Part 77

=374. Nitric Acid.=—Any nitric acid or nitrate which an agricultural product may contain may be leached out by treating the fine-ground material with cold water. From vegetable matters this extract is evaporated to a small bulk, filtered, if necessary, and tested for nitric acid by the usual treatment with ferrous sulfate and sulfuric acid. In the case of vegetable substances there will not usually be enough of organic matter to interfere with the delicacy of the reaction, but in animal extracts this may occur. Colored extracts should be decolorized with animal char (bone-black) before they are subjected to examination. It is not well to attempt to remove the organic matters, but, since they are more insoluble in water than the nitrates, the solution containing both may be evaporated to dryness and treated with a quantity of cold water insufficient for complete solution. The nitrates will be found in the solution obtained in a larger proportionate quantity than before.

=375. Amid Nitrogen.=—One or more atoms of the hydrogen in ammonia may be replaced by acid or basic bodies (alcohol radicles). In the former cases amids, in the latter amins result. In the ratio of displacement there are formed primary, secondary, and tertiary bodies determined by the number of hydrogen atoms replaced. The primary amids are the only ones of these bodies that are of interest in this connection.

The amids are easily decomposed, even on heating with water and the more readily with acids and alkalies, the amido radicle being converted into ammonia. A type of these reactions is given below.

CH₃.CO.NH₂ + H₂O = CH₂.CO.OH + H₃N.

On boiling an amid with hydrochloric acid, the ammonia is procured as chlorid whence it is easily expelled by heating with an alkali. In a body free of ammonia, an amid is easily detected by subjecting the substance containing it to the action of hot hydrochloric acid, filtering, neutralizing the free acid with sodium hydroxid, adding an excess thereof and distilling into an acid. In case the quantity of ammonia produced is very small it may be detected by the nessler reagent. Amids are soluble in a fresh, well washed preparation of cupric hydrate suspended in water. The hydrate also passes into solution forming a liquid of a deep blue color.

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.

=378. Qualitive Tests for Albumin.=—Albumin is one of the chief proteids and exists in both animal and vegetable substances. It is soluble in cold water and may therefore be separated from many of its nearly related bodies which are insoluble in that menstruum. In aqueous solutions its presence may be determined by the general reactions for proteid matters given above or by the following tests:

Precipitation by Heat.—Albumin is coagulated by heat. Vegetable albumins become solid at about 65° and those of animal origin at a somewhat higher temperature (75°). Some forms of animal albumin, however, as for instance that contained in the serum, coagulate at a lower temperature.

Precipitation by Acids.—Dilute acids also precipitate albumins especially with the aid of heat. Practically all the albumins are thrown out of solution by application of heat in the presence of dilute acids.

Mercuric Salts.—Acid mercuric nitrate and a mixture of mercuric chlorid, potassium iodid and acetic acid completely precipitate all albuminous matters.

The yellow or red color produced on heating albumin with the mercuric nitrate is known as Million’s reaction.

=379. Qualitive Test for Peptones and Albuminates.=—When peptones and albuminates are dissolved in an excess of glacial acetic acid and the solution treated with sulfuric acid a violet color is produced and also a faint fluorescence.

Separation of Peptones and Albumoses.—In a solution of peptones and albumoses the latter may be precipitated by saturating the solution with finely powdered zinc or ammonium sulfate.

Action of Phosphotungstic Acid.—All proteid matters in aqueous, alkaline or acid solutions, are precipitated by sodium phosphotungstate in a strongly acid solution. Acetic, phosphoric, or sulfuric acid may be used for producing the required acidity, preference being given to the latter.

Action of Trichloracetic Acid.—In the precipitation of albumin by trichloracetic acid, there is formed a compound of the two bodies which to 100 parts of albumin has 26.8 parts of the trichloracetic acid.

The different albuminoid bodies obtained by precipitation behave in a similar manner. There are formed flocculent precipitates insoluble both in dilute and concentrated acids in the cold and also at a high temperature, with the exception of the hemialbumose compound.

Albumin peptone, however, gives with the acid named in concentrated solution a precipitate easily soluble in an excess of the reagent. In the analysis of cow’s milk but not of human milk, this acid can be used for the estimation of the albuminoid substances. With both kinds of milk it can be used for the estimation of the albumin after the removal of the casein.

After precipitation of the albuminoid bodies, the milk sugar can be estimated by polarizing the filtrate and, volumetrically after removal of the excess of the acid by evaporation. By means of trichloracetic acid it is possible to separate albumin peptone from mucus and mucus peptone. A similar reaction is also produced by dichloracetic acid, but the reaction with this last agent is less delicate than with the other. Neither mucus nor albumin is precipitated by chloracetic acid.

=380. Action of Albumins on Polarized Light.=—Many of the albumins and albuminates, when in solution, strongly deflect the plane of polarized light to the left.