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to reduce completely a measured volume of alkaline copper solution, the end point being internally indicated by the reduction of methylene blue to methylene white by a minute excess of reducing sugar. Methylene blue is a thiazine dye, intensely colored and of oxidation reduction potential well suited to the titration of reducing sugars. A few drops of a 1-percent solution of this dye impart an intense blue color to the reaction mixture, which, in the absence of air, is almost instantly reduced to the leuco base by an excess of reducing sugar. The methylene white is rapidly reoxidized by air, particularly at the high alkalinity of the reaction mixture. Hence it is essential that air be excluded during the titration. This is accomplished by causing an uninterrupted current of steam to issue from the neck of the flask in which the analysis is performed.

Lane and Eynon determined the weight of each sugar required to reduce the copper completely. These weights, which vary with the nature of the sugar and with its concentration, constitute table of factors (tables 85 and 86), from which the proper one may be selected when the titer is known. The concentration of sugars is then

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(1) STANDARD METHOD OF TITRATION.-Ten or twenty-five milliliters of mixed Soxhlet reagent is measured into a flask of 300- to 400-ml capacity, and treated cold with almost the whole of the sugar solution required to effect reduction of all the copper, so that if possible not more than 1 ml is required later to complete the titration. The approximate volume of the sugar solutions required is ascertained by a preliminary incremental titration. The flask containing the cold mixture is heated over an asbestos-gauze plate. After the liquid has begun to boil, it is kept in moderate ebullition for 2 minutes, and then, without removal of the flame, 3 to 5 drops of the methylene blue indicator are added, and the titration is completed in 1 additional minute, so that the reaction liquid boils altogether for about 3 minutes without interruption.

The visual end point coincides with the disappearance of cupric ions and hence is the same as that marked by the ferrocyanide and the electrometric test. It has the advantage over the ferrocyanide test that the search for the end point can be completed without interruption of the titration.

Like all volumetric reducing-sugar methods, this method produces best results if almost all the sugar required is added in the early stages of the analysis. Usually the sugar concentration is not known with sufficient certainty to do this. Consequently, Lane and Eynon advise performing a preliminary titration in order to determine the approximate volume of solution required. This is most conveniently accomplished by adding an initial volume of 15 ml of the sugar solution to the measured volume of copper solution, boiling for about 15 to 20 seconds, and then adding further increments of sugar until the blue color of the copper solution has nearly disappeared. This point can be fairly judged within 1 or 2 ml of sugar solution. At this point the methylene blue is added and the titration completed dropwise, the period of operation occupying as nearly 3 minutes as possible.

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In the analysis of solutions of the hexoses, this incremental method is nearly as reliable as the standard method, but with solutions of the disaccharides and solutions containing sucrose, it is desirable to repeat the titration by the standard method.

The outlet of the burette should be fitted with a rubber tube connected with a glass tube bent twice at right angles in order that the solution shall not be heated by the reaction mixture. Glass stopcocks cannot be used as they invariably jam under the fluctuating temperature conditions.

Soxhlet's modification of Fehling's solution is used for the method. The solution is made by mixing exactly equal volumes of (1) a copper sulfate solution containing 69.28 g of CuSO4.5H2O per liter, (2) a solution containing 346 g of Rochelle salt and 100 g of sodium hydroxide per liter.

Pure copper sulfate ordinarily contains somewhat more than the theoretical amount of water. Hence the authors recommend that a standardization of the solution be performed by titration against a standard invert-sugar solution and that any small adjustment of the copper solution be made to correspond with the tables by addition of copper sulfate or water. Twenty-five milliliters of solution (1) should contain 440.9 mg of copper.

The standard solution of invert sugar may be prepared as follows: A solution of 9.5 g of pure sucrose is treated with 5 ml of hydrochloric acid (sp gr 1.19), made up to about 100 ml, left at room temperature for several days (e. g., about 1 week at 12° to 15° C or 3 days at 20° to 25° C) and then made up to 1 liter. The acidified 1-percent solution of invert sugar is very stable and retains its titer unchanged for months. A known volume of the standard solution is neutralized with sodium hydroxide and suitably diluted immediately prior to use. The experimental factor determined by titration, of the standard solution is

Titer Xmg of sugar per 100 ml =F'.

100

F' is then compared with F, the tabulated factor, and the copper solution is adjusted accordingly. If the deviation of the experimental from the tabulated factor is small, it has been the practice of this Bureau to apply the small correction to the tabulated factors rather than to make numerous adjustments of the copper solution.

Carry out the titration as described, find in the table the factor corresponding to the titer and, if necessary, apply to the factor the correction previously determined. Estimate the sugar by

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The Lane and Eynon factors are given in tables 85 and 86, pages

590-91.

(2) APPLICATIONS OF THE METHOD.

Determination of reducing sugars in raw sugars containing more than about 0.4 percent of reducing sugars.-A solution of the sample containing 5, 10, or 25 g per 100 ml is titrated against 10 ml of Fehling solution, the percentage of reducing sugar (as invert sugar) being found from table 85. Preliminary treatment with normal lead ace

tate and potassium oxalate is generally unnecessary except for very low-grade raw sugars.

The sucrose content may be found from the invert-sugar content before and after hydrolysis, which is carried out as described below.

Determination of reducing sugars in refined or raw sugars containing less than about 0.4 percent of reducing sugars. Twenty-five grams of the sample is dissolved with a quantity of neutralized standard invertsugar solution containing 0.1 g of invert sugar, and the whole is made up to 100 ml. A convenient procedure is to wash 25 g of the sample into a 100-ml flask containing the requisite quantity of neutralized invert-sugar solution. After solution and completion to volume, the solution is titrated against 10 ml of Fehling solution. The percentage of invert sugar in the sample is found by deducting 0.1 from the percentage of invert sugar in the solution and multiplying the remainder by 4, or it may be found directly from table 87, page 592.

Determination of invert sugar in molasses. If the sucrose in the sample is to be determined polarimetrically, it is convenient to weigh 65 g of the sample and dilute to 250 ml, using 50 or 100 ml of this solution for determination of reducing sugars, as described below.

Cane molasses.-A solution containing 13 g of the sample is treated with 25 ml of a 10-percent solution of normal lead acetate, made up to 250 ml, shaken and filtered. Fifty milliliters of the filtrate is treated with 5 ml of a 10-percent solution of potassium oxalate, a little alumina cream is added, and the solution is made up to 250 ml, shaken, and filtered. The filtrate, which is free from lead and calcium salts, represents a 1.04-percent solution of the sample and is titrated against 10 ml of Fehling solution. The percentage of invert sugar in the sample is found from table 85 by interpolation between the values given in the columns headed "no sucrose" and "1 g of sucrose per 100 ml."

NOTE. For purposes of interpolation, a 1.04-percent solution of cane molasses may be taken as containing 0.3 percent of sucrose.

Beet molasses.-One hundred milliliters of a solution of the sample (26 g in 100 ml) is treated with 30 ml of a 10-percent solution of normal lead acetate, shaken, and filtered. The filtrate, representing a solution of the sample of 20 g in 100 ml, is treated with enough solid potassium oxalate (e. g., about 2 g) to remove lead and calcium and filtered. The filtrate is titrated against 10 ml of Fehling solution, the percentage of invert sugar in the sample being found from table 85, column headed "10 g of sucrose per 100 ml.” If the sample contains more than 1 to 1.5 percent of invert sugar, the deleaded and decalcified solution should be further diluted with distilled water before titration.

NOTE. A 20-g sample of beet molasses may be taken as containing 10 g of

sucrose.

Determination of sucrose in cane or beet molasses.-Fifty milliliters of the clarified, deleaded, and decalcified 1.04-percent solution of cane molasses obtained as above is treated with 15 ml of 1.0 N hydrochloric acid, diluted to about 150 ml, heated to boiling, and kept boiling for 2 minutes. The solution is then cooled, neutralized with sodium hydroxide, and made up to 200 ml. The solution, which now represents a 0.26-percent solution of the sample, may be titrated against 10 ml of Fehling solution, the invert-sugar content being found from table 85,

column headed "no sucrose." The sucrose content of the sample is calculated from the difference between the invert-sugar content before and after hydrolysis. Sucrose in beet molasses may be determined by a similar method.

Determination of reducing sugars in starch, dextrin, and glucose.For most purposes it is sufficient to assume that the reducing sugars in starch dextrin and starch sugar consist of dextrose. This is determined by titrating a solution of the sample of suitable concentration against 10 or 25 ml of Fehling solution and finding the dextrose content of the solution from table 85 or 86.

If it is desired to determine dextrose and maltose in starch products, the procedure devised by Morris [37] may be used.

Determination of lactose in sweetened condensed milk.-In the analysis of sweetened condensed milk, the determination of lactose by Fehling solution is somewhat affected by the sucrose present. The effect of the sucrose is to reduce the volume of lactose solution required, and it may be allowed for by adding the requisite correction to the burette reading. Tables 85 and 86 give the values for lactose itself, and table 88 shows the burette corrections, in milliliters, for sucrose-lactose ratios of 3:1 and 6:1. At any given part of table 88 the correction is practically proportional to the sucrose-lactose ratio, and the proportionality holds up to a ratio of about 10:1.

(b) SCALES METHOD

(1) GENERAL.-Scales [38] devised an iodometric semimicro method of analysis which requires the use of samples of less than 20 mg of monosaccharides or less than 30 mg of diaccharides. The entire analysis is conducted in a single reaction vessel. Scales confined his experimental analyses to dextrose and concluded that each milliliter of iodine represented a constant weight of sugar irrespective of the concentration. He prescribed a 3-minute period of boiling over a free flame or an electric hot plate.

Isbell, Pigman, and Frush [39] employed a modification of the method for determining the reducing powers of a wide variety of sugars. They found that greater precision of analysis was obtained if the period of boiling was increased to 6 minutes. Shaffer and Somogyi (see p. 169) showed that relatively large differences in reaction rates occurred between certain sugars. Hence in order that a uniform method of analysis might be adopted, the time of reaction must be adapted to the most slowly reacting sugar. If the rapidly reacting sugars, dextrose and levulose, are analyzed, it is suitable to shorten the period of boiling to the 3 minutes prescribed by Scales.

Isbell, Pigman, and Frush were unable to verify Scales' statement. that each milliliter of iodine represented a constant weight of glucose, irrespective of the concentration of sugar, but found the function

f=1.0511+0.0021C+0.000086C2,

in which ƒ is the factor for glucose and Cis the titer in milliliters of 0.04 N iodine. The factors for glucose are shown in tables 22 and 23. The various sugars under strictly comparable conditions reduce different quantities of copper. It may be observed by comparing the reducing values given in table 24 that epimeric sugars give approxi

323414°-42-14

TABLE 22. Factors for calculating glucose by the Scales method

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mately like reducing values-that is, the configuration of carbon 2 does not materially influence the reducing power. The similarity of epimeric sugars probably arises from the rapidity with which they revert to the common enolic form. Since the groups about carbons 3, 4, and 5 are held more firmly, sugars which differ in the configuration of these carbons are not interconvertible in the alkaline solution and exhibit characteristic properties. Sugars in which the hydroxyl on carbon 3 is trans to the hydroxyls on carbons 4 and 5 give the highest reducing powers, while sugars which have cis hydroxyls on carbons 3 and 4 give lower reducing values. The differences in the values for lactose, maltose, cellobiose, and turanose show that the configuration of the nonreducing component in the disaccharide molecule influences the reducing power and that the reducing power of the disaccharide may be higher or lower than that of the reducing sugar component. If the glycosidic union is on carbon 3, as in turanose, the molecular reducing power is less than that of the corresponding monosaccharide. If the glycosidic union is on carbon 4, the molecular reducing power is about 1.4 that of the corresponding monosaccharide; and if the glycosidic union is on carbon 6, the molecuTABLE 23.-Factors for various sugars for use with the modified Scales method 1

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1 The factors given in this table are the averages of the values obtained in measurements conducted in this laboratory over a period of 8 years. Each value represents not less than 3 determinations. The individual determinations by the same operator did not vary over 1 percent, but the results of different operators varied by as much as 2 percent.

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