THE INFLUENCE OF ETHYLENE GLYCOL UPON SOME REACTIONS* BY L. MABEL YOUNG AND H. M. TRIMBLE
Ethylene glycol is known to act as a preservative in food preparations. Some direct studies have been made, too, upon its inhibiting action toward the growth of bacilli, molds and yeasts.l The question of its effect upon certain related reactions was taken up as a part of a series of studies with this interesting substance which are being carried out in this laboratory. All experiments were performed at 3ooC. The ordinary chemicals used were all of C.P. quality, Ethylene glycol was prepared by distilling prestone or dynamite grade glycol as previously described2 and its purity was established by suitable tests. The urease used was a fresh preparation manufactured from Jack bean meal by the Arlington Chemical Company. The invertase was a preparation “for analysis” from the Digestive Ferments Company. Fleischmann’s yeast purchased upon the open market was used in the fermentation experiments. A preparation of yeast grown in the laboratory was used in a few of the experiments. All glassware was cleansed with cleaning solution, washed well with distilled water and dried in a hot air bath before use. Effect of Ethylene Glycol upon Activity of Urease A number of semi-quantitative experiments upon the decomposition of urea by urease showed that the rate is diminished by the addition of even small quantities of glycol, that this effect increases with increasing concentration of this Substance, and that about 60 per cent by volume will almost stop the reaction. Altering the concentrations of urea and of urease within the limits imposed by experimental conditions, the concentration of glycol being held constant, did not greatly alter the relative rates. The results, however, were erratic, and the results of parallel experiments often showed poor agreement. It was found that the difficulties could be practically eliminated by working with solutions which were heavily buffered, though this imposed serious restrictions upon the range of concentrations which could be covered. Buffer solutions were prepared for more accurate experiments by mixing 50 volumes of molar K H 2 P 0 4with 9 volumes of molar K,HPOd. Five cc. of the buffer solution was pipetted into a 2 5 0 cc. Soxhlet flask and I cc. of glycol added. Three cc. of freshly prepared urea solution of the desired concentration were next added. The flask was then set in the thermostat
* Contribution from the Chemistry Department, Oklahoma A. & M. College, Stillwater, Oklahoma. Fuller: Ind. Eng. Chem., 16, 624 (1924). ?Trimble: Ind. Eng. Chem., 23, 1 6 j (1931).
INFLUENCE O F ETHYLENE GLYCOL UPON SOME REACTIONS
83 1
TABLEI The Decomposition of Urea by Urease Part
I
Reaction Mixture Same as control except I . 083 grams of glycol added and the whole made up to IO cc.
Control 0.03 grams urea 0 ,O O I grams urease preparation 5 cc. buffer solution Water to make a volume of I O cc. Time Minutes
Reaction Mixture Per cent decomposed
Control Per cent decomposed
Relative Activity
16.75
5.00
.3Q
40
28.25
8.75
.31
60 80
37.00 43.75
11.50
.31
13.IO
.30
20
IO0 I20
14.00 14.50
48.50
52.30
Average
Part Control 0.06 grams urea 0 .O O I grams urease preparation 5 cc. buffer solution Water to make a volume of I O cc. Time Minutes
.28 .28
.30
2
Reaction Mixture Same as control except I . 083 grams of glycol added and the whole made up to I O cc. Reaction Mixture Per cent decomposed
Control Per cent decomposed
Relative Activity
20
7.30
2.77
40
14.00
5.25
.38 .38
6.80 8.30
.37 .37
IO0 I20
22.25 25.60 28.50
9.00 9.10
.35
60 80
18.60
Average
.32
.36
Part 3 Control 0.I 2 grams urea o O O I grams urease preparation 5 cc. buffer solution Water to make a volume of I O cc. Time Minutes
Control Per cent decomposed
Reaction Mixture Same as control except I . 083 grams of glycol added and the whole made up to I O cc. Reaction Mixture Per eent decomposed
Relative Activity
20.30
2.75 5.25 7.20
.38 .38 .36
25.50
8.80 9.80
.35 .31
120
29.25 31.00
10.50
.34
150
31.50
I1
20
7.25
40
14.00
60 80 IO0
.oo
Average
.35
.36
832
L. MABEL YOUNG AND
n. M.
TRIMBLE
weighed out in the form of pellets and gently macerated with such a quantity of distilled water as would give ,001gram in I cc. and brought to temperature. Then I cc. of this suspension was pipetted into each flask, the flask was shaken, stoppered, and returned to the thermostat; noting the time of starting the reaction. Control experiments were run in each set, using exactly the same quantities of the other materials, but substituting water for the glycol. The hydrogen ion concentration was followed by indicator methods in a number of cases. It was found to remain constant throughout a t pH = 7 . 3 0 . At suitable intervals the reactions in successive flasks were stopped by adding 35 cc. of saturated Na&OI solution. The flasks were then placed in a hot water bath kept a t a temperature just under the boiling point of water and, by passing air through the mixture for two hours the ammonia was driven over into an excess of 0 . I 134 normal H2SO4 to which had been added 50 cc. of distilled water. No trace of ammonia could be detected in the flasks after that period of aeration. Kjeldahl connecting bulbs were used in order to keep any spray from entering the bottles of acid. I n order to find the amount of ammonia driven out of the reaction flasks the excess H2S04 was then titrated with NaOH which had been standardized against standard HCl. Phenolphthalein was the indicator used. This method followed in general that of Rocliwood and H ~ s a . ~ Repeated experiments were performed with solutions of three compositions, with their corresponding controls. All results for each of the sets were plotted, and the best curve drawn through the points. From these curve5 the per cents decomposed a t intervals of 20 minutes were read. Experiments were not continued for longer times than two hours because of the danger of losing ammonia from the flasks through absorption by the rubber stoppers. The agreement was good, considering the difficulty of reproducing a biological material such as the enzyme here used. The data are presented in Table I. The fourth column headed “Relative Activity” is obtained by dividing the values of column 3 by the corresponding values of column 2. I t serves as a measure of the inhibition brought about by added glycol. Obviously glycol present to the extent of IO^^ by volume reduces the activity of urease, under the conditions of the experiment, to approximately one-third the value which it has in an aqueous solution at the same concentration. Effect of Ethylene Glycol upon Fermentation The second reaction studied was the fermentation of sugars by Fleischmann’s yeast. Secondary ammonium phosphate was used as additional nutrient material. The apparatus used in studying the rate of fermentation is shown in Fig. I . A stirrer projected into the flask through a mercury seal, so that no loss of gases was possible. The mixture was stirred a t a constant rate throughout the entire course of the experiment. I n one hole of the stopper in the side arm of the flask a manometer tube filled with water was placed. In the other hole of the stoppei was an outlet tube at the end of which was a short length J. Am. Chem. SOC.,45, 2678 (1923).
INFLUENCE O F ETHYLENE GLYCOL UPON SOME REACTIONS
833
of rubber tubing which could be opened or closed by a screw clamp. The reaction mixture of sugar, (NH&HP04, HZS04,and ethylene glycol (or water) for a given experiment, 7 5 cc. in volume, was placed in the SoxNet flask. There were 5 cc. of 1.083 N.HzS04in each 7 5 cc. of the solution. One cake of Fleischmann’s yeast was put in suspension with 1 5 0 cc. of water
W
I
FIG.i Reaction Apparatus for Fermentation
and brought to 30OC. 2 5 cc. of the yeast suspension was then added through the side arm, the outlet tube tightly closed, and after five minutes the increase of pressure set up by the evolution of COn was measured by reading the displacement of the column of liquid in the manometer on a millimeter scale. The outlet tube was then quickly opened and the pressure in the two arms of and allowed to come to temperature. The urease preparation was now the tube equalized. The tube was then closed and the pressure again allowed to build up. This releasing of the pressure never consumed more than 1 5
L. MABEL YOUNG AND H. M. TRIMBLE
834
seconds a t most. Yeast from the same cake was always used for an experiment with glycol present and for its control. In order to find the quantity of carbon dioxide evolved during the fermentation it was assumed that the perfect gas law holds. Since COS is not a perfect gas, some error is involved in so considering it, but the error is, we believe, within the limits of the experimental error of the work itself. We have, then: plv = nlRT and p2v = n2RT
and (n2 - nl) =
V
Ap RT
where (n2 - nl) is the number of mols of COa liberated in unit time, R is the gas constant expressed in cc. millimeters of mercury, v is the volume of the space above the solution in the flask, T is the absolute temperature, and Ap is the increase in pressure per unit of time, expressed in millimeters of mercury. From the numbers of mols so obtained the volumes of carbon dioxide evolved for the various times, reduced to standard conditions, were calculated. A summation of these quantities, making allowance for the periods during which the pressure was being released, gave the quantities of gas liberated a t the various times. Readings of the manometer were taken every five minutes, but for convenience the tables were made using twenty-five minute intervals Table I1 shows the results obtained for representative experiments. All curves for both sucrose and glucose were found to follow the same general form. While these results could not be exactly reproduced, due to the variable nature of yeast, yet other experiments gave results closely parallel to these.
TABLEI1 The Fermentation of Glucose with Fleischmann's Yeast The reaction mixtures in each of the following experiments were made up using the quantities of the reacting substances indicated. In the control mixtures water was substituted in each case for equal volumes of glycol. The average results of the experiments are given. Part 0.7 5 grams glucose
Water to make a volume of Reaction Mixture
Per cent glycol
Time
Vol. co2 evolved
0.00
25min.
13.98 cc. 27.98 '' 40.27 " 53.47 " 62.26 "
50
''
75
'I
I00
'I
I25
('
cc. yeast suspension
yoglycol below indicated, by volume
0.50 grams (NH4)zHP04 5 cc. 1.083N. HzS04
Control
I 25
100
cc.
Per cent glycol
Vol. COZ evolved
Relative activity
20
4.29 cc. 9.78 ' I 14.39 '( 18.42 "
.35 .36 .35
22.15
''
.31
.3G
INFLUENCE O F E T H n E N E QLYCOL UPON SOME REACTIONS
83 5
TAFUZ I1 (continued) Part Per cent glycol 0.00
Time 25
min.
SO
9.42 CC.
Per cent glycol so
20. SO
7s IO0 125
2
Vol. co1 evolved
I' I'
30.79 40.31 46.97
Vol. C02 evolved
1.29 cc.
'
2 .os
.IO
2.54 2.96 3.22
" It
Relative activity
'I
I4
,082 .074 .069
The Fermentation of Sucrose with Fleischmann's Yeast The same concentrations of the reacting substances were used as in the glucose experiments, the sucrose being substituted for an equal weight of glucose. Part 3 Per cent glycol 0.00
Control Time 25
min.
SO
75 IO0
I25
'I
VOl. con evolved
5.68 cc. 16.40 " 25.88 '( 34.56 ' I 41.73
Reaction Mixture Per cent Vol. COZ glycol evolved 20
Relative activity
3.43 cc. 9.98 " 14.30 '' 17.69 "
.60 .60 '55
20.38
.49
"
.SI
Part 4 Per cent glycol 0.00
Time
50
I'
5.62 cc. 13.36
75
'l
20.79 " 27.25
"
"
32.84
' I
25
min.
Vol. COS evolved
IO0 125
Per cent glycol so
Vol. COZ evolved
Relative activity
1.40cc.
'25
1.77
I'
'
"
'093 ,075 ,062
1.94 2.04 2.04
"
I3
Part 5
75
0.00cc. 0.00
I'
0.16 " 0.16 '' 0.16 "
As shown by the last columns of the table, part I , the activity of yeast in bringing about fermentation of glucose is reduced to approximately 1/3 its value by addition of 2 0 % glycol by volume. With 5 0 % glycol present, (part 2) the activity is reduced to about 1/7 of its value in the absence of glycol and this inhibition increases with time until, after 1 2 5 minutes, it has nearly doubled.
83 6
L. MABEL YOUNG AND H. M. TRIMBLE
In the fermentation of sucrose (part 3 ) 20% of glycol by volume reduces the activity of the yeast to about 2 / 3 of the value which it has in the aqueous solution, a t the start. After 1 2 j minutes the activity has been reduced to about half its normal value. With 50% glycol (part 4) the inhibition approaches that found with the same relative amount when fermenting glucose. With 7 5 % glycol (part 5 ) inhibition of fermentation is complete within the limits of experimental error, Effect of Ethylene Glycol upon Inversion of Sucrose Because of its accuracy and convenience the polariscope was used to determine the rate of inversion of sucrose. The rate of inversion of sucrose with invertase was studied, first without and then with added glycol. The polariscope tubes were of the water jacketed form, and were supplied with water from a thermostat a t 30’. They were washed first with water, then with alcohol, and finally with distilled water, then rinsed several times with the solution whose rate of inversion was to be studied. One hundred cc. of the reaction mixture in each case was brought to 30’C. in the water bath. The sucrose solution had been made acid to litmus with acetic acid in order to establish a suitable pH. This is not highly important, however, for it has been found that for the action of invertase the pH may vary over quite a large range without detrimental effects. After the solution had been brought to temperature 5 cc. of invertase solution, also a t 30’C. was added and the solution introduced into the polariscope tubes. Measurements of the angle of rotation were made at suitable intervals for 90 to 774 minutes in various experiments, depending upon the velocity of the reaction in each case. A sodium fed flame was used as light source. I t is most convenient to express velocities of inversion in terms of K, the reaction rate a t zero time, or the velocity constant. For a reaction of the first order, according to which this reaction is most readily formulated, we have:
or (2)
K
=
’ t
log, (At
+ A,)
- log,
(A
+ A,)
where A, A, and A, are, respectively, the specific rotations a t zero time, a t time t and at complete inversion. Specific rotations were calculated by means of the equation (A)$ =
v, where (A); lg
is the specific rotation at time t, a is
the angle of rotation as measured, g is the number of grams of sucrose dissolved in v cc. of water, and 1 is the length of the polarimeter tube in decimeters. The equation giving the specific rotation of invert sugar is (A); = - ( 2 7 . 9 0 - 0.32 t)4from which we find that the specific rotation a t Woodman: “Food .4nalysis,” z j o (rgzq).
INFLUENCE O F ETHYLENE GLYCOL UPON SOME REACTIONS
83 7
total inversion at 30°, A, is -18.30. This value was also found by direct measurement. Obviously the term log, (A A.,) in equation ( 2 ) is constant. If, then, log, (A, A.,) be plotted as ordinate against t as abscissa, we have a straight line of which K, the velocity constant is the slope. In finding K the point formula for a straight line was employed. The difference of log, (A, - Q) for two different times was divided by the difference of the times. The average of all such approximate values of K for a given experiment was taken as its characteristic velocity constant. The plot of values of log, (A, A.,) against t always approximated closely a straight line. The velocity constants for any set of curves showed good agreement. The average of all the values for a given set was taken as the representative velocity constant.
+
+
+
P
01
.01
I
9m,.>:&.*
Ea
so
FIG.2 Inversion of Sucrose by Invertase in presence of Glycol
TABLE I11 The Inversion of 3 Per Cent Sucrose with Invertase Per cent glycol by volume
K per minute
0.00
-0.109
5.00
-0.048
20.00
-0.027
35.00
-0.013
50.00
-0,005
75.00
-0,002
Table I11 gives the average velocity constants for the inversion of solutions containing 3 per cent sucrose by weight together with a little acetic acid, as catalyzed by invertase, for various quantities of added ethylene glycol. The results are also set forth in Fig. 2 . The degree of inhibition for various per cents of added glycol, by volume, will be clear from this curve. Six separate experiments were run for each concentration of glycol.
838
L. MABEL YOUNG AND H. M. TRIMBLE
Turning, now, from reactions catalyzed by enzymes we studied the effect of added glycol upon the rates of inversion of sucrose in the presence of sulfuric and hydrochloric acids. All solutions contained 3% by weight of sugar. Solutions of sucrose and of acid were prepared in such volumes as when mixed would give I O O cc. of solution of the concentrations desired. They were separately brought to temperature, mixed and immediately introduced into the polariscope tubes. Four experiments were run for each con-
FIG.3 Inversion of Sucrose catalyzed by 0.50 N HCl I . No glycol K = -0.0103 L. 5 per cent glycol K = -o.oogo 3. 20 per cent glycol K = -0.m80 4. 35 per cent glycol K = - O . O I Z O 5. 50 per cent glycol K = -0.0168
centration of glycol with normal and half normal sulfuric and hydrochloric acids, two each for . z normal sulfuric and hydrochloric acids. Space will not permit the presentation of all the data, but those for the rate of inversion catalyzed by 0.50 normal HCl may be taken as typical. They are presented graphically in Fig. 3 and the corresponding velocity constants, taken from Table IV, are given. The data for all these experiments are presented in Table IV, where the velocity constants for various concentrations of glycol and acid are given. They are represented graphically in Fig. 4, where K is plotted against per cent of glycol.
INFLUENCE O F ETHYLENE GLYCOL UPON 60ME REACTIONS
80
IO
EO
40
o
io
so
so
40
FIG.4 Inversion of Sucrose by Acids in presence of Glycol I. 0.2 N HzSOI 4. 0.50 N HC1 2 . 0.2 N HC1 5. 1.00 N H ~ S O I 3. 0.4928 N HzSOI 6. 1.00 N HC1
TABLE IV The Inversion of 3 Per Cent Sucrose Catalyzed by Acid Per cent glycol by volume
0.20
N. HiSOi
N. HC1 per min.
0.20
K per min.
K
0
-0.0030
-o.0040
5
-o.0060
20
-0.0036 -o.0030
35
-0.003 2
-0.0063
50
-0.0040
-0.0068
Per cent glycol by volume
0.4928 N.
HzSOi K per minute
0
-0.0042
5
-0.0065
20
-0.0045
35
-0.005 I
50
-o.0060
Per cent glycol by volume
1.00
N. HzS04
K per min.
-0.0050
0.50 N.
HCI
K per min. -0.o 103 -o.0090 -0.0080 -0.0120
-0.0168 N. HCl
1.00
K per min.
0
-0.0130
-0.0240
5
-0.012 9
-0.0267
20
-0.0140
-o.0300
35
-0.01
70
-0.03 IO
50
-0,0220
-0.0500
839
so
840
1,. MABEL YOUNG AND H. M. TRIMBLE
The strange nature of the results will be apparent from the nature of the curves. When our first experiments revealed these peculiarities we returned and repeated them with great care, making every effort to remove all sources of error. We feel great confidence in the essential correctness of our results. In confirmation of their essential correctness we have the work of Ganguli and MalkanL5 In an article published since this work was completed they have set forth the results of a study of the catalysis of the inversion of sucrose by 0.1normal HCl as modified by added ethylene glycol. In general their curves resemble those found by us in these experiments. We are quite a t a loss to account for the effect of ethylene glycol upon the rate of inversion of sugar in the presence of acids. One of us has proved that, in concentrations as great as 5 mols per liter, it does not react appreciably with HCl or H2S04. The concentrations of acid were as high as 3 mols per liter and the time as long in some experiments as five weeks. The esterification with acetic acid, on the other hand proceeded regularly and normally. I n another study made in this laboratory, whose results are soon to be published, no sign of esterification was found with either HCl or HzS04. The acid concentration ran as high as molar with as much as I O mols of glycol present per liter. The hydrogen electrode method was used in making the measurements. Determinations of hydrogen ion concentrations using both the hydrogen electrode and the quinhydrone electrode have failed to reveal any such alteration of hydrogen ion concentration with concentration of sucrose or glycol as would be required to explain these irregularities. Compound formation between sucrose and glycol, it seems, would show more regular alterations with increasing concentrations of glycol, if this is the factor which is responsible. I t has been found by Scatchard6 and others that the hydrogen ion concentration of a solution changes as inversion proceeds with HCl as catalyst. The change is small, however, not more than I O millivolts a t the most. Scatchard, in fact, is inclined to attribute it to an alteration in boundary potential as shown by the potential of the cell used. Pennycuick' states that the hydrogen ion activity increases regularly during inversion by I to 3 per cent, according to the strength of the acid. It should be noted that both these workers inverted very much more sucrose in their experiments than we did. We, too, have found indications of a slight increase in hydrogen ion activity as the inversion of sucrose progresses, using the quinhydrone electrode technique. The course of inversion in these solutions, however, did not in any sense parallel this change. I t does not seem possible that any change in hydrogen ion concentration which is possible in these solutions could have produced the irregularities which we have found. Neither does it seem reasonable to attribute the irregularities to alterations in the degree of hydrolysis of the sucrose or alterations in the viscosity of the solutions.
J? Phys. Chem., 35, 2368 (1931). J. Am. Chem. SOC.,48,2026 (1926).
' J. Am. Chem. SOC.,48,6 (1926).
INFLUENCE O F ETHYLENE GLYCOL UPON SOME REACTIONS
841
Probably explanation of such results as those which we have found in all the cases which we have studied must wait until a better understanding of the nature of the combination between a catalyst and its substrate has been gained.
Summary The effect of ethylene glycol upon the rate of destruction of urea by urease, upon the rate of fermentation of sugars by yeast, upon the rate of inversion of sucrose by invertase and upon the rate of inversion of sucrose by acids has been studied. I t is found that enzyme activity is inhibited by the addition of ethylene glycol, and this inhibition is nearly complete if as much as 7 5 per cent by volume be added. The inversion of sucrose with acids as catalysts is sometimes promoted, sometimes retarded by the addition of glycol. Xovcnaber 24, 1931.
