THE INDIVIDUAL GROUPS OF PROTEINS 505 



The Calibration of the Apparatus. 



Since the principle of the method depends upon knowing the volume of 

 the air space in each bottle and the pressure difference in the manometer 

 when a volume of oxygen is evolved in each of the bottles, the apparatus 

 must be calibrated. The second bottle acts simply as an automatic correction 

 for changes of vapour tension, temperature and surface tension brought about 

 during the manipulations. Instead of knowing the volume of the bottles it 

 suffices for most purposes, provided that the same volumes of blood and reagents 

 are always used, to determine a constant, K, which represents the number 

 of cmm. of gas (v) set free in the flask for each mm. difference of level in the 

 manometer (/) ; 



K = - or v = K/ 



i.e. the number of mm. pressure produced multiplied by the constant of the 

 apparatus gives the volume in cmm. of gas evolved in the apparatus. 



The constant may be determined by Hberatirig a known volume of gas 

 in one of the vessels and observing the pressure difference for this volume 

 as described by Barcroft and Burn. 1 



A definite volume of gas is most easily liberated in the apparatus by 

 preparing oxygen from hydrogen peroxide and *iN permanganate according 

 to the equations : 



2 KMnO 4 + 3H 2 SO 4 = K 2 SO 4 + 2 MnSO 4 + sH 2 O + 50. 

 5 H 2 2 + 5 = 5 2 + 5H 2 Q 



from which it is calculated that 



316 gm. KMnO 4 = 5 x 22*2 litres of oxygen 



or 

 00316 gm. KMnO 4 = ni c.c. of oxygen at o and 760 mm. 



20 volume per cent, hydrogen peroxide is used, 40 c.c. being diluted to 

 1000 c.c. i c.c. of this solution should give off about *2 c.c. of oxygen. 



Before use the diluted hydrogen peroxide solution must be titrated as 

 follows : 



50 c.c. of the diluted solution are acidified with sulphuric acid and 

 titrated with the 'iN permanganate until a pink colour results. 8-10 c.c. 

 of permanganate should be required; if less than this volume be used, 

 the peroxide solution is too weak and a fresh one must be prepared. 



If 8-5 c.c. of permanganate be used, then the volume of oxygen liberated 



from i c.c. of dilute peroxide will be HIL -3, or -1887 c.c. at o and 



760 mm. This is the volume at N.T.P. which will be liberated when these 

 quantities are put into the apparatus. 



The two taps are opened to the air. It is important to pay careful 

 attention to this operation so as to avoid breaking the apparatus. The two 

 flasks are removed; the stoppers are carefully greased. Into one of the 

 flasks (right) is put i c.c. of peroxide + 2 c.c. of -oiN sulphuric acid; into 

 its cup in the stopper is put 0-2 c.c. of *iN permanganate and the opening 

 is covered with a piece of filter paper. Into the other flask (left) is put i 

 c.c. of peroxide + 2 c.c. of 'oiN sulphuric acid ; into its cup in the stopper is 

 put 0-2 c.c. of water and the opening covered with filter paper. 



The bottles are carefully replaced. 



The apparatus is hung up with the flasks in a water-bath of constant 

 temperature for 5 minutes, with the taps open to the air. 



The taps are closed to the air, both flasks being placed in contact with the 



manometer. 



1 J. Physiol., Vol. 45. 



