543 



carboxyl group is made inactive, (lie action appears to be equally 

 strong as in other bi-basic oxy-acids (as apple acid and tartaric 

 acid). In the same way it appears that when two or three groups 

 are made inactive at the same time, the action lias become as great 

 as in the then comparable compounds. 

 Now the question rises. 



1. is it also possible to prove such a diminution of the number 

 of free Ca-ions in less complicated systems than such biochemical 

 ones by the addition of citrate'? 



2. do the substituted citrates shoiv there a similar diminution of 

 .activity as in rennet- coagidation? 



The best way to answer these questions — the determination of 

 the concentration of the free Ca-ions in the original solutions — 

 is unfortunately barred, because we do not know a method as yet 

 to determine the concentration of free Ca-ions potentio-metrically. 

 It is, therefore, necessary to have recourse to indirect methods. The 

 most natural proceeding is to determine how much calcium is held 

 in solution by addition of citrate, when a substance that precipitates 

 the calcium as insoluble compound (e.g. oxalate, fluoride, pyrophos- 

 phate, soap etc.) is added to a diluted solution of a calcium salt. 

 The solubility product of this reaction must be chosen so that the 

 action of the citrate manifests itself so as to be easily measured. 

 If this solubility product is known, the percentage of free calcium 

 ions is known at least at this small concentration, while it is known 

 how much Ca remains in solution. ') 



The purest results will be obtained by an analytical determination 

 by weight of the quantity of the calcium that has been precipitated 

 or that has remained in the solution, as this can be carried out 

 without appreciably diluting the calcium solution. I shall perform 

 this experiment later on with citrate and with substituted citrates. 

 But in order to get a preliminary rough idea, a titration can also 

 be used, though this has the objection of appreciably diluting the 

 original solution. 



Mr. D. P. Ross van Lennep, who assisted me in my experiments 

 on the influence of substituted citrates on rennet coagulation, pointed 

 out to me that the soap- titration of calcium after Clark (as it is 

 used in the determination of the hardness of water) might render 

 us good services here. ') He carried out a number of experiments 



') The question in how far hydrolytic decomposition complicates the matter, will 

 be treated later. 



'j A drawback of this method is that the titration does not take place with 

 water, but with 56-volurae percentage alcohol, which changes the surroundings 



