VOLUMETRIC METHODS.
109. Classification.—Among the volumetric methods will be given those which are in common use or such as have been approved by the practice of analysts. Since the use of mercuric salts is now practiced to a limited extent, only a brief study of that process will be attempted. With the copper methods a somewhat extended description will be given of those depending on the use of copper sulfate, and a briefer account of the copper carbonate process.
In the copper sulfate method two distinct divisions must be noted, viz., first an indirect process depending first upon the reduction of the copper to a suboxid, the subsequent action of this body on iron salts, measured finally by titration with potassium permanganate; and second, a direct process determined either by the disappearance of the blue color from the copper solution, or by the absence of copper from a drop of the solution withdrawn and tested with potassium ferrocyanid. This last mentioned reaction is one which is found in common use. The volumetric methods are not, as a rule, as accurate as the gravimetric, depending on weighing the resultant metal, but they are far more rapid and well suited to technical control determinations.
110. Reduction of Mercuric Salts.—The method of determining sugar by its action on mercuric salts, is due to Knapp.[70] The method is based on the observation that dextrose and other allied sugars, will reduce an alkaline solution of mercuric cyanid, and that the mercury will appear in a metallic state.
The mercuric liquor is prepared by adding to a solution of ten grams of mercuric cyanid, 100 cubic centimeters of a solution of caustic soda of 1.145 specific gravity, and making the volume to one liter with water. The solution of sugar to be titrated, should be as nearly as possible of one per cent strength.
To 100 cubic centimeters of the boiling solution, the sugar solution is added in small portions from a burette and in such a way as to keep the whole mass in gentle ebullition.
To determine when all the mercuric salt has been decomposed, a drop of the clear boiling liquid is removed and brought into contact with a drop of stannous chlorid solution on a white surface. A brownish black coloration or precipitate will indicate that the mercury is not all precipitated. Fresh portions of the sugar must then be added, until no further indication of the presence of mercury is noted. The approximate quantity of sugar solution required to precipitate the mercury having thus been determined, the process is repeated by adding rapidly, nearly the quantity of sugar solution required, and then only a few drops at a time, until the reduction is complete.
One hundred cubic centimeters of the mercuric cyanid solution prepared as directed above, will be completely reduced by
| 202 | milligrams | of | dextrose, |
| 200 | ” | ” | invert sugar, |
| 198 | ” | ” | levulose, |
| 308 | ” | ” | maltose, |
| 311 | ” | ” | lactose. |
By reason of the unpleasant odor of the boiling mercuric cyanid when in presence of a reducing agent, the process should be conducted in a well ventilated fume chamber. With a little practice the process is capable of rapid execution, and gives reasonably accurate results.
111. Sachsse’s Solution.—The solution of mercuric salts proposed by Sachsse, is made by dissolving eighteen grams of mercuric iodid in twenty-five cubic centimeters of an aqueous solution of potassium iodid. To this solution are added 200 cubic centimeters of potash lye, containing eighty grams of caustic potash. After mixing the solution, the volume is completed to one liter. The sugar solutions used to reduce this mixture, should be more dilute than those employed with the mercuric cyanid, and should not be over one-half per cent in strength. The end of the reduction is determined as already described. After a preliminary trial, nearly all the sugar necessary to complete reduction, should be added at once, and the end of the reduction then determined by the addition of successive small quantities. One hundred cubic centimeters of the mercuric iodid solution prepared as directed above, require the following quantities of sugar to effect a complete reduction:
| 325 | milligrams | of | dextrose, |
| 269 | ” | ” | invert sugar, |
| 213 | ” | ” | levulose, |
| 491 | ” | ” | maltose, |
| 387 | ” | ” | lactose. |
By reason of the great difference between the reducing power of dextrose and levulose in this solution, it has been used in combination with the copper reduction method, to be described, to determine the relative proportion of dextrose and levulose in a mixture.[71]
It is now known that copper solutions require slightly different quantities of dextrose, levulose, or invert sugar to effect complete reduction, but the variations are not great and in the calculation above mentioned, it may be assumed that these differences do not exist.
Instead of using stannous chlorid as an indicator, the end of the reaction may be determined as follows: A disk of filtering paper is placed over a small beaker containing some ammonium sulfid. A drop of the clear hot solution is placed on this disk, and if salts of mercury be still present a dark stain will be produced; or a drop of the ammonium sulfid may be brought near the moist spot formed by the drop of mercury salt. An alkaline solution of zinc oxid may also be used.
The methods depending on the use of mercuric salts have, of late, been supplanted by better processes, and space will not be given here to their further discussion.
112. The Volumetric Copper Methods.—The general principle on which these methods depend, is found in the fact that certain sugars, notably, dextrose, (glucose), levulose, (fructose), maltose and lactose, have the property of reducing an alkaline solution of copper to a lower state of combination, in which the copper is separated as cuprous oxid. The end of the reaction is either determined by the disappearance of the blue color of the solution, or by the reaction produced by a drop of the hot filtered solution, when placed in contact with a drop of potassium ferrocyanid acidified with acetic.
The copper salt which is found to give the most delicate and reliable reaction, is the tartrate. The number of volumetric processes proposed and which are in use, is very great, and an attempt even to enumerate all of these can not be made in this volume. A few of the most reliable and best attested methods will be given, representing if possible, the best practice in this and other countries. The rate of reduction of the copper salt to suboxid, is influenced by the rate of mixing with the sugar solutions, the temperature, the composition of the copper solution and the strength of the sugar solution.
The degree of reduction is also modified by the rate at which the sugar solution is added, and by the degree and duration of heating, and all these variables together, make the volumetric methods somewhat difficult and their data, to a certain extent, discordant. By reason, however, of the ease with which they are applied and the speed of their execution, they are invaluable for approximately correct work and for use in technical control.
113. Historical.—It is not the purpose in this paragraph to trace the development of the copper reduction method for the determination of reducing sugars, but only to refer to the beginning of the exact analytical application of it.
Peligot, as early as 1844, made a report to the Society for the Encouragement of National Industry on methods proposed by Barreswil and Fromherz for the quantitive estimation of sugar by means of copper solution.[72] These methods were based on the property of certain sugars to reduce alkaline copper solution to a state of cuprous oxid first announced by Trommer.[73] This was followed by a paper by Falck on the quantitive determination of sugar in urine.[74]
In 1848 the methods, which have been proposed, were critically examined by Fehling, and from the date of his paper the determination of sugar by the copper method may be regarded as resting on a scientific basis.[75]
Since the date mentioned the principal improvements in the process have been in changing the composition of the copper solution in order to render it more stable, which has been accomplished by varying the proportions of copper sulfate, alkali and tartaric acid. For the better keeping of the solution the method of preserving the copper sulfate and the alkaline tartrates in separate flasks and only mixing them at the time of use has been found very efficacious.[76] For testing for the end of the reaction by means of an acetic acid solution of potassium ferrocyanid the filtering tube suggested by the author, the use of which will be described further on, has proved quite useful. Pavy has suggested that by the addition of ammonia to the copper solution the precipitated suboxid may be kept in solution and the end of the reaction thus easily distinguished by the disappearance of the blue color.[77] Allen has improved on this method by covering the hot mixture with a layer of paraffin oil whereby any oxidation of the suboxid is prevented.[78]
The introduction and development of the gravimetric process depending on securing the reduced copper oxid in a metallic state as developed by Allihn, Soxhlet, and others, completes the resumé of this brief sketch of the rise and development of the process.
114. Action of Alkaline Copper Solution on Dextrose.—The action to which dextrose and other reducing sugars are subjected in the presence of a hot alkaline copper solution is two-fold in its nature. In the first place there is an oxidation of the sugar which is transformed into tartronic, formic and oxalic acids, the two latter in very small quantities. At the same time another part of the sugar is attacked directly by the alkali and changed to complex products among which have been detected lactic, oxyphenic and oxalic acids, also two bodies isomeric with dioxyphenolpropionic acid. When the sugar is in large excess melassic and glucic acids have also been detected. The glucic acid may be regarded as being formed by simple dehydration but becomes at once resolved into pyrocatechin and gluconic acid according to the reaction C₁₂H₁₈O₉ = C₆H₆O₂ + C₅H₁₂O₇. The gluconic acid also is decomposed and gives birth to lactic and glyceric acids according to the formula C₆H₁₂O₇ = C₃H₆O₃ + C₃H₆O₄. The glyceric acid also in the presence of a base is changed into lactic and oxalic acids. Between lactic acid and pyrocatechin, existing in a free state, there is produced a double reciprocal etherification in virtue of which there arise two ethers isomeric with hydrocaffeic acid, C₉H₁₀O₄. One of these bodies is an acid and corresponds to the constitution
- CH₃
- /
- O ── CH
- / \
- C₆H₄ CO₂H (2)
- \
- OH (2)
and the other is of an alcoholic nature corresponding to the formula
- CO₂ ── CHOH ── CH₃ (1)
- /
- C₆H₄
- \
- OH₂ (2)
Of all these products only oxyphenic and lactic acids and their ethers and oxalic acid remain unchanged and they can be isolated. All the others are transformed in an acid state and they can only be detected by operating in the presence of metallic oxids capable of precipitating them at the time of their formation.[79]
115. Fehling’s Solution.—The copper solution which has been most used in the determination of reducing sugars is the one proposed by Fehling as a working modification of the original reagent used by Trommer.[80]
Following is the formula for the preparation of the fehling solution:
| Pure crystallized copper sulfate CuSO₄.5H₂O, | 34.64 | grams: |
| Potassium tartrate, | 150.00 | ” |
| Sodium hydroxid, | 90.00 | ” |
The copper sulfate is dissolved in water and the potassium tartrate in the aqueous solution of the sodium hydroxid which should have a volume of about 700 cubic centimeters. The two solutions are mixed and the volume completed to a liter. Each cubic centimeter of this solution will be reduced by five milligrams of dextrose, equivalent to four and a half milligrams of sucrose.
The reaction which takes place is represented by the following molecular proportions:
| C₆H₁₂O₆ | = | 10CuSO₄.5H₂O |
| Dextrose. | Copper sulfate. | |
| 180 | 2494 |
Fehling’s solution is delicate in its reactions but does not keep well, depositing cuprous oxid on standing especially in a warm place exposed to light. The fehling liquor was soon modified in its constitution by substituting 173 grams of the double sodium and potassium tartrate for the neutral potassium tartrate first used, and, in fact, the original fehling reagent contained forty grams of copper sulfate instead of the quantity mentioned above. Other proportions of the ingredients are also given by many authors as fehling solution.
116. Comparison of Copper Solutions for Oxidizing Sugars.—For the convenience of analysts there is given below a tabular comparison of the different forms of fehling liquor which have been proposed for oxidizing sugars. The table is based on a similar one prepared by Tollens and Rodewald, amended and completed by Horton.[81] The solutions are arranged alphabetically according to authors’ names:
1. Allihn:
34.6 grams copper sulfate, solution made up to half a liter; 173 grams potassium-sodium tartrate; 125 grams potassium hydroxid (equivalent to 89.2 grams sodium hydroxid) solution made up to half a liter.
2. A. H. Allen:
34.64 grams copper sulfate, solution made up to 500 cubic centimeters; 180 grams potassium-sodium tartrate; 70 grams sodium hydroxid (not less than 97° NaOH), solution made up to half a liter.
3. Bödeker:
34.65 grams copper sulfate; 173 grams potassium-sodium tartrate; 480 cubic centimeters sodium hydroxid solution, 1.14 specific gravity; 67.3 grams sodium hydroxid; fill to one liter; 0.180 gram grape sugar reduces according to Bödeker, 36.1 cubic centimeters of the copper solution = 0.397 gram copper oxid. The same quantity of milk sugar reduces, however, only twenty-seven cubic centimeters copper solution = 0.298 copper oxid.
4. Boussingault:
40 grams copper sulfate; 160 grams potassium tartrate; 130 grams sodium hydroxid.
5. Dietzsch:
34.65 grams copper sulfate; 150 grams potassium-sodium tartrate; 250 grams sodium hydroxid solution, 1.20 specific gravity; 150 grams glycerol.
6. Fleischer:
69.278 grams copper sulfate dissolved in about half a liter of water, add to this 200 grams tartaric acid; fill to one liter with concentrated sodium hydroxid solution; twenty cubic centimeters copper solution = forty cubic centimeters sugar solution, that contain in every cubic centimeter five milligrams grape sugar.
7. Fehling:
40 grams copper sulfate; 160 grams di-potassium tartrate = 600-700 cubic centimeters sodium hydroxid solution, 1.12 specific gravity, or from 54.6 to 63.7 grams sodium hydroxid, fill to 1154.4 cubic centimeters.
8. Gorup-Besanez:
34.65 grams copper sulfate; 173 grams potassium-sodium tartrate; 480 cubic centimeters sodium hydroxid solution, 1.14 specific gravity; equal 67.3 sodium hydroxid. Fill to one liter.
9. Grimaux:
40 grams copper sulfate; 160 grams potassium-sodium tartrate; 600-700 cubic centimeters sodium hydroxid solution, 1.20 specific gravity, equal to 92.5-107.9 grams sodium hydroxid. Fill to 1154.4 cubic centimeters. Ten cubic centimeters of this solution are completely decolorized by 0.050 gram glucose.
10. Holdefleis:
34.632 grams copper sulfate in one liter of water; 125 grams potassium hydroxid, equivalent to 89.2 grams sodium hydroxid; 173 grams potassium-sodium tartrate. Fill to one liter.
11. Hoppe-Seyler:
34.65 grams copper sulfate; 173 grams potassium-sodium tartrate; 600-700 cubic centimeters sodium hydroxid solution, 1.12 specific gravity; equal to 63.0-73.5 grams potassium hydroxid. Fill to one liter. One cubic centimeter is reduced by exactly 0.005 gram grape sugar.
12. Krocker:
6.28 grams copper sulfate; 34.6 grams potassium-sodium tartrate; 100 cubic centimeters sodium hydroxid solution, 1.14 specific gravity. Fill to 200 cubic centimeters. In 100 cubic centimeters of this solution is contained 0.314 gram copper sulfate, which is reduced by 0.050 grape sugar.
13. Liebermann:
4 grams copper sulfate; 20 grams potassium-sodium tartrate; 70 grams sodium hydroxid solution, 1.12 specific gravity. Fill to 115.5 cubic centimeters.
14. Löwe:
15 grams copper sulfate; 60 grams glycerol; 80 cubic centimeters sodium hydroxid, 1.34 specific gravity; 160 cubic centimeters water. Fill to half a liter.
15. Mohr:
34.64 copper sulfate; 150 grams di-potassium tartrate; 600-700 cubic centimeters sodium hydroxid solution, 1.12 specific gravity, equal to 70.5-82.3 grams sodium hydroxid. Fill to one liter.
16. Märcker:
35 grams copper sulfate, solution made up to one liter: 175 grams potassium-sodium tartrate; 125 grams potassium hydroxid, equivalent to 89.2 grams sodium hydroxid, solution made up to one liter.
17. Maumenè:
375 grams copper sulfate; 188 grams potassium-sodium tartrate;
166 grams potassium hydroxid. Fill to nine liters.
18. Monier:
40 grams copper sulfate; 3 grams stannic chlorid; 80 grams cream of tartar; 130 grams sodium hydroxid. Fill to one liter.
19. Neubauer and Vogel:
34.639 grams copper sulfate; 173 grams potassium-sodium tartrate; 500-600 grams sodium hydroxid solution, 1.12 specific gravity. Fill to one liter.
20. Pasteur:
40 grams copper sulfate; 105 grams tartaric acid; 80 grams potassium hydroxid; 130 grams sodium hydroxid.
21. Possoz:
40 grams copper sulfate; 300 grams potassium-sodium tartrate; 29 grams sodium hydroxid; 159 grams sodium bicarbonate, allow to stand six months before use. Fill to one liter. One cubic centimeter equals 0.0577 gram dextrose. One cubic centimeter equals 0.0548 gram cane sugar.
22. Rüth:
34.64 grams copper sulfate; 143 grams potassium-sodium tartrate; 600-700 cubic centimeters sodium hydroxid solution, 1.12 specific gravity. Fill to one liter.
23. Rodewald and Tollens:
34.639 grams copper sulfate, solution made up to half a liter; 173 grams potassium-sodium tartrate; 60 grams sodium hydroxid, solution made up to half a liter.
24. Schorlemmer:
34.64 grams copper sulfate; 200 grams potassium-sodium tartrate; 600-700 cubic centimeters sodium hydroxid solution, 1.20 specific gravity. Fill to one liter.
25. Soxhlet:
34.639 grams copper sulfate, solution made up to half a liter. 173 grams potassium-sodium tartrate; 51.6 grams sodium hydroxid, solution made up to half a liter.
26. Soldaini:
3.464 grams copper sulfate; 297 grams potassium bicarbonate. Fill to one liter.
27. Violette:
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.
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 = - 0.00004801x + 0.02876359x².
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 0.02876x² - 1.000048x + 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 715 parts | + | 5Fe₂(SO₄)₃ 2000 parts | + | 5H₂SO₄ 490 parts | = | 10CuSO₄ 1595 parts |
| + | 10FeSO₄ 1520 parts | + | 5H₂O 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 1520 parts | + | K₂Mn₂O₈ 316.2 parts | + | 5H₂SO₄ 784 parts | = | 5Fe₂(SO₄)₂ 2000 parts |
| + | 2MnSO₄ 302 parts | + | K₂SO₄ 174.2 parts | + | 8H₂O 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 360 parts | = | 5Cu₂O 715 parts | = | 10FeSO₄ 1520 parts | = | K₂Mn₂O₈ 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 Vₜ = V₀ [1 + 3β(t-t₀)].
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 = | P-Vₜ d | . | |
| 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 cuprous oxid. | Milligrams dextrose. | Milligrams cuprous oxid. | Milligrams 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 + 3.3x + 0.0041x², 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. Milligrams | Invert Sugar. Milligrams | Dextrose. Milligrams | Levulose. Milligrams | Galactose. Milligrams | Arabinose. Milligrams |
|---|---|---|---|---|---|
| 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]