(j) CREYDT RAFFINOSE FORMULA Creydt [29] has shown that, in the absence of other optically active substances, sucrose and raffinose can be estimated by the Clerget method. This estimation depends upon the fact that for the solution of two unknown quantities two equations are sufficient. One of these equations states that the direct polarization is the sum of the rotations of the two constituents, and the other states that the invert polarization is the sum of the rotations of the products of hydrolysis. Assume that the analysis is conducted at 20° C and that the normal weight of the sample is contained in 100 ml. Let the sample contain S percent of sucrose and R percent of anhydrous raffinose. Since the specific rotation of anhydrous raffinose is +123.2, while that of sucrose is +66.5, the raffinose in the mixture will have 1.852 times as great a rotatory power as an equal weight of sucrose. The direct polarization then will be Let the invert solution contain 13 g of sample and the reading in the presence of 0.634 N hydrochloric acid be multiplied by 2. Raffinose, upon inversion, yields a mixture of levulose and melibiose, the resultant rotation of which was, according to early measurements, 0.5124 times the rotation of the unhydrolyzed raffinose. Browne and Gamble [30], revising this ratio, found the value 0.514. Osborn and Zisch [31] found a slightly lower value, but nevertheless accepted and used the value 0.514. The structure of the Creydt formula is such that small variations in the raffinose inversion factor have little effect upon the calculated sucrose or raffinose percentages. The invert polarization of the raffinose constituent is then 0.514X1.852R=0.952R. invert reading of inverted sucrose varies slightly with the method of inversion. If we accept the Schrefeld method, the basic value of the negative constituent of the Clerget divisor is -33.00, while if we employ the Jackson and Gillis method of inversion at 60° C, the divisor is 33.18; if the method of overnight room-temperature inversion is employed, the value becomes -33.29. It is therefore necessary to derive three slightly different Creydt formulas. The If we assume that the basic value of the divisor is 133.00 at 20° C, P' -0.3300S+0.952R, whence, upon elimination of R from eq 40 and 41 (41) The derivation is quite similar when the other values of the divisor are used, the resulting formulas differing only in the values of the denominator, as appears in columns b and c in table 77, p. 563. These formulas are valid only at 20° C and for 13 g of dry substance taken for the invert polarization. The direct and invert polarizations of sucrose vary with temperature, P=S(1-0.0003 (t—20))=S(1.006-0.0003) P' S(-0.3300+0.005(t−20))=S(−0.4300+0.005t). Browne and Gamble [30] have shown that the change of direct polarization of raffinose with temperature is very nearly the same as that of sucrose. The direct polarization of a mixture of sucrose and raffinose is then P=S(1.006-0.0003t)+1.852R(1.006–0.0003t). (44) The accepted value of the temperature correction of the negative constituent of the Clerget divisor is +0.5t. Browne and Gamble have measured the change of polarization of invert raffinose with temperature, and have found that a solution of raffinose having a direct polarization of +1.00 would have an invert polarization of +0.478+ 0.0018t. The invert polarization of a mixture of sucrose and raffinose would then be P' S(-0.4300+0.005t)+1.852R (0.478+0.0018t). (45) This equation is strictly valid only for 13 g of dry substance taken for inversion, and a further correction of -0.000794 (m—13) must be applied to the parenthetical coefficient of S. Simultaneous solution of eq. 44 and 45 and the introduction of the concentration correction yield P(0.478+0.0018t) - P' (1.006-0.00031) S= (46) (47) Equation 46 is cumbersome to handle in ordinary analytical work. In order to facilitate its application, the respective coefficients have been computed and assembled in table 77, p. 563. Equation 46 may be expressed in the form aP-bP' S=bc+0.000794(m—13)’ (48) and the coefficients a, b, c, and be may be calculated for various temperatures. The term "c" is valid only for 13 g of dry substance, and for any variation of concentration the correction +0.000794 (m-13) as determined by Jackson and McDonald [15], or +0.000676 as used by Herzfeld, must be applied to the denominator. The Creydt formula, as is shown by its derivation, is valid only in the absence of other optically active substances than sucrose and raffinose. Beet products contain, in addition to these sugars, optically active nitrogenous substances, the presence of which vitiates the formula with respect to precision. It is, however, extensively used and the errors of analysis are tolerated. For strict accuracy the doubleenzyme method or, in certain localities, the double-acid method of Osborn and Zisch [31] is required. (k) WORK OF ZERBAN AND COLLABORATORS Zerban and Gamble [32] have summarized the work of The New York Sugar Trade Laboratory in a series of articles [33] and have applied the principles to a study of the analysis of crude products. The solutions previously analyzed contained known amounts of sucrose in mixture with invert sugar; reversion products of invert sugar; the amino compounds asparagine and aspartic acid, which are the principal substances of this nature found in cane products [34]; and salts. The four inversion methods employed were: (a) The official invertase method of the Association of Official Agricultural Chemists [18] p. 470, (b) Jackson and Gillis method II [3, p. 184], (c) Jackson and Gillis method IV [3, p. 187]; and (d) Schrefeld's modification of the Herzfeld plain acid method [10]. The inversions were carried out mostly at room temperature, but in some of the work higher temperatures were employed; 55° C in method (a), 60° C in methods (b) and (c), and 67° to 69.5° C in method (d). The results of these investigations may be summarized briefly as follows: 1. The solution used for the direct polarization must have the same dry-substance concentration as the solution used for inversion. 2. The Clerget divisor must be based on the dry-substance concentration and not on the sucrose concentration or on the invert reading alone. 3. It is preferable whenever possible to carry out the inversions at room temperature, because at high temperatures slight variations in the time used may have an appreciable effect on such reactions as the destruction of invert sugar in the presence of strong acid, on the hydrolysis of inversion products, and on the interaction between invert sugar and amino compounds. 4. The invertase method is the only one of the four methods compared which may be depended upon to give reliable sucrose results. 5. The sucrose result by Jackson and Gillis method II is increased by reversion products hydrolyzed under the conditions of the analysis. 6. The sucrose result by Jackson and Gillis method IV is increased by the hydrolysis of the reversion products in the same way as in method II, but aspartic acid or asparagine lowers the sucrose result considerably. 7. Accordingly, the difference between the sucrose result by Jackson and Gillis method II and that by the invertase method gives a relative measure of the reversion products hydrolyzed by hydrochloric acid under the conditions of the analysis. 8. The difference between the sucrose result by Jackson and Gillis method II and that by method IV gives a relative measure of the amino compounds present. 9. The plain acid method may give any kind of result, high, low, or correct within the limits of error, depending on the relative proportions of levulose, reversion products, and amino compounds present. 10. In the case of mixtures of known amounts of sucrose with a practically sucrose-free low-purity product, containing 13.74 percent of ash on the basis of dry substance, the salts, as such, had no noticeable effect on the Clerget divisor for any of the four methods investigated, provided the divisor was based on the dry-substance concentration. The four methods which had been applied to artificial mixtures containing known quantities of sucrose were then used in the analysis of various blackstraps and refinery sirups. The inversions were carried out at a temperature of 26° to 30° C, and the polariscopic readings were made at the standard temperature of 20° C. The results, which are averages of many analyses calculated in accordance with rules 1 and 2 given above, are shown in table 18. TABLE 18.-Comparsion of sucrose found by four Clerget methods Without exception, Jackson and Gillis method II gave higher figures than either the invertase method or Jackson and Gillis method IV. This shows that all the samples contained reversion products as well as amino compounds. The quantity of the reversion products, expressed as sucrose, in the refinery blackstraps varies from 0.64 to 1.32 percent, averaging 1.12 percent; and the quantity of amino compounds from 0.40 to 0.89 percent, averaging 0.61 percent. In the filtered sirups there are found 0.10 to 1.03 percent, averaging 0.57 percent, of reversion products, and 0.11 to 0.66 percent, averaging 0.29 percent, of amino compounds, all again expressed as equivalent sucrose. Expressed as asparagine or aspartic acid, the content of amino compounds in blackstraps and refinery sirups is, according to data given by Ambler [35] and by Browne [36], very much higher, in the neighborhood of 2 to 2.5 percent. Leaving out Jackson and Gillis method II, and comparing the results of the other three methods for all the three groups of products analyzed, it is found that the plain acid method gives the highest average results, 0.10, 0.26, and 0.15 percent, respectively, higher than Jackson and Gillis method IV. The average figures by this lastnamed method again exceed those of the invertase method by 0.28, 0.51, and 0.47 percent, respectively, owing to the differential effect of reversion products and of amino compounds. The application of the results, previously obtained with artificial mixtures, to the analysis of actual cane products has thus shown that Jackson and Gillis methods II and IV, used in conjunction with the invertase method, give valuable indications with regard to the relative quantities of reversion products and of amino acids present in such products. 3. INVERTASE METHODS (a) PREPARATION OF INVERTASE The enzyme, invertase, which can be prepared simply and abundantly from yeast, hydrolyzes sucrose under suitable conditions to invert sugar. The invertase prepared from "top" or baker's yeast inverts sucrose to invert sugar and also hydrolyzes raffinose to a mixture of levulose and melibiose, while a similar preparation from "bottom" or brewer's yeast contains also the enzyme, melibiase, and hence possesses the additional power to hydrolyze melibiose to a mixture of dextrose and galactose. It is thus possible, as Hudson and Harding showed [37], to devise analytical processes for the determination of sucrose and raffinose in their mixtures or in crude beet-sugar products which contain them. The two stages of the hydrolysis may be expressed as follows: Sucrose (invertase present)→ invert sugar. I. Raffinose (invertase present)→ levulose+melibiose. II. Melibiose (melibiase present)→ galactose+dextrose. Reynolds [38] has given detailed directions for the preparation, purification, and concentration of highly active invertase and melibiase solutions. For the preparation of invertase (free from melibiase), 10 pounds of baker's yeast are broken up and mixed with 5 liters of water. Two liters of toluene are added and the mixture is stirred at frequent intervals throughout the first 24 hours. The autolysis is allowed to continue for 7 days. The extract is then filtered by gravity on large fluted filters, yielding about 5 liters of filtrate. The residue is mixed with 2 liters of water and filtered. The two filtrates are combined and show usually an activity of k=0.028 (see p. 150) The 7 liters of filtrate are transferred to the ultrafilter, described below, concentrated to 1 liter, and washed with 1 liter of water. The concentrated extract is removed from the ultrafilter, diluted again to 7 liters, acidified with 14 ml of glacial acetic acid, and allowed to stand overnight. This produces the precipitation of flocculent material which is removed by slow filtration through paper. As soon as a sufficient quantity of solution has filtered, it is transferred to the ultrafilter and its concentration continued simultaneously with the filtration from the flocculent precipitate. The extract is concentrated to a volume of 800 ml. In an instance cited by Reynolds, the activity of one such preparation was k=0.22. For the preparation of invertase-melibiase 2.5 gallons of beer yeast is filtered on a large Büchner funnel to remove the wort. About 5,500 g of compressed yeast containing about 23 percent of solids is obtained. This is placed in a jar and 3 liters of toluene added. In a few hours the yeast becomes liquid and autolysis is continued for 7 days, the liquid being agitated once or twice each day. The extract is filtered by gravity on large fluted filters, yielding in 48 hours about 3 liters of filtrate. The residue is mixed with 1,200 ml of water, allowed to stand overnight, and again filtered. The combined filtrates should have an approximate volume of 4,200 ml and an activity of k=0.0818. The crude extract is ultrafiltered to a volume of 600 to 700 ml and washed with 1 liter of water. It is then diluted to 4 liters, treated with 8 ml of glacial acetic acid, and allowed to stand overnight, filtration through fluted paper funnels being started the next morning. Ultrafiltration is started as soon as sufficient filtrate has accumulated and is continued until the volume has been reduced to about 600 ml, whereupon it is washed with 3 liters of distilled water. The final extract is, if necessary, filtered again through paper. Reynolds found in one instance an invertase activity of k=0.554 and a melibiase activity of k=0.024. During ultrafiltration the solution is stirred with a motor-driven stirrer; otherwise the enzyme is liable to concentrate and precipitate at the membrane. Water should flow |