April, 1927
HYDROLYSIS
OF SUCROSE A N D RAFFINOSE
1019
[CONTRI~UTION FROM THE CARBOENDRATE LABORATORY, BUREAU OF CHEMISTRY, UNITEDSTATES DEPARTMENT OF AGRICULTURE]
CLERGET-INVERTASE HYDROLYSIS CONSTANTS OF SUCROSE AND RAFFINOSE' BY H. S. PAINEAND R. T. BALCH RECEIVED NOVEMBER 5, 1925
PUBLISHED APRIL7, 1927
Recent advances in methods of sugar analysis depending upon enzymic hydrolysis emphasize the importance of the relation between the polarizations before and after enzymic action as a useful physical constant, and render apparent the need for its precise evaluation. I n addition to a procedure2 for the determination of sucrose by invertase inversion which can be operated in the same amount of time as the rapid form of the acid-inversion Clerget method, the writers have recently described a procedureS for simultaneous determination of sucrose and raffinose in such products as beet sugar-house liquors. The latter method depends upon the action of the enzymes invertase and melibiase. As invertase hydrolyzes sucrose with production of invert sugar and causes hydrolytic cleavage of raffinose with production of fructose and melibiose, accurate data on the Clerget hydrolysis constants of invertase, with respect to both sucrose and raffinose, are highly desirable. Clerget invertase-inversion constants for a few sucrose concentrations have been reported by a number of investigators, but no systematic measurements covering a wide range of sucrose concentration have been made. Revious Data on Clerget Constants The belief that the presence of hydrochloric acid, used as a hydrolytic agent for sucrose, accounted for the variability of the Clerget constant, which would entirely disappear if invertase were employed in its place, was erroneously held until 1912, even though it was known long before that the specific rotation of fructose in pure water varies with its concentration. Tolman4 strongly expressed this view and Hudson, in 1910,5 apparently took it for granted and assumed that the value which he determined, namely, 131.7 for a 7% solution a t 20°, held for all concentrations below a 0.5 N weight solution. Ogilvie,6 in 1911, applying the yeast method of O'Sullivan and Tompson' to the analysis of beet molasses polarizing approximately 50' Ventzke, determined the Clerget constant Presented before the Sugar Division, at the 69th Meeting of the American Chemical Society, Baltimore, Maryland, April 6-10, 1925. Paine, J . Assoc. OfficialAgr. Chem., 8, 362 (1925). a Paine and Balch, Ind. Eng. Chem., 17, 240 (1925). Tolman, THIS JOURNAL, 24, 523 (1902). Hudson, J . Ind. Eng. Chem., 2, 143 (1910). Ogilvie, J . Soc. Chem. I d . , 30, 62 (1911). ' O'Sullivan and Tompson, J . Chem. SOL,59,46 (1891).
1020
H.
S. PAINE A N D R . T. BALCH
Vol. 49
to be 131.6. These two values agree closely with those of the authors, namely, for 6.5 g. in 100 cc., our inversion constant is 131.64, and for 7 g. in 100 cc., 131.68 instead of 131.6 and 131.7, respectively, as found by Ogilvie and by Hudson. Saillards was apparently the first to disclose the fact that even with invertase hydrolysis of sucrose, the Clerget constant varies with the concentration. Saillard'sg results are summarized in the relation, constant = 130.74 0.0664 G where t = sucrose concentration. Browne,lo in 1916, likewise pointed out the necessity of taking into consideration the concentration of sucrose present in determining the constant used in calculating the percentage of sucrose from the polariscopic readings before and after hydrolysis with invertase. No data were presented but he established the formula 5' = [lo0 ( P - I ) ] / 132 ( 0.0065 [132 - ( P - I ) ] ]for 20'. His constant may also be expressed as, constant = 131.14 0.0660 m , where m = g. of solids ira 100 cc. Jackson and Gillis,ll in 1920, reported the value -32.00' for the negative constituent of the Clerget constant based upon the Bates and Jackson conversion factor, which is equivalent to -31.97' Ventzke, on the Herzfeld-Schonrock standardization of the sugar scale. This value was determined from results obtained with mixtures of invert sugar and acid by extrapolation to zero acid. Sazavsky,12 in 1924, determined the negative constituent to be -31.97' Ventzke apparently on the Herzfeld-Schonrock scale, which likewise does not agree with the authors' results nor with the results of Zerban13 calculated from the most recent data on the specific rotation of fructose and dextrose, namely, -32.09. Zerban14also obtained experimentally the value 132.10 * 0.025 a t a sucrose concentration of 13.0 g. in 100 cc. Sufficient evidence was obtained to warrant the adoption of the constant 132.1 for this concentration by the Association of Official Agricultural Chemists in 1925.
+
+
Preparation and Testing of Purified Sucrose An approximately 50% solution of the highest grade commercial granulated sugar was clarified with salt-free alumina cream and decolorizing carbon. After the addition of diatomaceous earth, i t was filtered through hardened filter paper and concentrated in a vacuum to 78-80% sucrose a t a temperature not exceeding 35" (usually under 32"). This sirup was slowly stirred during crystallization and after several hours the mother 8 Saillard, Eighth International Congress of Applied Chemistry, 25, 541 (1912) ; Cir. Hebd. Synd. Fabr. Sucr. France, 1915. 9 Saillard, J . Fabr. Sucre, 57, No. 2 (1916). Browne, J . Assoc. Oficial Agr. Chem., 2, 140 (1916). l1 Jackson and Gillis, U.S. Bur. Standards Sci. Paper, No. 375, 1920. l 2 Sazavsky, Z . Zuckerind. Cechodooak. Rep., 48, 259 (1924). l 3 Zerban, THIS JOURNAL, 47, 1104 (1925). Zerban, J . Assoc. 0-ficial Agr. Chem., 8 , 384 (1926).
-4pri1, 1927
1021
HYDROLYSIS O F SUCROSE AND RAFFINOSE
liquor was separated from the crystals by centrifuging. The sugar was washed in the centrifuge with neutral dilute alcohol and finally with neutral 95% alcohol. It was dried by exposure to the air for a day and was then placed in a low-temperature vacuum oven through which a slow current of dry air was passed. Five lots of sucrose were prepared. Their composition is shown in Table I. Three of these lots were of nearly the same degree of purity. The percentage of ash was determined by incinerating 5 g. of sugar in a platinum dish. Copper-reducing substances were determined by a modification of the Soldaini method.
TABLE I ANALYSISOF SUCROSE USEDFOR DETERMINING CLERGETINVERSION CONSTANTS Preparation no. Source and treatment 1 Highest grade commercial sucrose recrystallized once from water after treatment with carbon 2 Same as 1, but recrystallized twice from water 3 Same as 2
Ash,
7
0
Invert sugar,
7
0
0.007 < 0 . 0 1
Water,
%
0.003
.002
<
