The hydrogen does not escape but combines in the nascent state with the
excess of phenylhydrazin to form anilin and ammonia.
The precipitation is accomplished as follows:
One part by weight of the sugar, two parts of phenylhydrazin
hydrochlorate, and three parts of sodium acetate are dissolved in
twenty parts of water and gradually heated on the water-bath.
The osazone slowly separates in a crystalline form and it is freed from
the mother liquor by filtration, and purified by solution in alcohol
and recrystallization. The crystals are composed of yellow needles,
which are difficultly soluble in water and more easily in hot alcohol.
The crystals are not decomposed by a dilute acid but are destroyed by
the action of strong acids.
_Dextrosazone._—The crystals melt at from 204° to 205°, reduce fehling
liquor, and dissolved in glacial acetic acid are slightly left rotating.
_Levulosazone._—This body has the same properties as the dextrose
compound.
_Maltosazone._—This substance melts at 206° with decomposition. It is
left rotating. Its structure is represented by the formula C₂₄H₃₂N₄O₉.
_Galactosazone._—This substance, C₁₈H₂₂N₄O₄, has the same centesimal
composition as the corresponding bodies produced from dextrose and
levulose. It is distinguished from these compounds, however, by its low
melting point, _viz._, 193°.
The above comprise all the phenylosazones which are important from
the present point of view. Sucrose, by inversion, furnishes a mixture
of dextros- and levulosazones when treated with phenylhydrazin, while
starch and dextrin yield the dextros- or maltosazone when hydrolyzed.
Lactose yields a mixture of dextros- and galactosazones when hydrolyzed
and treated as above described.
The reactions with phenylhydrazin are approximately quantitive and it
is possible that methods of exact determination may be based on them in
the near future.[138]
=173. Other Qualitive Tests for Sugars.=—The analyst may sometimes
desire a more extended test of qualitive reactions than those given
above. The changes of color noticed on heating with alkalies may
often be of advantage in discriminating between different sugars. The
formation of definite compounds with the earthy and other mineral bases
may also be used for qualitive determinations. One of the most delicate
qualitive tests is found in the production of furfurol and this will be
described in the following paragraphs.
=174. Detection of Sugars and Other Carbohydrates by Means of
Furfurol.=—The production of furfurol (furfuraldehyd) as noted in
paragraph =150=, is also used quantitively for the determination of
pentose sugars and pentosans.
Furfurol was first obtained from bran (_furfur_), whence its name,
by treating this substance with sulfuric acid, diluted with three
volumes of water, and subjecting the mixture to distillation. Its
percentage composition is represented by the symbol C₅H₄O₂, and its
characteristics as an aldehyd by the molecular structure C₄H₃O,C-HO.
Carbohydrates in general, when treated as described above for bran,
yield furfurol, but only in a moderate quantity, with the exception of
the pentoses.
Mylius has shown[139] that Pettenkofer’s reaction for choleic acid is
due to the furfurol arising from the cane sugar employed, which, with
the gall acid, produces the beautiful red-blue colors characteristic of
the reaction.
Von Udránszky[140] describes methods for detecting traces of
carbohydrates by the furfurol reaction, which admit of extreme
delicacy. The solution of furfurol in water, at first proposed by
Mylius, is to be used and it should not contain more than two and
two-tenths per cent, while a solution containing five-tenths per cent
furfurol is found to be most convenient. The furfurol, before using,
should be purified by distillation, and, as a rule, only a single drop
of the solution used for the color reaction.
The furfurol reaction proposed by Schiff[141] appears to be well suited
for the detection of carbohydrates. It is made as follows:
Xylidin is mixed with an equal volume of glacial acetic acid and the
solution treated with some alcohol. Strips of filter paper are then
dipped in the solution and dried. When these strips of prepared paper
are brought in contact with the most minute portion of furfurol,
furoxylidin is formed, C₄H₃OCH(C₈H₈NH₂)₂, producing a beautiful red
color. In practice, a small portion of the substance, supposed to
contain a carbohydrate, is placed in a test tube and heated with a
slight excess of concentrated sulfuric acid. The prepared paper is then
placed over the mouth of the test tube so as to be brought into contact
with the escaping vapors of furfurol.
The furfurol reaction with α-naphthol for some purposes, especially
the detection of sugar in urine, is more delicate than the one just
described. This reaction was first described by Molisch[142], who,
however, did not understand its real nature.
The process is carried on as follows: The dilute solution should
contain not to exceed from 0.05 to one-tenth per cent of carbohydrates.
If stronger, it should be diluted. Place one drop of the liquid in a
test tube with two drops of fifteen per cent alcoholic solution of
α-naphthol, add carefully one-half cubic centimeter of concentrated
sulfuric acid, allowing it to flow under the mixture. The appearance
of a violet ring over a greenish fringe indicates the presence of a
carbohydrate. If the substance under examination contain more than a
trace of nitrogenous matter, this must be removed before the tests
above described are applied.
If the liquids be mixed by shaking when the violet ring is seen, a
carmine tint with a trace of blue is produced. If this be examined with
a spectroscope, a small absorption band will be found between D and E,
and from F outward the whole spectrum will be observed. One drop of
dextrose solution containing 0.05 per cent of sugar gives a distinct
reaction by this process. It can be used, therefore, to detect the
presence of as little as 0.028 milligram of grape sugar. This test has
been found exceedingly delicate in this laboratory, and sufficiently
satisfactory without the spectroscopic adjunct.
The furfurol reaction is useful in detecting the presence of minute
traces of carbohydrates but is of little value in discriminating
between the different classes of these bodies.
It is not practical here to go into greater detail in the description
of qualitive reactions. The analyst, desiring further information,
should consult the standard works on sugar chemistry.[143]
=175. Detection of Sugars by Bacterial Action.=—Many forms of bacteria
manifest a selective action towards sugars and this property may in the
future become the basis of a qualitive and even quantitive test for
sugars and other carbohydrates. Our present knowledge of the subject
is due almost exclusively to the researches of Smith, conducted at the
Department of Agriculture.[144] Dextrose is the sugar first and most
vigorously attacked by bacterial action, and by proper precautions the
whole of the dextrose may be removed from mixtures with sucrose and
lactose.
The development of other forms of micro-organisms which will have the
faculty of attacking other and special forms of carbohydrates is to be
looked for with confident assurance of success.