September15,1933
INDUSTRIAL AND ENGINEERING CHEMISTRY
be obtained easily in a c. P. state and are inexpensive enough to be used for standardization purposes. Toluene has a kauri butanol value of nearly 102 and heptane has a value of only 26; thus mixtures of these two will cover the entire range of any ordinary lacquer thinner marketed and used today. Figure 5 shows the curve for the 16.6 per cent solution of bush dial kauri gum, kauri butanol values being plotted against percentages of toluene in a toluene-heptane mixture. Once such a curve is obtained for any kauri solution, any solvent may be titrated with this solution and then expressed in terms of per cent toluene or toluene number by reference to this curve. I n this way, variations due to differences in kauri solutions will be eliminated and a standard value obtained which should be easily reproducible in any laboratory, provided other points, such as the amount of kauri solution used and the temperature of test, are standardized. The authors propose that the kauri butanol solution become a testing medium only, standardized by a primary standard, the toluene-n-heptane blend. The solvent power of a paint and lacquer solvent will be expressed in comparable and re-
309
producible toluene numbers. A similar method of using a primary standard (isooctane and n-heptane blend) has been adopted and successfully used by the petroleum and automotive industries for determining the antiknock value of gasolines since 1929 (5).
(1)
BIRLIOGRAPHY Am. Paint Varnish Mfrs. Assoc., Sci. Sect., Circ. 313,390 (1927).
(2) Ibid., Circ. 318,492 (1927). (3) Edgar, G., IND.Eao. CHEM.,19, 145 (1927). (4) Fauser, E., Am. Paint Varnish Mfrs. Assoc., Sci. Sect., Circ. 291, 275 (1926). ( 5 ) Hosking, J. R., Rec. trav. chim., 48,622-36 (1929). (6) Kiehl, S. R., Am. Paint Varnish Mfrs. Assoc., Sci. Sect., Circ. 319,595 (1927),
(7) Paint and Varnish Superintendents' Club., Am. Paint Varnish Mfrs. Assoo., Sci. Sect., Circ. 378, 145 (1931). (8) Stewart, J. R., Ibid., Circ. 378, 143 (1931).
RECEIVED May 17, 1933. Presented before the Division of Paint and Varnish Chemistry at the 85th Meeting of the American Chemical Society, Washington, D. C., March 26 to 31, 1933.
Effect of Certain Preservatives on the Determination of Sucrose by the Invertase Method N
CHARLESF. POE,MARYCOOLEY,AND N. F. WITT D e p a r t m e n t of Chemistry, University of Colorado, Boulder, Colo.
K
JELDAHL in 1881 (6) proposed the use of invertase in lieu of hydrochloric acid as a hydrolytic agent of sucrose for use in the Clerget method. The first investigators to apply invertase t o the determination of sucrose in food products appear to be Ling and Baker (8) who used the enzyme to analyze molasses and other sugar products. Invertase is replacing hydrochloric acid for the inversion of sucrose because the former is more selective, having no effect on a number of acid-hydrolyzable substances of the nonsugar group. Since invertase is susceptible to a number of chemicals which retard or prevent its action, it was decided to investigate the effects of different food preservatives on the determination of sucrose by the invertase method. The effects of the common food preservatives, other than alcohol (1, 5 , IO), on the determination of sucrose have been little investigated, although a number of studies have been conducted relating to the effects of preservatives and other chemicals upon the enzyme before it has been used as a hydrolyzing agent for sucrose, including the researches of Euler and coworkers (2, 3, d ) .
thought advisable to test the particular sample of invertase preparation used in this research-for the optimum pH. The results are presented in Table I, which shows that the activity was fairly satisfactory for pH values from 3 to 6. The most efficient pH, however, was between values of 4 and 5. TABLEI. EFFECTOF PH TIME p H 3 Min. 0 5 15 25 40 60
ON ACTIVITY OF INVERTASE (10 grams sucrose per 100 cc.) P H 3.5 ~ H 4 . 0~ H 4 . 5P H 5.0 ~ H 6 . 0~ H 7 . 0~ H 8 . 0
38.5" 3 8 . 5 38.5 38.5 38.5 38.5 38.5 23.7 27.6 36.6 27.1 2 7 . 6 2 5 . 3 23 6 7 . 6 7.2 7.3 9 . 3 32.0 8.5 9.0 0.6 0.8 0.4 2 . 8 27.2 2.5 2.3 3 . 1 22.3 2 . 9 2 . 7 2 .0 -1.0 -1.2 -6.1 16.1 -5.8 -5 6 - 5 . 2 -4.8 -5.0 10.1 7 . 6 7 . 0 7 . 6 7 . 7 -7.0 -7.2 80 -9.4 -9.5 -9.6 -8.9 5.1 -9.3 -9.0 120 1 1 . 0 1 1 . 0 1 0 . 1 1.0 1 1 . 1 1 0 . 2 1 0 . 4 300 -11.2 -11.3 -11.2 -11.3 -11.3 -10.8 600 - 1 1 . 3 0 Polariscope readings, 200-mm. tube.
38.5 38.3 37.0 35.2 33.0 30.1 27.4 22.1 2.1 -6.8
Different amounts of the various preservatives were added to sucrose solutions and the rates of inversion were determined with invertase. The pH of each solution, excepting those to which formaldehyde was added, was adjusted with PROCEDURE acetic acid or sodium hydroxide to a value between 4.5 and 5. Ten-gram amounts of sucrose were weighed into 100-cc. flasks No alkali was added to the flasks containing formaldehyde and dissolved in about 70 cc. of water, the desired amount of because the preservative would have been changed; the pH preservative was added, and the pH was adjusted. The invertase value for the 0.1 per cent solution was 4.5 and for the 4 per was added, and the volume was made up t o 100 cc. The polari- cent solution, 3.25. The rates of hydrolysis for sugar soluscope reading was taken immediately and like readings were made at stated intervals, the temperature being kept at 25" C. tions containing the various preservatives, as compared to a As each portion was removed, a small amount of alkali was control containing no preservative, are given in Tables 11, added t o stop the action of the invertase and to complete the 111,and IV. mutarotation. Complete inversion did not take place with the sugar According to Nelson and Bloomfield (9) the optimum pH solutions containing certain preservatives. It therefore for the action of invertase is between 4.5 and 5. It was remained to be determined whether or not this retardation
ANALYTICAL EDITION
310
was due to the action of the preservative on the rotation of the sucrose or the sugars produced by inversion. TABLE11. EFFECTOF PRESERVATIVES ON ACTIVITY OF INVERTASE (10 grams sucrose per 100 cc.) 0.1 0.5 1.0 2.0
3.0
FORMALDEHYDE
0 5 15 25 40 60 120 300 600
38.5b 25.6 11.2 1.5 -6.5 . . ~ -9.0 -11.4 -11.2 -11.4
38.5 38.5 25.7 28.0 11.8 12.4 2.2 3.1 -6.0 -5.4 -8.6 -8.4 -11.3 -9.0 -11.4 -10.8 -11.3 -11.2
38.5 29.6 15.6 9.6 4.1 1.2 -4.6 -6.8 -8.1
38.5 28.8 14.2 4.8 1.1 -4.5 -8.0 -8.6 -9.6
38.5 30.8 16.2 10.8 6.7 3.1 -3.6 -6.1 -6.8
38.5 32.0 20.8 16.0 8.9 6.2 3.1 1.2 -4.2
SODIUM BISULFITE
38.5 38.5 38.5 38.5 38.5 38.5 38.5 5 25.0 24.6 23.8 24.5 25.0 25.2 24.8 10.0 10.4 10.0 9.9 10.2 9.2 9.0 15 25 1.2 0.9 1.0 1.2 1.0 1.8 2.1 -6 fi -6 2 -6 n --A R --A 2 --A 7 40 -6.8 - s : i -816 -8:s -912 -8.8 60 120 -11.3 -11.2 -11.3 -11.5 -12.0 -13.0 -14.4 -11.3 -11.5 -11.7 -12.5 -13.2 -14.4 300 -11.2 0 Grams per 100 cc. Bugar solution. b Polarisoope readings, 200-mm. tube. 0
-s:iJ
-s;Q
TABLE111. EFFECTOF PRESERVATIVES ON ACTIVITY OF INVERTASE TIME Min.
(10 grams sucrose per 100 cc.) 0.25 0.5 1 2 3 CONTROL GRAM^ GRAM GRAM GRAMS GRAMS BORIC ACID
0
15
25 40 60 120 300 600
38.5b 28.2 12.1 2.4 -5.2 -9.0 -10.6 -11.3 -11.4
38.5 27.6 13.0 2.8 -5.6 -7.8 -9.6 -10.7 -11.2
38.5 28.0 13.4 3.0 -4.8 -7.0 -7.9 -9.6 -10.8
38.5 27.2 14.0 4.2 -1.6 -3.2 -7.8 -8.6 -10.4
38.5 28.2 14.8 9.0 3.0 0.0 -5.0 -7.2 -8.1
38.5 30.2 15.2 10.2 5.8 1.4 -2.6 -5.1 -5.2
38.5 26.2 16.0 8.9 2.8 -2.8 -6.2 -9.0 -9.2
38.5 27.1 16.8 8.6 4.2 0.6 -4.8 -6.8 -7.1
38.5 26.8 18.8 10.2 6.2 1.7 -1.4 -5.2 -5.4
BORAX
38.5 38.5 38.5 25.5 26.0 25.3 12.2 12.0 11.6 2.1 1.6 1.2 -4.6 -5.0 -5.0 -7.0 -8.8 -7.6 -10.2 -9.6 -10.0 -10.8 -11.2 -11.4 -10.9 -11.3 -11.3 a Grams per 100 cc. sugar solution. b Polariscope readings, 200-mm. tube. 0 5 15 25 40 60 120 300 600
ON ACTIVITY OF TABLEIV. EFFECTOF PRESERVATIVE INVERTASE
TIME Min. 0 5 15 25 40 60 120 300 600
(10 grams sucrose per 100 cc.) 0.1 0.5 1 2 3 4 CONTROLGRAM^ GRAM GRAM GRAMSGRAMSGRAMS 38.5 38.5 25.5 26.0 10.8 11.4 0.4 1.8 -6.0 -5.6 -x I) -9.4 -10.8 -10.4 -11.4 -11.0 -11.4 -11.3
38.5s 24.6 10.6 1.0 -5.8 -9.2 -10.6 -11.4 -11.3
of dextrose and levulose and the maximum amounts of each preservative were separately dissolved in water and diluted up to 100 cc. After adding alkali to correct the mutarotation, readings were taken a t stated intervals. The results for any solutions which caused a change in rotation are given in Table V.
4.0
CONTROL GRAM" GRAM GRAM GRAMSGRAMSGRAMS
TIME Min.
38.5 25.8 11.6 2.1
-6.4
-8.4 -10.3 -10.9 -11.4
TABLEV. EFFECTSOF PRESERVATIVES ON ROTATION OF SUCROSE, DEXTROSE, AND LEVULOSE 4 GRAMSOF FORMALDEHYDE^ 10 grams of 5 grams of 5 grams of TIME sucrosea dextrosea levulosea Min 15.5bDC 0 38.5b?E -. .. 20.2 5 38.6 -26.2 19.4 15 38.6 -25.8 18.1 25 38.9 17.2 -25.8 40 39.4 17.2 -25.7 60 39.5 -25.2 17.1 120 39.9 40.0 -25.0 300 16.9 -25.0 16.7 40.0 600 -25.0 16.6 40.0 1440 a Amount of preservative and sugar in 100 cc. b Readings of blank Containing sugars alone. 0 All readings in a 200-mm. tube. ~
38.5 26.4 12.2 2.6 -4.2 -8.1 -10.6 -11.4 -11.3 a Grams per 100 0 0 . sugar solution. b Polariscope reading, 200-mm. tube.
38.5 38.5 27.0 27.2 12.4 12.8 3.0 3.0 -4.3 -4.8 -8.0 -8.1 -9.9 -10.2 -10.8 -10.4 -10.7 -10.3
38.5 27.4 13.0 3.4 -4.0 -7.8 -9.0 -9.1 -9.0
Accordingly, a series of experiments was conducted using the different sugars and the preservative without the invertase. Ten-gram amounts of sucrose and 5-gram amounts
4 GRAM0 O F SODIUM BISULFITE^ 5 grams of dextrosea 15.5b!c 16.2 13.2 12.1 11.9 11.8 11.8 11.8 11.7 11.6 of solution.
3 GRAM^ OP BORAX" 5 grams of levulose' -26.5brC -21.4 -21.3 -21.3 -21.4 -21.4 -21.3 -21.3 -21.3 -21.4
Formaldehyde was the only preseryative which affected the rotation of sucrose. Little effect was noted on the rotation of levulose or dextrose by this preservative. Therefore, the retardation caused by the higher percentages of formaldehyde, as shown in Table 11, must have been due to the detrimental action of the formaldehyde on the invertase. The sodium benzoate and sodium salicylate had no ill effect on the rotation of any of the sugars. When invertase was present sodium benzoate seemed to slow up slightly the action of invertase in the higher percentages. Sodium bisulfite with dextrose decreased the normal rotation considerably. This would account for the increased negative rotation recorded for solutions of sucrose containing invertase and sodium bisulfite (Table 11). This observation is in accord with results obtained by Tomoda and Taguchi (112) in work with sulfites and different sugars, Borax diminished the rotation of levulose. This in part would account for the apparent decreased activity of invertase as shown in Table 111. Table I11 also indicates that the action of invertase was retarded by boric acid, and Table V shows that boric acid alone had no effect on the sugars tested. Therefore, the retardation was due to the effect of the preservative on the invertase. Several investigators (7, 11) have found that borax decreases the rotation of dextrose and levulose, while boric acid has no effect on these sugars. Taking into account the amount of each preservative ordinarily used in food products (usually less than 0.25 per cent), it appears that the preservatives studied would have no ill effect on the determination of sucrose by the invertase method.
LITERATURE CITED
SODIUM BENZOATE
0 5 15 25 40 60 120 300 600
Vol. 5, No. 5
Bourquelot and Bridel, J. pharm. chim., 9, 321 (1914). Euler and Myrback, 2. physiol. Chem., 125, 297 (1923). Euler and Svanbwg. Ibid., 105,187 (1919). Euler and Svanberg, Fermentforschung,4, 29 (1920). Hudson and Paine, J. Am. Chem. SOC.,32, 1350 (1910). (6) Kjeldahl, Meddelelser Cark3berg Lab., 1, 337 (1881). (7) Levy and Doisy,J . Biol. Chem., 84, 749 (1929). (8) Ling and Baker, J. SOC.Chem. Znd., 17, 111 (1898). (9) Nelson and Bloomfield, J . Am. Chem. SOC.,46, 1025 (1924). (10) O'Sullivan and Thompson, J. Chem. SOC.,57, 834 (1890). (11) Rimbach and Weber, 2. physik. Chem., 51,477 (1905). (12) Tomoda and Taguchi, J. SOC.Chem. Ind. Japan, 33, 434 (1930). (1) (2) (3) (4) (5)
RECEIVED February 13, 1933.
