THE DETECTION AND ESTIMATION OF REDUCING SUGARS.

BY STANLEY R. BENEDICT.

(From the Shefield Laboratory of Physiological Chemistry, Yale University.)

(Received for publication, March 23, IgoT.)

THE DETECTION OF SUGARS.

Of the extremely large number of methods proposed for the detection of reducing sugars there are very few which may be regarded as specific for sugars alone. With one or two possible exceptions, these tests indicate only the presence or absence of reducing substances, and are inapplicable to the detection of sugars when other reducing substances are present. The fact that many of the sugars are powerful reducing agents in alkaline solutions, while they exert, at most, slight action in neutral or acid solutions, is very commonly recognized, and application is made of this fact in almost every test which has been pro- posed for the detection of these substances.

Gaud, Framm and others1 have made studies of the effect of alkali upon a number of the sugars. The substances formed appear to be oxidation products, possibly preceded by a dehy- dration and decomposition. The destructive action of alkalies upon glucose is a common matter of reference throughout the literature upon the estimation of sugars, and numerous sugges- tions have been made to avoid this difficulty by means of the substitution of ammonium hydroxide for potassium or sodium hydroxide, and keeping the temperature somewhat below the boiling point during the reaction, with other devices of a similar nature.2

The following experiments were undertaken to throw light upon the mechanism of the reducing action of the sugars in alka-

1 See De Bruyn and Ekenstein: Rec. Trav. Chem., xvi, p. 274, 1897;

Framm: Arch. f. d. ges. Physiol., Ixiv, p. 57 5,18g6, and Gaud: Compt. rend. de 1’Acad. des xi., cxix, p. 650, 1894.

2 See Gaud: 106. cit.

101

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102 Detection and Estimation of Sugars

line solution as well as to test the relative destructive action of certain of the alkalies upon various carbohydrates. A I per cent solution of dextrose was boiled for about aminute with one- half its volume of a IO per cent potassium hydroxide solution, the resulting solution cooled to room temperature and added to an equal volume of ordinary Fehling’s solution, also at room temperature. No change occurred in the mixture in the cold, and only a very faint reduction was obtained upon boiling. Clearly the sugar had been practically completely oxidized, so far at least as its power of affecting Fehling’s solution was con- cerned. This result was rather what might have been expected, inasmuch as we are here dealing with one of the most readily oxidized of the carbohydrates.

In the next experiment a solution of lactose was substituted for the dextrose. Upon adding an equal volume of Fehling’s solution to the cooled solution a heavy reduction of the copper occurred almost instantaneously in the cold.’

Five cubic centimeters of a I per cent solution of dextrose were now heated to boiling with about 0.5 gram of sodium carbonate, the resulting solution cooled, and mixed with an equal volume of Fehling’s solution. An almost instantaneous reduction of the copper compound occurred, as indicated by a heavy precipita-

r The only references which I have been able to find relating to the for- mation of a compound capable of reducing Fehling’s solution in the cold as a result of warming sugars with alkali, are in Kiihne’s Lekrbuck der physiologischen Ckemie (p. 5 18, 1868), in the work of Worm Mtiller and Hagen (Arch. f. d. ges. Pkysiol., xxii, p. 39 I, ISSO), and of Emmerling and Loges (Arch. f. d. ges. Pkysiol., xxiv, p. 184,r881). Ktihne states that dex- trose solutions, heated to about 70~ C. with sodium or potassium hydroxide solution will yield a solution capable of reducing Fehling’s solution in the cold. He suggests this fact as evidence that the sugars themselves do not reduce directly and suggests this procedure as a test for dextrose in the urine. Worm Miiller and Hagen discuss the bearing of this fact upon Trommer’s test for dextrose in the urine. Emmerling and Loges come to the conclusion that the reducing substance formed is acetone alcohol, CHsCO-CHaOH. At the time my work was done the statement of Kiihne on this subject was unknown to me, and inasmuch as neither he nor Worm Mtiller made comparisons of the action of the other alkalies and other sugars, nor gave a full theoretical discussion or analytical appli- cation of the fact which they stated, I have thought it desirable to give a rather complete discussion of my results.

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Stanley R. Benedict 103

tion of the yellow cuprous oxide. A solution of lactose substi- tuted for the dextrose in the above experiment yielded a similar result. In other words, a I per cent solution of glucose boiled for a few moments with IO per cent potassium hydroxide solu- tion almost completely loses its reducing power. A similar treat- ment of a lactose solution yields a solution containing some sub- stances capable of reducing Fehling’s solution in the cold. Boil- ing either lactose or dextrose with sodium carbonate yields a solution which reduces Fehling’s in the cold. It is particularly to be noted here that the potassium hydroxide solution destroys the reducing power of the dextrose, while the carbonate, under similar conditions, does not. This fact will be referred to later. The compound formed under these conditions is remarkably strong in its reducing power. Ammoniacal silver solutions are reduced instantaneously and completely in the cold. The prod- uct formed either from dextrose or lactose is capable of reducing Barfoed’s reagent upon boiling, even when the acidity is consider- ably greater than that called for in Barfoed’s formula.

These results indicate that the sugars themselves are not directly responsible for the strong reducing action of their alka- line solutions. They also admit of some valuable analytical applications. The first of such applications would be appar- ently to afford a differentiation of lactose from dextrose, the former substance in I per cent solution forming a compound upon boiling with alkali which will reduce Fehling’s fluid in the cold, while dextrose under similar conditions is destroyed. This appli- cation can be made, and by means of such a method it is possible to detect lactose in presence of dextrose, or to distinguish between dextrose and lactose solutions. The procedure is as follows: To about S-IO cubic centimeters of the solution to be tested is added one-half volume of IO per cent potassium hydroxide solu- tion and the resulting mixture boiled vigorously for about one and one-half minutes. The solution is then cooled and added to an equal volume of Fehling’s fluid. The immediate formation of a heavy precipitate which is bright yellow and fills the entire body of the solution indicates lactose. A slight precipitate is to be disregarded. The solution used for this test should contain between 0.5 and I per cent of the sugar. It is not a par- ticularly delicate reaction and will not give positive results where

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104 Detection and Estimation of Sugars

the amount of lactose is under 25 per cent of the total quantity of sugar present.

A much wider analytical application of these facts is possible. Upon them we may base a reaction which is apparently specific for sugars. Very few, if any other substances will, upon boiling with alkalies, form substances capable of reducing Fehling’s solu- tion in the cold. In just so far as this is true, this method of detecting sugars is specific. The procedure is as follows: To 5-8 cubic centimeters of the fluid to be tested in a test tube, is added about one-half gram of sodium carbonate. Heat the solution to boiling and continue the boiling for about one-half minute. Cool and add an equal volume of Fehling’s solution. If sugar is present a reduction will occur more or less promptly, one per cent or more giving a practically instantaneous reaction, smaller amounts requiring from three to five minutes. As thus carried out this method will detect sugar in solutions containing 0.1 per cent, or more, of sugar. Slightly warming the tube will increase the delicacy to about 0.05 per cent. These figures apply to relatively pure solutions of the sugars.

While this method is not nearly so sensitive as the usual Fehling’s method, it may become of material use in establishing the presence or absence of sugars in mixtures containing other reducing substances, and is delicate enough for many purposes.

Attention was called above to the fact that sodium hydroxide solutions destroy the reducing properties of glucose much more rapidly than does sodium carbonate solution.’ Yet the alkalin- ity given by sodium carbonate is amply sufficient for the glucose to exhibit its full reducing action. Considering the bearing of these facts upon sugar detection it will be evident that where only very small amounts of dextrose are present, or where some

l It is not to be inferred from this statement that a distinction is implied between the action of the hydroxides and carbonates in any respect other than may be accounted for by the difference in the degree of alkalinity represented by the two substances. If we use very dilute alkali solutions, stronger sugar solutions, or if the heating be more mild, it is perfectly pas- sible, as Ktihne and Worm Mtiller have shown, to obtain a solution which will still exhibit reducing properties. If, however, we endeavor to use hydroxide solutions sufficiently dilute to avoid the destruction of even minute quantities of dextrose, a solution is obtained which is utterly unfitted for ordinary qualitative or quantitative sugar work.

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Stanley R. Benedict 105

substance is present which temporarily interferes with the reduc- tion of the copper solution, a reagent in which the alkalinity is secured by means of sodium carbonate, instead of hydroxide, shouldgivemoresatisfactoryresults. In order to test this hypoth- esis the following work has been done.

Solutions of the following composition were prepared : SOLUTION A.

Crystallized copper sulfate. . . . . . . . . . . . . . _ . . 69.3 gm. Distilled water to . . . . . . . . . . . . 1000 cc.

SOLUTION B. Pure Rochelle salt . . . . . . . . . . . . . . . . . . . . . . . . 346 gm. Anhydrous sodium carbonate . . . . . . . . . . . . . . . . . . . . 200 “ Distilled water to . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 cc.

For use, these solutions are mixed in equal proportions, and the resulting mixture diluted with an equal volume of distilled water. When further diluted with an equal volume of distilled water, heated to vigorous boiling and allowed to cool spontane- ously, this solution should not show the slightest turbidity. The reagent thus prepared was tested regarding its power of detecting sugars as compared with Fehling’s fluid, under the following conditions. When distilled water solutions of dextrose were used and the solution boiled as in the usual procedure, it was found possible to obtain in most cases a perceptible reaction with Fehling’s fluid’ when the sugar present amounted to 0.001

per cent. The result here was in many instances uncertain and marked the full limit of the test as I applied it. The method of procedure was to add to 3 cubic centimeters of Fehling’s fluid in a test tube an equal volume of the solution to be tested, the resulting mixture being heated to vigorous boiling, which was continued for about one-half minute. The solution was then allowed to cool to room temperature, and unless precipitation had already occurred, it was again heated to boiling and allowed to cool, this process being sometimes repeated. Worm Muller and Hagen state that Fehling’s solution will indicate the presence of glucose in solutions containing 0.0008 per cent. I have been absolutely unable to obtain any reaction with 0.0008 per cent solutions of glucose with the procedure above described, while solutions of 0.001 per cent often gave negative results.

1 The Fehling’s fluid used was made up according to Soxhlet’s formula. 2 Arch. f. d. ges. Physiol., xxii, p. 383, 1880.

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106 Detection and Estimation of Sugars

With the reagent in which the carbonate is substituted for the hydroxide, according to the formula given above, and follow- ing an exactly similar method of procedure to that described for Fehling’s solution, I have obtained absolutely positive results with solutions containing o.oooo5 per cent of glucose. A dis- tinct yellowish precipitate is obtained with glucose solutions of this dilution, especially upon cooling. Further dilutions were not tested. It thus appears that the substitution of carbonate for hydroxide yields a solution many times as delicate for the detection of sugars as we have in Fehling’s fluid. A compari- son made with any dilute solution-for instance, about 0.01 per cent solutions of dextrose-will speedily convince anyone of the greater merits of the carbonate reagent.

The urine, containing as it does substances which either par- tially reduce or else inhibit the reduction of Fehling’s fluid, offers peculiar difficulties in the detection of small amounts of sugar. It is, therefore, of special interest to determine whether the carbonate solution possesses any advantages over Fehling’s fluid for the detection of dextrose in the urine. Fehling’s solution will usually indicate dextrose in the urine when present Up to, or in excess of, 0.1 per cent. Smaller amounts give abso- lutely no indication of their presence, save possibly by confusing changes in the color of the solution. When the amount is as small as o. I per cent, the results are often very uncertain. With the reagent in which the carbonate is substituted for the hydrox- ide, it is perfectly possible to detect as small amounts of dextrose in the urine as from 0.015 to 0.02 per cent. Even these small quantities yield results which are almost invariably far more positive than can be obtained with Fehling’s fluid in the presence of ten times the amount of sugar.

The procedure for the detection of sugar in the urine is as follows. Solutions A and B, made according to formula given above, are mixed in equal proportions. The resulting solution is then diluted by the addition of three times its volume of dis- tilled water. To about 6 cc. of this reagent in a test tube are added from 7 to g (not more) drops of the suspected urine. The mixture is heated to vigorous boiling for about one-fourth to one- half minute, and allowed to cool spontaneously to room tempera- ture. This process may be repeated if desired, though it is usu-

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Stanley R. Benedict 107

ally unnecessary. In the presence of sugar a precipitate will form which is often greenish or bluish green to begin with (in case the amount of sugar present is small), and usually becomes yellowish upon standing. This precipitate generally forms at or below the boiling temperature if the sugar present exceeds 0.06 per cent ; with smaller amounts it forms slowly, usually only upon cooling. With larger amounts of sugar the reaction is obtained very readily upon reaching the boiling temperature. The precipitate is then generally reddish or yellowish in color. The results obtained in this test, even with the smaller amounts of sugar are extraordinarily definite, and according to my expe- rience leave no room for uncertain interpretation. Normal urine will not, under these conditions, produce even the slightest tur- bidity in the reagent. Professor Mendel, of this laboratory, has suggested that the greenish precipitate obtained with urines containing small amounts of sugar, may be a compound of copper with the sugar, rather than a reduction product. While this may be the case, it seems more probable to the writer that the precipitate represents, if not a simple cuprous oxide or hydrox- ide, a compound of some constituent of the urine with reduced copper oxide. This appears the more likely, because, in the absence of the urine, sugar solutions of equal dilution give a definite reduction product of either the red oxide or the yellowish hydroxide. This latter compound, it may be remarked, is more usually obtained as a product of the reduction of the carbonate- copper solution, than is the case with Fehling’s fluid, particularly when only small amounts of sugar are present, owing probably to the less strongly dehydrating action of the carbonate solution.

Whatever may be the nature of the precipitate obtained as a result of boiling the carbonate copper solution with sugar-con- taining urines, it appears to be a most delicate and satisfactory method for the detection of dextrose in‘this fluid.

It is interesting to note in this connection the modification of Fehling’s test for sugar in the urine proposed by Worm Muller,l in which he suggests the use of solutions of sodium hydroxide more dilute than in ordinary Fehling’s fluid, small amounts of copper solution, and the continued heating of the solution well

1 Worm Miiller: Arch. f. d. ges. Physiol., xxvii, p. 107, 1882;

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Detection and Estimation of Sugars

below the boiling point. This modification is capable of detect- ing about 0.03 per cent of dextrose in the urine. Worm Muller himself suggested that the superior delicacy of this test over the ordinary Fehling’s method is due to the fact that the sugar reduces at a lower temperature than the reducing substances normally contained in the urine. In view of the facts brought out in the earlier portions of this paper it would be inferred that there are other factors here as well as the one he suggested. Thus it seems probable that ordinarily the interfering substances in the urine (chief among which is apparently creatinin, as pointed out by MacLean’) will inhibit the reducing action of the sugar long enough for the strong alkali to completely destroy the small amount of carbohydrate present. Lower temperature and weaker concentration of alkali would tend to prevent this destruction, so that eventually the glucose would have its normal reducing action. Similar results are apparently obtained where carbon- ate is substituted for the hydroxide. The procedure is not troublesome as in Worm Mtiller’s test, and the results are more delicate and conclusive.

Thus we see that the carbonate solution has the following advantages over Fehling’s solution. It is many times more delicate for the detection of sugars in pure solutions. This is just what we should expect from theoretical considerations. Feh- ling’s fluid, containing as it does, a substance which is strongly destructive to glucose, should not be used so long as we can sub- stitute something which is more effective and has not this destruc- tive action. The carbonate solution yields more definite results than does Fehling’s fluid, since there are fewer substances which interfere with its action than is the case with the other fluid. This can be shown by comparative tests with the urine, and fur- ther it can be most beautifully exemplified by comparisons of the reducing action of chloroform upon the two solutions. A solution of chloroform in water will reduce Fehling’s solution most copiously, even slightly below the boiling point, whereas its action is very slight upon the carbonate solution and only occurs upon prolonged boiling. There are many other substances which reduce Fehling’s solution more readily than the carbonate

i MacLean: Bio-them. Journ., i, p. I I I, 19 06.

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Stanley R. Benedict 109

solution, and thus interfere with the use of the former solution as a test for sugars to a greater degree than is the case with the carbonate solution; but chloroform is the most striking example. Regarding the stability of the carbonate solution the following should be stated. For delicate work in sugar detection, either in pure solutions or in the urine, the solutions making up the reagent should be freshly mixed and diluted. If it is desired to keep the mixed reagent on hand the two solutions should be mixed in equal proportions and to every liter of the undiluted mixture should be added from five to ten grams of sodium hydrox- ide. This small amount of alkali serves to prevent decomposition and does not affect the delicacy of the reagent to any great extent. Such a mixture will remain for weeks more delicate as a reagent than Fehling’s solution and is not nearly so caustic. It should be remembered, however, that the best results are only above obtained with freshly mixed solutions diluted according to the directions.

A few copper solutions containing carbonates have been pro- posed in the literature. The best known of these is Soldaini’s solution,’ which contains 3.464 grams of crystallized copper sul- fate and 297 grams of potassium bicarbonate dissolved in a liter of water. Ost2 later modified this solution by substituting for a portion of the bicarbonate, a nearly equal weight of the normal carbonate, as well as increasing the amount of copper sul- fate present. These solutions are open to serious objections to which the solution suggested in this paper is not. Soldaini’s solution is more difficult to prepare, a portion at least of the cop- per always being left undissolved as the carbonate, which must be filtered off. Furthermore the boiling with the test solution must be continued for some time, often continuously for ten or fifteen minutes before smaller amounts of sugars indicate their presence by even the slightest apparent reduction. Thereason for this fact appears to be that bicarbonate is capable of holding considerable amounts of cuprous oxide in solution, or else of pre- venting the reduction of cupric compounds by dextrose. This is readily proved by the following experiment. If to a portion of

l Soldaini: Chem. Centralbl., p. 389, 1889. 2 Ost: Ber. d. deutsch. chenz. Gesellsch., xxiii, I, p. 1035, r89o.

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110 Detection and Estimation of Sugars

the carbonate copper solution described earlier in this paper a considerable amount of bicarbonate of sodium is added, it will be found that the resulting solution shows no reduction even with large amounts of sugar except upon very long continued boiling, while a similar solution to which the bicarbonate has not been added gives a heavy precipitate even before the boiling point is reached. Thus it will be seen that the bicarbonate solu- tions are open to marked objection in the fact that they require very prolonged boiling to obtain any result, and are, therefore, scarcely applicable to qualitative work. Results are obtained with the solution above recommended almost instantaneously, unless the amount of sugar present is very small, in which case it may require from four to five minutes.

A METHOD FOR THE VOLUMETRIC ESTIMATION OF SUGARS.

Owing to the depth of color of the precipitate obtained upon the reduction of Fehling’s solution by means of sugars, the application of Fehling’s process to the volumetric determina- tion of sugar solutions is very difficult Even with long prac- tice it is almost impossible to obtain satisfactory results by the use of this method. Various modifications of the original Feh- ling’s fluid have been proposed with a view to render it more applicable to volumetric work. Most of these are based upon an attempt to keep the cuprous oxide formed in solution. In Pavy’s solution ammonia is added for this purpose. The objec- tions to the latter method are the great ease with which the dis- solved cuprous compound undergoes oxidation-hence the abso- lute necessity of entirely excluding air if accurate results are to be obtained-and furthermore the rapidity with which the ammo- nia boils out of the solution. In Gerrard’s method potassium cyanide is used as the solvent for the reduced copper compound. As ordinarily performed this method requires two titrations and must be carried out quite rapidly in order to avoid reoxidation of the reduced copper. The potassium cyanide solution used is unpleasant to work with and is quite unstable.

To the writer it seemed that a more satisfactory method of sol- ving the difficulties offered by Fehling’s solution in sugar titra- tion would be to obtain some modification of the solution, which should yield upon reduction not the red cuprous oxide but some

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Stanley R. Benedict III

colorless insoluble compound. In this case we should have the reduced copper compound in such a form as would prevent its reoxidation, while it would not obscure the end point of the reaction.

The well known insolubility of cuprous compounds of the hal- ogens led to the attempt to obtain the precipitation of the reduced copper as one of the haloid salts, through the addition of con- siderable quantities of chlorides, bromides, or iodides to Feh- ling’s solution. No satisfactory results were obtained with these substances. Closely related in chemical behavior to the halo- gens are the simple and complex cyanides, and the attempt was next made to see whether any of these substances would yield the desired results. Addition of potassium ferrocyanide to Fehling’s solution causes, upon reduction, the precipitation of a white compound, which, however, upon continued boiling becomes dark in color. This substance was, therefore, unsatisfactory except in a certain combination referred to later. If potassium sulfocyanide be added, in small amounts, to Fehling’s fluid it produces no appreciable change in the reduction product. If, however, it be added in considerable excess, the cuprous oxide formed will be held in solution. After the writer had found that carbonate copper solutions are much less destructive in their action upon dextrose than is the corresponding hydroxide solu- tion, as has been shown earlier in this paper, it was only natural to try the above suggested compounds in connection with this solution. It was speedily found that in this case potassium sul- focyanide yielded a very different result from that obtained with Fehling’s solution. After addition of potassium sulfocyanide to the carbonate solution it was found that upon reduction a chalk- white compound of cuprous sulfocyanide was produced, instead of the red oxide. This suggested at once the employment of this solution for volumetric purposes.

This fact was discovered by the writer early in November, 1906. A somewhat hasty review of the literature at that time, preliminary to working up the method, failed to reveal the fact that potassium sulfocyanide had ever been previously applied to sugar estimation, or even that the fact above stated had been mentioned in the literature. About January I, 1907, a paper

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II2 Detection and Estimation of Sugars

appeared by Bang,l describing a volumetric method for sugar estimation, depending upon the use of potassium sulfocyanide. In this paper Bang referred to the fact that he had pointed out in an earlier communication2 that addition of potassium sulfocy- anide to alkaline copper solution which contained no hydroxide, caused the precipitation of white cuprous sulfocyanide instead of the red suboxide. He had also suggested in this earlier paper a method for the estimation of dextrose, based upon this fact. Bang’s earlier paper had completely escaped my attention in the review of the literature on this subject, owing very probably to the fact that its title was in nowise concerned with sugar analy- sis, being “Ueber die Verwendung der Zentrifuge in der Quan- titativen Analyse. ” The first method Bang proposed was based upon an attempt to estimate the excess of potassium sulfocy- anide remaining in the solution after the reduction of the copper had taken place. This method appears to the writer obviously unsatisfactory. The cuprous sulfocyanide formed is not com- pletely insoluble in even a small excess of potassium sulfocyanide, and unless some excess of this salt is present a portion of the cop- per is precipitated as the suboxide. It would thus appear that determination of the residual potassium sulfocyanide would not yield very satisfactory results. Bang recognized the unsat- isfactory nature of his earlier method and in his second paper substitutes one which he considers much better. His method in this case, briefly outlined, is as follows: The copper solution employed is a modified Soldaini’s solution, to every liter of which is added two hundred grams of potassium sulfocyanide. To a measured volume of this solution is added a measured volume of the sugar solution to be determined, the mixture then being boiled for three minutes. (No precipitation takes place owing to the large excess of sulfocyanide present.) Then the solution is cooled and the excess of cupric copper determined by titration with standard hydroxylamine solution to a colorless solution. The results obtained appear to be very satisfactory. There are at least two objections to Bang’s method. The boiling copper solution cannot be titrated with the sugar solution directly to

1 B&&em. ZeitscJw., ii, p. 271, 1906 (December). 2 Festschrift fiir Hammarsten, 1906 (Upsala L&karef&enings Forhand-

lingar. Ny Foljd, Elfte Bandet. Supplement).

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Stanley R. Benedict 113

a colorless solution because of the employment of the bicarbon- ate solutionof Soldaini and Ost. This solution, as is mentioned earlier in this paper, requires continued boiling before complete reduction takes place, which renders impracticable any attempt to titrate directly to an end point. The second objection to Bang’s method is the employment of hydroxylamine for his final titration, a substance which even in form of its salts, is unstable, and not commonly available.

In the use of the writer’s method for the volumetric estimation of sugar three solutions are required. which are made up accord- ing to the following formulae: SOLUTION A.

Crystallized copper sulfate. . . . . . . . . . . . 69.30 gms. Distilled water to . . . . . . . . . . . . . . . . . . . . 1000 cc.

SOLUTIONB. Crystallized Rochelle salt . . . . . . . . . . . . . . . . . . . . . . . . . 346 gms. Pure anhydrous sodium carbonate . . . . . . . . . . . . . . 200 I‘ Distilled water to . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 cc.

SOLUTION C. Potassium sulfocyanide . . . . . . . . . . . . . . . . . . . . . . . . . 200 gms. Distilled water to . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 cc.

For use these solutions are mixed in equal proportions in the order indicated. To every 30 cubic centimeters of the solution thus obtained are added from 2.5 to 5 grams of pure anhydrous sodium carb0nate.l The amount of this substance added should roughly correspond to the dilution to which the solution will be

1 Regarding the addition of sodium carbonate in this connection the following should be stated. The alkalinity secured by the addition of the indicated portion of Solution B, is not great enough to give the most satis- factory end point. This lack of alkalinity might be overcome either by the addition of the solid carbonate as above suggested, or through the substitution of a greater amount of potassium carbonate in the formula of Solution B. (Solution B is practically saturated with sodium carbonate, which is, it will be remembered, much less soluble than the correspond- ing potassium compound.) I prefer the method above detailed for secur- ing the increased alkalinity. Sodium carbonate is much commoner than the potassium salt, keeps anhydrous better and is usually more available. The addition of a small portion of the dry salt, which may be roughly measured, is certainly no great trouble. Furthermore, the formula of Solution B, it will be noticed, coincides with the one found most satis- factory for qualitative work, as detailed earlier in this paper, so that such a solution is readily available for either qualitative or quantitative work.

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114 Detection and Estimation of Sugars

subjected during the titration, i. e., for titrating dilute sugar solutions add greater quantity of carbonate and vice versa. The solutions are mixed in a beaker of suitable capacity, the requisite quantity of carbonate added, and the mixture heated to boiling over a gauze until the carbonate completely dissolves. Thirty cubic centimeters of this mixture (equivalent to IO cubic centimeters copper sulfate solution) are equal to approximately 0.073 gram of pure dextrose.

The titration is carried out as follows-the sugar solution is run in from a burette rather rapidly-(not so rapidly as to inter- fere markedly with continuous vigorous boiling) until a heavy, chalk-white precipitate is formed and the color of the fluid begins to lessen perceptibly. The last portions should be run in in quantities of from two to ten drops (depending on depth of color remaining and the relative strength of the sugar solution), with a vigorous boiling of about one-fourth minute between each addi- tion. The end point of the reaction is the complete disappear- ance of the blue color. This point is sharp and satisfactory. The precipitate obtained is chalk-white and is rather an aid than a hindrance to the determination of the end point. While potas- sium sulfocyanide could be added in large enough excess to retain the precipitate in solution, I can see no advantage in such a procedure, whereas it has the disadvantage of permitting some reoxidation if the titration be carried on too slowly.

It may be of interest to mention a simple devise used by the writer with much success to prevent the annoying bumping of the solutions during the process of titration. This consists in the introduction into the titration beaker of a medium sized piece of pure, previously well washed, absorbent cotton. By stirring this cotton about as the titration proceeds, it is possible to entirely prevent the bumping which otherwise may become very troublesome. Glass wool may be used in place of cotton but is not much more satisfactory and is considerably less economical.

In order to test the accuracy and reliability of this method a solution of dextrose in distilled water was prepared of approx- imately one per cent. The sugar content of this solution was determined by Allihn’s gravimetric method, duplicate analyses being made in every case. Using this solution, the value of the copper solution used in the above method was computed from

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Stanley R. Benedict II.5

the results of three titrations, in whichwere employed IO, 20 and 30 cubic centimeters of the copper sulfate solution, respectively. The results of these titrations were exactly concordant, giving the value of one cubic centimeter of copper solution as equiv- alent to .00727 gram of dextrose. On the basis of this stand- ardization the strength of about twenty-five sugar solutions was determined, duplicate titrations being made in every case. The actual amount of sugar present in each solution was also determined by Allihn’s gravimetric method and the results thus obtained compared with the results of the titrations. Where the solution titrated was too strong for determination directly by Allihn’s method, it was diluted with a measured volume of water and then analyzed.

Duplicate analyses were invariably entirely concordant, the re- sults differing either not at all or only by such slight error as is experienced in reading the ordinary burettes. The titrated solu- tions varied in strength from 2.2 to 0.1 per cent of dextrose. To some of the solutions were added small portions of the common inorganic salts, such as might occur as impurities in ordinary sugar solutions. The results were not affected by the presence of these substances.

The values obtained for the various solutions were in every case in very close agreement with the results found by Al- lihn’s method. So far as could be determined, the value of the copper solution in terms of dextrose remained absolutely con- stant for strong or dilute solutions. In fact, in the case of many of the dilute solutions, i. e., under 0.3 per cent, the results were more closely in agreement with the actual sugar content (known in these cases because obtained by exact dilution of stronger solu- tions) than could be obtained by Allihn’s method, which, while giving very exact results and duplicates with solutions of about five-tenths of one per cent, is not so satisfactory for more dilute, or stronger solutions.

Attention is called to the fact that the value of the copper solution remains the same for dilute as for stronger solutions. This is not the case with many of the solutions employing the alkali hydroxides. I believe this fact finds ready explanation in the less strongly destructive action of the carbonates than the hydroxides, as pointed out earlier in this paper.

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116 Detection and Estimation of Sugars

A table is appended giving results of five typical titrations, carried out in exact accordance with the directions given above.

GRAMS OF Gmcos~ IN 10 cc. OF SOLUTION. NO. B ,Atpoh$s

x4 . Average from

Allihn’s Method. Write$F,tpion Average ?f Results

by Writer s Method.

1 0.0280 0.0270 0.0280 0.0277 0.0260 0.0274

-- 2 0.0530 0.0530 0.0528 0.0528

0.0531 0.0528

3 0.1021 0.1016 0.1020 0.1021 0.1012 0.1022

4 0.1810 0.1814 0.1815 0.1817 0.1819 0.1820

5 0.0112 0.0107 0.0117 0.0115 0.0102 0.0113

It should be stated here that where the sugar solution to be titrated contains less than three-tenths of one per cent, the writer has found the following method more expeditious and satisfactory, as avoiding extreme dilution during the titration. A measured amount (25 cc.) of the sugar solution to be deter- mined is run into 30 or 60 cubic centimeters of the copper titra- tion solution, the mixture boiled for from five to seven minutes and the residual copper determined with a dextrose solution of known strength, of approximately I per cent. While not an essential procedure, this method is expeditious and yields ac- curate results. In ordinary titrations the writer has used the mixed fluid in quantities of 30 or 60 cubic centimeters, the former volume being sufficient for sugar solutions of I per cent or less.

In cases where it is not convenient to standardize the copper solution against sugar solutions, of known strength, it is possible to obtain quite satisfactory results through the employment of an exact weight of pure crystallized copper sulfate. Where this method is employed the copper sulfate should be freshly recrystallized, dried upon blotting or filter paper and accurately weighed out upon an analytical balance, 69.30 grams being dis- solved in water and the volume made up to exactly one liter. Ten cubic centimeters of this solution are equivalent to approx-

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Stanley R. Benedict

imately .073 gram of dextrose. This process was tried upon three occasions by the writer and the results varied but slightly from those obtained through standardization of the solution with sugar solutions of known strength, though for absolute accuracy the latter process is of course to be preferred.

Some titrations have been made to test the applicability of this method to the determination of dextrose in the urine.’ The results obtained show a quite satisfactory approximation to the actual sugar content, although, just as should be expected, they are not exact, the variation usually being from five-hun- dredths to two-tenths of one per cent of the actual sugar content, depending upon the relative amount of dextrose present. Solu- tions containing larger percentages of sugar yield more correct results.

In conclusion it may be stated that the writer has met with certain substances (notably traces of chloroform) which are cap- able of causing a portion of the copper to precipitate as the red oxide, even in presence of the sulfocyanide. While impurities which will do this would be only rarely encountered, and although the interference with the end point is usually not marked, it is desired to offer an alternative formula for Solution C, which will obviate this slight difficulty entirely. The formula for this solu- tion is as follows :

Potassium ferrocyanide . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 gms. Potassium sulfocyanide . . . . . . . . . . . . . . . . . . . . . . . . . 125 “ Anhydrous sodium carbonate . . . . . . . . . . . . . . . . . . . . . . . 100 “ Distilled water to . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 cc.

The use of this solution does not change the value of the copper in terms of dextrose, and may be used entirely in place of the other formula if desired.

This method has not yet been tested regarding the other re- ducing sugars except to find that they also yield the white precipitate as a result of their action on the solution. There is no apparent reason why there should be any difficulty in such an application.

l Rudischand Celler (Journ. Am. Med. Assoc., Jan. 26, IgoT, p. 324) de- scribe a method for determination of sugar in the urine through the use of ordinary Fehling’s fluid to which is added a large amount of potassium sulfocyanide, thus preventing precipitation of cuprous oxide when reduc- tion occurs.

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Stanley R. BenedictOF REDUCING SUGARS

THE DETECTION AND ESTIMATION

1907, 3:101-117.J. Biol. Chem. 

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