Chapter VI: Preface: To Volume Third (6)

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]

TABLE SHOWING THE QUANTITY OF COPPER REDUCED BY DIFFERENT SUGARS.

Copper. Invert Sugar. Dextrose. Levulose. Galactose. Arabinose.
Milli- Milli- Milli- Milli- Milli- Milli-
grams. grams. grams. grams. grams. grams.
50 15.2 15.6 14.7 17.4 17.0
55 16.6 17.0 16.1 19.1 18.6
60 18.0 18.5 17.5 20.8 20.3
65 19.4 19.9 18.9 22.5 21.9
70 20.8 21.4 20.3 24.2 23.5
75 22.3 22.9 21.7 25.9 25.1
80 23.7 24.4 23.0 27.7 26.7
85 25.2 25.8 24.3 29.3 28.3
90 26.6 27.3 25.7 31.1 29.9
95 28.1 28.8 27.1 32.8 31.5
100 29.5 30.3 28.5 34.5 33.1
105 31.0 31.8 29.9 36.2 34.7
110 32.4 33.3 31.2 38.0 36.3
115 33.9 34.8 32.6 39.7 37.9
120 35.3 36.3 34.0 41.4 39.5
125 36.8 37.8 35.4 43.1 41.1
130 38.2 39.3 36.8 44.8 42.8
135 39.7 40.8 38.2 46.5 44.4
140 41.1 42.3 39.6 48.3 46.0
145 42.6 43.8 41.0 50.0 47.6
150 44.0 45.3 42.5 51.8 49.3
155 45.5 46.8 43.9 53.6 50.9
160 47.0 48.3 45.3 55.4 52.6
165 48.5 49.8 46.7 57.2 54.3
170 50.0 51.4 48.1 59.0 55.9
175 51.5 52.9 49.5 60.8 57.5
180 53.0 54.5 51.0 62.7 59.2
185 54.5 56.0 52.5 64.5 60.9
190 56.0 57.6 54.0 66.4 62.7
195 57.5 59.2 55.5 68.3 64.4
200 59.1 60.8 57.0 70.3 66.2
205 60.7 62.4 58.6 72.3 68.0
210 62.4 64.1 60.2 74.3 69.8
215 64.1 65.8 61.8 76.3 71.6
220 65.8 67.5 63.5 78.3 73.5
225 67.5 69.2 65.2 80.3 75.4
230 69.3 70.9 66.9 82.4 77.3
235 71.1 72.7 68.7 84.5 79.3
240 72.9 74.5 70.6 86.6 81.3
245 74.8 76.4 72.5 88.9 83.4
250 76.7 78.4 74.4 91.2 85.5
255 78.6 80.5 76.5 93.5 87.6
260 80.5 82.8 78.8 95.9 89.8
265 82.5 85.1 81.1 98.3 92.2
270 84.7 87.5 83.5 100.7 94.6
275 87.1 89.9 85.9 103.3 97.1
280 89.7 92.4 88.6 106.1 99.6
285 92.3 94.9 91.3 109.0 102.3
290 95.1 97.6 94.2 112.0 105.1
295 98.0 100.4 97.2 115.1 107.9
298 100.0 102.5 99.0 117.0 109.5

VOLUMETRIC METHODS BASED UPON THE USE OF AN AMMONIACAL COPPER SOLUTION.

=129. Pavy’s Process.=—The well-known solubility of cuprous oxid in ammonia led Pavy to adopt a copper reagent containing ammonia in the volumetric determination of reducing sugars.[95] In Pavy’s process an alkaline copper solution is employed made up in the usual way, to which a sufficient quantity of ammonia is added to hold in solution all the copper when precipitated as cuprous oxid. The solution used by Pavy has the following composition: One liter contains

Crystallized copper sulfate 34.65 grams
Potassium-sodium tartrate 173.00 ”
Caustic potash 160.00 ”

For use 120 cubic centimeters of the above reagent are mixed with 300 of ammonia of specific gravity 0.88, and the volume completed to one liter with distilled water. Twenty cubic centimeters of this reagent are equivalent to ten milligrams of dextrose or invert sugar when added in a one per cent solution.

In the use of ammoniacal copper solution, care must be taken that all the liquids employed be entirely free of oxygen and that the contents of the flask in which the reduction takes place be in some way excluded from contact with the air. Pavy secured this by conducting the reduction in a flask closed with a stopper carrying two holes; one of these served for the introduction of the burette carrying the sugar solution and the other carried a tube dipping into a water seal by means of a slit rubber tube, which would permit of the exit of the vapors of steam and ammonia, but prevent the regurgitation of the water into the flask.

The complete decoloration of the copper solution marks the end of the reaction. The usual precautions in regard to the length of the time of boiling must be observed.

It is easy to see that in the Pavy process the quantity of ammonia in the solution is rapidly diminished during the boiling and this has led to the suggestion of other methods to exclude the air. Among these have been recommended the introduction of a current of hydrogen or carbon dioxid. One of the best methods of procedure is that proposed by Allen, who recommends covering the copper solution by a layer of paraffin oil (kerosene).[96]

=130. Process Of Peska.=—Peska has also independently made use of Allen’s method of covering the solutions with a layer of paraffin oil and finds it reliable.[97] The copper reagent employed by him has the following composition:

Crystallized copper sulfate 6.927 grams
Ammonia, twenty-five per cent strength 160.00 cc.

The copper sulfate is dissolved in water, the ammonia added, and the volume completed to half a liter with distilled water. A second solution containing half a liter is made by dissolving 34.5 grams of potassium-sodium tartrate and ten grams of sodium hydroxid and completing the cool solution to half a liter with distilled water. In all cases the water used in making up the above solutions must be freshly boiled to exclude the air.

For the titration, fifty cubic centimeters of each of the above solutions are taken, mixed and covered with a layer of paraffin oil half a centimeter in depth. The reduction is not accomplished at a boiling temperature, but at from 80° to 85°. The manipulation is conducted as follows:

The mixed solutions are placed in a beaker, covered with oil, and heated to 80°. The temperature is measured by a thermometer which also serves as a stirring rod. The sugar solution is run down the sides of the beaker from a burette of such a shape as to be protected from the heat. After each addition of the sugar solution the mixture is carefully stirred, keeping the temperature at from 80° to 85°. The first titration is made to determine approximately the quantity of sugar solution necessary to decolorize the copper. This done, the actual titration is accomplished by adding at once the total amount of sugar solution necessary to decolorize, less about one cubic centimeter. Any sugar solution adhering to the side of the beaker is washed down by distilled water, the contents of the beaker well stirred, and the temperature kept at 85° for two minutes. The rest of the sugar solution is then added in quantities of one-tenth of a cubic centimeter until the decoloration is completed. The total time of the final titration should not exceed five minutes. The sugar solution should be as nearly as possible of one per cent strength. If a lower degree of strength be employed a larger quantity of the sugar is necessary to reduce a given quantity of copper.

In the case of dextrose, when a one per cent solution is used, eight and two-tenths cubic centimeters, corresponding to 80.2 milligrams of dextrose, are required to reduce 100 cubic centimeters of the mixed reagent. On the other hand, when the sugar solution is diluted to one-tenth of a per cent strength 82.1 milligrams are required.

With invert sugar slightly larger quantities are necessary, the reducing power being as 94.9 to 100 as compared with dextrose. With a one per cent strength of invert sugar it is found that eighty-four milligrams are required to reduce 100 cubic centimeters of the mixed reagent and when the strength of the invert sugar is reduced to one-tenth per cent 87.03 milligrams are required.

=131. Method Of Allein and Gaud.=—Allein and Gaud have proposed a further modification of the ammonia process which consists essentially in the suppression of rochelle salt and fixed caustic alkali and the entire substitution therefor of ammonia. Ammonia acts with much less vigor upon sugars than the caustic alkalies, and it is therefore claimed that the decomposition of the sugar due to the alkali is reduced to a minimum when ammonia is employed.[98] The copper solution is made as follows:

Dissolve 8.7916 grams of electrolytic copper in ninety-three grams of concentrated sulfuric acid diluted with an equal volume of water. Complete the resulting solution to one liter with concentrated ammonia. Ten cubic centimeters of this solution are equal to fifty grams of dextrose.

It is recommended that the reduction be accomplished in an atmosphere of hydrogen, but it is apparent that the use of kerosene is permissible in this case, and on account of its greater simplicity it is to be recommended as the best means of excluding the oxygen. The reduction is accomplished at a temperature of about 80°.

It is also proposed to reoxidize the copper by substituting a current of air for the hydrogen at the end of the reaction, and thus use the same copper a number of times. The danger of loss of ammonia, and the difficulty of determining when the oxidation is complete, render this regeneration of the reagent undesirable.

=132. Method of Gerrard.=—The method of Gerrard does not depend upon the use of ammonia, but the principle involved is the same, _viz._, the holding of the separated cuprous oxid in solution and the determination of the end of the reaction by the disappearance of the blue color. As first proposed by Gerrard, the copper sulfate solution is made of double the strength usually employed and to each 100 cubic centimeters thereof, before use, three and three-tenths grams of potassium cyanid are added. This is sufficient to hold the precipitated cuprous oxid in solution.[99]

The original method of Gerrard is found difficult of execution and the author, in conjunction with Allen, has lately modified it and reduced it to a practical working basis.[100]

In the new method the ordinary fehling solution is employed and it is prepared for use in the following way: Ten cubic centimeters of the fehling solution, or half that quantity of each of the component parts kept in separate bottles, are placed in a porcelain dish with forty cubic centimeters of water and brought to the boiling-point. To the boiling liquid is added, from a pipette, a five per cent solution of potassium cyanid until the blue color just disappears, or only a very faint tint of blue remains, avoiding any excess of the cyanid. A second portion of the fehling solution equal to that first employed is added, and to the boiling mixture the solution of sugar is added, from a burette, until the blue color disappears. The contents of the dish should be kept boiling during the addition of the sugar solution. The volume used will contain fifty milligrams of dextrose. The sugar solution should be of such a strength as to contain no more than half a per cent of reducing sugar.

The principle of the preparation of the solution may be stated as follows: If to a solution of copper sulfate, potassium be added until the blue color disappears, a double cyanid of copper and potassium cyanid is formed according to the following reaction: CuSO₄ + 4KCN = Cu(CN)₂.2KCN + K₂SO₄. This double cyanid is a salt of considerable stability. It is not decomposed by alkalies, hydrogen or ammonium, sulfid. With mineral acids it gives a whitish, curdy precipitate. With fehling solution the same double cyanid is formed as that described above. If, however, fehling solution be present in excess of the amount necessary to form the double cyanid of copper, this excess can be used in the oxidation of reducing sugar and the colorless condition of the solution will be restored as soon as the excess of the fehling is destroyed. The double cyanid holds in solution the cuprous oxid formed and thus complete decoloration is secured.

=133. Sidersky’s Modification of Soldaini’s Process.=—In all cases where the sugar solutions are not too highly colored, Sidersky finds that the method of reduction in a large test tube, as practiced by Violette, is applicable with the copper carbonate solution.[101] For more exact work it is preferred to determine the quantity of copper reduced by an indirect volumetric method. The sugar solution, properly clarified and the lead removed if subacetates have been used, is made of such a volume as to contain less than one per cent of reducing sugars. In a flask or large test tube are placed 100 cubic centimeters of the copper solution, which is boiled for a short time and the sugar solution added, little by little, from a pipette, at such a rate as not to stop the ebullition. The boiling is continued for five minutes after the last addition of the sugar. The vessel is taken from the flame and 100 cubic centimeters of cold water added, the whole brought on an asbestos felt and the cuprous oxid washed with hot water until the alkaline reaction has disappeared. The residual cuprous oxid is dissolved in a measured quantity of set sulfuric acid, semi- or fifth-normal, a few particles of potassium chlorate added, and the mixture boiled to convert any cuprous into cupric sulfate. The reaction is represented by the following formula:

3Cu₂O + 6H₂SO₄ + KClO₃ = 6CuSO₄ + KCl + 6H₂O.

The residual sulfuric acid is titrated with a set alkali in excess, ammonia being preferred.

The solution of ammonia is made by diluting 200 cubic centimeters of commercial aqua ammonia with 800 of water. Its strength is determined by adding a little copper sulfate solution as indicator and then the set solution of sulfuric acid until the blue color disappears. The copper sulfate secured from the cuprous sulfate as described above is cooled, and a quantity of the ammonia, equal to twenty-five cubic centimeters of the set sulfuric acid, added. The excess of the ammonia is then determined by titration with the sulfuric acid, the disappearance of the blue color being the indication of the end of the reaction. The number of cubic centimeters of the set sulfuric acid required to saturate the ammonia represents the equivalent of cuprous oxid originally present. One cubic centimeter of normal sulfuric acid is equivalent to 0.0317 gram of metallic copper.

To determine the weight of invert sugar oxidized, multiply the weight of copper, calculated as above described, by the factor 0.3546.[102] For a general application of this method of analysis the relative quantities of copper reduced by different quantities of sugar must be taken into consideration.

While, as has already been stated, the copper carbonate process has heretofore been applied chiefly to the detection of invert sugar, it has merits which justify the expectation that it may some time supplant the fehling liquor both for volumetric and gravimetric work. Large volumes of the reagent can be prepared at once and without danger of subsequent change. The action of the reagent on the hexobioses and trioses is far less vigorous than that of the alkaline copper tartrate, and the end reactions for volumetric work are, at least, as easily determined in the one case as the other.

=134. Method Depending on Titration of Excess of Copper.=—Instead of measuring the quantity of copper reduced, either by its disappearance or by reducing the cuprous oxid to a metallic state, Politis has proposed a method of analysis depending on the titration of the residual copper.[103] The reagents employed are:

(1) A copper solution containing 24.95 grams of crystallized copper sulfate, 140 grams of sodium and potassium tartrate, and twenty-five grams of sodium hydroxid in one liter:

(2) A solution of sodium thiosulfate containing 24.8 grams of the salt in one liter:

(3) A solution of potassium iodid containing 12.7 grams of iodin in one liter.

The reaction is represented by the formula

2CuCl₂ + 4KI = Cu₂I₂ + 4KCl + I₂.

The analytical process is carried out as follows: In a 100 cubic centimeter flask are boiled fifty cubic centimeters of the copper solution, ten cubic centimeters of about one-tenth per cent reducing sugar solution are added, the boiling continued for five minutes, the flask filled to the mark with boiling water and its contents filtered. Fifty cubic centimeters of the hot filtrate are cooled, slightly acidified, potassium iodid solution added in slight excess; and the iodin set free determined by titration with sodium thiosulfate. The quantity of iodin obtained corresponds to the unreduced copper remaining after treatment with the reducing sugar. The number of cubic centimeters of thiosulfate used subtracted from twenty-five will give the number of cubic centimeters of the copper solution which would be reduced by five cubic centimeters of the sugar solution used.

_Example._—In the proportions given above it was found that eleven cubic centimeters of thiosulfate were required to saturate the iodin set free. Then 25 - 11 = 14 cubic centimeters of copper solution reduced by five cubic centimeters of the sugar solution. Since one cubic centimeter of the copper solution is reduced by 0.0036 gram of dextrose the total dextrose in the five cubic centimeters = 0.0036 × 5 = 0.0180 gram.

The above method does not seem to have any practical advantage over those based on noting the disappearance of the copper and is given only to illustrate the principle of the process. While the titration of the iodin by sodium thiosulfate is easily accomplished in the absence of organic matter, it becomes difficult, as shown by Ewell, when organic matters are present, as they always are in the oxidation of a sugar solution. Ewell has therefore proposed to determine the residual copper by a standard solution of potassium cyanid, but the method has not yet been developed.[104]

GRAVIMETRIC COPPER METHODS.

=135. General Principles.=—In the preceding pages the principles of the volumetric methods of sugar analysis by means of alkaline copper solution have been set forth. They depend either on the total decomposition of the copper solution employed by the reducing sugar, or else on the collection and titration of the cuprous oxid formed in the reaction. In the gravimetric methods the general principle of the process rests upon the collection of the cuprous oxid formed and its reduction to metallic copper, the weight of which serves as a starting point in the calculations of the weight of reducing sugar, which has been oxidized in the solution.

The factors which affect the weight of copper obtained are essentially those which influence the results in the volumetric method. The composition of the copper solution, the temperature at which the reduction is accomplished, the time of heating, the strength of the sugar solution and the details of the manipulation, all affect more or less the quantity of copper obtained. As in the volumetric method also, the kind of reducing sugar must be taken in consideration, dextrose, levulose, invert sugar, maltose and other sugars having each a definite factor for reduction in given conditions. It follows, therefore, that only those results are of value which are obtained under definite conditions, rigidly controlled.

=136. Gravimetric Methods of the Department of Agriculture Laboratory.=—The process used in this laboratory is based essentially on the methods of Maercker, Behrend, Morgen, Meissl, Hiller and Allihn.[105] Where dextrose alone is present, the table of factors proposed by Allihn is used and also the copper solution corresponding thereto.

For pure invert sugar, the tables and solutions of Meissl are used. For invert sugar in the presence of sucrose, the table and process proposed by Hiller are used.

The reduction of the copper solution and the electrolytic deposition of the copper are accomplished as follows:

The copper and alkali solutions are kept in separate bottles. After mixing the equivalent volume of the two solutions in a beaker, heat is applied and the mixture boiled. To the boiling liquid the proper volume of the cold sugar solution is added. This must always be less than the amount required for complete reduction. The solution is again brought into ebullition and kept boiling exactly two minutes. A two-minute sand glass is conveniently used to determine the time of boiling. At the end of this time an equal volume of freshly boiled cold water is added, and the supernatant liquor at once passed through a gooch under pressure. The residual cuprous oxid is covered with boiling water and washed by decantation until the wash water is no longer alkaline. It is more convenient to wash in such a way that, at the end, the greater part of the cuprous oxid is in the gooch. The felt and cuprous oxid are then returned to the beaker in which the reduction is made. The gooch is moistened with nitric acid to dissolve any adhering oxid and then is washed into the beaker. Enough nitric acid is added to bring all the oxid into solution, an excess being avoided, and a small amount of water added. The mixture is again passed under pressure through a gooch having a thin felt, to remove the asbestos and the filtrate collected in a flask of about 150 cubic centimeters capacity. The washing is continued until the gooch is free of copper, when the volume of the filtrate should be about 100 cubic centimeters. The liquid is transferred to a platinum dish holding about 175 cubic centimeters and the flask washed with about twenty-five cubic centimeters of water. From three to five cubic centimeters of strong sulfuric acid are added and the copper deposited by an electric current.

=137. Precipitating the Copper.=—When no more nitric acid is used than indicated in the previous paragraph, it will not be necessary to remove it by evaporation. The platinum dishes containing the solutions of the cuprous oxid are arranged as shown in the figure for the precipitation of the copper by the electric current. Each of the supporting stands has its base covered with sheet-copper, on which the platinum dishes rest. The uprights are made of heavy glass rods and carry the supports for the platinum cylinders which dip into the copper solutions. The current used is from the city service and is brought in through the lamp shown at the right of the figure. This current has a voltage of about 120. After passing the lamp it is conducted through the regulator shown at the right, a glass tube closed below by a stopper carrying a piece of platinum foil, and above by one holding a glass tube, in the lower end of which is sealed a piece of sheet platinum connected, through the glass tube, with the lamp. The regulating tube contains dilute sulfuric acid. The strength of current desired is secured by adjusting the movable pole. A battery of this kind easily secures the precipitation of sixteen samples at once, but only twelve are shown in the figure. The practice here is to start the operation at the time of leaving the laboratory in the afternoon. The next morning the deposition of the copper will be found complete. The wiring of the apparatus is shown in the figure. The wire from the regulator is connected with the base of the first stand, and thence passes through the horizontal support to the base of the second, and so on. The return to the lamp is accomplished by means of the upper wire. This plan of arranging the apparatus has been used for two years, and with perfect satisfaction.

Where a street current is not available, the following directions may be followed: Use four gravity cells, such as are employed in telegraphic work, for generating the current. This will be strong enough for one sample and by working longer for two. Connect the platinum dish with the zinc pole of the battery. The current is allowed to pass until all the copper is deposited. Where a larger number of samples is to be treated at once, the size of the battery must be correspondingly increased.

=138. Method Used at the Halle Station.=—The method used at the Halle station is the same as that originally described by Maercker for dextrose.[106] The copper solution employed is the same as in the allihn method, _viz._, 34.64 grams of copper sulfate in 500 cubic centimeters, and 173 grams of rochelle salt and 125 grams of potassium hydroxid in the same quantity of water. In a porcelain dish are placed thirty cubic centimeters of copper solution and an equal quantity of the alkali, sixty cubic centimeters of water added and the mixture boiled. To the solution, in lively ebullition, are added twenty-five cubic centimeters of the dextrose solution to be examined which must not contain more than one per cent of sugar. The mixture is again boiled and the separated cuprous oxid immediately poured into the filter and washed with hot water, until the disappearance of an alkaline reaction. For filtering, a glass tube is employed, provided with a platinum disk, and resembling in every respect similar tubes used for the extraction of substances with ether and alcohol. The arrangement of the filtering apparatus is shown in Fig. 44. In the Halle method it is recommended that the tubes be prepared by introducing a platinum cone in place of the platinum disk and filling it with asbestos felt, pressing the felt tightly against the sides of the glass tube and making the asbestos fully one centimeter in thickness. This is a much less convenient method of working than the one described above. After filtration and washing, the cuprous oxid is washed with ether and alcohol and dried for an hour at 110°, and finally reduced to metallic copper in a stream of pure dry hydrogen, heat being applied by means of a small flame. The apparatus for the reduction of the cuprous oxid is shown in Fig. 45. The metallic copper, after cooling and weighing, is dissolved in nitric acid, the tube washed with water, ether and alcohol, and again dried, when it is ready for use a second time. The percentage of dextrose is calculated from the milligrams of copper found by Allihn’s table.

=139. Tables for Use in the Gravimetric Determination of Reducing Sugars.=—The value of a table for computing the percentage of a reducing sugar present in a solution, is based on the accuracy with which the directions for the determination are followed. The solution must be of the proper strength and made in the way directed. The degree of dilution prescribed must be scrupulously preserved and the methods of boiling during reduction and washing the reduced copper, followed. The quantity of copper obtained by the use of different alkaline copper solutions and of sugar solutions of a strength different from that allowed by the fixed limits, is not a safe factor for computation. It must be understood, therefore, that in the use of the tables the directions which are given are to be followed in every particular.

=140. Allihn’s Gravimetric Method for the Determination of Dextrose.=—_Reagents_:

I. 34.639 grams of CuSO₄.5H₂O, dissolved in water and diluted to
half a liter:

II. 173 grams of rochelle salts } dissolved in water and diluted
125 grams of KOH, } to half a liter.

_Manipulation_: Place thirty cubic centimeters of the copper solution (I), thirty cubic centimeters of the alkaline tartrate solution (II), and sixty cubic centimeters of water in a beaker and heat to boiling. Add twenty-five cubic centimeters of the solution of the material to be examined, which must be so prepared as not to contain more than one per cent of dextrose, and boil for two minutes. Filter immediately after adding an equal volume of recently boiled cold water and obtain the weight of copper by one of the gravimetric methods given. The corresponding weight of dextrose is found by the following table:

ALLIHN’S TABLE FOR THE DETERMINATION OF DEXTROSE.

(A) = Milligrams of copper.
(B) = Milligrams of dextrose.

(A) (B) (A) (B) (A) (B) (A) (B) (A) (B)
10 6.1 46 23.9 82 41.8 118 60.1 154 78.6
11 6.6 47 24.4 83 42.3 119 60.6 155 79.1
12 7.1 48 24.9 84 42.8 120 61.1 156 79.6
13 7.6 49 25.4 85 43.4 121 61.6 157 80.1
14 8.1 50 25.9 86 43.9 122 62.1 158 80.7
15 8.6 51 26.4 87 44.4 123 62.6 159 81.2
16 9.0 52 26.9 88 44.9 124 63.1 160 81.7
17 9.5 53 27.4 89 45.4 125 63.7 161 82.2
18 10.0 54 27.9 90 45.9 126 64.2 162 82.7
19 10.5 55 28.4 91 46.4 127 64.7 163 83.3
20 11.0 56 28.8 92 46.9 128 65.2 164 83.8
21 11.5 57 29.3 93 47.4 129 65.7 165 84.3
22 12.0 58 29.8 94 47.9 130 66.2 166 84.8
23 12.5 59 30.3 95 48.4 131 66.7 167 85.3
24 13.0 60 30.8 96 48.9 132 67.2 168 85.9
25 13.5 61 31.3 97 49.4 133 67.7 169 86.4
26 14.0 62 31.8 98 49.9 134 68.2 170 86.9
27 14.5 63 32.3 99 50.4 135 68.8 171 87.4
28 15.0 64 32.8 100 50.9 136 69.3 172 87.9
29 15.5 65 33.3 101 51.4 137 69.8 173 88.5
30 16.0 66 33.8 102 51.9 138 70.3 174 89.0
31 16.5 67 34.3 103 52.4 139 70.8 175 89.5
32 17.0 68 34.8 104 52.9 140 71.3 176 90.0
33 17.5 69 35.3 105 53.5 141 71.8 177 90.5
34 18.0 70 35.8 106 54.0 142 72.3 178 91.1
35 18.5 71 36.3 107 54.5 143 72.9 179 91.6
36 18.9 72 36.8 108 55.0 144 73.4 180 92.1
37 19.4 73 37.3 109 55.5 145 73.9 181 92.6
38 19.9 74 37.8 110 56.0 146 74.4 182 93.1
39 20.4 75 38.3 111 56.5 147 74.9 183 93.7
40 20.9 76 38.8 112 57.0 148 75.5 184 94.2
41 21.4 77 39.3 113 57.5 149 76.0 185 94.7
42 21.9 78 39.8 114 58.0 150 76.5 186 95.2
43 22.4 79 40.3 115 58.6 151 77.0 187 95.7
44 22.9 80 40.8 116 59.1 152 77.5 188 96.3
45 23.4 81 41.3 117 59.6 153 78.1 189 96.8