fact that the invert sugar which is present as an impurity has its rotation increased negatively in the invert reading in the presence of the hydrochloric acid. Browne [25] has shown by applying the simple acid Clerget method to pure synthetic mixtures of invert sugar and sucrose that gross inaccuracies can be caused by the changed rotation of the added invert sugar in the two polarizations.

TABLE 17.-Errors of analysis of sucrose caused by invert sugar

Jackson and Gillis sought to avoid this source of error by adding to the solution for direct polarization a neutral salt in such quantity that its effect on the invert sugar just equalled that of hydrochloric acid. The effects of added reagents on the rotation of invert sugar have been described on page 137. It was shown that 10 ml of 6.34 N hydrochloric acid in 100 ml increased negatively the rotation of the negative constituent of the divisor by 1.25° S. From the list of neutral salts it can be computed that 2.312 g of sodium chloride in 100 ml produces the same change of rotation. If, therefore, this weight of salt is added to the solution for direct polarization, the invert sugar present as an impurity will have the same rotatory power in both polarizations. But the addition of salt has now diminished the rotatory power of the normal sucrose solution from 100° to 99.38° S. The total change in rotation of sucrose is then from 99.38 to -33.18, and the Clerget divisor becomes 132.56 at 20° C. This is the basis of the Jackson and Gillis method IV described on page 155.

Nitrogenous substances consisting mainly of amino acids and their internally compensated salts occur in both beet and cane molasses. Many of these are optically active but possess one rotatory power in acid solution and quite a different one in neutral or alkaline solution. In order to compensate for this source of error Saillard [26] proposed that the invert solution be neutralized with sodium or potassium hydroxide. The method was elaborated in greater detail by Jackson and Gillis, who proposed that ammonia be used for neutralization because it had less destructive action on invert sugar than the more caustic alkalies. The neutralization of 10 ml of 6.34 N hydrochloric acid with ammonia produces 3.392 g of ammonium chloride; this causes an increased value of the negative constituent of the Clerget divisor, making it -33.84. This method of compensation was designed for beet products which were free from invert sugar. The basic value of the Clerget divisor becomes 133.84 for inversion at 60° C, or 133.94 for room-temperature inversion. This was designated Jackson and Gillis method III.

Many samples of sugar products contain both invert sugar and amino acids. Jackson and Gillis in method II sought to compensate for the altered rotation of these substances by neutralizing the solution

323414°-42-11

for invert polarization with ammonia and adding 3.392 g of ammonium chloride to the solution for direct polarization. This diminishes the rotation of sucrose from 100° to 99.43° S. The basic value of the divisor according to this method is 133.27.

Method III has had such limited use that it will not be described in the present Circular. While it is capable of eliminating the errors which otherwise would be introduced by the altered rotations of aminoacids, it still serves no useful purpose, because quite invariably products which contain aminoacids also contain raffinose. Such products, therefore, contain three unknown quantities and cannot be analyzed accurately by processes which yield but two equations. The method, however, has been utilized by Osborn and Zisch, whose procedure is described in a later paragraph (p. 160).

Saillard [27] has emphasized the fact that molasses contains sodium and potassium salts of organic and inorganic acids which diminish the rotation of sucrose and increase the levorotation of invert sugar. These two effects are incompletely compensated, the effect on invert sugar being the greater. His proposed remedy is to determine the ash content of each sample, and in a control test to determine the Clerget divisor by adding to pure sucrose and to the invert-sugar solution sodium or potassium chloride in amount equivalent to the ash in the sample. Thus for beet molasses no less than four polarizations and an ash analysis are required for a single Clerget test. Saillard's proposal has not been put into extensive practice, primarily because of the prohibitive labor involved. Moreover, the procedure rests on a questionable basis, because the assumption is made that sodium and potassium chlorides produce the same effects as the alkali salts of all other acids, regardless of the nature of the anion. In other words, Saillard unwittingly assumes the constancy of "molecular depression," which numerous previous experiments had shown was lacking. Nevertheless, it is quite possible that Saillard's procedure diminishes the error caused by alkali salts in the sample, and it would be desirable to investigate the effects upon the rotations of sucrose and invert_sugar of the particular alkali salts which are known to occur in molasses. The special determination of the divisor for each test recommended by Saillard appears to be an unnecessary complication, for we have complete equations for the effects of sodium chloride upon sucrose and invert sugar and of potassium chloride upon invert sugar. If the procedure should prove of value, these equations could be solved for any desired concentration of salt.

Zerban and Gamble [28] have studied the question by preparing and analyzing known solutions of sucrose mixed with low-purity products of high ash content and observed no noticeable effect on the Clerget divisor, provided the divisor was based on the dry-substance concentration. (See conclusion 10, p. 145).

The ingenious suggestion has been made by R. J. Brown, who pointed out that the data of Jackson and Gillis showed that the effect of a given quantity of salt was approximately twice as great on invert sugar as on sucrose, and that in the absence of invert sugar in the sample, if the concentration of salt in the invert polarization is half as great as in the direct, the effect is completely compensated in the two polarizations. It is then merely necessary to use the normal weight for the direct polarization and the half-normal weight for the invert polarization.

(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(1.006-0.00031)

P' S(-0.3300+0.005(t-20))=S(-0.4300+0.005).

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(1.006-0.00031)+1.852R (1.006-0.00031).

(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)

(1.006-0.00031) [0.908-0.0032t+0.000794 (m-13)]

R=

P-S
1.852

(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

S=

aP-bP'
be+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.