Kov.,
1911
T H E J O C R S A L OF I A - D U S T R I A L AND E L Y G I - Y E E R I ~ Y GC H E M I S T R 1 7 .
ranges of strength, say I t o 7 0 0 , I to 8 0 0 , I to 900, etc. By this is meant I gram of pancreatin to 7 0 0 cc. of milk. After it is found between what wide ranges the strength of the pancreatin lies, a second series of tests are made a t intermediate values until the exact strength of the pancreatin is found. In carrying out the test it was found necessary t o make the milk slightly alkaline with sodium bicarbonate to prevent the pancreatin from curdling the milk. This coagulation is probably due to the presence of an enzyme in pancreatin similar t o rennin. The materials required for the test are as follows: 0 . 5 gram pancreatin added to sufficient distilled water t o make 5 0 cc. of solution. goo cc. of milk containing 1.8 grams of sodium bicarbonate. z grams of rennin (I : 3 0 , 0 0 0 in IO minutes or equivalent) and I cc. of 6 per cent. acetic acid (U.S. P.) added t o jo cc. of distilled water. After warming the milk, place exactly j o cc. in a cylindrical tube of about IOO cc. capacity. Prepare several such tubes and place in a water bath maintaining the temperature a t 40 degrees C. Add t o the tubes of milk the following amounts of the pancreatin solution : cc. 8.33 7.69 7.14 6.66 6 25
(1 : 600) (1 : 6 5 0 ) (1 : 700) (1 : 750) (1 : 800)
In each case note the exact time when the pancreatin is added, mix well and after digesting fifteen minutes place j cc. of the digested milk in a test tube, add 3 cc. of the rennin solution and shake well. N o precipitate indicates that the casein has all been peptonized and that the pancreatin is stronger 'than the strength tested. For example if there was no precipitation a t I : 7 0 0 , but there was a precipitation a t I : 7 5 0 , then i t would be necessary to run more digestions between I .: 7 0 0 and I : 7 j o . Make a fresh solution of pancreatin and use the following amounts : cc. 7.04 6.94 6.84 6.75
(1 : 710) (1 : 720) ( 1 : 730) (1 : 740)
In this manner i t can be determined quite accurately how many times its own weight of milk a given sample of pancreatin will peptonize. I n order to get accurate results the test must be carried out strictly in accordance with directions. As stated above acid will precipitate peptonized milk, therefore, just enough acid is added t o the rennin solution so that 3 cc. of this solution mill neutralize the sodium bicarbonate in 5 cc. of the peptonized milk. Then the rennin will do its work, for it will not form the'precipitate in an alkaline solution. Attention must be called to the fact that pancreatin in a neutral solution deteriorates quite rapidly. Therefore this solution should be made up the last thingi;o it can be added immediately t o the milk. The amount of pancreatin solution suggested is SUEcient for testing the strengths as indicated.
823
It must be borne in mind that all tests of this nature are purely arbitrary ; consequently a slight variation in the carrying out of such a test would give different results. The method gives accurate results to within two or three per cent. which is closer than pepsin can be tested on egg albumen and equal to the accuracy of the starch methods for testing diastases. If this proposed test is carried out as indicated it will undoubtedly prove very satisfactory and will make it possible for one to know the exact proteolytic activity of a sample of pancreatin. In testing various commercial samples of pancreatin by the above test the following results were obtained: No. 1 : 280 1 : 120 1 : 130 1 : 670 1 : 8.50 1 : 1450
1 : 1750 1 : 1200 1 : 960
It will be noted that there is a wide variation in the tryptic value of commercial. pancreatins. The writer suggests if the above test meets with favor in the hands of other workers, it may serve as the basis for the official assay of the U . S. P. From the above examination of commercial pancreatins it would appear that a requirement of I to 800 or I to 1000would be a fair standard. CCIENTIFIC D B P A R T X E S T .
PARKE.DAVIS8; Co3re.%ru. DETROIT.
THE ACCELERATING ACTION OF HC1 UPON THE STARCHCONVERTING PROPERTIES OF PANCREATIN AND MALT.' B Y -4.Z l n l b l E R M A N
Received August 28, 1 9 1 1
A pancreatin of such strength that one part will convert 2 j parts of corn starch can be so accelerated that one part will convert 12j parts of corn starch and 2 5 0 parts of potato starch in j minutes. This acceleration of the starch converting action of pancreatin is accomplished by extremely small quantities of HC1, which are added when making the starch paste. The proportion of acid used is so small that it absolutely does not convert any starch into glucose, €or the proportion necessary to do this would destroy the pancreatin. The temperature a t which the conversion takes place is important ; when the pancreatin is added to the starch paste the latter should be a t 135' F. The pancreatin is added in solution a t 70' F. (no higher), so that the temperature after the addition of the pancreatin to the starch paste is about I z 5 O F. (not lower). The quantity of HC1 necessary is of one per cent., absolute acid, of the *weight of the starch. The starch paste consists of one part of starch to 16 parts of water, the HCl being added to the water before the addition of the starch. The mixture is then 1 Paper read before the Pharmaceutical Division. June meeting of the American Chemical Society. Indianapolis.
T H E J O U R N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y .
824
gelatinized and cooled t o 135' F. for the addition of the pancreatin dissolved in water. Infusion of malt very readily undergoes acid fermentation and the acid products thus formed accelerate the action of the malt upon the starch. It is for this reason t h a t malt infusion is less active upon starch when made neutral. The accompanying table shows the action of pan-
Nov., 1911
creatin and malt upon starch, under different conditions. The following tests were made with two different specimens of pancreatin, one converting 2 5 parts of corn starch and the other 35 parts of corn starch b y the U. S. P. test. The reactions are termed converted when there is no longer any reaction with iodine: Is:
3.8
i;
5
0
*a&%
'6
.
U?#AJ Q
Z Z ? 1
g h
P
Quantity of pancreatin Accelerated one part, HCI 0.242
Potato 250
210
After
5 minutes
2
Accelerated one p a r t , HC1 0 . 2 1
Corn 125
105
After 5 minutes
3
I n neutral solution one p a r t
Potato 50
42
After 5 minutes
4
In neutral solution one part
Corn 25
21
After 5 minutes
5
In alkaline solution one p a r t , sodium bicarbonate 3l/a parts
6
Corn 25
After 10 minutes
' / a hr.
1/2
hr. 20.74 1 br. 23.63 4 hrs. 27.00 1/2 hr. 12.60 1 hr. 13.27 4 hrs. 14.33
I h r . 64.22 4 hrs. 67.68 1/2 hr. 65.20 1 hr. 70.00 4 hrs. 79.80 I/? hr. 49.40 1 br. 56.24 4 hrs. 64.30 1/2 hr. 60 .OO 1 hr. 63.20 4 hrs. 73 .OO
1/2
hr.
1 hr. 4 hrs.
8.56 8.82 9.62
hr. 1 hr. 4 hrs.
9.06 9.45 10.56
l/2
Corn 25
I n a!kaline solution one part, sodium bicarbonate 1 6 . 6 p a r t s
Corn 125
21
105
After 5 minutes
After 4 hours
1/2
1/2
hr.
J
hr. 4 . 0 7 1 hr. 4 . 4 0 4 hrs. 4 . 6 0
1/2
1 hr. 42.00
4 hrs. 45.85
4 hrs. In alkaline solution one part, sodium birarbonate 1 . 6 p a r t s
Corn 125
105
After 4 hours
1/2
hr. 43.15
23.56
4 hrs. 50 30 hr. 1 hr. 4 hrs. 22.44 br.
hr. 1 hr. 4 hrs.
1 br.
23.56
hr. 1 hr. 4 hrs.
I/Z
1 hr. 45.00
l/2
1 hr. 8
hr. 40.78
II
hr. 55.89 The proportion 1 hr. 64.22 of acid is e x 4 hrs. 67.88 tremely small I/? hr. 3 2 . 6 6 a n d does not 1 hr. 3 5 . 0 0 c o n v e r t any 4 hrs. 40.00 starch into sugar 1/2hr. 9.87 1 hr. 11.26 It is added t o 4 hrs. 22.85 the starch and l/Zhr. 6 . 0 0 water b e f o r e 1 hr. 6 . 3 3 gelatinizing 4 hrs. 6 . 8 2 1/2
In alkaline solution one p a r t , sodium bicarbonate 0 . 2 6 p a r t
7
21
117.0 1 hr. 134.86 4 hrs. 142.12 l/2 hr. 68.46 1 h r . 73.50 4 hrs. 83.79
a hr 55.89
4 hrs. 22.44
1/2
4.32 4.50 5.01
hr.
1 hr. 4 hrs. 10.07 l/z hr. 1 hr. 4 hrs. 10.07
.-d -.-z
B '5 ."g Quantity of malt in a n infusion 1 in 4. 9 Accelerated one part, HCl 0.0058
8 !-
P Potato 6
5 04
After 5 minutes
Corn 3
2.52
After 5 minutes
10 Accelerated one part. HC1
0.0029
11 Neutral one p a r t
Potato 2l/,
1 .89
Neutral one part
Corn 1 . 1 2
0 , 9 5 1 After 5 minutes
Corn 1.12
0.951
12
13 Alkaline one p a r t . sodium bicarlionate 0.16 p a r t
14 Alkaline one part. sodium carbonate 0 . 1 6 p a r t Potato 1 . 1 2
0.951
After 5 minutes
After 10 minutes
After 5 minutes
'
hr. 1.658 1 h i . 1.764 4 hrs. 2.024 1/2 hr. 1.108 1 hr. 1.175 4 hrs. 1.126 h i . 0.580 1 hr. 0 605 4 hrs. 0.667
hr. 65.80 1 hr. 7 0 . 0 4 hrs. 80 35 1/2 hr. 58.63 1 hr. 62.00 4 hrs. 6 4 . 8 8 I/* hr. 61.00 1 h r . 63.70 4 hrs. 68.10
hr. 0.519 1 hr. 0 , 5 7 0 4 hrs. 0.625
h r . 54.76 1 hr 60.00 4 hrs 65 77
I/*
1/2
h r . 0.589 0.628 4 h r s . 0 682
1/2
1 hr.
hr. 32.90 No. 10 continued for 24 1 h r . 3 5 . 0 0 hours yielded 3 . 2 5 maltose
l
1/2
1/2hr. 62.20 1 hr. 66.07 4 hrs 71.72
4 hrs. hr. 1 hr. 4 hrs. 1/2 hr. 1 hr. 4 hrs.
40.10 22.00 23.31 24.33 11.51 12.00 13.24
'/z hr. 10.30 1 hr 11.10 4 hrs. 12.50 hr. 11.69 1 hr. 12.45 4 hrs 1 5 . 5 0
4
I/z
.
NOV.,191 I
T H E JOURNAL OF I1VDUSTRIAJ.L A N D E N G I N E E R I N G C H E M I S T R Y . COMPARATIVE DIASTATIC ACTIONOH P A N C R E A T l N
Pancreatin, 1 to 25 and 1 to 35 starch test LT. S. P. Corn starch ........................................... Water.. Hrdrochlori Pancreatin. 1 to 2.5 Converts in 5 minutes Temp. 135' F., one panc. to 125 starch
....
...........................
...............................................
Water Hydrochloric acid, N/10. .............................. Pancreatin, 1 to 25.. .................................. Converts in 5 minutes Temp. 135' F.. one panc. t o 125 starch Corn starch.. ......................................... Water. ............................................... Hydrochloric acid. "10 ......................... Pancreatin, 1 t o 25.. .................................. Converted in 5 minutes
Hydrochloric acid, N/10. . . . . . . . . . . . . . . . . . . Pancreatin, 1 to 25.. Converted in 5 minutes Temp. 13.5' F., one panc. to 175 starch Corn starch ...........................................
..................................
.......................................... c acid, N/lO.. . . . . . . . . . . . . . . .......... Pancreatin, 1 t o 2 5 . . . . . . . . . . . . . . ..............
3 . 7 5 grams 60 cc. 1 cc. 0.030 gram
1.875 grams 60 cc. 0 . 5 cc. 0.015 gram
3 . 7 5 giams 60 cc. 0 . 5 cc 0.021 gram
3 . 7 5 grams 60 ec. 2/9 cc. 0.021 gram
3 . 7 5 grams 60 cc. '/a cc. 0.021 gram
Converts in LO minutes
.................. ............................................. ..............................
Corn starch.. ... Water... Hydrochloric acid, N/10.. Pancreatin, 1 t o 3 5 . . Converted in 5 minutes Temp. 135' F..one panc. t o 175 starch Cornstarch ........................................... Water.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrochloric acid. N/10.. Pancreatin, 1 to 2 5 . . . . . . . . Converted in 35 minutes Temp. 135' F.. one panc. t o 125 starch Potato starch..
..................................
3.75 grams 60 cc. 2.5 cc. 0.021 gram
..................... .....................
1 ,875 grams 60 cc. , 2 cc. 0.015 gram
....................................... .............. ................................ ....................
3 . 7 5 grams 60 cc. 1 cc. 0.015 gram
..
Converted in 5 minutes Temp. 135' F..one panc. to 2 Potato starch Wnter.. Hydrochloric acid, N/lO. Pancreatin, 1 to 25. ................................... Converted in 5 minutes Temp. 135" F.. cne panc. t o 250 starch Potato starch .........................................
................ ............. .............................................. ...............................
.....
Pancreatin, 1 to 3 5 . . Converted in 5 minutes Temp. 135' F.,one panc. to 350 starch
AND
MALT.
Temp. 104' F.,one panc. t o 25 starch Malt infusion 1 in 6 acidity (as lactic acid) 0.35 per cent. Corn starch.. Water. Malt infusion. .......................................... Converts in 5 minutes Temp. 13.5' F..one malt to 2'/4 starch
.......................................... .................................................
.........................................
Corn starch.. a'ater. ................................................ Malt infusion, made neutral ........................ Converts in 5 minutes Temp. 135' F., one malt to 1 . I 5 starch Corn starch.. .......................................... Water. ................................................ Hydrochloric acid, N/lO., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Malt infusion.. .................................. Converts in 5 minutes
................... :.
Hydrochloric acid, N/lO.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Converts in 5 minutes Temp. 135' F., one malt to 5 7 / 1 ~starch Corn starch ............................................ Water.. Hydrochloric acid, N/lO.. . . Mall infusion., Converts in 10 minutes Temp. 135' F., one malt t o 57/,0 starch Potato starch. .......................... W a t e r . . . ............................................. Malt infusion.. Converted in 5 minutes Temp. 1.35' F..one malt t o 4'/1 starch Potato starch.. . . . . . . . . . . . Water. . . . . . . . . . . . Hydrochloric acid .................... Malt infusion.. .................... Converted in 5 mi Temp. 135' F..one malt to 5%0 starch Corn starch..
7 grams 120 cc. 2 cc. 0.021 gram
... 3 . 7 5 grams Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 cc. Sulphuric acid, N/lO.. ................................. 1 cc. Pancreatin, 1 t o 25..................................... 0,030 gram Converted in 50 minutes Temp. 135' Corn starch ...... 3.75 grams Water . . . . . ...... 60 cc. Tartaric acid, A'/lO,. .................................. 3 cc. Pancreatin, 1 to 25..................................... 0 . 3 0 gram Converted in 30 minutes one panc. t o 125 starch ............................... 3 . 7 5 grams ............................... 60 cc. 25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0,030 gram Boric acid ( G . 3 grams to liter) . . . . cc. 3 Converted in 30 minutes Temp. 135" F., one panc. to 125 starch Corn starch.. ......................................... 3 . 7 5 grams \T-ater. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 cc. Paucreatin, 1 to 2 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.15 gram Converts in 5 minutes
3.75 grams 60 cc. 20 cc.
3 . 7 5 gram3 60 cc. 1 cc. 4 cc.
3 . 7 5 grams 60 cc. 4 cc. 2 cc.
.............................................. .........................................
3 . 7 5 grams 60 cc. 1/2 cc. 4 cc.
.........................................
5.75 grams 60 cc. 5 cc.
...
3 75 grams 60 cc. 2 cc. 4 cc.
..........................................
3 . 7 5 grams 60 cc. 2 cc. 0.015 gram
3.75 grams 60 cc. 10 cc.
Malt infusion ................................ Converted in 10 millutes
~
3.75 grams 60 cc. 1 cc. 4 cc.
3 . 7 5 grams Corn starch.. .. ............ 6 0 cc. Water. ........... 1 cc. Tartaric acid, N/10.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 cc. Malt iniusion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Converted in 10 minutes one malt to 57/10 starch Temp. 135O F.. 3.75 grams Corn starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 cc. Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... 2 cc. Boric acid (6.3 grams to one lite 4 cc. Malt infusion., . . . . . . . . . . . . . . .......... Converted in 10 minutes Temp 135' F., one malt to 57/10 starch
........
826
T H E J O U R S A L OF I . Y D C S T R I A L A21’D ENGISEERI.YG C H E A J I I S T R Y .
The starch paste was prepared using 33/a grams starch t o 60 cc. water. The commercial starch contains about 16 per cent. moisture. The pancreatin was added to the starch paste when the temperature of the latter was 130’ F., which fell t o 1 1 0 ’ F., a t which temperature the digestion was continued. One gram anhydrous starch equals I.0 5 5 grams maltose. These tables by comparison indicate that by acceleration of pancreatin it can be made a cheaper starch converter than malt is. The malt infusion was added to the starch paste when the temperature of the latter was 130O F., which fell to 110’ F., a t which temperature the’ digestion was continued. The reducing substances in the malt infusion were estimated and calculated as maltose 0.105 gram to I cc. of malt infusion, and subtracted from the total reduction by the copper, t o obtain the actual amount of’maltose formed from the starch b y the malt. LABORATORY DIGESTIVE FERMENTS COMPANY, CHICAGO.
____--
DETERMINATION OF MALIC ACID.’ R y P. B. DUNBAR AND R.F. RACON. Received September 21, 1911.
INTRODUCTION.
The method described herein is based on the fact first observed by Walden* that under certain conditions uranium salts produced a very marked increase in the specific rotation of I-malic acid. Walden noticed that a similar but less marked effect is produced by uranium salts on d-tartaric acid, d-methyl tartrate, I-quinic, and 1-mandelic acids, the increased rotation being in the same direction as the rotation of the aqueous solution of the substance. He concluded that the effect is limited t o the active oxyacids. As stated b y Walden, the requisites of an agent which is to be used for the detection of malic acid by optical means are: ( I ) That the direction of rotation shall be constant, and ( 2 ) that the increase in rotation shall be as large as possible. This, of course, applies also t o the quantitative estimation of malic acid. In addition, the agent in question should have little or no influence on any other substances which may occur in conjunction with malic acid. It has long been known that certain substances such as the oxygen compounds of b o r o n , ~arsenic, antimony, molybdenum, and tungsten4 have the power of increasing the specific rotation of the oxy-acids. Uranium salts, however, are the only ones which fulfil the requirements just stated with any degree of satisfaction. The authors have recently had the privilege of reading a n article by P. A. Yoder, entitled “A Polariscopic 1 Presented at the Indianapolis meeting of the American Chemical Society. Rer. d . chem. Ges , [ 3 ] 30, 2889 (1897). Biot, Mkm. de l’acad. r o y . sci., 16, 229 (1838); Ann. chim. phys.. [31 11, 82 (1884); 29, 341, 430 (1850); 69, 229 (1860). Pasteur. Ann. chim. hhys., [ 3 ] 69, 243 (1860). Gernez, through Landolt’s “Optical Rotation of Organic Substances,” translated by Long. p. 248.
Nov., 1911
Method for the Determination of Malic Acid and its Application in Cane and Maple Products.”‘ This paper was presented a t the forty-second meeting of the American Chemical Society, in July, 1910, and contains the results of a large amount of work. The method outlined depends on the use of a uranium salt, but in other respects it differs materially from the one here proposed. With this exception, no one appears to have attempted the quantitative determination of malic acid by treatment with uranium. T H E METHOD I N BRIEF.
The method, in brief, consists in treating a portion of the neutralized solutionz containing malic acid with uranyl acetate and polarizing it. The algebraic difference between the reading so obtained and that of the untreated solution is multiplied by the factor 0.036 to obtain the percentage of malic acid present. With the exception of d-tartaric acid, none of the common optically active substances interfere with the determination, and consequently they need not be removed. Uranyl acetate slightly decreases the rotation of sugars. This may give rise to an error in the estimation of small amounts of malic acid in the presence of large amounts of invert sugar. Hence, when the rotation of the original solution is negative and the approximate amount of sugar or malic acid is unknom-n, or when more than I O per cent. of reducing sugars and less than 0 . 2 j per cent. of malic acid are present, it is necessary to precipitate the malic acid and treat the filtrate with uranyl acetate also. In this case the polarization of this filtrate, instead of that of the untreated solution, is subtracted from the polarization of the solution containing both malic acid and uranyl acetate, and the difference is multiplied by 0.036. DETAILS O F THE NETHOD.
Dilute a measured volume of the solution, usually I O cc., with quite a large volume of water, add phenolphthalein, and titrate v i t h standard alkali to a decided pink color. Transfer another measured. portion of the solution (7j cc. is a convenient volume) to a I O O cc. graduated flask, and add enough standard alkali, calculated from the above titration, t o neutralize the acidity. A slight excess of alkali is not objectionable. If the solution is dark colored, add j or I O cc. of alumina cream. Dilute to the mark, mix thoroughly, and filter if necessary through a folded filter. ( I ) Treat about 2 5 cc. of the filtrate with powdered uranyl acetate, adding enough of the salt so that a small amount remains undissolved after two hours. Two and one-half grams of urahyl acetate will usually be sufficient, except in the presence of large amounts of malic acid. In ’case all the uranium salt dissolves more should be added. Allow the mixture t o stand for two hours, shaking frequently. Filter through a folded filter until clear and polarize if possible in a 2 0 0 mm. tube. If the solution is too dark t o read in a zoo mm. tube, a I O O or 50 mm. tube may be used. THISJOURNAL. 3, 563 (1911). If no mineral acids are present it is not necessary t o neutrali~ethr malic arid. 1
2
,
’
