But at these low temperatures the acid penetrates with less rapidity into the cells, 

 in which accordingly the enzyme can become more or less active producing some 

 indoxyl. Hence, in the acid extraction at low temperature, it is advisable to rub 

 the leaves down in a mortar, immersed in the acid liquid. 



In particular at boiling temperature and when using an extraction liquid of 

 an acidity of 2 to 3 cc. of normal oxalic acid, it is easy to obtain a quite un- 

 decomposed isatan solution from the growing woad-leaves, even of the youngest 

 still neutrally reacting meristemes. In consequence of the boiling temperature, 

 aided by the perfect surrounding of the cells with the dilute acid, the isatase 

 is destroyed simultaneously with the dying of the protoplasm, by which decom- 

 position of isatan is quite excluded. As the extraction continues, there is an inter- 

 change between the feabler acidity within (0.5 cc. normal pCt), and the stronger 

 acidity without the young cell, and at the end of the experiment, a solution of 

 isatan of 0.5 to 2 cc. of normal acid per 100 cc. of juice in obtained, when the 

 weight of the leaves used, equals that of the extraction liquid. 



More acid used in the boiling than the said percentage causes isatan decomposi- 

 tion, by which not only indoxyl but also brown products of decomposition originate. 



Oxalic acid can be replaced by other acids and by acid salts. Thus I obtained 

 good results with dilute sulphuric acid and phosphoric acid, and with a saturated 

 solution of boric acid, at room temperature. Acetic acid causes a feebler decom- 

 position than oxalic acid. When the appearance of brown products of decom- 

 position during the boiling is taken as a criterion for the decomposition, I found 

 that 12 cc. of normal acetic acid added to 100 cc. of juice (ca. 0.8 weight percentage), 

 is about proportioned to 5 cc. of normal oxalic acid (=0.3 weight percentage). 

 Acid salts act like acids. Kaliumbioxalate and biphosphate can only be used in 

 strongly diluted solutions. With a cold saturated solution of kalium bitartrate 

 the extracting may be operated at boiling temperature without decomposition; 

 only by prolonged boiling a little indigo-blue is produced. I prefer, however, the 

 extraction with oxalic acid. Therewith the solutions remain clear and of a light 

 yellow and can very easily be filtered 1 ) ; after filtering, the remaining leaf-matter 

 is soft, but by no means slimy, and can quite well be pressed dry, so that, in 

 consequence of the high water percentage of the leaves, a quantity of extract 

 is obtained nearly twice as much as the original volume of the oxalic-acid solution. 



If with the thus obtained isatan solution enzyme experiments are to be per- 

 formed, the acid must be removed, which is best done by boiling with chalk 2 ). 

 As the reaction of the chalk is slightly alkaline it should be very finely divided, 

 as larger particles form a little indigo on their surface. After filtering off the 

 oxalate and the superfluous chalk, a liquid results somewhat brownisch indeed, 

 but not so much as to be hurtful to the enzyme experiments. 



This liquid cannot be evaporated to dryness without being decomposed, even 

 not at room temperature, because during the concentration the acidity increases. 

 To neutralize the syrupic matter is troublesome. 



') If the woad-leaves are boiled with more acid than 2 to 3 cc. normal per TOO cc. 

 of the juice, the decoction grows slimy and gives trouble in filtering. 



2 ) Neutralizing without endangering the subsequent enzyme action, can also be 

 done with lead-, mangan-, magnesia-, or baryta-carbonate, but I prefer chalk. 



