TABLE 24.-Relative molecular reducing power1 (modified Scales method)

1 Ratio of the reducing power of the sugar to the reducing power of glucose.

lar reducing power is about 1.2 that of the corresponding monosaccharide. It may be observed from the results given in table 25 that the molecular reducing powers of the pentoses are slightly lower and of the heptoses slightly higher than the molecular reducing powers of the corresponding hexoses.

TABLE 25.-Molecular reducing power for configurationally related substances

The configurations of carbons 2, 3, 4, and 5 are indicated by plus and minus signs according to whether the OH lies to the right or left when the formula is written in the conventional manner. For example, the configuration of d-glucose is indicated by +++. In the case of the pentoses, the sugar has been classified with the configurationally related hexose. Thus d-xylose, d-lyxose, l-arabinose, and l-ribose are placed with groups having the (+) configuration for carbon 5.

(2) METHOD FOR MAKING SUGAR DETERMINATIONS.

Reagents. (1) Dissolve 16 g of copper sulfate (CuSO4.5H2O) in 125 to 150 ml of water. Dissolve sodium citrate, 150 g of 2NазC&H5O7. 11H2O or 124 g of Na,C,H,O7.2H2O; 130 g of anhydrous sodium carbonate; and 10 g of sodium bicarbonate in about 650 ml of water, while warming them slightly to accelerate solution. Cool and combine the two solutions while stirring, make up to 1 liter, and filter. (2) 0.04 N solution of iodine containing 4 percent of potassium iodide.

(3) 0.04 N sodium thiosulfate solution containing 0.1 g of sodium carbonate.

(4) Hydrochloric acid solution containing 60 ml of concentrated HCl per liter.

(5) Acetic acid solution containing 24 ml of glacial acetic acid per liter.

Procedure.-To 10 ml of a solution containing 10 to 20 mg of a monosaccharide (or about 30 mg of a disaccharide) and contained in a 300 ml Erlenmeyer flask, add from a fast-draining pipette 20 ml of the copper reagent. Stopper the flask with a two-hole rubber stopper and place over an electric heater or gas flame so regulated that the solution comes to boil in 4% minutes. Allow the solution to boil for 6 minutes and then cool it in an ice-water bath for 4 minute while keeping the solution in gentle circular motion. Remove the flask from the ice-water bath, draw up 25 ml of 0.04 N iodine solution into a pipette, and while holding the pipette, pour into the flask from a graduate 100 ml of the acetic acid solution, mix gently, and add the iodine solution from the pipette. Then pour 25 ml of the hydrochloric acid solution down the walls of the flask and into the solution. Mix with a gentle circular motion and titrate the excess iodine with 0.04 N sodium thiosulfate, using starch as the indicator. Subtract the back titration from the iodine originally added and multiply this value by the sugar factor to give the milligrams of sugar in the sample. For best results each worker should determine his own factors by applying the method to known quantities of sugars. If a large number of determinations is to be made, charts in which the factors are plotted against the titrations may be constructed so that the factor for any titration may be obtained readily.

(c) SCHOORL METHOD FOR INVERT SUGAR IN CANE MOLASSES [40]

Solutions. Soxhlet solution, p. 170. Deleading solution. Dissolve 7 g of Na2HPO4.12H2O and 3 g of K2C2O4.H2O and make to 100 ml. Procedure. Dissolve 6 g of molasses in water in a 250-ml volumetric flask and defecate with 15 ml of a 10-percent neutral lead acetate solution. Make to volume and filter. Transfer 50 ml of the filtrate to a 100-ml flask and add 5 ml of deleading solution. Make to volume and filter. Pipette 50 ml of the filtrate containing 0.6 g of molasses into a 300-ml Erlenmeyer flask and add accurately 50 ml of Soxhlet reagent. Add one or two fragments of washed and ignited pumice, and place the flask on a wire gauze resting on an asbestos card with a central hole 6.5 cm in diameter. Heat to boiling in 4 minutes and continue the boiling for exactly 2 minutes. Cool rapidly without agitation and add 25 ml of KI solution (20 g in 100 ml) and 35 ml of H2SO, (1 volume of concentrated acid to 5 volumes of water). Titrate

the liberated iodine with 0.1 N thiosulfate, using 3 to 4 ml of a 1-percent starch solution as indicator. Determine the blank titration, using 50 ml of water instead of the sugar solution. Deduct the titer of the test sample from that of the blank, and multiply the result by 6.357 to obtain the number of milligrams of copper reduced. Refer the weight of copper to table 96, p. 602, and read the percentage of invert sugar. The table is applicable only to a 0.6-g sample of molasses. A linear interpolation yields accurate results.

(d) METHODS FOR SMALL PERCENTAGES OF INVERT SUGAR IN SUCROSE

(1) OFNER METHOD [41].-(Official method of the Czechoslovakian Republic.)

Reagent. Dissolve 5.0 g of CuSO4.5H2O, 10.0 g of anhydrous Na2CO3, 300 g of pulverized Rochelle salt, and 50 g of Na2HPO4.12H2O in about 900 ml of water at room temperature, warming slightly at the end of the solution, if necessary. When completely dissolved, it is advisable to heat for 2 hours on the water bath to destroy mold spores. Cool and fill to 1 liter. Treat with active carbon or kieselguhr and filter. Preserve the solution in a dark place.

Sodium thiosulfate.-Dissolve 4.00 g of crystals and make to a volume of 500 ml or, preferably, prepare a stock solution containing in 500 ml, 20.0 g of crystals and 1 ml of N NaOH or 0.1 g of Na2CO3. Dilute 100 ml to 500 ml, as required. Standardize in the usual way or titrate against the following iodine solution.

Iodine solution.-Dissolve 2.05 g of pure iodine in about 10 g of iodate-free KI dissolved in a few milliliters of water. Make to a volume of 500 ml and preserve in a dark place.

Starch solution.-Rub 2.5 g of soluble starch and about 10 mg of red mercuric iodide in a little water. Dissolve the starch in about 500 ml of boiling water.

Approximately N hydrochloric acid.

Sodium phosphate solution.-Dissolve 100 g of Na2HPO4.12H2O and make to 1 liter. (For lead precipitation.)

Neutral lead acetate.-Dissolve 250 g of Pb(C2H3O2)2.3H2O and fill to 1 liter.

Procedure. Refined or affined sugars.-Dissolve 20 g of the sample in distilled water and make up to 100 ml. Transfer 50 ml of the solution to a 300-ml Erlenmeyer flask and add 50 ml of the copper solution. Mix well, add a knife point of pumice or talcum powder, and set on a wire gauze resting on an asbestos card having a central hole 6.5 cm in diameter. Heat to boiling with a Bunsen flame within 4 to 5 minutes and continue the boiling for exactly 5 minutes, diminishing the flame so that only the tip touches the gauze. Cool without agitation by immersion in cold water. Pour from a graduated cylinder 15 ml of 1.0 N hydrochloric acid down the wall of the flask, and add immediately a carefully measured volume of the iodine solution (burette or calibrated pipette). The volume of iodine will vary from 5 to 20 ml, according to the amount of copper reduced, but must always be added in excess. After the first few milliliters of iodine have flowed in, the remainder must be added with continuous agitation. Stopper the flask and allow the iodine to react for 2 minutes with occasional agitation. Add 5 ml of the starch solution and titrate the excess of iodine with thiosulfate. Deduct the volume

of the excess iodine from the volume added. One milliliter of iodine solution is equivalent to 1 mg of invert sugar. Ten grams of pure sucrose have, under the conditions of analysis, a reducing power equivalent to 1 ml of iodine; hence deduct 1 ml from the volume of iodine required in the test for reoxidation of the copper. In general, deduct 0.1 ml of iodine for each gram of sucrose in the sample.

Pure sugar sirups.-Prepare a solution containing a known weight of dry substance, preferably 7 to 10 g in 50 ml.

Raw sugar.-Transfer 52 g of the sample to a 200-ml volumetric flask. Dissolve in water, add 2 to 3 ml of neutral lead acetate. Make to volume and filter. Transfer 153.6 ml to a 200-ml flask and add 15 ml of sodium phosphate solution and make to volume. Add 1 g of active carbon, mix thoroughly and allow to stand 15 minutes. Filter and take 50 ml (10 g) of the filtrate for analysis. For the copper analysis take 20 ml of iodine. If the sample contains more than 0.15 percent of invert sugar, take 25 ml (5 g) for analysis. Deduct 0.1 ml from the volume of iodine for each gram of sucrose in the sample.

More impure after-products. Dissolve 52 g in water, add 4 to 5 ml of lead acetate, fill to 200 ml, and filter. Pipette 153.6 ml into a 200-ml flask, add 20 ml of sodium phosphate solution, make to 200 ml, mix, and filter. Add 1 g of active carbon, mix thoroughly, and allow to stand for 15 minutes. Filter and take for analysis 50 ml or some smaller volume that will contain not more than 15 mg of invert sugar.

Molasses and low-purity sirups.-Dissolve 26 g in water, add 10 ml of neutral lead acetate, fill to 200 ml, mix, and filter. Transfer 153.6 ml of the filtrate to a 200-ml volumetric flask, add 20 ml of sodium phosphate solution, fill to volume, mix, and filter. Add 4 g of active carbon, agitate thoroughly, allow to stand 15 minutes, and filter. Take 50 ml (5 g) of molasses for analysis. Conduct the analysis as described above, but continue the boiling for 7 instead of 5 minutes, since the impurities in low-grade products diminish the rate of reduction of copper. Since 5 g of molasses contains about 2.5 g of sucrose, deduct 0.25 ml from the iodine volume.

Low-grade products frequently contain substances, notably SO2, which are oxidized by iodine. Ofner, therefore, recommends a "cold" experiment in which the analysis is carried out as described above but with the omission of the boiling period. The iodine consumed in the cold analysis, together with the correction for sucrose, is deducted from the total iodine consumed in the analysis. (2) SPENGLER, TÖDT, AND SCHEUER METHOD [42].

Preparation of stock Müller's solution.-Dissolve 35 g of CuSO4.5H2O in 400 ml of boiling water and in a separate container 173 g of Rochelle salt and 68 g of anhydrous Na2CO3 in 500 ml of boiling water. Allow both solutions to cool and pour the second solution into the first with agitation. Add 1 to 2 teaspoonfuls of active carbon and after several hours' standing, filter with suction through a hardened filter paper. If copper compounds separate subsequently, the solution must be refiltered.

Procedure. Transfer 100 ml of a solution containing 10 g of sugar to a 300-ml Erlenmeyer flask, add 10 ml of the stock Müller solution, and heat for 10 minutes in a boiling-water bath. The bath must be heated so strongly that the introduction of the flask does not interrupt the boiling. Adjust the position of the flask so that the level of the

inner liquid surface is at least 2 cm below the outer. Cool rapidly to about room temperature without agitation and add 5 ml of 5 N acetic or tartaric acid and then 20 or 40 ml of 0.0333 N iodine solution. After the precipitated cuprous oxide is completely dissolved, titrate the excess of iodine with 0.0333 N thiosulfate, using a few milliliters of 1- to 2-percent starch solution as indicator.

Conduct a blank analysis with 100 ml of water in the same manner. Deduct the thiosulfate titer of the sugar analysis from the blank titer to determine the volume of 0.0333 N iodine used for reoxidation of the precipitated copper. One millititer of 0.0333 N iodine equals 1 mg. of invert sugar.

When a series of analyses is conducted, it is obviously necessary to determine the blank only occasionally.

The stability of the thiosulfate solution is increased by the addition of about 3 ml of normal sodium hydroxide per liter of solution.

A deduction of 2 ml of 0.0333 N iodine (equals 2 mg of invert sugar) is made to correct for the reducing power of 10 g of pure sucrose and, in general, in the same proportion for smaller weights of sample, for example, for 2.5 g of sucrose a deduction of 0.5 ml is made.

The authors recommend for many cases a "cold analysis," which is conducted in the manner described above except that the heating is omitted. This permits a correction for such impurities as sulfites, which otherwise would be reported as invert sugar.

Ten milliliters of Müller solution contains sufficient copper to oxidize 40 mg of invert sugar, but the authors recommend that samples be taken containing not over 30 mg. Thus if the invert sugar is in excess of 0.3 percent, a correspondingly smaller sample should be taken.

(3) LUFF-SCHOORL METHOD [43]. (For determination of invert sugar in cane sugars ranging from 0.3 to 4.0 percent.)

Preparation of copper solution.-Dissolve with gentle heating 17.3 g of CuSO4.5H2O and 115 g of citric-acid crystals in about 200 ml of water in a 1-liter volumetric flask. To this solution, after cooling, add with agitation 185.3 g of anhydrous Na2CO3 in about 500 ml of water. It is important that the second solution be added to the first and not the first to the second. Make to 1 liter, shake well with 2 g of washed and ignited kieselguhr, and filter with suction. The total alkalinity should be 1.78 N (phenolphthalein) and should be controlled.

Procedure.-Transfer to a 300-ml Erlenmeyer flask, 25 ml of the copper reagent and 25 ml of the sugar solution containing 5 g of the sample, or, if necessary, such smaller quantity as contains not more than 45 mg of invert sugar. Add a few fragments of pumice, and place the flask on a wire gauze resting on an asbestos card having a central hole 6.5 cm in diameter. Fit the flask with a reflux air condenser, heat to boiling in 3 minutes, and continue the boiling for exactly 5 minutes. Cool at once without agitation in tap water. To the cooled solution add 15 ml of KI solution (20 g in 100 ml) and 15 ml of H2SO4 (25 g of concentrated acid in 100 ml,) in such a manner as to avoid loss by effervescence. Titrate the liberated iodine with 0.1 N thiosulfate, using about 1 ml of a 1-percent starch solution as indicator. Make a blank determination, using 25 ml of water in place of the sugar solution. Refer the difference between the volume of 0.1 N thiosulfate required by the blank and the sample under test