382
I S D USTRIAL A S D ENGIXEERISG CIIELL’IXTRY
is also of interest. The Lessing rings present the greatest surface, the glass tubes are intermediate, and the glass rods present the least. At first thought it seems logical, and is, in fact, frequently assumed, that greater surface produces better fractionation. The curves indicate that, in these experiments, the reverse is true. A careful consideration of the three packings will reveal that, while the Lessing ring offers greater surface, it also allows a more direct path for the vapors, permitting them to ascend without being forced into such intimate contact. By the same reasoning, the glass rods force the greatest contact, since they necessitate more drastic changes in direction of the vapors. Obviously, the Lessing rings are better adapted to high rates, and this is shown by the curves. It would therefore appear that greater surface of packing increases efficiency only if the effect of greater surface is not offset by losses in intimacy of contact. SHORTER COLUMNS-A second comparison of a series of shorter columns of the more popular designs was made in exactly the same way but a t only one rate of distillation. This series included Pear, Wurtz, LeBel-Henninger, Glinsky, and Vigreux. These are illustrated in Figure 4. A short packed column similar to 3a was also included for the pur-
\’d.19. No. 3
pose of correlating these result. 1% hose on the longer columns. Tests were also made on e !, ‘ I column without the insulation and controlled reflux, as +‘le,dumns are normally run. A straight tube without packing was also tested. The data on the shorter columns are shown in Figure 6. It will be noted that the Wurtz, Pear, and LeBel-Henninger are very little more efficient than a plain unpacked tube. The efficiency is actually less with the controlled reflux, showing that these columns owe what value they do have to the fractional condensation which takes place in them. The Glinsky and Vigreux are considerably better, as would be expected because in these a fairly intimate contact between liquid and vapor is effected. The Glinsky floods a t a low rate, but the Vigreux was found to have a high capacity of 2.5 cc. per minute distillate at a 9 : l reflux ratio. The Vigreux, however, a t the higher rates is much less efficient than the packed column. Acknowledgment
The authors wish to acknowledge the services of J. A. Alexander and F. A. Brill, Jr., who carried out a large part of the laboratory work.
Effect of Temperature on Bating‘ By Henry B. Merrill A. F. GALLUN & SONSC o . , MILWAUKEE, WIS.
The effect of temperature upon deplumping, digesvaried to give a n optimum HE effect of temperation of keratose, and digestion of collagen has been result, but i t by no means ture upon so complex studied. The rate of deplumping increases somewhat follows that the best results an operation as bating with temperature. Over the bating range, the rate of obtainable a t a given temis necessarily equally comdigestion of keratose is about doubled for a 10’ C. rise perature are as good as the plex. At least six different in temperature. In the same range, the rate of digesb e s t r e s u l t s obtainable at functions have been ascribed tion of collagen increases threefold between 15” and some higher or lower temperat o b a t i n g , 2 n a m e l y , (I) 25’ C. and nineteenfold between 25” and 35” C. ture. rendering the skin flaccid, The effect of temperature ( 2 ) adjuiting the hydrogenion concentration of the solution adhering to the skin, (3) on three of the important functions of bating will be conremoval of lime, (4) digestion of elastin fibers, ( 5 ) par- sidered separately in this paper. The functions considered tial digestion of collagen, and (6) digestion of keratose, or are (1) rendering the skin flaccid, or “deplumping,” ( 2 ) the degradation products of keratinous structures partially digestion of collagen, and (3) digestion of keratose. Of the broken down by lime. The first three functions, concerning remaining roles played by bating, the removal of lime and which there is no question, are performed chiefly by the adjustment of pH values are probably very little affected inorganic buffer salts contained in the bate. The last three, by temperature. The effect of temperature on elastin digesconcerning which there still exists considerable uncertainty, tion will be considered at a later time. are performed by the pancreatic enzymes. The effect Effect of Temperature on Deplumping of temperature upon bating, then, is the resultant of the separate effects upon some six independent processes, which I n contact with alkaline solutions, such as the lime liquors may be, and are, influenced to very different extents by the commonly used for unhairing, skins become very much same change in temperature. swollen, or “plumped.” The swelling of skins is (in the The effect of temperature upon bating is of much practical absence of appreciable quantities of neutral salts) a function importance. I n general, the tanner’s problem is to adjust of the pH values of the solutions with which they are in the three independent variables under his control (time of contact. When skins are bated the ammonium chloride, bating, temperature of bating, and quantity of bate used) or other buffer salt employed, reduces the pH value of the so as to give the best possible result. Too protracted a solution from about 12.5 to about 8. This fall in p H value bate, too strong a bate, or too warm a bate may cause over- is accompanied by a decrease in swelling of the skins, which bating and damage to stock. The most common method go from a plumped to a “fallen” condition. of control probably is to fix arbitrarily the temperature Temperature may influence this deplumping process, and concentration factors and vary the time factor to get either by affecting the degree of deplumping obtainable at the best results. Although this practice works, a knowledge equilibrium a t a given pH value or by affecting the rate of the effect of temperature is also much to be desired. For at which this equilibrium is established. The effect of a any one temperature the time and concentration may be change in temperature of bating upon both the rate and 1 Presented before the Division of Leather and Gelatin Chemistry the ultimate extent of deplumping was studied, using the at the 72nd Meeting of the American Chemical Society, Philadelphia, Pa., method described by Wilson and Gallun.3 Pieces about
T
September 5 to 11, 1926. t Wilson and Merrill, THIS JOURNAL, 18, 185 (1928).
3
TIXISJOURNAL, 16, 71 (1923).
INDUSTRIAL A N D ENGINEERING CHEMISTRY
March, 1927
one-half inch square were cut from the butt of a calfskin, after liming, unhairing, arid washing, but before bating. The thickness of these pieces was measured with a Randall and Stickney gage, allowing the plunger to rest on the skin for exactly two minutes before taking the reading. The pieces, suitably marked, were then placed in bottles containing 250 cc. of amnioniurn ch!oride solutions, with or wit'liout the addition of enzyme; the bottles were immersed in thermostats; and the digestion was allowed to proceed for a definite time. At the end of the experiment, the pieces were removed from the solutions and their thickness was remeasured. The difference between the initial and final thickness, divided by the former, gave the decrease in initial thickness, which was taken as a measure of cleplumping. From three t o five pieces were used for each t'est, and the results averaged. l3efoi.e studying the temperature effect, it mas necessary to determine the effect of ammonium chloride concentration, t,he effect of enzymes, and the time required to attain equilibrium a t some one temperature, in order t o fix experimental conditions. It was found that, as the concentration of ammonium chloride was increased, the degree of depluinping aleo increased until enough ammonium chloride was prefent to react with all the lime. Furt,her additions of ammonium chloride had very little effect upon pH value, and consequently none on the extent of deplumping. The presence of enzymes increases the deplumping, but only when they are present in quantities much greater than are ever used in bating, the effect being due, seemingly, to the actual destruction of a considerable portion of the skin. The effect of temperature on the rate of depluniping was studied by running series of experiments at different temperatures, making the time of digestion the independent variable in each series. The results of these tests are given in Table I. The concentration of ammonium chloride used lies in the range where further increases of concentration do not much affect the extent of deplumping. No enzyme was added in these experiments. of Temperature o n Rate of Deplumping (0.864 gram ammonium chloride per liter)
Table I-Influence
' I
HGUYS 0.5 1.0 2.0 4.0 17.0
D E C X E A S E I N I N I T I A L THICKNI?SS AT
,
c.
30' C.
P e r cent
Per cent
P e r cenl
I
9.9 15.8 15.5 20.3 22.2
22.7 22.0 27.2
23 0 29.7 28.2
29.6
29.4
250
...
...
3.50
c
Per cenl
I n general, it may be concluded that temperature has little effect on deplumping, when changes of a few degrees are involved. If, however, it were desired to change from a bate a t 30" C. to one 20 degrees lower, a somewhat longer time of bating would be required to effect complete deplumping, aside from other considerations.
2 3 4 5 6 Time digested (hours) Figure 1-Rate of Solution of Keratose (Keratose 2 Grams per Liter; Trypsin No. 6, 0.02 Gram per Liter) a t Diffe;ent Temperatures 1
Effect of Temperature on Rate of Digestion of Keratose The effect of temperature on the rate of a reaction is usually measured by determining the velocity constant of the reaction a t different temperatures. The quotient of the reaction velocity constants at temperatures 10 degrees apart is commonly taken as the temperature coefficient of the reaction for the particular interval in question. For most reactions the temperature coefficients, measured in the neighborhood of room temperatures, are between 2 and 3. It is not possible to obtain satisfactory values for the velocity constant of the hydrolysis of keratose in the presence of trypsin, because, generally speaking, the course of the reaction cannot be expressed in terms of any of the familiar
r
I
1
1
L
2.5.1 26.7 28.7 28.6 28.7
The speed with which complete deplumping is attained increases with the temperature. Thus, a t 30" and 35" C . virtually complete deplumping was reached in from 1 to 2 hours; at 25" C. a somewhat longer time was required; and a t 7 " deplumping was not complete in 17 hours. The difference in rate is not rery marked, however, except in passing from moderate to low temperatures. To determine the effect of temperature on the extent of deplumping at equilibrium, a series of experiments was carried out at temperatures increasing from 10" C. by increments of 5" C., the time of digestion being 18 hours in all cases. The results of these tests (Table 11) show that the extent of deplumping increases somewhat with increasing temperature. Here again the effect is not appreciable except when rather wide variations in temperature are in question. Table 11-Influence
383
of Temperature o n Deplumping (18 Hours) (0.864 gram ammonium chloride per liter) 10 15 20 25 30 35 40 Temperature, C. Decrease in thickness, per cent 2 2 . 9 2 1 . 8 2 3 . 8 2 4 . 9 2 2 . 6 25 4 24 4
1
b
2 3 4 5 Time digested (hours) Figure 2-Rate of Digestion of Keratose (Keratose 2 Grams per Liter; Trypsin No. 6, 0.02 Gram per Liter) a t D i f f e r k t Temperatures
equations of chemical kinetics. The reason for this probably is that the enzyme itself is destroyed during the progress of the reaction, so that the rate of decomposition of the substrate falls off more rapidly than would be predicted from the mass law. To measure the rate of hydrolysis of keratose it is, therefore, necessary to measure the time required for the digestion of some specified fraction of the total keratose present, under definite conditions of concentration of substrate and enzyme.
INDUSI'RIAL A N D ENGrINEERING CHEMISTRY
384
Wilson and Merril12,4showed that, for a fixed concentration of keratose, the time required for the digestion of a definite fraction of the substrate is inversely proportional to the concentration of enzyme. They have described a method for measuring the activity of different enzymes on keratose, the results being expressed in terms of l/hg, where h is the number of hours required for the digestion of 40 per cent of 2.0 grams of keratose, contained in 1 liter of solution of p H 8.0, at 40" C., and g is the concentration of enzyme in grams per liter. I n making the determination, g is fixed arbitrarily and h is measured by determining the fraction of the total keratose digested a t difTerent time
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Vol. 19, No. 3
time, however, the enzyme may have lost so much of its activity at 50" C. that the reaction practically stops, so that a very much longer time is required to digest 50 per cent of the substrate a t 50" C. than at 40" C. Different enzyme preparations undergo inactivation at very different rates, the less powerful preparations being, in general, more resistant to temperature inactivation than more highly purified materials. From this it follows that temperature coefficients found for one enzyme specimen cannot safely be used for others without further tests. These conclusions are fully supported by the following experimental results : The rate of digestion of keratose was determined by the Wilson-Merrill method, a t 5-degree intervals from 15 " to 60" C. The enzyme used, one of those employed in earlier experiments4 and designated as No. 6, was the same a t all temperatures. The percentage digested was plotted as a function of time (Figures 1 and 2) and the time required for the digestion of 20, 30, 40, and 50 per cent of the total substrate was read off from the curves for each temperature. l/hg was calculated for the different fractions to total digestion for each temperature. The values so obtained are collected in Table I11 (columns 2 to 5). Temperature coefficients were calculated by dividing l/hg for each temperature into l/hg for the temperature 10 degrees higher. These coefficients are collected in Table I11 (columns 6 to 9 ). Table 111-Effect of Stage of Reaction on Apparent Temperature Coefficient of Digestion of Keratose by Trymin"
4 5 6 Time digested (hours) Figure %-Rate of Digestion of Keratose (Keratose 2 Grams per Liter; Trypsin No. 9, 0.002 Gram per Liter) a t DifferLnt Temperatures 1
2
3
intervals, plotting the percentage digested against time and determining the time required for 40 per cent digestion by interpolation. The percentage of keratose digested is determined by precipitating undecomposed keratose a t its isoelectric point, filtering and weighing, and subtracting the weight found from that taken initially. For further details of the method, the papers of Wilson and Merrill already referred to may be consulted. T o determine the effect of temperature on the rate of digestion of keratose, the obvious procedure is to measure l/hg a t several different temperatures, employing the same enzyme. This course, however, involves certain difficulties, as the following considerations will show. The net effect of temperature on any enzyme reaction is the resultant of two independent and distinct effects. Like most reactions, the digestion of a protein by an enzyme increases in velocity with rising temperature. On the other hand, the enzyme undergoes a spontaneous inactivation, which is more rapid the higher the temperature. Whether an increase in temperature will accelerate or retard, an enzyme action depends upon which of these two processes is most affected. At lower temperatures the change in the rate of spontaneous inactivation with temperature is relatively small. At higher temperstures ib is relatively large. The result is that the velocity of most enzyme reactions passes through a maximum a t about 40" C. The inactivation of an enzyme naturally becomes more apparent the longer the period of digestion, from which it follows that the relative rate of digestion a t two different temperatures will be determined in magnitude, and even in sign, by the fraction of substrate the digestion of which is taken as the end point of the reaction. At 50" C., for example, the hydrolysis may start much more rapidly thaq at 40°, and the time required for 20 per cent digestion may be much shorter a t the higher temperature. After a short 4
J . A m Lealher Chem. Assoc , 21, 2, 50 (1926)
I
oc.
16 20 25 30 35 40 45 50 55 60
I
I 14.9 21.3 31.3 38.5 71.4 83.3 125.0 161.3 166.6 90.9
8.3 11.9 17.8 25.0 41.7
60.0
71.4 86.2 96.1 43.5
5.1 7.6 12.2 17.2 26.3 33.3 47.6 53.2 57.5 23.6
3.2 6.8 8.0 13.2 18.2 23.0 33.1 34.3 25.6 b
2.1 1.8 2.3 2.2 1.8 1.9 1.3 0.56
2.1 2.1 2.4 2.0 1.7 1.7 1.3
0.50
... ...
I
2.4 2.3 2.2
1 .Q
1.8 1.6 1.2 0.44
....
2.6 2.3 2.3 1.7 1.8 1.5 0.78
...
grams keratose per liter; 0.02 gram enzyme No. 6 per liter; pH, 8.0. b Reaction stops. a 2
I
-i
I
I
I
f
4
I
I
I
Time digested (hours) Figure &Rate of Solution of Keratose (Keratose, 2 Grams per Liter; Trypsin No. 2, 0.2 Gram per Liter) a t Different Temperatures
The temperature coefficient is dependent on the stage of the reaction chosen as the end point in measuring the activity of the enzyme. At lower temperatures, the coefficient found is larger, the nearer to completion the reaction is allowed to proceed. At higher temperatures the temperature coefficient becomes smaller as the reaction progresses. .For example, consider the interval 45" to 55" C.
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I N D U S T R I A L A N D ENGINEERING CHEMISTRY
March, 1927
For 20 per cent digestion, the time required is 1.3 times as long a t 45" as a t 55"; for 50 per cent digestion, i t takes only 0.78 times as long at the lower temperature. That the magnitude of the temperature coefficient of the hydrolysis of keratose is dependent upon the nature of the enzyme employed is shown by the results given in Figures 3 and 4 and in Table IV. The rate of digestion of keratose was determined a t intervals of 10 degrees with two other samples, in addition to No. 6. One of these samples was a very weak enzyme and the other a very strong one. No. 6 occupies an intermediate position. The temperature coefficient falls off, with increasing temperature, much faster in the case of the strongest enzyme. All these values refer to 40 per cent digestion of the substrate. I n the temperature range available for practical bating, however, neither the purity of the enzyme nor the stage of the reaction has much effect upon the temperature COefficient. Generally speaking, bating is carried out between 25" and 35" C., and it is safe to say that 40" is the maximum temperature that can be employed for the process, as collagen begins to hydrolyze rapidly a little above this temperature. Confining our attention to the temperature coefficients for the range from 15" to 40", we see that nearly the same result is obtained whether the fraction of keratose hydrolyzed is 20 or 50 per cent, and whether the enzyme be crude or refined. Throughout this range the temperature coefficients are in the neighborhood of 2 , which means that for a 10-degree rise in temperature a given amount of enzyme will hydrolyze a given amount of keratose in half the time, or half the amount of enzyme will hydrolyze the given amount of keratose in the same time. Thus, SO far as the digestion of keratose is concerned, i t is possible to bate a t any temperature below 40 " by making suitable adjustments in the quantity of enzyme used or in the time of bating, or in both. Coefficient of Hydrolysis of Keratose w i t h Table IV-Temperature Different Specimens of Trypsina (l/hg)T + I , J ( ~ / ~ ) T
TEMPERATURE
45 a
41.7 102.0 263.0 379.0
5.1 12.2 26.2 47.6
0.6
i::: 6.85
1
No. 9
No. 6
2.4
2.4 2.2 1.8 1.2
?::. . .
No. 2 2.2 2 . 7 (?) 1.9
...
2 grams keratose per liter; x grams trypsin per liter.
Effect of Temperature on Digestion of Collagen
The method proposed by Wilson and Merril14 for estimating the activity of enzymes on collagen consists in determining the quantity of enzyme required to hydrolyze 20 per cent of a 0.5 gram sample of collagen (in the form of hide powder), contained in 100 cc. of a solution having a p H value of 8.0, a t 40" C., in 3 hours. The fraction hydrolyzed is formed by determining nitrogen in the filtrate from the undecomposed collagen. Experiments are run with different quantities of enzyme, the percentage hydrolyzed is plotted as a function of enzyme concentration, and the concentration of enzyme required to hydrolyze 20 per cent in 3 hours is determined graphically. Results are expressed in terms of l/g, where g is the required enzyme concentration. This method was applied to the investigation of the effect of temperature on the rate of digestion of collagen by running series a t 15", 25", 35", and 45" C. The results of these experiments are given in Figure 5, the curves representing the weights of collagen digested, after subtraction of blanks, plotted as a function of enzyme concentration. When attempts were made to compute l / g for the different
385
temperatures, it was seen that a t 15" the digestion proceeds so slowly, and the shape of the curve is such, that at no concentration of enzyme m i l l 20 per cent of the substrate be digested in three hours. l/g was therefore computed for the digestion of 10 per cent of the collagen taken. The results (Table V) are quite different from those obtained with keratose. Instead of a fairly uniform, and rather low, temperature coefficient over the range available for bating, the temperature coefficient for the tryptic digestion of collagen varies widely between different temperatures, and for certain intervals is very high. Thus, from 15" to 25" C., the activity of trypsin on collagen increases threefold, but from 25" to 35" C. it increases nineteenfold. This is exactly the temperature range within which bating is usually done. Table \'-Effect
15 25 35 45
of Temperature on Hydrolysis of Collagena
0.750 0.230 0.012 0.002
1.33 4.16 83.30 500.00
3.1 19.2 6.0
...
grams collagen (as hide powder) per liter; x grams trypsin KO.6 per liter; pH, 8.0; digested for 3 hours. g = grams per liter of trypsin required for disestion of 10 per cent of the total collagen in 3 hours. a 5.0
The fact that collagen is so very much more readily hydrolyzed a t 35" than a t a temperature 10" lower cannot but have very imp o r t a n t bearings on bating. If, as 6o is generally supposed, the hydrolysis of collagen is to be a v o i d e d , $ 2 0 then it would seem b t o follow t h a t 40 lower temperatures, with a longer time of bating or a bate higher in enzyme, should 2 give better results ;2o than the temperatures f r e q u e n t l y 10 employed a t present. Under such c o n d i t i o n s , deplumping and 0.2 0.4 0.6 0.8 keratose digestion Enzyme (grams per liter) of Temperature on R a t e of would go on to the Figure +Effect Hydrolysis of Collagen same extent as a t the higher temperature, but the collagen should be less att y k e d . On the other hand, it is by no means certain that the digestion of a part of the collagen is a bad thing. For some kinds of leather it is probably the chief end attained by bating. I n such cases, a drop of 10" in temperature would necessitate a very large increase in enzyme concentration to maintain the desired rate of solution of the collagen. It would seem that bating tests at different temperatures should be capable of throwing much light upon the question as to the real function of the enzyme.
