'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 Niodine 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 Na,CO, 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 H2SO, (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

to table 97, p. 602, to ascertain the corresponding weight of invert sugar in the sample.

Standardization of thiosulfate.-Dissolve 25.5 g of Na2S2O3.5H2O and 0.2 g of Na2CO, and make to 1 liter. Standardize with pure K2Cr207.

Weigh accurately about 0.22 g of K2Cr2O, and dissolve in 450 ml of water in a 750-ml Erlenmeyer flask. Add successively 15 ml of KI solution (20 g in 100 ml) and 15 ml of concentrated HCI. Titrate the liberated iodine with thiosulfate, using 3 ml of a 1-percent starch solution as indicator.

(a) SOMOGYI MODIFICATION OF SHAFFER AND HARTMANN MICROMETHOD FOR DEXTROSE

Shaffer and Hartmann [44] elaborated in detail a method for the determination of sugar in blood, which was applicable to the analysis of samples containing from 0.07 to 2.2 mg of reducing sugar. The method was subsequently applied to other materials than blood and indeed has been found generally applicable to any material containing reducing sugar. It is particularly serviceable in instances in which it is necessary to conserve the supply of material.

Shaffer and Hartmann copper reagent was prepared by mixing copper sulfate, tartaric acid, and sodium carbonate, the last two substances reacting and releasing carbon dioxide. The solution also contained potassium iodate and potassium iodide in such concentration that it was 0.02 N with respect to iodine which was released upon acidification.

Somogyi [8], in a study of the method, found that the amount of copper reduced was in a high degree dependent upon the alkalinity of the solution. Within a narrow range, in which the ratio of Na2CO2 to NaHCO3 lay between 7:13 and 2:3 and the pH varied from 9.40 to 9.55, the highest copper values were obtained and the ratio of copper to sugar remained constant. In the Shaffer and Hartmann solution the ratio of carbonate to the bicarbonate formed by the reaction of tartaric acid with sodium carbonate was variable and depended upon the variable amount of carbon dioxide lost. Somogyi therefore modified the Shaffer and Hartmann solution.

Dissolve the Rochelle salt, sodium carbonate, and sodium bicarbonate in about 500 ml of water, and into this pour with stirring the copper sulfate dissolved in about 100 ml of water; then add the solution of the other constituents and dilute to 1 liter. (Only the potassium iodate need be weighed accurately.)

Measure 5 ml of the reagent into a large test tube (250 by 25 mm) and add 5 ml of the sugar solution containing not less than 0.1 mg and not more than 2.0 mg of dextrose. Mix by gentle shaking, cover the tube with a small funnel, bottle cap, or glass bulb, and keep it in a boiling-water bath for 15 minutes. Cool by placing in a shallow dish of water until the temperature is lowered to 35° or 40° C. Add with agitation 1 ml of 5 N H2SO, (or its equivalent) and see that all Cu2O is promptly dissolved. After about 2 minutes, titrate with 0.005 N sodium thiosulfate. A blank titration on 5 ml of the reagent is determined after heating with an equal volume of water.

The difference between the blank and the titration of a determination is equivalent to the copper reduced and thus to the sugar. The corresponding amounts of sugar are given in table 98, p. 603, which is a modified form of the table given in the original article.

Notes.-Details of the determination of sugar in 0.2 ml of blood are given in the reference cited.

A 0.005 N thiosulfate solution cannot be kept unchanged for more than a few days. It is advisable to keep a 0.1 N stock solution and prepare 1:20 dilutions as required.

Any agitation of the test tubes, from the beginning of heating in the water bath up to the addition of acid, should be avoided to minimize reoxidation of cuprous oxide by air.

It is undesirable to cool below 30° C. If the sample contains more than 1 mg of sugar, incomplete oxidation of copper may occur.

In order that the pH of the reagent may remain unaltered, it is important that the sample be neutralized with sodium hydroxide (not carbonate). Phenol red is a suitable indicator and renders the end point of the thiosulfate titration more distinct.

(b) BENEDICT MODIFICATION OF FOLIN AND WU METHOD [46, 47]

Reagents. Alkaline copper solution.-Dissolve 200 g of sodium citrate and 60 g of sodium carbonate in about 800 ml of water. Then dissolve 6.5 g of pure copper sulfate crystals in about 100 ml of water and add to the former solution with agitation. Add 9 g of ammonium chloride, dilute to 1 liter, and mix.

Tungstic acid color.-Dissolve 100 g of pure sodium tungstate in about 600 ml of water in a liter flask. Add 50 g of pure arsenic pentoxide, then 25 ml of 85-percent phosphoric acid and 20 ml of concentrated hydrochloric acid. Boil 20 minutes. After cooling this, add 60 ml of commercial formalin, 45 ml of concentrated hydrochloric acid, and 40 g of sodium chloride. Dilute to 1 liter and mix.

To 100 ml of the alkaline copper solution add 2.5 to 3.0 g of pure anhydrous sodium sulfite and preserve for use. This solution is not reliable after 1 month.

Procedure.-Transfer 2 ml of the sugar solution and 2 ml of the copper reagent to a Folin and Wu sugar tube. Into another tube transfer 2 ml of a standard dextrose solution containing 0.1 (or 0.2) mg per milliliter and 2 ml of copper reagent. Mix by side-to-side shaking and place the two tubes in boiling water for 5 mintues. Cool by immersion in cold water and add to each 2 ml of tungstic acid color reagent. After 1 to 2 minutes dilute to 25 ml, mix thoroughly, and compare in a colorimeter. The sugar in the unknown solution is calculated by the formula

Depth of column of standard

Depth of column of unknown 10 (or 20)=mg per 100 ml.