CH. VIII.] FORMOL TITRATION. 215 



the control and in all the observations made. The digestion mixture is usually 

 opalescent and pigmented. A simple control is obtained by boiling a portion 

 of the digest in a flask to destroy the enzyme, adding formol and phenol 

 phthalein and titrating to a definite pink. The control and all subsequent 

 titrations are brought to the same colour. But the best results are obtained 

 by use of the comparator of Cole and Onslow, constructed to hold large boiling 

 tub (see fig. 37). With this it is possible to titrate sharply to a definite P H . 

 The principle underlying the method of titration is described in Ex. 322. To 

 save material in this experiment only one buffer solution is used, instead of 

 the two in Ex. 322. 



A further point of interest in connexion with formol titrations is the 

 fact that the amount of soda required to make the digestion mixture alkaline 

 to P H = 8-3 (pink with phenol phthalein) increases during the course of diges- 

 tion, being especially marked in the early stages of tryptic digestion. This is 

 not observed in the experiment conducted by the method described below, 

 owing to the fact that the formol is added directly to the sample. The rela- 

 tionships between the amounts of alkali required before and after the addition 

 of formol with various proteins subjected to the action of different proteolytic 

 enzymes is being carefully studied, as it seems possible that they will throw 

 light on the nature of the groupings in the protein molecule that are liable 

 to attack by the different enzymes. 



Formol Solution. Dilute commercial formaldehyde (40 per cent.) with 

 an equal volume of distilled water. Add 10 drops of a 0-5 per cent, solution of 

 phenol phthalein in 50 per cent, alcohol for every 100 cc. of the solution and 

 titrate with o-i N. soda until a faint pink tinge is obtained. It may be 

 necessary to add a few more drops of the alkali from time to time owing to the 

 oxidation of the formaldehyde to formic acid. 



Casein Solution. Make a 10 per cent, solution of commercial casein accord- 

 ing to the directions given in Ex. 87 A (i.) to (vi.). Add toluol, shake well, 

 and place in a deep water bath or air incubator at 38 to 40 C. for 24 hours, 

 shaking at intervals. When all preparations for the titrations have been 

 made, there are added 25 to 50 cc. per litre of the pancreatic extract described 

 on p. 212. The bottle is well shaken, a sample immediately withdrawn for 

 titration, and the bottle replaced in the incubator or water bath. Further 

 samples are taken at intervals, the following making a suitable series : , \, i, 

 2, 6, 24 and 48 hours. 



Method of titration. Withdraw 20 cc. of the digestion mixture 

 with a pipette and transfer it to the tube (3) of the comparator 

 (fig. 37). Another portion of 20 cc. is placed in tube (2). In (i) 

 place 25 .cc. of a buffer solution of P H = 8-5 (see p. 28). In (4) place 

 about 25 cc. of water. Into tubes (i) and (3) measure 10 drops of 

 O'5 per cent, phenol phthalein by means of a dropping pipette 

 (fig. 5). To (3) add 5 cc. of the neutralised formol. To (2) add 5 cc. 

 of water to make the colour and opalescence comparable with that 

 of (3). Titrate (3) with 0-2 N. soda from a burette. The end point 

 is reached when the appearance seen on the ground glass screen at 

 Y is the same as that seen at X. If more than 2 cc. of the alkali are 

 required, the same volume of water should be added to tubes (i) and 



