BY MARGARET H. O'dWYER. 515 



Stutzer's Reagent was prepared aeeording to the directions given in Methods 

 of Analysis of the Association of Oflficial Agricultural Chemists of America 

 (1921). Each c.e. of the Reagent contained 0.01 gram of copper hydroxide. The 

 tannin salt solution used contained 10 per cent, tannic acid, and 10 per cent, 

 sodium chloride. 



Barnstein's method consists in adding to the hot aqueous extract 25 c.c. of 

 copper sulphate containing 60 g. of CUSO4.5H2O per litre. Then 25 c.c. of 

 dilute sodium hydroxide solution (12.5 : 1000) are slowly added while the solution 

 is stirred. The precipitate is then allowed to settle and further treated as in 

 Stiitzer's method. Lunge and Keane consider that the concentration of the copper 

 sulphate and of the sodium hydroxide solutions is chosen so that the whole of 

 the copper is not precipitated by the alkali, while the precipitate contains as 

 much effective cupric hydroxide as is used in Stiitzer's method. The time taken 

 in the preparation of Stiitzer's reagent, the making up of which is rather a 

 lengihy process, would also be saved by the use of this reagent. 



For the actual determinations: — 



(1) 20 c.c. of Stiitzer's reagent were added to an aqueous solution, formed 

 by boiling 1 gTm. of the material in 50 c.c. of water, according to the directions 

 given by the Assn. Offlc. Agric. Chemists of America in their methods of analysis 

 (1921), and the N determined in the precipitate by Kjeldahl's method. 



(2) 1 grm. of the material was boiled in 50 c.c. of water, and the hot solution 

 treated with 15 c.c. of the tannin salt solution, eentrifuged, washed with the re- 

 agent, ^nd Kjeldahled in order to determine the amount of nitrogen present. 



(3) 1 gTm. of the material was treated with 50 c.c. of H2O and the estima- 

 tion rnade according to the details given above (Barnstein's method). The N in 

 the precipitate was determined as before. 



• (4) 50 c.c. of the aqueous extract were evaporated down to 10 c.e. and 

 poured into 90 c.c. of alcohol 94 per cent., making the solution of the strength 

 of 85 per cent., heated to boiling, allowed to stand 3 hours, and filtered. From 

 the filtrate the alcohol was then distilled off and the N determined as usual in 

 the residue (Petrie, 1908). 



The protein content in each case was found by multiplying the amount of N 

 present by the factor 6.25 (vide These Proceedings, slvi., 1921, p. 244). 



The results obtained in the small number of grasses so far examined appear 

 to bear out Dr. Petrie's contention that Stiitzer's reagent precipitates some of the 

 non-protein nitrogen. This is a point of considerable importance. 



My thanks are again due to Professor Watt, Mr. Gr. Wright, and Dr. Petrie 

 and others for very valuable assistance in the prosecution of this work. 



[NOTE: — Since the writing of this paper, the author has been engaged in 

 research work under Dr. Schryver in the Bio-chemical laboratory of the Imperial 

 College of Science and Technology, South Kensington, London. In this laboratory 

 important work is being carried out on the proteins of plants (Chibnall and 

 Schry^'er, Biochem. Journ., 15 ''921, p. 60; Buston and Schryver, Biochem. 

 Journ., 15, 1921, p. 636) tht results of which should be applicable to grasses, 

 and I hope at some futu're date to have an opportunity of further examination 

 of Australian gTasses by methods other than those outlined in the paper. M.H.O'D.] 



