for this purpose. Gentle agitation of the urine with an equal vol- 

 ume of petroleum ether in a test-tube for 2 minutes suffices to re- 

 move all traces of thymol. 



