2. Preparation and properties of isatan. 



Indoxyl and isatan are very unstable and still at present most imperfectly 

 known substances, which only in acid solutions can easily be distinguished from 

 each other, in neutral solutions, without the use of of isatase, with much more 

 trouble, in alkaline solutions not at all, because in these isatan produces indoxyl. 



The reason why at first I thought that the woad must contain free indoxyl 

 and no compound of it, is the fact that in the extracts obtained from young 

 woad-leaves, rich in isatan, as well by decoction as by cold extraction, the isatan 

 is decomposed and an indoxyl solution is obtained. Now I admitted in the be- 

 ginning, that if in the woad, as was my leading theory, a glucoside was present, 

 which, in analogy to the indican, must be decomposed by an enzyme, at the 

 decoction no indoxyl but exclusively this glucoside would be obtained, because 

 by boiling the enzyme is suddenly destroyed. In this view I was supported by 

 the fact, that this indeed takes place with Indigofera and Polygonum, which by 

 decoction yield indican, by cold extraction indoxyl. 



But I began to doubt of the generality of this theory, when observing, that 

 Phajus grandiflorus, which belongs to the indican plants, nevertheless 1 ) produces 

 indoxyl at decoction. So this seemed also possible with the woad, though it was 

 clear, that the properties of the glucoside ought in this case to be quite different 

 from those of indican. 



But I was only put on the right way, by the experience, that it is possible 

 to obtain from the leaves of the woad, by the extraction with dilute acids a 

 solution, which remains unchanged at the air, although it yields with alkalies 

 much indigo-blue, while an equally acid indoxyl solution slowly oxidizes at the 

 air to indigo. I then clearly saw why I had before obtained indoxyl from the 

 woad. My experiments had been performed on a small scale; I had been able 

 with care to select growing leaves and buds only; but they contain much isatan 

 and so little acid, that the enzyme isatase can become active, so that by decoction, 

 as well as by cold extraction with water, and even with alcohol, the produce 

 indoxyl, though at the decoction and alcohol extraction mixed with much isatan, 

 which fact I only observed later. If I had used older leaves which contain more 

 acid, I should have found at once isatan quite free from indoxyl. 



The relative constancy of isatan in feebly acid solutions, even at boiling 

 temperature can be utilized for its preparation. 



Though the acidity during the extracting must befeeble yet it must be strong 

 enough to prevent the decomposition of the isatan by the isatase. To this end 

 an acidity of 1.6 to 3.2 cc. of normal oxalic acid per 100 cc. of the extraction 

 liquid, (o.i to 0.2 weight percentage) suffices, for the acidity of the older leaves 

 themselves amounts to about 1,5 cc. normal per 100 cc. of the juice, and this 

 is the very limit of acidity above which the isatan becomes inactive. If the extraction 

 is effected by boiling, this degree of acidity should be exactly observed. In cold 

 extraction, with oxalic acid, the isatan is much less subject to decomposition, 

 so that, below 50 C. solutions of i to 3 pCt. oxalic acid can safely be employed. 



') Indigofermentation. Kon. Akad. van Wetensch., Amsterdam, Proceedings of the 

 Meeting of Maart 1900, pag. 573. 



