INDUSTRIAL AND ENGINEERING CHEMISTRY
October 15, 1932
22410 X 760 X T X W 1000 X 273 X V X A p where T = absolute temperature of vapor bath W = weight of sample taken, mg. V = volume of inner jacket, ml. Ap = change of pressure within A , mm. M.W.
=
Collecting constants, the above equation becomes: M.W.=KX-
mination can be made in 20 minutes with accuracy comparable with the large Victory Meyer apparatus. TABLEI. TYPICAL MOLECULAR WEIQHTDETERMINATIONS COMPOUND B. I'. C. C~HioOz 134.7
T X W
AP
It is apparent that the constant, K , of any apparatus may be calculated, once the volume of the inner jacket is known. DISCUSSION The average accuracy of the method is within 2.5 per cent; with accurate estimation of pressure change, the error is usually Power. Table I illustrates some typical results obtained with the apparatus; some of the data were first trials of students in a class of advanced organic chemistry. As the sample can be weighed out in less than 15 minutes, and the vaporization requires less than 5 minutes, the entire deter-
367
(Constant for apparatus, 3.059) TEMP. WT. OF M. W. M. W. OF BATHSAMPLE AI' FOUNDCALCD.ERROR O C. Mg. Mm. 7% 140.0 140.0
6.605 6.624
95.0 94.0
87.8 89.0
90.1 90.1
-2.8 -1.2
CeHa
80.4
99.6 99.6
3.704 5.977
53.5 88.0
78.9 77.4
78.1 78.1
$1.0 -0.9
CClr
76.0
100.5 100.5
5.540 4.691
41.0 33.6
164.4 156.6
163.8 153.8
$0.4 +1.8
CHCla
61,2
100.5 100.5
6.074 6.087
57.3 57.5
121.1 121.0
119.4 119.4
$1.4 +1.3
CHsCOCHs
56.5
400.5 100.5
1.754 8 087
34.0 160.8
58.9 57.4
58.1 58.1
4-1.4 -1.2
I
LITERATURE CITED (1) Bleier and Kohn, Monatsh., 20, 505 (1899). (2) Chapin, J . Phys. Chem., 22, 337 (1918). (3) Lumsden, J . Chem. Soc., 83, 342 (1903). RECEIVEDJuly 15, 1932.
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Note on Measurement of Activity of Commercial Invertase H. C . GORE,The Fleischmann Laboratories, 158th St. and Mott Ave., New York, N. Y.
T
HE enzyme invertase is now on the market in extremely active form in aqueous solution preserved in glycerol. It is used in the food industries for the inversion of cane sugar, especially to prevent crystallization. Its action in cream centers and fondant coatings, in which the slow inversion caused by the invertase offsets the tendency of the sucrose to crystallize, has recently been described by Paine ( 5 ) . Our knowledge of the laws of its action is due to O'Sullivan and Tompson, Hudson, Nelson, Willstatter, and to many others; information on the solubility relations of sucrose, invert sugar, and water a t equilibrium has been published by Jackson (9). We still lack, however, a rapid, convenient, and exact method for measuring the activity of commercial invertase. The unimolecular reaction constant has been used extensively as a means of measuring invertase activity. Nelson (4) showed conclusively, however, that the reaction does not follow the unimolecular law, except under unusual conditions, but that the unimolecular constant values steadily increase during the course of the inversion. The method described below is offered as a contribution to the problem. It is based on the method used by Hudson and Paine (b), and the activity is expressed in a similar manner. It differs from it essentially in that an arbitrary reaction time has been selected Ammonia is used in terminating the inversion, as suggested by Bayliss (f), avoiding on the one hand the uncertainty incident to the use of dry sodium carbonate, and on the other the possibility of destruction of the sugar by use of excessive amounts of strong alkali. The sample of invertase is diluted fifty-fold with watere. g., 2 grams are diluted to 100 cc. Of the diluted sample, 5 cc. are placed in a flask and 50 cc. of sucrose solution added a t 20" C., with the time noted using a fast-running 50-cc. pipet. (The sucrose solution contains per liter 100 grams of cane sugar and 40 cc. of Walpole acetate buffer of 4.6 pH; 10.2 CC. of 1 N acetic acid and 9.8 cc. of 1 N sodium acetate diluted to 100 cc.) The solution is then incubated a t 20" C. for a 50-minute interval, when 0.5 cc. of strong
ammonia is added. The solution, after standing for a few minutes, is polarized a t 20' C. in a 2-decimeter tube. The initial polarization is determined by polarizing at 20" C, a mixture of 5 cc. of the diluted sample, 0.5 cc. of strong ammonia, and 50 cc. of the sugar solution, mixed in the order named. The activity K ~ iso evaluated by the formula
where a is the calculated range through which the polarization changes upon complete inversion by invertase, and x is the fall in polarization observed. The value a is calculated from the formula 100 where P' is the original polarization, P , corrected for the zero reading of the polariscope, and t is the temperature. Example: The initial reading was 34.8" V. The zero reading of the polariscope was 0.5" V. The reading after the 50minute digestion period was 18.0" V. 34.8 - 0.5 Then a = loo X 131.7 = 45.17' V. x = 34.8 - 18 = 16.8' V. and Kso = 0.20 Thus, the activity is simply the unimolecular reaction constant calculated at the 50-minute interval and multiplied by the dilution. LITERATURE CITED (1) Bayliss, "The Nature of Enzyme Action," Longmans, 1919. (2) Hudson and Paine, J . Am. Chem. Soc., 32 774 (1910). (3) Jackson and Silsbee, Bur. Standards, Tech. Paper 259 (1924). (4) Nelson and Vosburgh. J. Am. Chem. Soc , 39, 790 (1917). (5) Paine, "Successful Quality Control in Food Processing," McGrawHill, 1930. RECEIVED May 13,1932. Preaented before the Division of Sugar Chemktry at the 83rd Meeting of the American Chemical Society, New OrleanrJ, La., March 28 t o April 1, 1932.
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