If amids be added to a cold solution of potassium nitrate in sulfuric acid free nitrogen is evolved.
376. Ammoniacal Nitrogen.—This combination of nitrogen may be detected by distilling the sample, or an aqueous extract thereof, with magnesia or barium carbonate. The ammonia is collected in an acid and detected therein by the usual qualitive reactions.
377. Proteid Nitrogen.—There are a few general qualitive reactions for proteid nitrogen and some special ones for distinct forms thereof. Below will be given a few of those reactions which are of most importance to the agricultural analyst:
Conversion into Ammonia.—All proteid matters are converted into ammonia on boiling with strong sulfuric acid in presence of an oxygen carrier. Mercury is the substance usually selected to effect the transfer of the oxygen. Bodies which are found to be free of nitrates, ammonia and amids, are subjected directly to oxidation with sulfuric acid, and the ammonia produced thereby is distilled and detected in the manner already suggested. If nitrogen be present in the form of ammonia, amids and nitrates, the substance may be heated with an acid, hydrochloric or acetic, thrown on a filter, washed with hot dilute acid and the residue tested as above for proteid nitrogen.
Biuret Reaction.—When proteid matter is dissolved in sulfuric acid, the solution, made alkaline with potassium hydroxid and treated with a few drops of a solution of copper sulfate, gives a violet coloration. This is commonly known as the biuret reaction, because the substance C₂H₆N₃O₂, biuret, left on heating urea to 160° gives the coloration noted in the conditions mentioned.
It has been found by Bigelow, in this laboratory, that if a solution is to be examined containing a very small amount of a proteid or similar body, the copper sulfate solution should not contain more than four grams of CuSO₄.5H₂O in 100 cubic centimeters of water, and the test should first be made by adding to the solution one or two drops of this copper sulfate solution, and then a strong excess of potassium or sodium hydroxid. The test may be repeated, using from one-half to two cubic centimeters of the copper sulfate solution, according to the amount of proteid present. If too much of the copper sulfate solution be employed its color may conceal that of the reaction.
Heating to the boiling point sometimes makes the violet color more distinct.
If a solid is to be examined it is first suspended in water, and in this state treated in the same manner as a solution. If solution is not complete, the mixture should be filtered when the color produced may be observed in the filtrate.
Proteoses and peptones give a red to red-violet and other proteids a violet to violet-blue coloration.
Xanthoproteic Reaction.—Strong nitric acid produces a yellow coloration of proteid matter, which is intensified on warming. On treating the yellow mixture with ammonia in slight excess the color is changed to an orange or red tint.