The acid methods may be divided into two groups, the plain acid methods (p. 154 and 155) and the compensation methods. The compensation methods were devised, first, to eliminate errors in the analysis of products high in invert-sugar content arising from the increased rotation of the original invert sugar in the presence of acid, and second, to eliminate errors caused by the change of rotation of amino acids when highly acidified. Thus for the analysis of relatively pure samples high in invert sugar Jackson and Gillis method IV (p. 155) is to be recommended. For the analysis of samples containing both invert sugar and amino acids Jackson and Gillis method II (p. 154) is available. There is, however, in many products of the sugar industry a group of disaccharides or oligosaccharides which are not sucrose or raffinose but are hydrolyzable by acids. The hydrolysis of these substances introduces errors into the Clerget analysis which are obviously insurmountable except by the enzyme methods. (b) REVISED METHODS OF JACKSON AND GILLIS (1) GENERAL METHODS OF INVERSION.-The following methods of inversion were designed to avoid destruction of invert sugar subsequent to the completion of the inversion. There is, however, a variable amount of destruction during the process of inversion which is unavoidable. Each method, therefore, requires the use of its appropriate value of the Clerget divisor. These methods, which were determined by inversion of pure sucrose, are in general applicable to all products which do not contain considerable quantities of inorganic salts of weak acids. They can be applied to cane molasses but not to beet molasses. The latter should be inverted in a bath at 70° C, as described on page 155. (a) Pipette 50 ml of the solution into a 100-ml flask, add 20 ml of water and 10 ml of hydrochloric acid (d20° 1.1029 or 24.85° Brix at 20° C). Immerse in a water bath at 60° C. Agitate the solution continually for about 3 minutes, and allow it to remain in the bath for a total time of 9 minutes. Cool quickly. Basic value, 133.18. (b) Pipette 70 ml of the solution into a 100-ml flask, add 10 ml of hydrochloric acid (d20° 1.1029) and proceed as in (a). Basic value, 133.18. (c) Pipette 75 ml of the solution, add 10 ml of hydrochloric acid (d20° 1.1029) and proceed as in (a), but allow the solution to remain a total time of 9.5 minutes. Basic value, 133.18. (d) Pipette 50 ml and add 20 ml of water, or pipette 70 ml of the solution, into a 100-ml flask, add 10 ml of acid, d20 1.1029, and allow to remain 30.8 hours at 20° C, 14.6 hours at 25° C, or 7.1 hours at 30° C. Basic value, 133.28. (e) Walker method. Transfer 50 ml and add 25 ml of water or transfer 75 ml of the solution to a 100-ml flask and heat in a water bath to 65° C. Remove from bath, add 10 ml of hydrochloric acid (d20° 1.1029), allow to cool spontaneously for 15 minutes, and cool to the temperature of polarization. In the case of low-grade products containing an excess of basic lead acetate, add previous to heating, 1 ml (or 2 ml if excess of lead is large) of the acid used for inversion. Basic value, 133.18 (tentative). (2) METHOD I.-(Applicable to pure sucrose, or to sugar mixtures in which the impurities are unaffected optically by hydrochloric acid.) Reagents. Hydrochloric acid (d20° 1.1029 or 24.85 Brix). Prepare a normal solution of the sample or a solution of such fractional normality as the nature of the substance will permit. Defecate, if necessary, with basic lead acetate in the usual manner, making to volume at the temperature at which the observations are to be made, and filter. (If desired, the excess of lead may be removed at this point by the addition of pulverized potassium or sodium oxalate. However, it is necessary that the whole filtrate be treated by the deleading reagent, since the latter exerts an effect upon the rotation of invert sugar which should be as far as possible offset by a similar effect on sucrose.) Polarize the solution to obtain the direct reading and, if necessary, correct to the value which would have been obtained if 26 g of the sample were taken in 100 ml of solution. To obtain the invert polarization, invert as described on page 153. make to a volume of, 100 ml at the temperature at which the observations are to be made, and polarize. Correct the observed polarization to that of a normal solution. The algebraic difference between the two polarizations corrected for dilution gives the value P-P'. Determine by refractometer the dry-substance concentration of the original sample and calculate the weight of solids, m, taken for the invert polarization. Calculate the percentage of sucrose by the formula S= P-P Basic value +0.0794 (m-13)-0.53 (t-20) The basic value is 133.18 for inversion at 60° C or 133.28 for roomtemperature inversion. (3) METHOD II.-(General method applicable to all products.) Reagents. Hydrochloric acid (d20° 1.1029 or 24.85 Brix); ammonium hydroxide solution, 5 to 6 N; solution of ammonium chloride containing 226 g per liter; pulverized potassium or sodium oxalate. Ascertain by at least three concordant titrations in the presence of methyl orange the volume of the ammonia solution required to neutralize 10 ml of the hydrochloric acid. Prepare a normal solution of the sample or a solution of such fractional normality as the nature of the substance will permit. Defecate, if necessary, with basic lead acetate in the usual manner, making to volume at the temperature at which the observations are to be made. Filter. (If desired, the solution may at this point be freed from lead; but if this is done, the deleading reagent must be added to the whole filtrate. Finely pulverized potassium oxalate in minimum quantity is added until precipitation is complete. Filter. If this procedure is omitted, the lead is precipitated satisfactorily by the chlorides added later.) Pipette into two 100-ml flasks two equal volumes of the filtrate (50, 70, or 75 ml). For the direct polarization, add to 1 portion 15 ml of the ammonium chloride solution or 3.392 g of dry ammonium chloride. Make to volume at the temperature at which the observations are to be made; filter, if necessary, and polarize. For the invert polarization, add hydrochloric acid to the other portion and invert by one of the methods described on page 153. Cool quickly. After the solution has become quite cold add from a burette during continual shaking, the precisely determined volume of ammonia required to neutralize the acid. Adjust the temperature, make to volume, filter, if necessary, and polarize at carefully controlled temperature. Correct both polarizations to the normal weight of sample. Calculate the percentage of sucrose by the formula P-P' S= Basic value +0.0794(m-13)-0.53(t-20)' in which m is the weight of dry substance taken for invert polarization. The basic value of the divisor is 133.27 for 60° C inversion or 133.37 for room-temperature inversion. (4) METHOD IV.-(Applicable in the presence of invert sugar, but inapplicable in the presence of optically active non-sugars which change rotation with acidity. In principle sodium chloride equalizes the effect of hydrochloric acid on invert sugar present as an impurity.) Prepare a normal solution of the sample or a solution of such fractional normality as the nature of the substance will permit. Defecate, if necessary, with basic lead acetate in the usual manner, making to volume at the temperature at which the observations are to be made. Filter. (If desired, the excess of lead may be removed at this point. Add pulverized potassium or sodium oxalate to complete precipitation of lead. The deleading reagent should be added to the whole filtrate. If the deleading is omitted, the lead is satisfactorily removed by the chlorides subsequently added.) Pipette two 70-ml (or 50 ml+20 ml of water) portions of the clear filtrate into two 100-ml flasks. If preferred, 75-ml portions may be taken. To 1 portion add 2.315 g of sodium chloride or 7.145 ml of a saturated sodium chloride solution or 10 ml of a solution containing 231.5 g per liter; make to volume at the temperature at which the observations are to be made and polarize. To the other portion add hydrochloric acid and invert by one of the methods described on page 153. Cool and make to volume at the temperature at which the observations are to be made. Polarize. Correct both rotations to the normal weight of sample. Calculate the percentage of sucrose by the formula S= P-P' Basic value+0.0794 (m-13)-0.53(t-20)' in which m is the weight of dry substance taken for the invert polarization. The basic value is 132.56 for 60° C inversion or 132.66 for room-temperature inversion. (c) ACID METHODS OF THE ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS (1) DETERMINATION OF SUCROSE BY POLARIZATION BEFORE AND AFTER INVERSION WITH HYDROCHLORIC ACID.-(In the presence of much levulose, as in honeys, fruit products, sorghum sirup, cane sirup, and molasses, the optical method for sucrose, requiring hydrolysis by acid, gives erroneous results.) Direct reading.-Proceed as directed under "Direct reading," page 157. Invert reading.-Pipette a 50-ml portion of the lead-free filtrate into a 100-ml flask and add 25 ml of water. Then add, little by little, while rotating the flask, 10 ml of hydrochloric acid (sp. gr. 1.1029 at 20°/4° or 24.85° Brix at 20°C). Heat a water bath to 70°C and regulate the burner so that the temperature of the bath remains approximately at that point. Place the flask in the water bath, insert a thermometer, and heat with constant agitation until the thermometer in the flask indicates 67°C. (This preliminary heating period should require from 2% to 2% minutes). From the moment the thermometer in the flask indicates 67°C, leave the flask in the bath for exactly 5 minutes longer, during which time the temperature should gradually rise to about 69.5°C. Plunge the flask at once into water at 20°C. When the contents have cooled to about 35°C, remove the thermometer from the flask, rinse it, and fill almost to the mark. Leave the flask in the bath at 20°C for at least 30 minutes longer and finally make up exactly to volume. Mix well and polarize the solution in a 200-mm tube provided with a lateral branch and a water jacket, maintaining a temperature of 20°C. This reading must also be multiplied by 2 to obtain the invert reading. If it is necessary to work at a temperature other than 20°C, which is permissible within narrow limits, the volumes must be completed, and both direct and invert polarizations must be made at exactly the same temperature. Calculate sucrose by the following formula: in which S= 100(P-I) 143+0.0676 (m-13)-0.53t' S percentage of sucrose, P=direct reading, normal solution, I invert reading, normal solution, t=temperature at which readings are made, m=grams of total solids from original sample in 100 ml of the invert solution. Determine with the refractometer the total solids as percentage by weight, as directed on page 258, and multiply this figure by the density at 20°C, as obtained from table 113, page 626. Inversion at room temperature. The inversion may also be accomplished as follows: (1) To 50 ml of the clarified solution, freed from lead, add 10 ml of hydrochloric acid (sp. gr 1.1029 at 20°/4° or 24.85° Brix at 20°C) and set aside for 24 hours at a temperature not below 20°C; or, (2) if the temperature is above 25°C, set aside for 10 hours. Make up to 100 ml at 20°C and polarize. Under these conditions, the formula must be changed to the following: (2) DETERMINATION OF SUCROSE AND RAFFINOSE BY POLARIZATION BEFORE AND AFTER INVERSION WITH HYDROCHLORIC ACID.-(Of value chiefly in the analysis of beet products.) If the direct reading is more than 1° higher than the percentage of sucrose, as calculated by the formulas given on p. 156, raffinose is probably present. Calculate sucrose and raffinose by the following formulas: When the polarizations are made at 20° C, P=direct reading, normal solution, = R=percentage of anhydrous raffinose. The following formulas are applicable at all temperatures: in which P(0.478+0.0018t2)-I(1.006-0.000311) (0.908-0.0032t2) (1.006-0.000311) R_P(0.43–0.005t1⁄2)+I(1.006–0.0003t1) (1.681-0.0059t1⁄2) (1.006-0.000311) P= direct reading, normal solution, R= percentage of anhydrous raffinose, t1 temperature of the direct polarization, See also discussion of Creydt's raffinose formula on page 143. (d) INVERTASE METHODS OF THE ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS (1) DETERMINATION OF SUCROSE. Direct reading.-Dissolve the double-normal weight of the substance (52 g), or a fraction thereof, in water in a 200-ml volumetric flask; add the necessary clarifying agent, avoiding any excess; shake, dilute to the mark with water, mix well, and filter, keeping the funnel covered with a watch glass. Reject the first 25 ml of the filtrate. If a lead clarifying agent was used, remove the excess lead from the solution when sufficient filtrate has collected, by adding anhydrous sodium carbonate a little at a time, avoiding any excess; mix well and filter again, rejecting the first 25 ml of the filtrate. (Instead of weighing 52 g into a 200-ml flask, two 26-g portions may be diluted to 100 ml each and treated exactly as described. Depending on the color of the product, multiples or fractions of the normal weight may be used and the results reduced by calculation to the basis of 26 g in 100 ml.) Pipette one 50-ml portion of the lead-free filtrate into a 100-ml flask, dilute with water to the mark, mix well, and polarize in a 200-mm tube. The result, multiplied by 2, is the direct reading (P of formula given below), or polarization before inversion. (If a 400-mm tube is used the reading equals P.) If there is a possibility of mutarotation, allow sufficient time for its completion. Invert reading.-First determine the quantity of acetic acid necessary to render 50 ml of the lead-free filtrate distinctly acid to methyl 903232 O-50 - 12 |