=411. Preparation of the Sample.=—Animal products present many
difficulties in respect of the reduction thereof to a sufficiently
comminuted condition for analytical examination. In the case of bones,
the choppers used for preparing them for feeding to fowls are the most
efficient apparatus for reducing them to fragments. In this condition
they may be ground to a finer state in a sausage machine. The flesh of
animals may be reduced by this machine, with two or three grindings,
to a fairly homogeneous mass. Subsequent grinding in a mortar with
powdered glass or sharp sand may serve to reduce the sample to a finer
pulp, but is not usually necessary and should be avoided when possible.
The sample thus prepared serves for the estimation of water, ash
and fat by methods already described. The sample should be prepared
in quantities of considerable magnitude, the whole of any organ or
separate portion of the body being used when possible. In examining the
whole body the relative weights of blood, bones, viscera, muscle, hide
and other parts should first of all be ascertained and noted.
=412. Treatment of Muscular Tissues for Nitrogenous Bodies.=—For
the present purpose a brief sketch of the method of separating the
nitrogenous bodies in the muscular tissues of the body is all that
can be attempted. For methods of examining the different organs and
parts of the body in greater detail, standard works on physiological
chemistry may be consulted.[387]
_Extraction with Cold Water._—A noted quantity of the finely divided
tissues is mixed with several volumes of ice-cold water and well rubbed
occasionally for several hours, the temperature meanwhile being kept
low. The mixture is poured into a linen bag and the liquid portion
removed by gentle pressure. The residue in like manner is treated with
fresh portions of cold water until it gives up no further soluble
matters. An aliquot portion of the extract is concentrated to a small
bulk and serves for the determination of total nitrogen. The methods of
separating and estimating nitrogenous bodies in flesh soluble in water
will be given in considerable detail further on.
_Extraction with Ammonium Chlorid and Hydrochloric Acid._—The residue,
after exhaustion with cold water, is extracted with a solution of
ammonium chlorid containing 150 grams of the salt in a liter. This
method of extraction is entirely similar to that with water just
described. Globulins and myosin pass into solution by this treatment.
The residual mass is washed as free as possible of the solvent and
is then further extracted with dilute hydrochloric acid containing
four cubic centimeters of the fuming acid in a liter. The treatment
with dilute acid is continued until no further substance passes
into solution. This is determined by neutralizing a portion of the
extract with sodium carbonate, or by the direct addition of potassium
ferrocyanid. In either case absence of a precipitate indicates that no
nitrogenous matters are present in the solution.
_Extraction with Alkali._—The residue from the acid extraction is
washed with water until the acid is removed and then extracted in a
similar manner with a dilute solution of sodium or potassium hydroxid
containing not to exceed two grams of the caustic to the liter. When
this residue is finally washed with water and a little acetic acid,
it will be found that practically all the purely albuminous bodies
contained in the tissues have been extracted with the exception of any
fibrin, which the blood, present in the tissues at the commencement of
the extraction, may have contained. The extract should be acidified
with acetic as soon as obtained.
_Extraction with Boiling Water._—The residual matter boiled for some
time with water will part with its collagen, which, when transformed by
the heat into glutin, passes into solution.
The sarcolemma, membranes, elastic fibers and keratin remain
undissolved.
=413. Contents of the Several Extracts.=—By the systematic treatment of
muscular tissues in the manner just described, the nitrogenous bodies
they contain are separated into five classes, _viz._:
_Cold Water Extract._—This contains serum albumin, serum globulin,
muscle albumin, myosin, mucin and peptone.
_Ammonium Chlorid Extract._—This solution contains the globulins and
also in many cases some myosin and serum globulin.
_Hydrochloric Acid Extract._—When the extractive matter removed by
hydrochloric acid, thrown out by sodium carbonate and well washed with
water, has a neutral reaction, it consists of syntonin, when acid, of
an albuminate.
_Alkali Extract._—The acid albumin of the animal tissue is found in the
alkaline solution and may be thrown out by making the solution slightly
acid.
_Insoluble Residue._—The fifth class contains the insoluble nitrogenous
bodies mentioned above.
=414. General Observations.=—Only a brief résumé of the methods of
treating animal tissues for nitrogenous bases is given above, since
a more elaborate discussion of these principles and methods would
lead too far away from the main purpose of this manual. For practical
purposes, the most important of these bodies are those soluble in water
and the methods of treating these will be handled at some length.
Unfortunately, the methods of determining the exact qualities of these
bodies are not as satisfactory in case of animal as in vegetable
nitrogenous bodies. The flesh bases, soluble in water, contain a much
larger percentage of nitrogen than is found in true proteid bodies,
and therefore the multiplication of the weight of nitrogen found
therein by 6.25 does not give even a near approximation of the actual
quantities of the nitrogenous bodies present in the sample.
Some of the flesh bases contain more than twice as much nitrogen as
is found in proteids, and in such cases 3.12, and not 6.25, would be
the more correct factor to use in the computation. When possible,
therefore, these bodies should be precipitated and weighed after
drying, but this is not practicable in many instances. The sole
resource of the chemist in such cases is to determine the nature of the
body as nearly as possible by qualitive reactions, then to determine
the total nitrogen therein and multiply its weight by the corresponding
factor. The principal flesh bases have the following percentages of
nitrogen and the approximate factors for calculating analytical data
are also given:
Name of base. Formula. Per cent Factor.
nitrogen.
Glutin C₁₃H₂₀N₄O₅ 17.95 5.57
Carnin C₇H₈N₄O₂ 31.11 3.21
Kreatin C₄H₁₉N₃O₂ 32.06 3.12
Kreatinin C₄H₇N₃O₂ 37.17 2.69
Sarkin C₅H₄N₄O 41.18 2.43
=415. Composition of Meat Extracts.=—The meat extracts of commerce
contain all the constituents of meat that are soluble in warm water.
The parts which are soluble in warm water and not in cold are found
in the cold aqueous solution as suspended or sedimentary matters.
Among the nitrogenous bodies present are included albumin, albumose
and peptone among the proteids, carnin, kreatin, kreatinin, sarkin and
xanthin among the non-proteids, and inosinic and uric acids and urea
among other nitrogenous bodies. Among the non-nitrogenous bodies are
found lactic and butyric acids, inosit and glycogen. Among mineral
bodies occurs the phosphates and chlorids of the common bases. In
addition to these bodies, meat extracts may also contain gelatin and
other decomposition products of proteid matter. Since meat extract is
supposed to be prepared by the digestion of the meat free of bones
and put in cold water or in warm water not above 75°, the presence
of gelatin would indicate a different method of preparation, _viz._,
either by boiling water or water heated above the boiling point under
pressure. In a properly prepared extract, the percentage of gelatin is
very small.
Approximately one-tenth of the whole nitrogen present is in the form
of albumoses and only a trace as peptones. By far the greater part of
the nitrogen exists as flesh bases (kreatin, etc.). The composition of
three meat extracts, numbers one and two solid and number three liquid,
is given in the subjoined table.[388]
No. 1. No. 2. No. 3.
Per cent. Per cent. Per cent.
Total nitrogen 9.28 9.14 2.77
Nitrogen as albumin trace 0.08 trace
” ” albumose 0.96 1.21 0.70
” ” peptone trace trace none
” ” flesh bases 6.81 5.97 1.56
” ” ammonia 0.47 0.41 0.09
” in compounds insoluble in
sixty-six per cent alcohol 0.21 0.33 0.25
” ” other bodies 0.83 1.14 0.17
=417. Analysis of Meat Extracts.=—The analysis of a meat extract
should include the determination of the water, ash and total nitrogen.
After multiplying the nitrogen which exists as proteids by 6.25 and
adding together the percentages of all the ingredients, ash, water,
etc., including ammonia, the sum is to be subtracted from 100 and the
difference entered as non-nitrogenous organic matter. The nature of
this conglomerate has already been explained.
_Water._—It is advisable to determine the water in a partial vacuum
(=20=) or in an atmosphere of hydrogen (=23-25=).
The water may also be determined in solid extracts by placing about
five grams of the material in a flat bottom tin foil dish about
fifty-five millimeters in diameter and twenty millimeters deep. The
material is dissolved in enough warm water to fill the dish a little
over one-half and the liquid is then absorbed by adding a weighed
quantity of fibrous asbestos or of dry fragments of pumice stone.
The asbestos is to be preferred because of the fact that it may be
subsequently cut into small bits for the determination of the gelatin.
The dish thus prepared is dried to constant weight in a steam-bath
or vacuum oven. The weight of the dish and of the added absorbent,
together with that of the material employed and of the dried dish and
its contents, give the data for calculating the percentage of water.
The contents of the dish are used as described further on for the
determination of gelatin. In liquid extracts the water is determined in
an entirely analogous manner, using about twenty grams of the material
and omitting the solution in water.
In solid extracts, the part insoluble in cold water is determined
separately.
_Ash._—The ash is determined by ignition at the lowest possible
temperature, best in a muffle (=28-32=). The ash should be examined
qualitively. Where a quantitive analysis is desired, larger quantities
of the extract are incinerated and the constituents of the ash
determined in the usual way.[389]
_Total Nitrogen._—Since nitrates are not present unless added in
the manufacture, the total nitrogen is best determined by moist
combustion.[390]
_Nitric Nitrogen._—The extract should be tested for nitrates and if
present they are determined in the manner already described.[391]
_Ammoniacal Nitrogen._—When ammonia is present it is determined by
distillation with magnesia.[392]
Since boiling with magnesia may cause the distillation of more
ammonia than is present as ammonium salts, the plus being due to the
decomposition of some other nitrogenous compounds, Stutzer replaces the
magnesia with barium carbonate.[393]
_Proteid Nitrogen Insoluble in Sixty-Two Per Cent Alcohol._—The aqueous
solution is treated with strong alcohol until the mixture contains
about sixty-two per cent of the reagent. The precipitate produced is
separated by filtration, washed with sixty-two per cent alcohol and the
nitrogen therein determined.
_Albumose Nitrogen._—This is secured by saturating the aqueous solution
with zinc or ammonium sulfate. The separated albumoses are skimmed from
the surface, thrown in a filter, washed with a saturated solution of
zinc sulfate and the nitrogen determined therein by moist combustion.
In the filtrate from the above separation, peptone is detected
qualitively by adding a few drops of dilute solution of copper sulfate
(biuret reaction).
_Kreatin, Kreatinin and Other Flesh Bases._—The clear, aqueous solution
of the extract is acidified with sulfuric, mixed with a solution of
sodium phosphotungstate and allowed to stand for about six days. The
precipitate is collected, washed with a solution of the precipitant,
and the nitrogen therein determined. The nitrogen found, less that due
to ammonia, represents the total nitrogenous matter precipitated by
the phosphotungstic acid. From this quantity is deducted the nitrogen
in the proteids, precipitated by sixty-two per cent alcohol and by
ammonium or zinc sulfate, and the remainder represents the nitrogen in
flesh bases.
The nitrogen thrown out by the phosphotungstic acid is deducted from
the total nitrogen, and the remainder represents the nitrogenous bodies
not precipitable by the reagent named.
This method of separating the nitrogenous matters in meat extracts is
based on the observation that these bodies contain at most only a small
quantity of peptones, so small as to be safely negligible.[394]
_Quantities used for Analysis._—In conducting the separations above
noted, it will be found convenient to use in each case about five
grams of the solid or twenty of the liquid extract. In the nitrogen
determinations, the weight of the sample should be inversely
proportional to its content of nitrogen.
=417. Preparation of the Phosphotungstic Reagent.=—The phosphotungstic
reagent is conveniently prepared as follows:
Dissolve 120 grams of sodium phosphate and 200 of sodium tungstate in
one liter of water and add to the solution 100 cubic centimeters of
strong sulfuric acid. When the reagent is prepared for general purposes
it is customary to acidify with nitric, but in the present instance,
inasmuch as the precipitate is used for the determination of nitrogen,
it is evident that sulfuric should be substituted for nitric acid.
In all cases the analyst must be assured of the strong acidity of
the reagent, and in addition to this the solutions of proteid matter
to which the reagent is added must first be made strongly acid with
sulfuric.
=418. Zinc Sulfate as Reagent for Separating Albumoses from
Peptones.=—When the albumoses are separated from the peptones, by
precipitation with ammonium sulfate, there may be danger of some of
this reagent adhering to the albumose, and in this way the quantity of
nitrogen obtained on analysis may be increased. To avoid an accident of
this kind Bömer replaces the ammonium by zinc sulfate.[395]
Since the precipitation of the albumoses by saturated saline solutions
depends on their hydrolytic power, the substitution of another salt
for ammonium sulfate capable of strongly attracting water, may be made
if that salt does not possess any objectionable property. Crystallized
zinc sulfate will dissolve in less than its own weight of cold water
and is therefore well suited for the purpose in view.
In the case of a meat extract, the precipitation is accomplished as
follows: Fifty cubic centimeters of the extract, freed from all solid
matter by filtration and containing about two grams of the soluble
proteids, are saturated in the cold with finely powdered zinc sulfate.
The separated albumoses collect on the surface and are skimmed off,
poured on a filter and washed with cold saturated zinc sulfate
solution. The filter and its contents are used for the determination of
nitrogen by moist combustion.[396]
The filtrate from the precipitated albumoses gives no biuret reaction,
and, therefore, as in the use of ammonium sulfate, is free of albumin.
The biuret reaction is applied to the zinc sulfate filtrate as follows:
The filtrate is greatly diluted with water and freed of zinc by means
of a saturated solution of sodium carbonate. The filtrate free of zinc
is evaporated on the steam-bath, made strongly alkaline with sodium
hydroxid and treated with a few drops of a two per cent copper sulfate
solution, added successively.
Another advantage possessed by the zinc sulfate is found in the fact
that in the filtrate from the separated albumoses the peptones and
other flesh bases can be thrown out by phosphotungstic acid. Before the
application of the reagent, the filtrate should be made strongly acid
by adding about an equal volume of dilute sulfuric acid (one part of
acid to four of water.)
The nitrogen in the precipitate thus obtained is determined by moist
combustion in the manner already suggested.
If the proteid matters contain salts of ammonium it is probable
that a difficultly soluble double sulfate of zinc and ammonium,
(NH₄)₂SO₄.ZnSO₄.6H₂O, will be found in the precipitate. Ammonium salts,
if present, should therefore be removed by distillation with magnesia.
It is better, however, to throw down the ammonia with the first zinc
precipitate, distil this with magnesia and determine the amount of
nitrogen derived from the ammonia compounds. In a second sample, the
total nitrogen is determined by moist combustion and the difference
between the two results gives that due to albumoses.
=419. Examination for Muscular Tissue.=—Some samples of meat extracts
contain small quantities of finely ground muscular tissue. For
detecting this the extract is treated with cold water and the insoluble
residue examined with a microscope. If muscular tissue be found, about
eight grams of the extract or twenty-five of the fluid preparation, are
treated with cold water, the insoluble matter collected upon a filter,
washed with cold water, and the nitrogen determined in the residue. The
percentage of nitrogen multiplied by 6.25 gives the quantity of muscle
fiber proteids present. The filtrate from the above determination
is acidified with acetic, boiled, any precipitate which is formed
collected and the nitrogen therein determined. The nitrogen obtained
multiplied by 6.25 gives the quantity of coagulable albumin present.
An aliquot portion of the filtrate is used for the determination of
nitrogen and the percentage therein found, deducted from the total
nitrogen of the sample, gives a remainder which may be used as a
representative of the whole of the nitrogen present in the form of
albumin and muscular tissue.
=420. Estimation of Gelatin.=—The tin foil dish and its contents
used for the determination of water, as above described, are cut
into small pieces, placed in a beaker and extracted four times with
absolute alcohol. After the removal of the alcohol, the residue is
extracted with ice water containing ten per cent of alcohol, in
which a small piece of ice is kept to avoid a rise of temperature.
The beaker should be shaken during the extraction, which should last
for about two minutes. Where large numbers of samples are treated
at once, any convenient form of shaking machine may be employed. At
least two extractions with ice water must be made. The residue is then
collected upon a filter and washed with ice water until the washings
are completely colorless. The residue on the filter is replaced in
the beaker, boiled with water, well washed on the filter with boiling
water, the filtrate and washings concentrated and the nitrogen therein
determined.
The principle of this determination is based on the fact that gelatin
is almost completely insoluble in ice water while serum peptones and
albumin peptones are almost completely soluble in that reagent. On
the other hand, the flesh bases and the proteids present are almost
completely removed by the preliminary treatment with alcohol and ice
water or are left undissolved by the hot water. The solution in boiling
water, therefore, contains practically nothing but gelatin.[397]
In a later article, Stutzer modifies the method given above as
follows:[398]
Of dry and moist extracts from five to seven grams and of liquid
extracts from twenty to twenty-five grams are used for the
determination and placed in tin foil dishes, as described above. In
case of solid extracts, a sufficient quantity of warm water is added to
completely dissolve them, the solution being facilitated by stirring.
In case the solution is too thin it should be concentrated before going
further. It is treated with a sufficient amount of dust-free ignited
sand to completely absorb it, and the dish and its contents are then
dried to a constant weight. The dried contents of the dish are rubbed
up in a mortar, the dish cut into fine bits, and all placed in a
beaker. The solid syrphete[399] is extracted four times with 100 cubic
centimeters of absolute alcohol, the alcohol in each case being poured
through an asbestos filter for the purpose of collecting any matters
suspended therein. In a large flask are placed 100 grams of alcohol,
300 grams of ice and 600 grams of cold water, and the flask is placed
in a large vessel and packed with finely divided ice. Four beakers
marked _b, c, d, e_ are also placed in ice and the beaker containing
the syrphete, left after extraction with absolute alcohol as above
mentioned, is marked _a_ and also placed in pounded ice. The extraction
with cold alcoholic water proceeds as follows:
In beaker _a_ are poured 100 cubic centimeters of the mixture in the
large flask, its contents are stirred for two minutes and then the
liquid portion poured off into beaker _b_ to which, at the same time,
a piece of ice is added. In beaker _a_ are poured again 100 cubic
centimeters from the large flask, treated as above described, and the
liquid extract poured into beaker _c_. In like manner the extraction
in beaker _a_ is continued until each of the beakers has received
its portion of the extract. By this time the liquid over the sand in
beaker _a_ should be completely colorless. The filtration of the liquid
extract is accomplished as follows:
In a funnel of about seven centimeters diameter is placed a perforated
porcelain plate about four centimeters in diameter which is covered
with asbestos felt with long fiber. Three filters are prepared in this
way. On the first filter are poured the contents of beaker _b_. After
the liquid has passed through, the sand and other residue in beaker _a_
are transferred to the filter and the beaker and residue washed with
the alcoholic ice water from the large flask. The filtration should
be accomplished under pressure. On the second filter are poured the
contents of beaker _c_. On the third filter the contents of beakers
_d_ and _e_. The washing with alcoholic ice water from the large flask
is continued in each instance until the filtrate is colorless. At the
same time the asbestos filter, which was used in the first instance for
filtering the absolute alcohol extract, is washed with the alcoholic
ice water mixture from the large flask. At the end the sand remaining
in beaker a together with all the asbestos filters are brought
together into a porcelain dish, boiled two or three times with water,
the aqueous solution filtered and the filtrate concentrated and used
for the estimation of the nitrogen. The quantity of nitrogen found
multiplied by 6.25 represents the proteid matter in the gelatin of the
sample.
The object of the multiple filters, described above, is to accelerate
the process, and they are required because the gelatin quickly occludes
the filter pores. For this reason the asbestos filters are found
to operate better than those made of paper. It should be mentioned
that the residue of the peptones insoluble in alcohol may contain,
in addition to gelatin, also small quantities of albumoses. From the
quantity of albumose nitrogen found, it is understood that the nitrogen
in the form of coagulable albumin, determined as described in the first
process mentioned above, is to be deducted, since these coagulable
albumins are insoluble in alcohol.
=421. Estimation of Nitrogen in the Flesh Bases Soluble in
Alcohol.=—About five grams of the dry extract, ten grams of the
extract containing water or twenty-five grams of the liquid extract
are placed in a beaker and enough water added in each case to make
about twenty-five cubic centimeters in all. Usually no water need be
added to the liquid extracts. Very thin peptone solutions should be
evaporated until the content of water is reduced to seventy-five per
cent. The solution, prepared as above indicated, is treated slowly
with constant stirring with 250 cubic centimeters of absolute alcohol,
the stirring continued for some minutes and the vessel set aside for
twelve hours, at the end of which time the precipitate is separated by
filtration and washed repeatedly with strong alcohol. Leucin, tyrosin
and a part of the flesh bases are dissolved by alcohol. The alcohol is
removed by distillation and the residue dissolved in water. Any flocky
residue which remains on solution with water is removed by filtration,
the nitrogen determined therein and the quantity thereof added to the
albumose nitrogen found, as hereafter described.
The volume of the aqueous solution is completed with water to half a
liter. One hundred cubic centimeters of this solution are used for the
determination of total nitrogen, and another 100 cubic centimeters
for the determination of ammoniacal nitrogen by distillation with
barium carbonate. A part of the ammonia may have escaped during the
preliminary distillation of the alcohol and therefore the amount found
may not represent the whole amount originally present. The use of the
above determination is principally to ascertain the correction to be
made in the amount of total nitrogen found in the first 100 cubic
centimeters of the solution.
=422. Treatment of the Residue Insoluble in Alcohol.=—The residue
insoluble in alcohol is washed from the filter into the beaker in
which the first solution was made. The aqueous mixture is warmed
on a water-bath until the alcohol adhering to the precipitate is
completely evaporated, when the contents of the beaker are poured upon
a filter free of nitrogen. A small part of the albumose, by reason of
the treatment with alcohol, tends to remain undissolved, and it is
advisable to collect this albumose upon a filter, wash it well with hot
water and estimate the nitrogen therein. The quantity of nitrogen thus
found is to be added to the albumose nitrogen determined as described
later on.
The total filtrate obtained from the last filtration is made up to
a volume of half a liter, of which fifty cubic centimeters are used
for the determination of total nitrogen, fifty cubic centimeters for
the determination of gelatin, albumose and peptone, and 100 cubic
centimeters for the residual peptones. The albumose, together with the
gelatin and peptones carried down with it, is precipitated with zinc or
ammonium sulfate solution, and its per cent calculated from the amount
of nitrogen found in the precipitate. The true peptone is determined by
subtracting the quantity of nitrogen determined as albumose from the
total nitrogen in solution.
The rest of the liquid, _viz._, 300 cubic centimeters, is evaporated to
a small volume and tested qualitively for true peptones as follows:
To separate the albumose and gelatin a concentrated liquor is treated
with an excess of finely divided ammonium sulfate so that a part of
the salt remains undissolved. The separated albumose, gelatin and
undissolved ammonium salts are collected on a filter, the filtrate
mixed with a few drops of dilute copper sulfate solution and a
considerable quantity of concentrated soda or potash lye added. Care
should be taken that the quantity of copper is not too great, otherwise
the peculiar red coloration will be obscured by the blue color of the
copper solution.
=423. Pancreas Peptone.=—The filtrate obtained as described above, by
treating the portion of the material insoluble in alcohol with warm
water, contains in addition to the albumose and gelatin the whole of
the pancreas peptone which may be present. To separate this peptone,
100 cubic centimeters of the aqueous solution are evaporated in a
porcelain dish until the volume does not exceed ten cubic centimeters.
When cool, at least 100 cubic centimeters of a saturated cooled
solution of ammonium sulfate solution are added, the mixture thoroughly
stirred, the precipitate collected upon a filter and washed with a cold
saturated solution of ammonium sulfate. The contents of the filter
are dissolved in boiling water, the filter thoroughly washed and the
filtrate and washings evaporated in a porcelain dish with the addition
of barium carbonate until, on the addition of new quantities of barium
carbonate, no further trace of ammonia can be discovered. The residue
is extracted with water, the barium sulfate and carbonate present
separated by filtration, well washed and the nitrogen determined in the
evaporated filtrate and washings in the usual way and multiplied by
6.25 to determine the quantity of pancreas peptone.
=424. Albumose Peptone.=—A part of the albumose peptone which may be
present is determined in conjunction with the other bodies mentioned
above. The chief quantity is found in the solution of the residue
insoluble in alcohol in the following manner:
Fifty cubic centimeters of the solution of this residue in hot water
are mixed with an equal volume of dilute sulfuric acid, one volume
of acid to three of water, in the cold, and a solution of sodium
phosphotungstate added until it produces no further precipitate. The
precipitate is washed with dilute sulfuric acid and the nitrogen
determined therein. The nitrogen thus found is derived from the
albumose, pancreas peptone and gelatin. The quantity of nitrogen in the
pancreas peptone and gelatin, as above described, is subtracted from
the total quantity found in the phosphotungstic acid precipitated, and
the remainder represents the nitrogen due to the albumose.
=425. Nitrogen in the Form of Flesh Bases Insoluble in Alcohol.=—This
is determined by subtracting the quantity of nitrogen, determined by
the phosphotungstic acid method already described, from the total
quantity of nitrogen found in the precipitate insoluble in alcohol and
soluble in water.