(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.
Figure 43. Apparatus for the Electrolytic Deposition of Copper.
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.
Figure 44. Apparatus for Filtering Copper Suboxid.
Figure 45. Apparatus for Reducing Copper Suboxid.
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.
141. Meissl’s Table for Invert Sugar.—Invert sugar is usually the product of the hydrolysis of sucrose. The following table is to be used when the hydrolysis is complete, i. e., when no sucrose is left in the solution. The solution of copper sulfate and of the alkaline tartrate are made up as follows: 34.64 grams of copper sulfate in half a liter, and 173 grams of rochelle salt and 51.6 grams sodium hydroxid in the same volume. The quantity of sugar solution used must not contain more than 245 nor less than ninety milligrams of invert sugar.
In the determination twenty-five cubic centimeters of the copper solution and an equal volume of the alkaline tartrate are mixed and boiled, the proper amount of sugar solution added to secure a quantity of invertose within the limits named, the volume completed to 100 cubic centimeters with boiling water, and the mixture kept in lively ebullition for two minutes. An equal volume of recently boiled cold water is added and the cuprous oxid at once separated by filtration on asbestos under pressure, and washed free of alkali with boiling water. The metallic copper is secured by one of the methods already described.
Table for Invert Sugar by Meissl and Wien.[107]
- (A) = Milligrams of copper.
- (B) = Milligrams of invert sugar.
142. Table for the Determination of Invert Sugar (Reducing Sugars) in the Presence of Sucrose.—The method adopted by the Association of Official Agricultural Chemists is essentially that proposed by Meissl and Hiller.[108] Prepare a solution of the material to be examined in such a manner that it contains twenty grams of the mixed sugars in one hundred cubic centimeters, after clarification and the removal of the excess of lead. Prepare a series of solutions in large test tubes by adding one, two, three, four, five etc. cubic centimeters of this solution to each tube successively. Add five cubic centimeters of the mixed copper reagent to each, heat to boiling, boil two minutes and filter. Note the volume of sugar solution which gives the filtrate lightest in tint, but still distinctly blue. Place twenty times this volume of the sugar solution in a 100 cubic centimeter flask, dilute to the mark, and mix well. Use fifty cubic centimeters of the solution for the determination, which is conducted as already described, until the weight of copper is obtained. For the calculation of the results use the following formulas and table of factors of Meissl and Hiller:[109]
| Let Cu = | the weight of the copper obtained; |
| P = | the polarization of the sample; |
| W = | the weight of the sample in the fifty cubic |
| centimeters of the solution used for determination; | |
| F = | the factor obtained from the table for conversion |
| of copper to invert sugar; | |
| Cu | = approximate absolute weight of invert sugar = Z; |
| 2 | |
| 100 | |
| Z × —— = | approximate per cent of invert sugar = y; |
| W | |
| 100P | = R, relative number for sucrose; |
| P + y | |
| 100 - R = | I, relative number for invert sugar; |
| Cu | = per cent of invert sugar. |
| W |
Z indicates the vertical column, and the ratio of R to I, the horizontal column of the table, which are to be used for the purpose of finding the factor (F) for calculating copper to invert sugar.
Example:—The polarization of a sugar is 86.4, and 3.256 grams of it (W) are equivalent to 0.290 gram of copper. Then:
| Cu | = | 0.290 | = 0.145 = Z |
| 2 | 2 |
| Z × | 100 | = 0.145 × | 100 | = 4.45 = y |
| W | 3.256 |
| 100P | = | 8640 | = 95.1 = R |
| P + y | 86.4 + 4.45 |
100 - R = 100 - 95.1 = 4.9 = I
R : I = 95.1 : 4.9
By consulting the table it will be seen that the vertical column headed I = 150 is nearest to Z, 145, the horizontal column headed 95: 5 is nearest to the ratio of R to I, 95.1: 4.9. Where these columns meet we find the factor 51.2, which enters into the final calculation:
| CuF | = | .290 × 51.2 | = 4.56 the true per cent of invert sugar. |
| W | 3.256 |
Meissl and Hiller’s Factors for the Determination of
More Than One Per Cent of Invert Sugar.
| Ratio of sucrose to invert sugar = |
Approximate absolute weight of invert sugar = Z. | ||||||
|---|---|---|---|---|---|---|---|
| I = 200 | I = 175 | I = 150 | I = 125 | I = 100 | I = 75 | I = 50 | |
| R : I. | mg. | mg. | mg. | mg. | mg. | mg. | mg. |
| 0 : 100 | 56.4 | 55.4 | 54.5 | 53.8 | 53.2 | 53.0 | 53.0 |
| 10 : 90 | 56.3 | 55.3 | 54.4 | 53.8 | 53.2 | 52.9 | 52.9 |
| 20 : 80 | 56.2 | 55.2 | 54.3 | 53.7 | 53.2 | 52.7 | 52.7 |
| 30 : 70 | 56.1 | 55.1 | 54.2 | 53.7 | 53.2 | 52.6 | 52.6 |
| 40 : 60 | 55.9 | 55.0 | 54.1 | 53.6 | 53.1 | 52.5 | 52.4 |
| 50 : 50 | 55.7 | 54.9 | 54.0 | 53.5 | 53.1 | 52.3 | 52.2 |
| 60 : 40 | 55.6 | 54.7 | 53.8 | 53.2 | 52.8 | 52.1 | 51.9 |
| 70 : 30 | 55.5 | 54.5 | 53.5 | 52.9 | 52.5 | 51.9 | 51.6 |
| 80 : 20 | 55.4 | 54.3 | 53.3 | 52.7 | 52.2 | 51.7 | 51.3 |
| 90 : 10 | 54.6 | 53.6 | 53.1 | 52.6 | 52.1 | 51.6 | 51.2 |
| 91 : 9 | 54.1 | 53.6 | 52.6 | 52.1 | 51.6 | 51.2 | 50.7 |
| 92 : 8 | 53.6 | 53.1 | 52.1 | 51.6 | 51.2 | 50.7 | 50.3 |
| 93 : 7 | 53.6 | 53.1 | 52.1 | 51.2 | 50.7 | 50.3 | 49.8 |
| 94 : 6 | 53.1 | 52.6 | 51.6 | 50.7 | 50.3 | 49.8 | 48.9 |
| 95 : 5 | 52.6 | 52.1 | 51.2 | 50.3 | 49.4 | 48.9 | 48.5 |
| 96 : 4 | 52.1 | 51.2 | 50.7 | 49.8 | 48.9 | 47.7 | 46.9 |
| 97 : 3 | 50.7 | 50.3 | 49.8 | 48.9 | 47.7 | 46.2 | 45.1 |
| 98 : 2 | 49.9 | 48.9 | 48.5 | 47.3 | 45.8 | 43.3 | 40.0 |
| 99 : 1 | 47.7 | 47.3 | 46.5 | 45.1 | 43.3 | 41.2 | 38.1 |
143. Table for the Estimation of Milk Sugar.—The solutions to be used for this table are the same as those employed in the preceding table for the estimation of invert sugar. The milk sugar is supposed to be in a pure form in solution before beginning the analysis. The method to be employed for milk will be given in the part devoted to dairy products.
In the conduct of the work twenty-five cubic centimeters of the copper solution are mixed with an equal quantity of the alkaline tartrate mixture, and from twenty to one hundred cubic centimeters of the sugar solution added, according to its concentration. This solution should not contain less than seventy nor more than 306 milligrams of lactose. The volume is completed to 150 cubic centimeters with boiling water and kept in lively ebullition for six minutes. The rest of the operation is conducted in the manner already described. From the weight of copper obtained the quantity of milk sugar is determined by inspecting the table. It is recommended to use such a weight of milk sugar as will give about 200 milligrams of copper.
Table for Determining Milk Sugar.
- (A) = Milligrams of copper.
- (B) = Milligrams of milk sugar.