=382. Total Nitrogen.=—Any one of the methods heretofore described for
the estimation of total nitrogen in soils or fertilizers is applicable
for the same purpose to agricultural products. One among these,
however, is so superior in the matter of convenience and certainty, as
to make it preferable to any other. The moist combustion of the sample
with sulfuric acid with subsequent distillation of the ammonia produced
is the process which is to be recommended.[349]
The usual precautions for securing a representative sample should be
observed, but no further directions are needed. In all cases hereafter,
where the estimation of nitrogen is enjoined, it is understood that the
moist combustion process is to be used unless otherwise stated.
=383. Estimation of Ammoniacal Nitrogen.=—If the distillation of
ammonia be accomplished with the aid of magnesia alba or barium
carbonate it may be safely conducted on the finely ground materials
or, in case of animal bodies, in as fine a state of subdivision as
may be conveniently secured. Since the salts of ammonia are easily
soluble in water they may be all obtained in aqueous solution, and the
distillation of this solution with magnesia gives correct results.
Experience has shown that the stronger alkalies, such as sodium and
potassium hydroxids, cannot be safely used in the distillation of
ammonia from mixtures containing organic nitrogenous materials because
of the tendency of these bodies to decomposition, in the circumstances,
yielding a portion of their nitrogen as ammonia. Barium carbonate acts
with less vigor on non-ammoniacal nitrogenous matters than magnesia,
and in some cases, as pointed out further on, may be substituted
therefor with advantage. There is no danger of failing to obtain a part
of the ammonia on distillation with magnesia provided the latter does
not contain more than a trace of carbonate.[350]
When no easily decomposable organic nitrogenous matters are present,
the distillation may be conducted with the stronger alkalies in the
manner prescribed.[351] All the necessary details of conducting the
distillation are found in the preceding volumes of this work.
=384. Estimation of Amid Nitrogen.=—In bodies containing no ammonia,
or from which the ammonia has been removed by the method described in
the preceding paragraph, the nitrogen in the amid bodies is converted
into ammonia by boiling for about an hour with five per cent sulfuric
or hydrochloric acid. The ammonia thus produced is estimated in the
usual manner after distillation over magnesia free of carbonate. The
free acid is exactly neutralized with sodium or potassium carbonate
before the addition of the magnesia. The results are given in terms of
asparagin. The reaction which takes place in the decomposition of the
amid body is indicated by the following equation:
Asparagin. Sulfuric Aspartic Ammonium
acid. acid. sulfate.
2C₄H₈N₂O₃ + 2H₂O + H₂SO₄ = 2C₄H₇NO₄ + (H₄N)₂SO₄.
Half of the nitrogen contained in the amid body is thus obtained as
ammonia.
It is advisable to calculate all the amid nitrogen in agricultural
products as asparagin.
=385. Sachsse’s Method.=—A method for the determination of amid
bodies by liberation of free nitrogen has been described by Sachsse
and Kormann.[352] It is based on the reaction which takes place when
amid bodies are brought into contact with nitrites in presence of an
acid. The mixture of the reagents by which the gas is set free is
accomplished in the apparatus shown in Fig. 103. The vessel _A_ has a
capacity of about fifty cubic centimeters and carries a stopper with
three perforations for the arrangement shown.
[Illustration: FIG. 103.—APPARATUS FOR AMID NITROGEN.]
[Illustration: FIG. 104.—SACHSSE’S EUDIOMETER.]
About six cubic centimeters of a concentrated aqueous solution of
potassium nitrite are placed in _A_ and the lower parts of the tubes
_a_ and _b_ are filled with water to a little above _e_ in order to
exclude the air therefrom. Dilute sulfuric acid is placed in one of
the funnels and an aqueous solution of the amid in the other. The air
is displaced from the empty part of _A_ by introducing the sulfuric
acid, a little at a time, whereby nitrous acid and nitric oxid are
evolved. This operation is continued until all the air has been driven
out through _c d_, the open end of _d_ being kept in the liquid in the
dish shown in Fig. 104. The eudiometer in which the evolved nitrogen
is measured is shown in Fig. 104, and should have a capacity of about
fifty cubic centimeters, and be graduated to fifths. It is filled
with the solution of ferrous sulfate contained in _B_ by sucking at
_g_, after which the clamp _h_ is replaced, the cock _f_ closed, and
the free end of _d_ placed in the lower end of the eudiometer. The
solution of the amid is run slowly into the generator _A_, Fig. 103,
together with small additional quantities of the sulfuric acid when
the evolution of gas becomes slow. From time to time _h_ is opened
and fresh quantities of the ferrous solution allowed to flow into the
eudiometer. Any trace of the amid remaining in the funnel is washed
into _A_ with pure water, with care to avoid the introduction of air.
When the liquid in _A_ assumes a permanent blue color the decomposition
is complete. The residual gas is driven out of _A_ by filling with
water. The tubes _d_ and _h_, after all the nitric oxid is absorbed,
are removed from the eudiometer which is transferred to a cylinder
containing water and immersed therein until the two liquid surfaces
are at the same level and the volume of the nitrogen observed. After
correction for temperature and pressure, the weight of the nitrogen is
calculated. Twenty-eight parts by weight of nitrogen correspond to 150
of pure asparagin, 181 of tyrosin and 131 of leucin.[353] This method of
procedure is difficult of manipulation and is apt to give results that
are too high. It cannot be preferred to the more simple and accurate
processes already described.
=386. Preparation of Asparagin.=—In case the analyst desires to prepare
a quantity of asparagin for comparative purposes it may be easily
accomplished in the following way: A sufficient quantity of pease
or beans is sprouted in a dark place and allowed to grow until the
reserve food of the seed is exhausted. The young sprouts are gathered,
shredded and subjected to strong pressure. The juice thus obtained is
boiled to coagulate the albumin, and thrown on a filter. The filtrate
is evaporated to a thin sirup and set aside to allow the asparagin
to separate in a crystallized form. If the crystals at first formed
are colored they may be dissolved, decolorized with bone-black, and
recrystallized. Instead of the above method the young shoots may be
shredded, extracted with hot water and the extract treated as above. A
larger yield of the asparagin is obtained by the latter process than by
the one mentioned above.[354]
=387. Detection and Estimation of Asparagin and Glutamin.=—Of all
the amid bodies asparagin is the most important from an agricultural
standpoint, because of its wide distribution in vegetable products.[355]
Asparagin is easily obtained from the aqueous extracts of plants by
crystallization.[356] In addition to its crystalline characteristics
asparagin may be identified by the following tests. Heated with
alkalies, including barium hydroxid, asparagin yields ammonia. Boiled
with dilute acids it forms ammonium salts. A warm aqueous solution
dissolves freshly prepared copper hydroxid with the production of a
deep blue color. Sometimes, on cooling, crystals of the copper compound
formed are separated. Asparagin crystallizes with one molecule of
water. Glutamin gives essentially the reactions characteristic of
asparagin, but crystallizes without water in small white needles.
Asparagin is easily detected with the aid of the microscope by placing
sections of vegetable tissues containing it in alcohol. After some
time microscopic crystals of asparagin are separated. The presence of
large quantities of soluble carbohydrates seriously interferes with the
separation of asparagin in crystalline form.
For the detection of glutamin the liquid containing it is boiled with
dilute hydrochloric acid, by which ammonia and glutamic acid are
formed. On the addition of lead acetate to the solution the glutamic
acid is thrown out as a lead salt, in which, after its decomposition
with hydrogen sulfid, the characteristic properties of glutamic acid
can be established.
The above process is chronophagous and also uncertain where the
quantity of glutamin is very small and that of other soluble organic
matters very large. A much better process, both for the detection
of glutamin and asparagin, is the following, based on the property
possessed by mercuric nitrate of precipitating amids.
The aqueous extract containing the amid bodies is mixed with lead
acetate until all precipitable matters are thrown out and the mixture
poured into a filter. To the filtrate is added a moderately acid
solution of mercuric nitrate. The precipitate produced is collected
on a filter, washed, suspended in water, decomposed with hydrogen
sulfid and again filtered. The amid bodies (glutamin, asparagin, etc.)
are found in the filtrate and can be detected and estimated by the
processes already described. A reaction showing the presence of an amid
body is not a positive proof of the presence of asparagin or glutamin,
since among other amids, allantoin may be present. This substance is
found in the sprouts of young plants and also in certain cereals, as
shown by researches in this laboratory.[357] Allantoin, glutamin, and
asparagin, when obtained in solution by the above process, may be
secured, by careful evaporation and recrystallization, in well defined
crystalline forms. Asparagin gives lustrous, rhombic prisms, easily
soluble in hot water, but insoluble in alcohol and ether.
Allantoin is regarded as a diureid of glyoxalic acid and has the
composition represented by the formula C₄H₆N₄O₃. It crystallizes in
lustrous prisms having practically the same solubility as asparagin.
Glutamin is the amid of amidoglutaric acid. It crystallizes in fine
needles. Its structural formula is represented as
CO.NH₂
/
C₃H₅(NH₂)
\
CO₂H.
=387. Cholin and Betain.=—Cholin is a nitrogenous base found in both
animal and plant tissues. Its name is derived from the circumstance
that it was first discovered in the bile. It is found in the brain,
yolk of eggs, hops, beets, cottonseed and many other bodies. When
united with glycerolphosphoric acid it forms lecithin, a compound of
great physiological importance. From a chemical point of view, cholin
is oxyethyltrimethyl-ammonium hydroxid,
OH
/
C₂H₄ ; (C₅H₁₅NO₂).
\
N(CH₃)₃.OH
It is crystallized with difficulty and is deliquescent. Its most
important compound, from an analytical point of view, is its platinum
salt C₅H₁₄ONCl₂PtCl₄. This salt crystallizes in red-yellow plates and
is insoluble in alcohol.
Betain, C₅H₁₁NO₂, is the product of the oxidation of cholin.
In this laboratory the bases are separated from cottonseed and from
each other by the process described below.[358]
About five pounds of fine-ground cottonseed cake are extracted with
seventy per cent alcohol. The material should not be previously treated
with dilute mineral acids because of the danger of converting a part
of the cholin into betain. The alcohol is removed from the filtered
extract and the residue dissolved in water. The aqueous solution is
treated with lead acetate until no further precipitation takes place,
thrown on a filter, the lead removed from the filtrate with hydrogen
sulfid and the liquid evaporated to a viscous syrup. The sirup is
extracted with alcohol containing one per cent of hydrochloric acid.
The solution thus obtained is placed in a deep beaker and the bases
precipitated by means of an alcoholic solution of mercuric chlorid. The
complete separation of the salts requires at least two weeks.
The double salts of the bases and mercury thus obtained, after freeing
from the mother liquor, are recrystallized from a solution in water
and from the pure product thus obtained the mercury is removed after
solution in water, by hydrogen sulfid. The filtrate, after separating
the mercury, contains the bases as chlorids (hydrochlorates). The
solution of the chlorids is evaporated slowly in (pene) vacuo to a
thick sirup and set over sulfuric acid to facilitate crystallization.
The hydrochlorates are obtained in this way colorless and in
well-shaped crystalline forms.
In a quantitive determination, a small amount of the fine meal is
extracted at once with one per cent hydrochloric acid in seventy per
cent alcohol, the salts obtained purified as above and weighed.
The following process serves to determine the relative proportions of
cholin and betain in a mixture of the two bases.
A definite weight of the chlorids, prepared as directed above, is
extracted by absolute alcohol. This treatment dissolves all the cholin
chlorid and a little of the betain salt. The alcoholic solution is
evaporated and again extracted with absolute alcohol. This process is
repeated three times and at the end the cholin chlorid is obtained free
of betain. In a sample of cottonseed cake examined in this laboratory
the two bases were found present in the following relative proportions,
_viz._, cholin 17.5 per cent, betain 82.5 per cent. Thus purified the
cholin is finally precipitated by platinum chlorid. For a description
of the special reaction, by means of which cholin and betain are
differentiated, the paper cited above may be consulted.
These bodies have acquired an economic interest on account of their
occurrence in cottonseed meal, which is so extensively used as a
cattle food. It is evident from the relative proportions in which they
occur that the less nocuous base, betain, is the more abundant. It is
possible, however, that the base originally formed is cholin and that
betain is a secondary product.
Experience has shown that it is not safe to feed cottonseed meal to
very young animals, while moderate rations thereof may be given to
full-grown animals without much expectation of deleterious results. In
the case of toxic effects it is fair to presume that a meal has been
fed in which the cholin is relatively more abundant than the betain.
=389. Lecithin.=—Lecithin is a nitrogenous body, allied both to the
fats and proteids and containing glycerol and phosphoric acid. Its
percentage composition is represented with some accuracy by the formula
C₄₂H₈₆NPO₉, or according to Hoppe-Seyler, C₄₄H₉₀NPO₉. It appears to
be a compound of cholin with glycerolphosphoric acid. It is widely
distributed both in animal and vegetable organisms, in the latter
especially in pease and beans.
From a physiological point of view, lecithin is highly important as the
medium for the passage of phosphorus from the organic to the inorganic
state, and the reverse. This function of lecithin has been thoroughly
investigated in this laboratory by Maxwell.[359]
In the extraction of lecithin from seeds (pease, beans, etc.) it is not
possible to secure the whole of the substance by treatment with ether
alone.[360]
The extraction of the lecithin may, however, be entirely accomplished
by successive treatments for periods of about fifteen hours with pure
ether and alcohol. This is better than to mix the solvents, since, in
this case, the ether having the lower boiling point is chiefly active
in the extraction. When the extraction is accomplished by digestion
and not in a continuous extracting apparatus the two solvents may be
mixed together and thus used with advantage. After the evaporation of
the solvents, the lecithin is ignited with mixed sodium and potassium
carbonate whereby the organic phosphorus is secured without loss in an
inorganic form. Where greater care is desired, the method described for
organic phosphorus in soils may be used.[361] The inorganic phosphorus
thus obtained is estimated in the usual way as magnesium pyrophosphate.
For analytical purposes, the extraction of lecithin from vegetable
substances is conducted in this laboratory as follows:[362] The
fine-ground pea or bean meal is placed in an extraction apparatus
and treated continuously with anhydrous ether for fifteen hours. The
ether in the apparatus is replaced with absolute alcohol and the
extraction continued for six hours longer. The alcoholic extract is
evaporated to dryness and treated with ether. The part of the lecithin
at first insoluble in ether becomes soluble therein after it has been
removed from the vegetable tissues by alcohol. Moreover, any trace of
inorganic phosphorus which may have been removed by the alcohol, is
left undissolved on subsequent treatment with ether. The ether extract
from the alcohol residue is added to that obtained directly, the ether
removed by evaporation, and the total lecithin oxidized and the residue
used for the estimation of phosphorus as already described.
In determining the lecithin in eggs, the procedure employed for
vegetable tissues is slightly changed.[363] The whole egg, excluding
the shell, is placed in a flask with a reflux condenser and boiled for
six hours with absolute alcohol. The alcohol is then removed from the
flask by evaporation and the residue treated in like manner with ether
for ten hours. The ether is removed and the dry residue rubbed to a
fine powder, placed in an extractor and treated with pure ether for
ten hours. The extract thus secured is oxidized after the removal of
the ether by fusion with mixed alkaline carbonates and the phosphorus
determined in the usual way.
=390. Factor for Calculating Results.=—The percentage of lecithin is
calculated from the weight of magnesium pyrophosphate obtained by
multiplying it by the factor, 7.2703.[364] This factor is calculated
from the second formula for lecithin given above, in which the
percentage of phosphorus pentoxid, P₂O₅, is 8.789.
_Example._—In fifty-four grams of egg, exclusive of the shell, is found
an amount of organic phosphorus yielding 0.0848 gram of magnesium
pyrophosphate. Then 0.0848 × 7.2703 = 0.61652 and 0.61652 × 100 ÷ 54 =
1.14. Therefore the percentage of lecithin in the egg is 1.14.
=391. Estimation of Alkaloidal Nitrogen.=—The alkaloids contain
nitrogen in a form more difficult of oxidation than that contained in
proteid or albuminoid forms. It is doubtful whether any of the nitrogen
in alkaloids becomes available for plant nutrition by any of the usual
processes of fermentation and decay to which nitrogenous bodies are
submitted in the soil. Likewise, it is true that it is not attacked by
the digestive processes in any way preparatory to its assimilation as
food by the animal tissues. Alkaloidal nitrogen is therefore not to be
regarded as a food either for the animal or plant.
For the general methods of estimating alkaloids the reader is referred
to standard works on plant chemistry and toxicology. The alkaloids of
interest in this manual are those which are found in tobacco, tea,
coffee and a few other products of agricultural importance. The best
methods of isolating and estimating these bodies will be given in the
part of the volume devoted to the special consideration of the articles
mentioned.