Effect of Reversion Products and Amino Compounds on Sucrose Determinations in Cane Products F. W. ZERBANAND C. A. GAMBLE,New York Sugar Trade Laboratory, 80 South St., New York, N. Y,
D
URING the years 1924 to 1930 the associate referee on polariscopic methods of the Association of Official Agricultural Chemists, assisted by various collaborators, made a critical comparative study of several modern methods of sucrose determination by double polarization (7). The solutions analyzed contained known amounts of sucrose in mixture with invert sugar, reversion products of invert sugar, amino compounds (asparagine and aspartic acid which are the principal substances of this nature found in cane products, S),and salts. The four inversion methods employed were: (a) the official invertase method of the A. 0. A. C. (8); (b) Jackson and Gillis method I1 (6); (c) Jackson and Gillis method IV (6); and (a) Schrefeld's modification of Herzfeld's plain acid method (3). The inversions were carried out mostly a t temperature, but in some of the work higher ternperatures were employed: 55" C. in method a; 60' C. in methods b and c; and 67" to 69.5' C. in method a. The results of these investigations may be summarized briefly as follows:
one solution containing several normal weights was prepared, clarified with dry lead subacetate, and filtered. For the invertase method, a portion of the filtrate was deleaded with dry ammonium dihydrogen phosphate, and equal parts of the final filtrate, each containing 6.5 grams of product in 100 d. of final volume, were used for the direct reading and for the inversion. The portions used for the three acid hydrolysis methods were prepared in the same way except that dry potassium oxalate was employed as a deleading agent. The inversions were carried out at a temperature of 26' to 30' C., and the polariscope readings were made at the standard temperature of 20' C. The results, calculated in accordance with rules 1 and 2 given above, are shown in Table I. TABLE 1.
FOUND BY FOURCLERQET METHODS
COMPARISON OF SUCROSI3
J. & G. S A M P L ~INVERTASEMETHODI1 %
J. & a. METHODI V
PLAIN ACID METHOD
%
%
30.96 38.50 38.19 36.48 33.61 29.97 38.14 35.27 36.14
31.02 38.55 38.26 36.70 33,81 30.19 38.19 35.18 36.24
% FILTERED SIRUPS
1 2 3 4 6 6 7 8 Av.
1. The solution used for the direct polarization must have
!he 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 haye an appreciable effect on such reactions as the destruction o! 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 0: the four methods compared which may be depended upon to give reliable sucrose results. 5. The sucrose result by Jackson and Gillis method I1 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 11. But aspartic acid or asparagine lowers the sucrose result considerably. 7. Accordingly, the difference between the sucrose result by Jackson and Gillis method I1 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 differencebetween the sucrose result b Jackson and Gillis method I1 and that by method I V gives a rehive measure of the amino compounds resent. 9. The plain acid mettod 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 per cent 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.
30.65 38.17 37.82 35.56 33.13 29.94 38.29 35.11 34.86
31.51 38.71 38.85 36.59 33 * 73 30.26 38.39 35.39 35.43
REFINERY BLACKSTRAPS
9 10 11 12 13 14
35.84 32 80 31.72 34.26 34.65 30.76 33.34
15 16 17 18 19 20 21 22 23
33.28 30.65 33.30 34.16 31.91 30.76 35.47 40.59 32.80 33.65
Av.
37.12 34.19 33.02 35.58 35.29 31.55 34.46
36.34 33.79 32.46 34.69 34.71 31.11 33.85
36.30 34.00 32.84 34.94 34.62 31.97 34.11
RAW SUQAR BLLCKSTRAPB
AV.
33.65 31.03 34.06 34.36 31.80 31.04 36.46 41.63 33.05 34.12
33.68 31.28 34.14 34.58 31.94 31.30 36.81 41.65 33.08 34.27
method I1 not UnfortunatelY, Jackson and be used on the raw sugar blackstraps because the inverted solutions turned so dark upon neutralization with ammonia that they could not be read accuratelyeven with a halfshadow angle of 15 degrees. However, in the two series where Jackson and Gillis method I1 could be applied, it gave the highest average results, followed in both cases by the plain acid method, then Jackson and ~ i l l method i~ IV, whereas the invertase method gave the lowest average values. Considering the products in these series individually, Jackson and Gillis method I1 gave the highest results of all four in eleven out of a total of fourteen cases, and in the remaining three, the plain acid method gave the maximum figures. This is in agreement with the statement made above that the plain acid method may give any kind of result, depending on the composition of the product. I n every single case Jackson and Gillis method I1 gave higher figures than either the invertase or Jackson and Gillis
The same four methods which had been applied to artificial mixtures containing known quantities of sucrose have now been used in the analysis of various blackstraps and refinery sirups. In order to obtain strictly comparable results, only 34
January 15, 1933
INDUSTRIAL AND ENGINEERING CHEMISTRY
method IV. This shows that all the fourteen 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 per cent, averaging 1.12 per cent; and the quantity of amino compounds from 0.40 to 0.89 per cent, averaging 0.61 per cent. I n the filtered sirups there are found 0.10 to 1.03 per cent, averaging 0.57 per cent, of reversion products, and 0.11 to 0.66 per cent, averaging 0.29 per cent, 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 (1) and by Browne (4), very much higher, in the neighborhood of 2 to 2.5 per cent. Leaving out Jackson and Gillis method 11, 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 per cent, respectively, higher than Jackson and Gillis method IV. The average figures by this last-named method again exceed those of the invertase method by 0.28, 0.51, and 0.47 per cent, respectively, owing to the differential effect of reversion products and of amino compounds. Upon making individual comparisons, it is found that Jackson and Gillis method IV gives a higher result than the invertase method in twenty-one cases. I n all of these the reversion products, expressed as sucrose, exceed the amino compounds, expressed the same way; in the remaining two cases, samples 7 and 19, the opposite is true, as actually shown in sample 7 by comparison with the result of Jackson and Gillis method 11. The plain acid method gives the highest result in nine-
35
teen cases, including all the raw sugar blackstraps, intermediate results in three, and the lowest result in one case, again proving the statement made above with regard to this method. The invertase method gives the lowest result in all samples but three, I n two of these exceptions, samples 7 and 19, this is due to the preponderance of amino compounds over the reversion products, as already explained. I n the last remaining case, sample 13, the acid method happens to give the lower result, but with a difference of only 0.03 per cent, which is well within the limit of error. 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 I1 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.
LITERATURE CITED (1) Ambler, 3. A.,Intern. Sugar J . , 29,439 (1927). (2) Assoc. Official Agr. Chem., Official and Tentative Methods, 3rd ed., p. 370 (1931). (3) Ibid., p. 372. (4) Browne, C. A., and Blouin, R. E., La. Sugar Expt. Station, Bull. 91, 93 (1907). ( 5 ) Jackson and Gillis, Bur. Standards, Sci. Paper 375,184 (1920). (6) Jackson and Gillis, Ibid., p. 187. (7) Zerban, J . Assoc. O$cciaZAgr. Chem., 8,384(1925);9,166(1926); 10,183(1927); 11,167(1928);12,158(1929);13,188(1930); 14,172 (1931). (8) Zerban, Orig. C m . 8th Intern. Congr. Appl. Chem., 8, 103 (1912). RECEIVEDJuly 19, 1932. Presented before the Division of Sugar Chemistry at the 84th Meeting of the American Chemical Society, Denver, Colo., August 22 t o 2 6 , 1932.
A Rapid Method for Distinguishing Bleached Sulfate from Bleached Sulfite RALPHW. SHAFFER,MacAndrews & Forbes Co., Camden, N. J.
N
0 STAIN appears to be known which clearly differentiates between bleached sulfate and bleached sulfite in pulp and paper. The following formula affords a simple method of indentification. One gram of sodium carbonate is dissolved in 175 cc. of distilled water, and to this solution 1 gram of c. P. brazilin is added, stirring until dissolved. The solution gives sharper differences if used fresh. If retained for future use, air should be excluded. The solution may be applied directly to the sample to be identified, or if the preparation of a microscope slide is desired, the procedure is as follows: One gram of pulp or paper is disintegrated by boiling for 10 minutes with distilled water. The fiber is screened and shaken up with 50 cc. of water. The micro-slide is prepared by placing 4 drops of the fiber suspension on the slide and allowing to dry. The slide is then plunged for an instant into the stain solution prepared as directed above. Excess stain solution is removed with hardened filter paper, a few drops of U.8. P. white paraffin oil are placed on the slide, and any excess oil is removed. Bleached sulfite is stained a wine-red color, and bleached sulfate (Kraft) is stained a purple color by the brazilin solution. If the analyst encounters a slide containing some fibers which appear doubtful as to identification, he should prepare
a duplicate slide and test it with Lofton-Merritt stain, in order to eliminate the possibility of reporting as bleached fibers some which in reality are partially bleached. If the Lofton-Merritt stain shows a sulfate reaction, and yet with brazilin the fibers react to produce a lighter shade than the deep purple of the fully bleached sulfate, the obvious conclusion would be that these fibers are partially bleached sulfate. Similarly, if the Lofton-Merritt stain shows a sulfite reaction, and yet the fibers react with brazilin to produce a lighter shade than the vivid wine-red of the fully bleached sulfite, the conclusion would be that these fibers are partially bleached sulfite. To emphasize the supersensitivity of this stain to all contamination, the following explanatory data are added: METHODOF DISINTEQRATION. The standards of the Technical Association of the Pulp and Paper Industry have been adopted in all other microanalyses, and a t the beginning of work on brazilin every effort was made to have the technic conform to those standards. This was found impossible, however, because the stain is so very sensitive. The slightest trace of acid, or addition or subtraction of alkali, makes the stain worthless, and, although the author has tried the T. A. P. P. I. method of disintegration many times for this stain, results have never been successful. An explanation is that all the sodium hydroxide is not washed out after boiling
