34.64 grams copper sulfate; 187.0 potassium-sodium tartrate; 78.0
sodium hydroxid made up to one liter. Ten cubic centimeters equal
0.050 gram dextrose. Ten cubic centimeters equal 0.0475 gram cane
sugar.
=117. Volumetric Method used in this Laboratory.=—The alkaline copper
solution preferred in this laboratory has the composition proposed by
Violette. The copper sulfate and alkaline tartrate solutions are kept
in separate vessels and mixed in proper proportions immediately before
use, and diluted with about three volumes of water. The reduction is
accomplished in a long test tube at least twenty-five centimeters in
length, and from thirty-five to forty millimeters in diameter.
The sugar solution employed should contain approximately one per cent
of reducing sugar. If it should have a greater content it should be
reduced with water to approximately the one named. If it have a less
content, it should be evaporated in a vacuum at a low temperature
until it reaches the strength mentioned above. A preliminary test
will indicate almost the exact quantity of the sugar solution to be
added to secure a complete reduction of the copper. This having been
determined the whole quantity should be added at once to the boiling
copper solution, the test tube held in the open flame of a lamp
giving a large circular flame and the contents of the tube kept in
brisk ebullition for just two minutes. The lamp is withdrawn and the
precipitated suboxid allowed to settle. If a distinct blue color remain
an additional quantity of the sugar solution is added and again boiled
for two minutes. When the blue coloration is no longer distinct, the
presence or absence of copper is determined by aspirating a drop or
two of the hot solution with the apparatus described below. This clear
filtered liquor is then brought into contact with a drop of potassium
ferrocyanid solution acidulated with acetic. The production of a brown
precipitate or color indicates that some copper is still present,
in which case an additional quantity of the sugar solution is added
and the operation continued as described above until after the last
addition of sugar solution no coloration is produced.
=118. The Filtering Tube.=—The filtering tube used in the above
operation is made of a long piece of narrow glass tubing with thick
walls. The length of the tube should be from forty to forty-five
centimeters. One end of the tube being softened in the flame is pressed
against a block of wood so as to form a flange. Over this flange is
tied a piece of fine linen.[82]
Instead of using a linen diaphragm the tube is greatly improved, as
suggested by Knorr, by sealing into the end of the tube while hot a
perforated platinum disk. Before using, the tube is dipped into a
vessel containing some suspended asbestos felt and by aspiration a thin
felt of asbestos is formed over the outer surface of the platinum disk.
By inverting the tube the water which has entered during aspiration is
removed. The tube thus prepared is dipped into the boiling solution in
the test tube above described and aspiration continued until a drop of
the liquor has entered the tube. It is then removed from the boiling
solution, the asbestos felt wiped off with a clean towel, and the drop
of liquor in the tube blown through the openings in the platinum disk
and brought into contact with a drop of potassium ferrocyanid in the
usual way. In this way a drop of the liquor is secured without any
danger of a reoxidation of the copper which may sometimes take place on
cooling.
[Illustration: FIGURE 42. APPARATUS FOR THE VOLUMETRIC ESTIMATION OF
REDUCING SUGARS.]
The careful analyst by working in this way with the volumetric method
is able to secure highly accurate results. The apparatus used is shown
in the accompanying illustration.
=119. Suppression of the Error Caused by the Action of the Alkali on
Reducing Sugars.=—Three methods are proposed by Gaud for correcting
or suppressing the error due to the action of the alkali upon
reducing sugars. In the first place, the common method followed may
be employed, depending upon the use of an alkaline copper solution of
known composition and the employment of a reducing sugar solution of
a strength varying between one-half and one per cent. The error which
is introduced into such a reaction is a constant one and the solution
having been tested once for all against pure sugar is capable of giving
fairly accurate results.
In the second place, a table may be constructed in which the error
is determined for sugar solutions for varying strengths, _viz._,
from one-tenth of one per cent to ten per cent. If _y_ represent the
error and _x_ the exact percentage of reducing sugar present then the
correction may be made by the following formula;
_y_ = -0.00004801_x_ + 0.02876359_x_².
In order to use this formula in practice the percentage of reducing
sugar obtained by the actual analysis must be introduced and may be
represented by θ. The formula for correction then becomes 0.02876_x_² -
1.000048_x_ + θ = 0; whence the value of _x_ is easily computed.
In the third place, the error may be eliminated by substituting for
an alkali which acts upon the glucose one which does not, _viz._,
ammonia. At the temperature of boiling water ammonia does not have any
decomposing effect upon reducing sugars. It is important, however,
that the reduction take place in an inert atmosphere in order to
avoid the oxidation of the dissolved cuprous oxid and the temperature
need not be carried beyond 80°. The end of the reaction can be easily
distinguished in this case by the disappearance of the blue color. When
one reaction is finished the copper may be completely reoxidized by
conducting through it a current of air or oxygen for half an hour, when
an additional quantity of ammonia may be added to supply any that may
have evaporated, and a new reduction accomplished with exactly the same
quantity of copper as was used in the first. The solution used by Gaud
contains 36.65 grams of crystallized copper sulfate dissolved in water
and the volume completed to one liter with ordinary aqueous ammonia.[83]
=120. Permanganate Process for the Estimation of Reducing
Sugars.=—Dextrose, invert sugar, and other reducing sugars can also be
determined with a fair degree of accuracy by an indirect volumetric
process, in which a standard solution of potassium permanganate is
used as the final reagent.[84] The principle of the process is based
upon the observation that two molecules of dextrose reduce from an
alkaline cupric tartrate solution five molecules of cuprous oxid.
The five molecules of cuprous oxid thus precipitated when added to an
acid solution of ferric sulfate, will change five molecules of the
ferric sulfate to ten molecules of ferrous sulfate. The reaction is
illustrated by the following equation:
{ 5Cu₂O } + { 5Fe₂(SO₄)₃ } + { 5H₂SO₄ } = { 10CuSO₄ }
{715 parts} { 2000 parts } {490 parts} {1595 parts}
+ { 10FeSO₄ } + { 5H₂O }
{1520 parts} {90 parts}
The ten molecules of ferrous sulfate formed as indicated in the above
reaction, are reoxidized to ferric sulfate by a set solution of
potassium permanganate. This reaction is illustrated by the equation
given below:
{ 10FeSO₄ } + { K₂Mn₂O₈ } + { 5H₂SO₄ } = { 5Fe₂(SO₄)₂ }
{1520 parts} {316.2 parts} {784 parts} { 2000 parts }
+ { 2MnSO₄ } + { K₂SO₄ } + { 8H₂O }
{302 parts} {174.2 parts} {144 parts}.
By the study of the above equations it is seen that two molecules
of dextrose or other similar reducing sugar, are equivalent to one
molecule of potassium permanganate, as is shown by the following
equations:
{ 2C₆H₁₂O₆ } = { 5Cu₂O } = { 10FeSO₄ } = { K₂Mn₂O₈ }
{ 360 parts } {715 parts} {1520 parts} {316.2 parts}
It is thus seen that 316.2 parts by weight of potassium permanganate
are equivalent to 360 parts by weight of dextrose; or one part of
permanganate corresponds to 1.1385 parts by weight of dextrose. If,
therefore, the amount of permanganate required in the above reaction to
restore the iron to the ferric condition, be multiplied by the factor
mentioned above, the quotient will represent in weight the amount of
dextrose which enters into the reaction. The standard solution of
potassium permanganate should contain 4.392 grams of the salt in a
liter. One cubic centimeter of this solution is equivalent to five
milligrams of dextrose.
=121. Manipulation.=—The saccharine solution whose strength is to be
determined should contain approximately about one per cent of sugar.
Of this solution ten cubic centimeters are placed in a porcelain dish
together with a considerable excess of fehling solution. When no
sucrose is present, the mixture may be heated to the temperature of
boiling water and kept at that temperature for a few minutes until
all the reducing sugar is oxidized. There should be enough of the
copper solution used to maintain a strong blue coloration at the end
of the reaction. A greater uniformity of results will be secured by
using in all cases a considerable excess of the copper solution. When
sucrose or other non-reducing sugars are present, the temperature of
the reaction should not be allowed to exceed 80° and the heating may
be continued somewhat longer. At this temperature the copper solution
is absolutely without action on sucrose. The precipitated suboxid is
allowed to settle, the supernatant liquid poured off through a filter
and the suboxid washed thoroughly a number of times by decantation with
hot water, the washings being poured through the filter. This process
of washing is greatly facilitated by decanting the supernatant liquid
from the porcelain dish first into a beaker and from this into a third
beaker and so on until no suboxid is carried off. Finally the wash
water is poured through a filter-paper bringing as little as possible
of the suboxid onto the paper. The suboxid on the filter-paper and in
the beakers is next dissolved in a solution of ferric sulfate made
strongly acid with sulfuric; or in a sulfuric acid solution of ammonia
ferric sulfate which is more easily obtained free from impurities than
the ferric sulfate. When all is dissolved from the beakers the solution
is poured upon the suboxid which still remains in the porcelain dish.
When the solution is complete it is washed into a half liter flask and
all the vessels which contain the suboxid are also thoroughly washed
and the wash waters added to the same flask. The whole is rendered
strongly acid with sulfuric and made up to a volume of half a liter.
The process carried out as directed, when tested against pure sugar,
gives good results, not varying from the actual content of the sugar
by more than one-tenth per cent below or three-tenths above the true
content. The distinct pink coloration imparted to the solution by the
permanganate solution as soon as the iron is all oxidized to the ferric
state marks sharply the end of the reaction. In this respect this
process is very much to be preferred to the usual volumetric processes
depending upon the coloration produced with potassium ferrocyanid
by a copper salt for distinguishing the end of the reaction. It is
less convenient than the ordinary volumetric process by reason of the
somewhat tedious method of washing the precipitated cuprous oxid. When
a large number of analyses is to be made, however, the whole can be
washed with no more expenditure of time than is required for a single
sample. One analyst can, in this way, easily attend to fifty or a
hundred determinations at a time.
In the application of the permanganate method to the analysis of the
juices of sugar cane and sorghum it is directed to take 100 cubic
centimeters of the expressed juice and clarify by the addition of
twenty-five cubic centimeters of basic lead acetate, diluted with
water, containing enough of the lead acetate, however, to produce a
complete clarification. It is not necessary to remove the excess of
lead from the filtrate before the determination. Ten cubic centimeters
of the filtrate correspond to eight cubic centimeters of the original
juice. For percentage calculation the specific gravity of the original
juice must be known. Before the addition of the alkaline copper
solution, from fifty to seventy-five cubic centimeters of water should
be added to the clarified sugar juice and the amount of fehling
solution used in each case should be from fifty to seventy-five cubic
centimeters. The heating at 75° should be continued for half an hour
in order to insure complete reduction and oxidation of the sugar. The
sucrose can also be estimated in the same juices by inverting five
cubic centimeters of the clarified juice with five cubic centimeters
of dilute hydrochloric acid, by heating for an hour at a temperature
not above 90°. Before adding the acid for inversion, about 100 cubic
centimeters of water should be poured over the five cubic centimeters
of sugar solution. The washing of the suboxid and the estimation of the
amount reduced are accomplished in the manner above described.
This method has been extensively used in this laboratory and with very
satisfactory results. The only practical objection which can be urged
to it is in the time required for filtering. This fault is easily
remedied by adopting the method of filtering through asbestos felt
described in the next paragraph.
For the sake of uniformity, however, the copper solution should
be boiled for a few minutes before the addition of the sugar in
order to expel all oxygen, the sugar solutions should be made with
recently boiled water and the precipitation of the suboxid should be
accomplished by heating for just thirty minutes at 75°. At the end of
this time an equal volume of cold, recently boiled, water should be
added and the filtration at once accomplished.
=122. Modified Permanganate Method.=—The permanganate method as used
by Ewell, in this laboratory, is conducted as follows: After the
precipitate is obtained, according to the directions given in the
methods described, it is thoroughly washed with hot, recently boiled
water, on a gooch. The asbestos, with as much of the precipitate as
possible, is transferred to the beaker in which the precipitation was
made, beaten up with from twenty-five to thirty cubic centimeters
of hot, recently boiled water, and from fifty to seventy-five cubic
centimeters of a saturated solution of ferric sulfate in twenty-five
per cent sulfuric acid are added to the beaker and then poured through
the crucible to dissolve the cuprous oxid remaining therein. If the
precipitate be first beaten up with water as directed, so that no
large lumps of it remain, there is no difficulty in dissolving the
oxid in the ferric salt; while if any lumps of the oxid be allowed to
remain there is great difficulty. After the solution is obtained, it is
titrated with a solution of potassium permanganate of such a strength
that each cubic centimeter is equivalent to 0.01 gram of copper.
In triplicate determinations made by this method the precipitates
obtained required after solution in the ferric salt, 28.7, 28.9, 28.6
cubic centimeters of potassium permanganate solution, respectively.
For the quantities taken this was equivalent to an average percentage
of reducing sugars of 4.19. The percentage obtained by the gravimetric
method was 4.26.
The method seems to be sufficiently accurate for all ordinary purposes
and is extremely rapid.
The permanganate solution used should be standardized by means
of metallic iron, but in ordinary work it is also recommended to
standardize by check determinations of reducing sugars in the same
sample by the gravimetric method.
=123. Determination of Reducing Sugar by the Specific Gravity of the
Cuprous Oxid.=—Gaud proposes to determine the percentage of reducing
sugar from the specific gravity of the cuprous oxid. The manipulation
is carried out as follows:
In a porcelain dish are placed fifty cubic centimeters of the alkaline
copper solution and an equal quantity of water and the mixture
maintained in ebullition for two or three minutes. The dish is then
placed on a boiling water-bath and twenty-five cubic centimeters of a
reducing sugar of approximately one per cent strength added at once.
The reduction is thus secured at a temperature below 100°, which is an
important consideration in securing the minimum decomposing effect of
the alkali upon the sugar. The dish is kept upon the water-bath for
about ten minutes when the reduction is complete and the supernatant
liquor should still be intensely blue. The precipitate is washed by
decantation with boiling water, taking care to avoid the loss of any
of the cuprous oxid. The washing is continued until the wash waters
are neutral to phenolphthalein. The cuprous oxid is then washed into a
pyknometer of from twenty to twenty-five cubic centimeters capacity,
the exact content of which has been previously determined at zero. It
is filled with boiling water, the stopper inserted, and after cooling
the flask is weighed. Let _P_ be the weight of the pyknometer plus the
liquid and the precipitate, the total volume of which is equal to the
capacity of the flask at the temperature at which it was filled, that
is _V_ₜ = _V_₀ [1 + 3β(_t_-_t_₀)].
This formula is essentially that given in paragraph =51=, for
calculating the volume of a pyknometer at any temperature, substituting
for 3β, γ the cubical expansion of glass, _viz_., 0.000025.
The specific gravity of the dry cuprous oxid is Δ = 5.881 and let the
specific gravity of water at the temperature of filling, which can be
taken from any of the tables of the density of water, be _d_. The total
weight _p_ of the precipitated suboxid may then be calculated by the
following formula:
_P_-_V_ₜ_d_
_P_ = -----------.
1 - _d_
----
Δ
The density of water at 99°, which is about the mean temperature of
boiling water for laboratories in general, is 0.95934, and this may be
taken as the weight of one cubic centimeter for purposes of calculation
in the formula above.
In order to obtain exact results, it is important that the weight
_P_ be reduced to a vacuum. The weight of cuprous oxid not varying
proportionally to the weight of reducing sugar, it is necessary to
prepare a table showing the principal numerical values of the two, in
order to be able to calculate easily all the possible values, either
directly from the table or by appropriate interpolations. Following are
the chief values which are necessary for the calculation:
Milligrams Milligrams Milligrams Milligrams
cuprous oxid. dextrose. cuprous oxid. dextrose.
10 5.413 100 46.221
20 9.761 200 91.047
30 14.197 300 138.842
50 23.036 400 188.928
It is claimed by the author that the above method is both simple and
rapid and can be applied with an error of not more than one-thousandth
if the corrections for temperature and pressure be rigorously
applied.[85]
=124. The Copper Carbonate Process.=—While the copper solutions which
have been mentioned in previous paragraphs have only a slight action
on sucrose and dextrin yet on prolonged boiling even these bodies show
a reducing effect due probably to a preliminary change in the sugar
molecules whereby products analogous to dextrose or invert sugar are
formed. In order to secure a reagent, to which the sugar not reducing
alkaline copper solutions might be more resistant Soldaini has proposed
to employ a liquor containing the copper as carbonate instead of
as tartrate.[86] This solution is prepared by adding to a solution
of forty grams of copper sulfate one of equal strength of sodium
carbonate. The resulting copper carbonate and hydroxid are collected on
a filter, washed with cold water, and dried. The reaction which takes
place is represented by the following formula:
2CuSO₄ + 2Na₂CO₃ + H₂O = CuCO₃ + CuO₂H₂ + 2Na₂SO₄ + CO₂.
The dry precipitate obtained, which will weigh about fifteen grams, is
placed in a large flask with about 420 grams of potassium bicarbonate
and 1400 cubic centimeters of water. The contents of the flask are
heated on a steam-bath for several hours with occasional stirring
until the evolution of carbon dioxid has ceased. During this time the
liquid is kept at the same volume by the addition of water, or by
attaching a reflux condenser to the flask. The potassium and copper
compounds at the end of this time will be found dissolved and the
resulting liquor will have a deep blue color. After filtration the
solution is boiled for a few minutes and cooled to room temperature.
The volume is then completed to two liters. A more direct method of
preparing the solution, and one quite as effective, consists in adding
the solution of the copper sulfate directly to the hot solution of
potassium bicarbonate and heating and shaking the mixture until the
copper carbonate formed is dissolved. After filtering the volume is
made as above. The proportions of reagents employed are placed by
Preuss at 15.8 grams of crystallized copper sulfate and 594 grams of
potassium bicarbonate.[87] The soldaini reagent is extremely sensitive
and is capable of detecting as little as half a milligram of invert
sugar. The presence of sucrose makes the reagent more delicate, and it
is especially useful in determining the invert sugar arising during the
progress of manufacture by the action of heat and melassigenic bodies
on sucrose.
=125. The Analytical Process.=—As in the case of fehling solution a
great many methods of conducting the analysis with the soldaini reagent
have been proposed. The general principle of all these processes is
the one already described for the alkaline copper tartrate solution,
_viz._, the addition of the reducing sugar solution to the boiling
reagent, and the determination of the end of the reaction by the
disappearance of the copper.[88]
Practically, however, these methods have had no general application,
and the use of the soldaini reagent has been confined chiefly to the
determination of invert sugar in presence of a large excess of sucrose.
For this purpose the sugar solution is not added until the blue color
of the reagent has been destroyed, but on the other hand, the reagent
has been used in excess, and the cuprous oxid formed collected and
weighed as metallic copper. The weight of the metallic copper found,
multiplied by the factor 0.3546, gives the weight of invert sugar in
the volume of the sugar solution used. According to Preuss, the factor
is not a constant one, but varies with the quantity of invert sugar
present, as is seen in the formula _y_ = 2.2868 + 3.3_x_ + 0.0041_x_²,
in which _x_ = the invert sugar, and _y_ the metallic copper.[89]
=126. Tenth Normal Copper Carbonate Solution.=—In the study of some
of the solutions of copper carbonate, proposed for practical work,
Ettore Soldaini was impressed with the difficulty of dissolving so
large a quantity of carbonate in the solvent employed.[90] The solution
recommended by Bodenbender and Scheller,[91] in which forty grams
of the crystallized copper sulfate were used, failed to disclose
an equivalent amount of copper in the reagent ready for use. For
this reason a tenth-normal copper solution is prepared by Soldaini
containing the equivalent of 3.464 grams of copper sulfate in one
liter. The reagent is easily prepared by adding slowly the dissolved
or finely powdered copper salt to a solution of 297 grams of potassium
bicarbonate, and after complete solution of the copper carbonate
formed, completing the volume to one liter. With this reagent as little
as one-quarter of a milligram of reducing sugar can be easily detected.
For the quantitive estimation of sugar a solution of the above strength
is to be preferred to the other forms of the soldaini reagent by reason
of the ease of direct comparison with standard fehling solutions.
The analytical process is conducted with the tenth-normal solution,
prepared by Soldaini and described above, as follows: Place 100 cubic
centimeters of the reagent in each of several porcelain dishes heat
to boiling, and add little by little the sugar solution to one dish
until the blue color has disappeared. Having thus determined nearly the
exact quantity of sugar solution required for the copper in 100 cubic
centimeters of the reagent the whole of the sugar solution is added at
once, varying slightly the amounts added to each dish. The boiling is
continued for fifteen minutes, and the contents of the dishes poured
on filters. That filtrate which contains neither copper nor sugar
represents the exact quantity of sugar solution which contained fifty
milligrams of dextrose.
=127. Relation of Reducing Sugar to Quantity of Copper Suboxid
Obtained.=—The relation of the quantity of copper reduced to the amount
of sugar oxidized by the copper carbonate solution has been determined
by Ost, and the utility of the process thereby increased.[92] The
solution used should have the following composition: 23.5 grams of
crystallized copper sulfate, 250 grams of potassium carbonate, and 100
grams of potassium bicarbonate in one liter. Without an indicator the
end reaction is distinctly marked by the passage of the blue color into
a colorless solution. Ost affirms that this solution is preferable
to any form of fehling liquor because it can be kept indefinitely
unchanged; it attacks sucrose far less strongly, and an equal quantity
of sugar precipitates nearly double the quantity of copper. The boiling
requires a longer time, as a rule ten minutes, but this is a matter of
no importance, when the other advantages are taken into consideration.
The relations of the different sugars to the quantity of copper
precipitated are given in the table in the next paragraph.
=128. Factor for Different Sugars.=—For pure dextrose the relation
between sugar and copper reduced has been determined by Ost, and the
data are given in the table below. The data were obtained by adding
to fifty cubic centimeters of the copper solution twenty-five cubic
centimeters of sugar solutions of varying strength and collecting,
washing, and reducing the cuprous oxid obtained in a current of
hydrogen in a glass tube by the method described further on.
The boiling in all cases was continued, just ten minutes, although a
slight variation from the standard time did not produce so great a
difference as with fehling reagent. In the case of dextrose, when fifty
milligrams were used with fifty cubic centimeters of the solution,
the milligrams of copper obtained after six, ten and twenty minutes’
boiling were 164.6, 165.5, and 166.9 respectively.[93]
The data differ considerably from those obtained by Herzfeld, but
in his experiments the boiling was continued only for five minutes,
and this is not long enough to secure the proper reduction of the
copper.[94]