Nov. i8, 1918 Physiological Studies of Normal and Blighted Spinach 399 



water. Two grams of magnesium oxid, previously reduced to cream with 100 cc. of 

 water, were added to the flask and distilled, the distillate being received in an Erlen- 

 meyer flask containing Njio sulphuric acid. From the ammoniacal nitrogen found 

 by titration the ammonia nitrogen found in (2) was subtracted, giving the nitrogen of 

 the acid araids. 



That the distillates obtained at the distillation of the hydrolyzed and evaporated 

 spinach extracts with magnesium oxid actually represented ammonia was shown by 

 the preparation of chloroplatinates from the distillates in question. The platinum 

 double salts usually, though not always, showed a platinum percentage which was close 

 to the 43.93 required by the formula (NH4)2PtCl6. 



(4) The humin nitrogen, which resulted from the action of the boiling hydro- 

 chloric acid upon the aqueous spinach, and which was due in part to the presence in the 

 latter of diamino acids and monoamino acids, was estimated in the magnesium-oxid 

 residue remaining in the Kjeldahl flask from the acid amid estimation in (3). The 

 magnesium-oxid residue was completely decanted off on a Gooch crucible provided 

 with a linen cloth fllter, the filtrate being received in a beaker placed in Witt's Alter- 

 ing apparatus. The residue was now repeatedly (about 10 times) treated with small 

 quantities (25 cc.) of boiling hot water, and finally quantitatively filtered and washed 

 on the Gooch crucible (filtrates and washings being received in another beaker). 

 The magnesium-oxid cake with the aid of dilute sulphuric acid was then quantita- 

 tively transferred to a 500 cc. Kjeldahl flask and the nitrogen estimated according to 

 Kjeldahl 's method. 



(5) The basic nitrogen was estimated in the filtrates and washings from the 

 magnesium-oxid cake obtained in (4). The combined liquids, with the exception of 

 the first decantation, which in order to avoid the formation of a brown precipitate 

 was not concentrated, were evaporated on the water bath to a small volume. This 

 was added to the first decantation, cooled to 20° C, made up to 100 cc, and treated 

 with 5 gm. of sulphuric acid, 30 cc. of a solution containing 20 gm. of phosphotungstic 

 acid, and 5 gm. of sulphuric acid per 100 cc. After at least 24 hours, the precipitate 

 was filtered through an S. and S. filter and washed with about 200 cc. of a solution 

 containing 2.5 gm. of phosphotungstic acid and 5 gm. of sulphuric acid per 100 cc, 

 the washing being effected by rinsing the precipitate from the filter into a beaker and 

 returning to the filter three times. The washed precipitate was then Kjeldahlized 

 and tritrated, giving the proportion of the basic nitrogen. 



That diamino acids formed a part of the basic nitrogen was demonstrated in the 

 following way: Another portion of the phosphotungstic-acid precipitate obtained in 

 the manner outlined was treated with barium hydroxid in excess, and the barium 

 phosphotungstate filtered out and washed. Filtrate and washings were not treated 

 with carbon dioxid to remove the excess of bar3rta, the filtrate and washings from 

 barium carbonate being evaporated on the water bath to a small volume. This 

 concentrated solution gave the following tests: 



1. Phosphotungstic-acid solution gave immediately a heavy, white precipitate. 



2. Phosphomolybdicacid gave a yellow precipitate. 



3. MercToric chlorid gave a gray flocculent precipitate. 



4. Silver nitrate gave a grayish-white precipitate, soluble in excess of ammonia. 



5. The solution was distinctly alkaline. 



6. Addition of neutralized formaldehyde to the alkaline solution made it turn 

 acid, pointing to the presence of carboxyl and amino groups. 



(6) The monoamino acid nitrogen was estimated in the filtrate from the phospho- 

 tungstic-acid precipitate. To remove the excess of phosphotungstic and sulphuric 

 acids from the filtrate the latter was treated with barium hydroxid whose excess was 

 removed with carbon dioxid. Both tlie barium phosphotimgstate (plus barium sul- 

 phate) and the barium-carbonate precipitates were repeatedly washed with boiling 



