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A S A L Y TICAL EDITIOi1'
Vol. 2 , K O. 1
Determination of Liquefying Power of Malt Diastase' S. J 6 z s a and H. C. G o r e FLEISCHMAN LABORATORIES, S E W YORE,N. Y.
I
T IS generally recognized that two enzymes exist in malt, one of which liquefies starch paste, the other then saccharifying it. Up to the present time no exact method has existed for measuring liquefying power. This is a consequence of the peculiar nature of starch paste. The senior writer observed that cold starch paste flows in nonuniform layers, showing even in low concentration the structure of a slimy liquid. Stirring by hand did not break down this structure. A high-speed mixer, however, was effective. A uniform, remarkably stable, highly viscous liquid, very different from starch paste produced with slower stirring, resulted. By the use of this starch paste it has been possible readily to measure the liquefying power of diastase, with an excellent degree of precision. All of the starches triedpotato, corn, rice, wheat, and Canna edulis-gave this result. Potato starch was selected, however, for use in developing the liquefying power method here described, as it is readily obtainable in a state of high purity. Fluid starch pastes containing as much as 10 per cent of starch may be made by this method. Five per cent potato starch paste, however, had the most satisfactory concentration for use when the viscosity changes on liquefaction by diastase were determined by pipet. When a hot 5 per cent paste of potato starch is subjected to high-speed stirring, the lumpy, jelly-like mass breaks down in a few minutes to a comparatively thin limpid solution. On cooling, it thickens slightly and loses some of its transparency. If once more beaten in the cold with the high-speed stirrer, the viscosity again decreases. The paste now no longer possesses a nonuniform structure. The viscosity of such a starch paste remains substantially unchanged for many hours. After the development of the stable starch paste of constant initial viscosity, the next step was the determination of the relation between the viscosity of the starch paste and the percentage of starch liquefied a t any stage during the action of diastase. A sample of the starch paste was liquefied completely and boiled to destroy the enzyme, after which a series of mixtures with the original starch paste was prepared. The viscosity (outflow time) from the pipet of this series of mixtures was then determined. From the curve obtained by plotting the percentage declines in viscosity against the amounts of liquefied starch, the percentage of starch liquefied by diastase acting under the standard conditions could be determined. As the delivery rate of a viscous liquid depends in large measure on such factors as the size and shape of the tip and the diameter and length of the delivery tube, each pipet gives a curve of its own. However, the curves will be nearly identical with the one shown in Figure 1. Once the curve for a given pipet has been constructed determination of the rate of liquefaction of the starch paste by diastase can be made with great facility. It is only necessary to determine the outflow time after the diastase solution has been incubated with the starch paste for the time specified. The temperature must be carefully controlled. The results are obtained directly in terms of enzyme and substrate. The details of the method and examples of its use follow. The method consists in determining the weight of starch liquefied in 1hour by 1part of malt when 100 grams of starch 1 Received September 20, 1929. Presented before the Division of Sugar Chemistry a t the 78th Meeting of the American Chemical Society, Minneapolis, Minn., September 9 t o 13, 1929
paste containing 4.211 grams of dry starch is acted on by 10 cc. of malt infusion containing 10 mg. of malt a t 21' C. for 1 hour. Equipment (1) A high-speed mixer. This is the stirrer used for mixing drinks a t soda fountains. It consists of a Hamilton Beach Universal motor, with vertical shaft, provided with a silver-plated hexagonal lenticular button, 1inch a t its greatest diameter. The speed without load is about 11,000 revolutions per minute. (2) An enameled saucepan about 6 inches wide a t the bottom, 9 inches wide a t the top, and 5 inches deep. (3) A 100-mesh brass sieve. (4) Several precipitating jars, 2.5 inches (6.3 cm.) wide a t the bottom, 2 inches ( 5 cm.) wide a t the top, and 5 inches (10 cm.) high. (5) A 15-cc. pipet. (6) A 100-cc- water-jacketed pipet. The water jacket should contain a thermometer. The specifications of this pipet should be those given by the U. S. Bureau of Standards The outflow time for water should be within 55-57 seconds. The delivery tube of this pipet is marked 2 inches (5 cm.) below the bulb. With a n 81 per cent glycerol solution of specific gravity 1.2138 a t 20"/20° C., the time of drainage from mark to mark should be 210-250 seconds a t 21' C. The tip of the pipet should have a gradual taper of about 2 cm., with a well finished orifice. Pipets with longer, and especially with irregular, tapers should not be used. The inside diameter of the delivery tube is preferably about 4.5 mm. (7) A stopwatch recording a t least 0.2 second. (8) One or more accurate thermometers reading to 0.2" C. (9) Potato starch of highest obtainable purity. This should be of the B.K.M.F. grade, obtainable from Joseph Morningstar and Co., New York, N. Y., or of a n equally high grade. P r e p a r a t i o n of Standard S t a r c h Paste
Heat to boiling 1800 cc. of distilled water in an enameled tared saucepan. A stout glass stirring rod may conveniently be weighed with the saucepan. Weigh out a portion of potato starch of highest obtainable purity which contains 84.22 grams of dry matter. The required weight of starch containing 15.78 per cent moisture will be 100 grams. Mix the starch with about 200 cc. of water a t 45-50' C. and, while kept suspended in the water by vigorous stirring, add to the vigorously boiling water in the pan. To avoid burning, immediately remove the pan from the fire. Stir the thick mass which results with the high-speed mixer for 3 minutes. During the mixing the thick masses which sometimes adhere to the sides and bottom of the pan are dislodged by the glass rod. Now place the pan with the starch paste in cold water and stir with the glass rod several times during cooling. This is necessary to prevent the formation of surface films. IJ7hen cooled to about 24-25' C., add 50 cc. of Walpole's acetate buffer, pH 4.6 (102 cc. of normal acetic acid and 98 cc. of normal sodium acetate diluted to 1 liter). The resulting pH is about 5.0-5.2. Then make the weight of the starch paste up to 2000 grams with distilled water, and stir the paste with the high-speed mixer for 30-45 seconds to obtain uniform and thorough mixing. Then allow the paste to flow through the 100-mesh sieve for the purpose of removing any small lumps. If the technic has been correct a few small lumps only will be present. It is now ready for use. The starch paste prepared in this way, when mixed with 10 per cent of its weight of water and stirred for 1 minute, should give the same outflow time a t 21" C., within 10-15 seconds, as the glycerol solution specified.
11%-DCSTRIALA.VD E S G I S E E R I S G C H E X I S T R Y
January 15, 1930
If a different make of stirrer is used different results may be obtained. A few tests in which the time of stirring is varied, however, should show the proper conditions for the preparation of the starch paste. Determination of Liquefying Curve
Two lots of starch paste are prepared. (1) NON-LIQUEFIED STARCH PASTE-This is made up of a mixture of 6 lots, each consisting of 150 grams of original starch paste stirred for 1 minute a t 20.5" C. with 15 cc. of water. (2) FULLY LIQUEFIED STARCH-This is prepared by warming 900 grams of the original starch paste to 65" C. and adding 90 cc. of a comparatively strong infusion of malt (20 grams of distillers malt plus 200 cc. of water digested 1 hour a t room temperature and filtered). After standing for about 1 hour a t 65" C. the solution is heated to boiling and cooled, and the original weight (990 grams) is restored by adding water. 8 m n Z
A?,,;, d;,we/ied
J&ih
Jo
24
4"
50
60
90
80
0
800
____ .___ -
'
-
----
________-
clear filtrate to 1 liter and use 15 cc. in each determination. Weigh out 150 grams of the starch paste in a precipitating jar and adjust the temperature to 20.5" C. Then add 15 cc. of malt infusion, and immediately stir the starch paste and malt infusion for 1 minute with the high-speed stirrer. .4t once cool the mixture to 21 " C. and let stand for 1 hour at 21 O C. Record the outflow time. The initial outflow of time is determined in the same manner, using 15 cc. of water instead of the malt infusion. This should be repeated a t least once. EVALUATION OF RESULTS-The liquefying power is equal to the weight of starch liquefied divided by the weight of malt taken. As in the method described, 100 grams of starch paste 0'04211, rvhere is acted on by 10 mg. of malt, 15.P. = 0.01 is the percentage of starch liquefied. The average initial outflow time of the starch paste, stirred with water as specified, was 240.6. The final outflow time was 52 seconds, the relative outflow time, the total range within which the viscosity changed during liquefying thus being 188.6 seconds. Three samples of infusion of finely ground malt gave the results shown in Table 11.
.
_--
__ _____
27
-
---
-----
i
Table I1 OUTFLOWTIME FALLIN OCTFLOW TIME Seconds Seconds Per cent
MALT 1 Distillers 2
P a l e brewers
3
Brewers
117 7 118 4 134.6 132.2 136.4 133.2
122 9 122 2 106.0 108.4 104.2 107.4
65 1 64 7 56.2 57.5
55,2 56.9
The quantities of starch liquefied read directly from the curve (Figure 1) and the liquefying power of the samples were as shown in Table 111. -
my
a;. O m r h
Table I11 coo
2 000
000
A,7uy'iired
Figure 1
These lots are mixed, not using the high-speed stirrer, in varying proportions corresponding to different percentages of nonliquefied and fully liquefied starch, the temperatures adjusted to 21" C., and the outflow times recorded. The declines in outflow times, expressed in percentage, are plotted against milligram. of starch liquefied. The curve is given in Figure 1. Data obtained by the senior writer are given in Table I. Table I hION-
LIQUEFIED STARCH
FULLY LIQUEFIED STARCH
PASTE Grams
Grams
165 148 5 132.0 115.5 99.0 82.5 66.0 49.5 33.0 16.5 0
0 16.5 33.0 49.5 66.0 82.5 99.0 115.5 132.0 148.5 165.0
PASTE
MALT
STARCH
LIQUEFIED
Paste Per cent 0 10 20 30 40 50 60
io
so
90 >a0
Dry
Ma. 0 421 842 1263 1684 2106 2527 2948 3369 3790 4211
OUTFLOU'
RELATIVE OUTFLOW
TIMES TIMES Seconds 235.4 185.3 144.7 117.5 96.3 81.9 70.9 63.5 57.7 54.2 52.0
183 4 131.3 92.7 65.5 44.3 29.9 18.9 11.5 5,7 2.2
0.0
DECLISE IS
OUTFLOW
TIME Per cent 0.00 28,41 49 46 64.29 75.85 83,70 89.70 93.i 3 96 89 98.80 100,00
Figure 1 shows the percentage decline in outflow time plotted against percentage of starch paste and milligrams of dry starch liquefied. Application of Method to Determination of Liquefying Power of Malt
PREPARATION O F h 1 a L T ExTRAcT-Mix 20 grams Of the finely ground malt with 1 liter of water and allow to stand for 1 hour with occasional mixing. Then filter the infusion, rejecting the first 50 cc. of the filtrate. Dilute 50 cc. of the
STARCH Per cent
LIQUEFIED LIQUEFYING POWER
1
65.1 64.7
MR. 12s5 1275
0
56.2 57.5
1015 I050
3
55.2 56.9
993 1045
128,s
127,5 Av.127.0 101.5 105,o Av. 103,2 99.5 104.5 i l v 102.0
Discussion
The viscosity of the starch paste is remarkably sensitive to temperature. Although fairly permanent in its viscosity, fresh starch paste must be made up for each day's work. The viscosity of the starch paste is obviously of cardinal importance. We have had no difficulty in obtaining approximately the same viscosity in starch pastes made on different days, within 5 seconds. Thus 150 grams of starch paste were stirred as specified with water. The outflow time measured immediately after mixing (after cooling to 21" C.) was 234.6 seconds. After standing for 1 hour a t 21" C. it was 232.4 seconds. A second fresh 150-gram portion of the starch paste was then stirred for 1 minute with 15 cc. of mater and cooled to 21" C., after which its viscosity was determined. The outflow time was 236.2. This shows the constancy t o be expected. Sometimes, however, we have observed wider variations, probably due only to lack of exact temperature control. Although any type of viscometer may be used, we have chosen the pipet method as the simplest and most generally available. It is obvious that not only the viscosity a t the end of an hour can be measured but also viscosities a t the end of any desired period, in order to observe liquefying action as completely as may be desired. Once the method of preparation of the starch paste has been
AXALYTICAL E D I T I O S
28
Vol. 2, s o . 1
mastered and the liquefying curve established, determinations may be made with facility and exactness. Acknowledgment
Smith fellowship for Hungarian engineers, whose generosity made the journey to America possible. Bibliography
The senior author wishes to express his appreciation of the kindness of Jeremiah Smith, Jr., former Cnited States Deputy of the League of Nations to Hungary, donor of the Jeremiah
Oppenheimer and Kuho, "Die Fermente und ihre Wirkungen," Thieme, 1925. Sherman, Kendall, and Clark, J Chem soc , 8 2 , 1073 (1910) Sherman and Schlesinger, Ibid , 35, 1617, 1784 (1913).
Determination of Chlorides in Salt Brines' S. L. Neave ILLINOIS STATEWATERSURVEY, URBANA, ILL
I
N A cooperative study with the Illinois State Geological Survey of the oil wells of this region, some 200 complete mineral analyses had to be made on brines from these wells. The analyses were reported both in the ionic form and in hypothetical combinations. Difficulties arose in making the combinations because the concentrations of sodium and chloride so greatly exceeded those of the other ions that small, normally tolerable, errors in their determination seriously affected the other hypothetical salt combinations. The following procedure, adopted to attain the necessary accuracy, without prohibitive expenditure of time and effort, has been in successful use for the last five years. Analytical Procedure
The procedure requires (1) a preliminary titration to determine the approximate chloride content, and hence the dilution required by the sample, after which an accurate dilution is made and aliquots of it used for the determination both of chloride and of sodium and potassium; (2) a gravimetric determination of chloride, the precipitant being standard silver nitrate; and (3) a back titration of the excess silver nitrate in the filtrate. For the rough titration, dilute 10 cc. of sample to 100 cc. and treat 5 cc. of the mixture with 10 cc. 0.1 N silver nitrate in the presence of dilute nitric acid, the excess silver nitrate being titrated in the usual way with 0.1 N potassium thiocyanate using ferric alum indicator. Compute the approximate number of milligrams per liter of chloride ion, using the dilution factor 2000. Then prepare an accurate dilution in accordance with Table I and treat the aliquot of this (diluted if necessary to 50 cc. with distilled water) with 1 cc. concentrated nitric acid and 25 cc. 0.1 N silver nitrate (or 50 cc. for chloride contents over 64,000). Coagulate the precipitate by warming and vigorous agitation and filter on a weighed Gooch crucible, washing twice by decantation with hot 2 per cent nitric acid before transferring to the crucible, and then once or twice on the crucible with hot water. Dry the silver chloride for 24 hours at 115-125" C. Table I-Dilutions CHLORIDE (APPROXIMATE) Mg.per irter
Required by S a l t Brines DILUTION A L I Q ~ OTAKE.\ T FOR AXALYSIS REQUIRED
o-imn
1 7
Table 11-Sample Analytical D a t a GRAVIMETRICVOLUMETRIC AVERAGE M g per l r k r .ME. p e r tiler M g . per liter 67190 67072 67131 25362 25315 25338
ERROR P e r cent 0 18 0 14
Sources of Error
Perfect agreement between the results obtained by the two methods is not to be expected. The gravimetric procedure gives high results on account of adsorption by silver chloride of other salts. Thus, uThen increasing concentrations of pure sodium chloride are precipitated, the positive errors in the gravimetric results increase logarithmically in accordance with the adsorption isotherm. Some of the experimentally determined differences, with constant total volume and quantity of silver nitrate, are shown in Table 111. Table 111-Adsorption Errors i n Silver Chloride Precipitation 4gC1 PRECIPITATED Calcd. Found DIFFERE~CE Gram Gram -VIg 0.11516 0.11524 0.08 0.18362 0,18372 0.10 0.23799 0.23810 0.11 0,34100 0.34112 0.12 0.37801 0.37814 0.13 0.47995 0,48010 0.15 0.49017 0.49032 0.15
Furthermore, if the precipitates are reduced t o metallic silver and reweighed as such, these errors disappear. The volumetric method tends to give low values, as a result of both adsorption and end-point errors, though these influences can be minimized by producing the same weight of silver chloride (0.3 gram) in the standardization of the solutions as in the analysis. The average of the two methods has been found to closely approach the true value of the chloride (determined as metallic silver), and becomes identical with it when a number of determinations are averaged, as seen in Table IV.
cc.
cc. 50 25 50
Chloride C o n t e n t for T e n Analyses of a S o d i u m Chloride S o l u t i o n PROCEDCRB M g . per cc. Alean gravimetric 3 587 Mean volumetric 3 576
25 255
Average Gra5imetric (as Ag)
~
50 50
Titrate the filtrate and washings with 0.1 N potassium thiocyanate, with ferric alum indicator, and calculate the chloride from the silver nitrate used in the precipitation. The difference between the gravimetric and volumetric reReceived September 4 1929.
ExaniPLE
None .~
1606-3200 Pione 100-500 3200-8000 8000-16000 50-500 50-1000 16000-32000 50-1000 32000-64000 50-1000 64000-120000 50 cc. AgNOa required for this,
1
sults should not exceed 0.4 per cent of their average, and is usually much less. Persistent differences of 0.4 per cent or more indicate the presence of bromine or iodide in determinable amounts. Two random examples are shown in Table 11.
Table IV-Average
3 5815 3 581
Acknowledgment
The investigation here reported was conducted under the direction of A. M. Buswell, chief of the Illinois State Water Survey and a member of the Joint Committee on Standard Methods for the Examination of Water and Sewage.
