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.[342] In case the quantity
of ammonia produced is very small it may be detected by the nessler
reagent.[343] 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.[344]
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.[345]
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.[346]
The gyrodynats of some of the albumins and albuminates are given below:
Serum albumin [α]_{D} = -57°.3 to -64°.6.
Egg albumin [α]_{D} = -35°.5 to -38°.1.
Serum globulin [α]_{D} = -47°.8.
Milk albumin [α]_{D} = -76°.0 to -91°.0.
Our knowledge of the gyrodynatic numbers of the proteids and allied
bodies is too fragmentary to be of any great help in analytical work.
In practice, the rotatory power of these bodies becomes a disturbing
force in the determination of milk sugar.[347] A further study of this
property of certain proteids may lead to analytical processes for their
detection and determination, but no reliable methods for this can now
be recorded.
=381. Alkaloidal Nitrogen.=—Only a general statement can be made here
in respect of the detection of alkaloidal nitrogen in vegetable or
animal tissues. Alkaloids are not found in healthy animal tissues and
the description of methods for isolating and detecting ptomaines is
foreign to the purpose of this work. In vegetable tissues the presence
of alkaloids may be established by the following methods of examination.
The fine-ground tissues are made to pass a sieve of half millimeter
mesh and when suspended in water are acidified with sulfuric. The
mixture is then thoroughly extracted by shaking in a separatory funnel
with petroleum ether, benzene and chloroform, successively. Some
resins, glucosids and a few alkaloidal bodies not important here are
extracted by this treatment.
The residue is made distinctly alkaline with ammonia and treated
as above with the same solvents. In the solution obtained as last
mentioned nearly all the alkaloidal bodies found in plants are
contained.
All the alkaloids in a plant may be obtained by digesting the finely
divided material with dilute sulfuric acid. The acid solution thus
obtained is made nearly neutral with ammonia or magnesia, concentrated
to a sirup, and gums, mucilage, etc. thrown out by adding about three
volumes of ninety-five per cent alcohol. The alkaloids are found in the
filtrate. The alcohol is evaporated from the filtrate and the residue
tested for alkaloids by group reagents.[348] Potassium mercuric iodid
and phosphotungstic and molybdic acids are types of these reagents.
The same group reagents may also be applied to the extracts obtained
with petroleum ether, benzene and chloroform, in all cases, after the
removal of the solvents by evaporation.