400 Journal of Agricultural Research voi. xv. no. 7 



hot water. Filtrate and washings from barium carbonate were evaporated on the 

 water bath to small volume, filtered, washed, and finally made up to 100 cc., of which 

 20 cc. were Kjeldahlized to ascertain the quantity of nitrogen present. The remaining 

 80 cc. were formol -titrated, having previously been freed -from carbon dioxid and 

 phosphoric acid whose presence would interfere with formol titration {46). For 

 this purpose there were added to the 80 cc. of the solution 2 gm. of barium chlorid 

 which were dissolved by shaking, then i cc. of a 0.5 per cent of phenolphthalein 

 solution and enough of saturated barium hydroxid solution until a red color appeared. 

 Five cc. of NJs barium hydroxid were added in excess, made up to the mark with 

 water (usually 100 cc), shaken and filtered after a few minutes. Of this filtrate 

 definite quantities, usually of 40 cc. each, were neutralized with NJ^ hydrochloric 

 acid and formol titrated, the data of the titration being recalculated to the total soluble 

 nitrogen. In cases in which the solution was too dark for formol titration, it was 

 decolored by the formation in the solution of a precipitate of silver chlorid (or copper 

 sulphid). Ordinarily the solution was rendered acid with NI2 hydrochloric acid, 

 whereupon about 10 cc. of NJ2 silver-nitrate solution was gradually added while the 

 flask was constantly shaken. Inasmuch as the presence of silver wouM interfere 

 with the formol tritration, care was taken to insiue an excess of the chlorin ion in 

 the solution by adding to it about 5 cc. of 2 A^ barium-chlorid solution. The silver- 

 chlorid precipitate formed in the solution usually carries down the coloring substance, 

 so that the filtrate shows a yellowish light color and can then readily be formol titrated . 



(7) The PEPTiD NITROGEN was estimated in the aqueous spinach extract, on hydroly- 

 sis with 20 per cent hydrochloric acid for 8 hours. From the hydi'olyzed solution 

 ammonia and humin nitrogen were removed in the manner already described. The 

 filtrate and washings from magnesium-oxid residue were then evaporated on the water 

 bath, cooled, and made up to 100 cc, of which 20 cc. were Kjeldahlized to ascertain 

 the nitrogen present. The remaining 80 cc. were freed from carbon dioxid, phosphoric 

 acid, and coloring matter, as outlined above. Aliquots of the filtrate (made up to 

 100 cc.) usually portions of 40 cc. each, were then formol-titrated. From the amino 

 acid nitrogen found here, was subtracted the amino-acid nitrogen (minus tlie ammonia 

 present as such) which was found directly in the water extract of the spinach materials 

 by formol titration. 



(8) The residual solubi^e nitrogen made up of nitrogenous compounds other than 

 those given above constitutes the difference between the total water-soluble nitrogen 

 and the sum of the nitrogen found as ammoniacal nitrogen in (2), acid amid nitrogen 

 in (3), humin nitrogen in (4), basic nitrogen in (5), monoamino acid nitrogen in (6), 

 and peptid nitrogen in (7). 



The results obtained by the methods described are summarized in Table VIII. 



As will be seen, the first section of Table VIlI presents the results 

 expressed in percentage of the total soluble nitrogen of the spinach mate- 

 rials, while in the second and third sections the data are expressed in 

 percentage of the total nitrogen and of the oven-dried spinach material, 

 respectively. 



The total nitrogen of the spinach is given in Table III. The exam- 

 ination of the latter part of this table shows that, with one exception,^ 

 the nitrogen content of all the healthy spinach materials is higher than 

 the nitrogen content of the corresponding diseased materials. It is true 

 that investigation showed that the soil of the diseased spinach has a 



1 Compare No. s of Table III, first part, with No. 4 of the second part. 



