1910.] Quantitative Estimation of Hydrocyanic Acid, etc. 575 



and (4), both as regards evolution of HON and decline of electrical response. 

 In chloroform vapour the evolution of HON was greatest, and the abolition 

 of electrical response most rapid. In water vapour there was no evolution 

 of HCN and no diminution of electrical response, so that it appeared at 

 first sight as if the two kinds of effect, increasing in the first case, 

 decreasing in the second, were the associated consequence of the same 

 disturbance of protoplasm. 



But on further examination, more especially by quantitative experiments 

 in which the degree of anaesthetic action was graduated and the evolution 

 of hydrocyanic acid estimated, this view proved to be untenable. 



In order to make quantitative experiments it was necessary to use the 

 anaesthetic reagents in aqueous solution. By preliminary trials it was found 

 possible to do so in weak solution of sodium picrate, which, in the absence 

 of an anaesthetic, was found to have little or no toxic action upon the leaves. 

 A leaf immersed in "picrate fluid" — O'OS per cent, picric acid + 0-5 per 

 cent, sodium carbonate — remains alive for many days, with an undiminished 

 electrical response and without any evidence of HON evolution, whereas 

 a leaf immersed in the same solution +0"4c.c. chloroform per 100 c.c. loses its 

 electrical response and reddens the liquid, in a few hours at ordinary 

 temperatures (16° to 18°), in a few minutes at a temperature of 40°. Similar 

 results are obtained with " picrate fluid " +5 c.c. ether per 100 c.c, or 20 c.c. 

 ethyl alcohol per 100 c.c. 



The varying tints obtained, obviously dependent upon varying amounts of 

 hydrocyanic acid evolved, led me to make experiments with various 

 mixtures containing known small amounts of hydrocyanic acid, in order to 

 see whether a reliable colour scale could be obtained. Equal volumes of 

 picrate fluid and HON solution (titrated by AgNOs) at concentrations O'l, 

 0-01, 0-001, and 0"0001 per cent, gave tints which on appropriate dilution 

 were found to be reasonably concordant, and stable even when exposed to 

 direct sunlight. It was found necessary that the picrate should be taken in 

 great excess in relation to the cyanide, and that in any case, by reason of 

 the slowness of the reaction, comparisons of tint should be taken 24 hours 

 after mixture at ordinary temperatures, or after an hour or two in an 

 incubator at 40°. 



The Colour Scale. — The mixture adopted as standard is made from equal 

 volumes of picrate fluid and 0'002-per-cent. HON left for 24 hours in an 

 incubator at 40°. This mixture contains 10 milligrammes HON per litre, 

 and has a red colour of an intensity T 10, where T 1 denotes the tint 

 corresponding with 1 milligramme HCN per litre, or 1 millionth gramme 

 per 1 c.c. The colour T 10 is nearly matched by the colour of a 5-per-cent. 



