=178. Historical.=—As long ago as 1868 Wanklyn proposed to conduct the
combustion of organic bodies in a wet way, using potassium permanganate
as the oxidizing body.[147] About ten years after this he attempted
to extend the method so as to estimate the quantity of proteid matter
in a sample by treatment with an alkaline solution in presence of the
permanganate salt. One gram of the finely pulverized sample was treated
in a liter flask with one-tenth normal potash lye. After digestion for
some time, from ten to twenty cubic centimeters were taken for the
determination. According to the supposition of Wanklyn, pure albuminoid
matters thus treated yielded one-tenth of their weight of ammonia,
or about fifty per cent of the total nitrogen appeared as ammonia.
The ammonia content of the sample was determined by the colorimetric
process devised by Nessler. It is needless to add that the process of
Wanklyn proved to be of no practical use whatever, acting differently
on different albuminoid matters, and even on the same substance.
No other attempt was made to perfect the moist combustion process
until Kjeldahl[148] introduced the sulfuric acid method in 1883. The
simplicity, economy, and adaptability of this method have brought it
into general use. At first the process was only applied to organic
nitrogenous compounds in the absence of nitrates, but especially by
the modifications proposed by Asboth, Jodlbaur, and Scovell, it has
been made applicable to all cases, with the possible exception of a
few alkaloidal and allied bodies. The moist combustion process for
determining nitrogen is now generally employed by chemists in all
countries, not only for fertilizer control, but also for general work.
=179. The Method of Kjeldahl.=—The process originally proposed by
Kjeldahl is applicable only to nitrogenous bodies free of nitric
nitrogen. The principle of the process is based on the action of
concentrated sulfuric acid at the boiling-point in decomposing
nitrogenous compounds without producing volatile combinations and
the subsequent completion of the oxidation by means of potassium
permanganate. The original process has been modified by many analysts
but the basic principle of it has remained unchanged. It will therefore
prove useful here to describe the process as originally given.[149]
The weighed substance is placed in a small flask. With solid bodies
this is a very simple operation, but with liquids more difficult.
Liquids which are not decomposed, on heating, should be evaporated in
a thin glass dish which can be ground up and placed in the digestion
flask with the desiccated sample. The strongest sulfuric acid is added
in sufficient quantity to secure complete decomposition, not less than
ten cubic centimeters in any case. The acid must be free of ammonia and
be kept in such a way as not to absorb ammonia from the atmosphere of
the laboratory. To guard against danger of error from such an impurity
frequent control determinations should be made. In control experiments
one or two grams of pure sugar should be used as the organic matter. If
the acid employed contain traces of ammonia the necessary corrections
should be made in each analysis. The flask having been charged is
placed on a wire gauze over a small flame. The organic matter becomes
black and tar-like and soon there is a rapid decomposition attended
with the evolution of gaseous products among which sulfur dioxid is
found. To avoid danger from spurting, the digestion flask should be
placed in an oblique position. The flask should have, at least, a
capacity of 100 cubic centimeters and a long neck and be made of a
kind of glass capable of withstanding the action of the boiling acid.
Particles of the carbonized organic matter left on the sides of the
flask by the foaming of the mass at first are gradually dissolved by
the vapors of the boiling acid as the digestion proceeds. The action
of the sulfuric acid is not entirely finished when gases cease to be
given off but the digestion should be continued until the liquid in the
flask is clear and colorless or nearly so. Usually about two hours are
required to secure this result. When aided by the means mentioned below
the time of digestion can be very materially shortened. By adding some
fuming sulfuric acid, or glacial phosphoric acid, the dilution caused
by the formation of water in the combustion of the organic matter, can
be avoided. For albuminoid bodies it is hardly necessary to continue
the combustion until all carbonaceous matter is destroyed. The full
complement of ammonia is usually obtained after an hour’s combustion
even if the liquid be still black or brown, but with other nitrogenous
bodies the case is different so that upon the whole it is safest to
secure complete decoloration.
The temperature must be maintained at the boiling-point of the acid
or near thereto since at a lower temperature, for instance from 100°
to 150°, the formation of ammonia is incomplete. Since all organic
substances of whatever kind are dissolved by the boiling acid the
previous pulverization of the material need be carried only far enough
to secure a fair sample. Many substances give up practically all
their nitrogen as ammonium sulfate when heated with sulfuric acid as,
for instance, urea, asparagin, and the glutens. In most of the other
organic bodies fully ninety per cent of the nitrogen are likewise
secured as the ammonium salt. In the aromatic compounds, or even in
the form of amid in anilin salts, the nitrogen is more resistant to
the action of sulfuric acid. In the alkaloids where the nitrogen is
probably a real component of the benzol skeleton the formation of
ammonia is very incomplete. But even in the cases where the conversion
of the nitrogen into ammonia is practically perfect it is advisable
to finish the process by completing the oxidation with potassium
permanganate. The permanganate should be used in a dry powdered form
and added little by little to the hot contents of the digestion flask,
the latter being held in an upright position and removed meanwhile
from the lamp. When carefully performed there is no danger of loss
of ammonia although the oxidation is, at times, so vigorous as to be
attended with evolution of light. The permanganate must always be added
in excess and until a permanent green color is produced. The flask is
then gently heated for from five to ten minutes over a small flame, but
this is not important. The heating must not be too strong or else a
strong evolution of oxygen will take place with a consequent reduction
of the manganese compound. When this happens the liquid again becomes
clear and there is a loss of ammonia.
After cooling, the contents of the flask are diluted with water, the
green color giving place to a brown with a rise of temperature. After
cooling a second time the whole is brought into a distillation flask of
about three-quarters of a liter capacity and attached to a condenser
which ends in a vessel containing titrated sulfuric acid. About forty
cubic centimeters of sodium hydroxid solution of one and three-tenths
specific gravity are added and the stopper at once inserted to prevent
any loss of ammonia. To prevent bumping during the distillation some
zinc dust is added securing an evolution of hydrogen during the
progress of the distillation. In this case the bumping is prevented
until near the end of the operation when it begins anew, probably by
reason of the separation of solid sodium sulfate. After the end of
the distillation, the excess of acid remaining in the receiver is
determined by a set alkali solution and thus the quantity of ammonia
obtained easily calculated. Kjeldahl, however, preferred to titrate the
solution after adding potassium iodate and iodid, a mixture which in
the presence of a strong acid sets free a quantity of iodin equivalent
to the free acid present. The iodin thus set free is titrated by a set
solution of sodium thiosulfate using starch as an indicator. The merits
of this method are sharpness of the end reaction and the possibility of
using only a small quantity of the nitrogenous body for the combustion.
The sulfuric acid used in the receiver is made of the same strength as
the thiosulfate solution; _viz._, about one-twentieth normal. Thirty
cubic centimeters of this were found to be the proper amount for use
with substances taken in such quantities as to produce ammonia enough
to neutralize about half of it. The titration is carried on as follows:
A few crystals of potassium iodid are dissolved in the acid mixture
obtained after the distillation is completed, then a few drops of the
starch-paste and finally a few drops of a four per cent solution of
potassium iodate. The iodin set free is then oxidized by the addition
of the one-twentieth normal sodium thiosulfate solution until the blue
color disappears.
_Example_: Sulfuric acid used, 30 cc.
Equivalent to sodium thiosulfate, 30 cc.
Blank combustion required, 29.8 cc thiosulfate solution.
Combustion of 0.645 gram of barley 14.5 cc “ “
required, -------
Thiosulfate corresponding to barley, 15.3 cc.
In the computation it is more simple to multiply the corresponding
number of cubic centimeters of thiosulfate by seven, half the atomic
weight of nitrogen, and divide the product by the weight of the
substance taken, which will give the per cent of nitrogen therein.
15.3 × 7
Then ---------- × 100 = 1.66 = per cent of nitrogen in sample taken.
0.645
A more detailed description of the method of making the titration
follows: After the distillation is finished the condensing-tube is
rinsed with a little water, after which the sulfuric acid unneutralized
in the receiver is determined. It is advisable first to test the
reaction of the distillate with litmus paper before going any further;
for if at any time all the acid should be found neutralized it will
be necessary to add a sufficient quantity of one-twentieth normal
sulfuric acid before adding the potassium iodid, etc., otherwise the
determination will be irreparably lost. Add to the contents of the
flask ten cubic centimeters of the potassium iodid and two cubic
centimeters of the potassium iodate solutions, described further on,
and the sodium thiosulfate is then run in from a burette till the
fluid, which is constantly kept agitated by shaking the flask, shows
only a bare trace of yellow coloration from the iodin still present.
Starch solution is then added, and the blue color obtained is at once
removed by additional thiosulfate solution. When some experience has
been gained, the eye is able to discern, with great certainty, even the
slight coloration caused by only a small trace of free iodin.
In regard to the sensitiveness of the end reaction and the accuracy
of the result, this method of titration leaves nothing to be wished
for. The strength of the thiosulfate solution is determined in exactly
the same manner, and with starch as an indicator. For this purpose,
measure ten cubic centimeters of one-twentieth normal sulfuric acid
into an erlenmeyer, add 120 cubic centimeters of ammonia-free water,
ten cubic centimeters of potassium iodid solution, and two cubic
centimeters of iodate solution; add thiosulfate solution till the fluid
shows only the above-mentioned light yellow tint, then add starch,
and finally thiosulfate. In this way the strength of the thiosulfate
is ascertained, which of course must be occasionally re-determined,
under exactly the same conditions as with the nitrogen determinations
themselves, and every possible error is thereby excluded. That the
solution once decolorized within a short time again assumes a deep blue
color, is a matter of no concern, inasmuch as both solutions are added
in such a manner that the end reaction lies exactly at the point when
the starch iodid reaction distinctly disappears.
=180. Theory of the Reactions.=—As has been seen above the final
product of heating a nitrogenous organic compound with sulfuric acid
and an oxidizing body is ammonium sulfate. The various steps by which
this is obtained have been traced by Dafert:[150]
(1) The sulfuric acid abstracts from the organic matter the elements of
water:
(2) The sulfur dioxid produced by the action of the residual carbon on
sulfuric acid exercises a reducing effect on the nitrogenous bodies
present:
(3) From the nitrogenous bodies produced by the above reduction ammonia
is formed by the action of an oxidizing body:
(4) The ammonia formed is at once fixed by the acid as ammonium
sulfate. According to the theory of Asboth the hydrogen which is formed
during the action of sulfuric acid on organic matter, when in a nascent
state, also aids greatly in the production of ammonia. This idea is
based on the fact that with those bodies which afford a deficit of
hydrogen the formation of ammonia is imperfect.[151]
=181. Preparation of Reagents.=—(1) _Pure Sulfuric Acid._—As is well
known the so-called pure sulfuric acid in the market usually contains
ammonia, a fact which compelled Kjeldahl to determine the quantity
of nitrogen in the acid in every instance, and to make correction
for the same in the analysis. An acid absolutely free from this
impurity may, however, easily be prepared by the distillation of the
commercial article in a small glass retort holding easily about 400
cubic centimeters. To conduct this operation without danger it is only
necessary to arrange the apparatus so that the heavy fluid is heated
to boiling, not from the bottom of the retort, but from its sides,
and that the upper portion of the body and neck is kept sufficiently
warm so that the sulfuric acid fumes are not allowed to condense and
flow back into the retort. Both these ends are attained simply by
surrounding the retort with a piece of sheet iron, cylinder-shaped
beneath, and with an oval upper part, having an opening of about one
centimeter in diameter for the neck of the retort. To conduct the
distillation a burner is used with an arrangement for spreading the
flame. To avoid with certainty all bumping of the sulfuric acid and
the resulting danger therefrom, the lamp is so arranged that only the
products of combustion go up between the retort and its iron hood,
without allowing the flame itself to come into contact with the glass
vessel. The retort should be filled about half full, or with 200 cubic
centimeters of acid. By this device, without any danger whatever,
about one liter of sulfuric acid may be distilled in a day. The retort
will stand numerous distillations. Once begun, the distillation takes
care of itself; it is necessary to discontinue it when only the bottom
of the retort is covered with sulfuric acid, and to fill fresh acid
through a funnel when the retort has cooled off. The first twenty cubic
centimeters of the distillate going over are collected by themselves
and rejected. What comes over later is, as shown by experience,
absolutely ammonia-free, and can be used without any correction, for
the nitrogen determinations according to Kjeldahl. The acid is kept in
a stoppered bottle in a place not reached by ammonia fumes. The ten
cubic centimeter pipette used for measuring the quantity of sulfuric
acid required for each determination, is fastened in the perforated
rubber stopper with which the bottle is kept closed, and is itself
closed above by a small rubber tube with a plug of glass wool in it.
(2) _Potassium Permanganate._—Crystals of this salt are crushed
(not pulverized) with a pestle into small pieces of about one-half
millimeter size, which are kept in a long glass tube of about ten
millimeters diameter, closed with a stopper.
(3) _Ammonia-free Water._—Common distilled water cannot be used in the
determination of nitrogen according to Kjeldahl, since it contains
ammonia. It may be obtained free from the same by redistillation in a
large glass retort with the addition of a few drops of sulfuric acid.
All vessels used in the determination are rinsed out beforehand with
this water.
(4) _Ammonia-free Soda-lye_ is most conveniently prepared by adding 270
grams of common sodium hydroxid in sticks, little by little, to one
liter of distilled water which is kept continually boiling, by means of
a small flame, in a good-sized silver dish. The dish is kept covered
with a glass plate. Care has to be exercised not to add the alkali too
rapidly, nor in too large quantities at a time for in this case the
fluid will boil too violently at every addition of the alkali. After
the operation is finished the lye is at once siphoned into a glass
flask, and when cold, is poured into a glass-stoppered bottle.
(5) _One-twentieth Normal Sulfuric Acid_ is prepared from sulfuric acid
and water both absolutely ammonia-free, and is kept in a place where no
fumes of ammonia can reach it, in a well-stoppered glass bottle, the
stopper being smeared with vaseline.
(6) _Sodium Thiosulfate Solution._—This should be of the same strength
as the one-twentieth normal sulfuric acid. It is prepared by dissolving
the salt in ammonia-free water, and is compared with the acid, to
which has been added potassium iodid and iodate, using starch as an
indicator, in the manner described above. The solution is kept in a
well-stoppered bottle, in the dark. When the salt and water used are
perfectly pure, it will keep unchanged for a long time.
(7) _Potassium Iodid._—Dissolve five grams of chemically pure potassium
iodid in ammonia-free water and make the volume 100 cubic centimeters.
Ten cubic centimeters of this solution are used for each determination;
keep the solution in the dark and in a well-stoppered bottle.
(8) _Potassium Iodate._—Dissolve four grams of chemically pure
potassium iodate in ammonia-free water and make the volume 100 cubic
centimeters. Use two cubic centimeters of this solution for each
determination.
(9) _Starch Solution._—Digest pure starch for about a week with dilute
hydrochloric acid, wash perfectly free from chlorin by decantation, and
finally dry it between filter-paper. The starch is then dissolved in
water with the aid of heat. Such a solution will keep for an indefinite
time, if it be saturated with common salt. Ten grams of this starch are
dissolved in 1,000 cubic centimeters of ammonia-free water; use one or
two cubic centimeters for each determination.
=182. Kjeldahl Method as Practiced by the Holland Royal Experiment
Station.=—_Necessary Reagents_: 1. Phosphosulfuric acid, made by mixing
a liter of sulfuric acid of specific gravity 1.84 with 200 grams of
phosphoric anhydrid:
2. Alkaline sodium sulfid solution, made by dissolving 500 grams of
sodium hydroxid and six grams of sodium sulfid or eight and one-half
grams of potassium sulfid, in a liter of water: