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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 .
Nov., 1911
3. Volumetric Estimation of Orthocresol with Bromine. R . Clauser. dependent upon time of standing after dilution. Oeslerr. Chem. Z t g . . 11, 585 (1899): through (Abst. Analyst. 1900, 74). Compare Expts. 15-24 with Expts. 25-28. 4 A New Method for Analysis of Commercial Phenols. S. B . Schrwer. It now remained to ascertain whether or not a J. SOC.Chem. I n d . , 1899, 553. 5. Quant. Estimation of Resorcinol. Degener. J . braklische smaller amount of K I would counteract tendency for Chemie, [Z 1 20,322:through (Abst. Beilstein’s “Organische Chemie.” 2, 916). decomposition of tribromresorcinol and under what 6. Preparation of Iodo Derivatives of Phenols and Determination of conditions such would obtain. ‘ I t was found that Resorcinol. E. Richard. J . Pharm. Chim., [5] 15, 217-22 (1902): through J . SOC.Chem. I n d . , 1902, 423). this decomposition was prevented ; that solution must (Abst. and Tribromresorcinolbrom. Benedikt. 7. Tribromphenolbrom not stand too long after addition of K I before dilu- Berichle. 12, 1005-1006; through (Abst. J. Chem. S O C . ,1879, 36, 717). RESEARCH LABORATOXY, tion; that in diluted condition, time of standing might ELI LILLY& COMPANY, be somewhat prolonged without deleterious results ; INDIANAPOLIS. that after dilution and addition of KI, solution should not stand as long as I hour before titration (Expst. A NEW AND ACCURATE METHOD FOR DETERMINING THE 2 9 and 30-31, 32, 33-34, 3s). TRYPTIC VALUE O F PANCREATIN.‘ Several experiments were run varying the length B Y CLARENCE F. RAMSAY. of time bromine was allowed to react with resorcinol Received August 28,.1911. in order to study the length of time it would take K I At the present time very little attention is being to produce proper reversal under varying conditions. paid to pancreation as regards its proteolytic activity. In general, it may be said that KI should react a t This is probably due to the fact that no satisfactory least as long as bromine is permitted to react with test has been proposed by which it might be ascerresorcinol. Experiments 36, 37, 38, 39, 40, 41, 42, tained just how much proteid a given sample of pan43, 44, 45, 46, 47, 48 and 49. creatin will digest. It was found in Expt. 50 that reaction between broThe tryptic value of pancreatin should be taken mine and resorcinol would not be completed in 1 5 into account for it is more energetic than pepsin. secs.; in Expts. j r and 52 that solution should set a t I n the case of pepsin considerable time is necessary least 5 mins. after addition of K I when excess of bro- for proteid to be converted into peptone while trypsin mine is large; in Expt. 53 that reaction between bro- converts proteids rapidly into proteoses and peptone. mine and resorcinol solutions is much ‘more rapid It has been stated that .trypsin is so energetic that . when strong acid is used. i t will convert proteid beyond the stage of peptone The method of Degener would be open to consider- into leucin, tyrosin, and other amides. able error, due t o formation of the additive compound The U. S. P. method for determining the tryptic and to insufficient time and dilution for its decom- power of pancreatin is very indefinite and uncertain position. because of the manner of testing the end reaction. Richards’ iodometric method has several features It states that by adding a little nitric acid to a portion that condemn i t : Ist, solutions darken upon standing of the peptonized milk no coagulation should occur. so that end point is obscured; 2nd, even upon pro- As regards the word coagulation in this test there ionged standing results are much too low (Expts. is a difference of opinion. If we are to think of co63-73). agulation as the result obtained when adding rennin The following method is suggested for the estima- to milk it would be better to call this separation tion of resorcinol in commercial resorcinol: Its ac- a precipitation. The result depends altogether upon curacy may be noted by reference to Expts. 54-62. the amount of acid added. If a minute quantity Weigh out I ,4563 g. resorcinol. Dissolve in dis- of acid is added, no precipitate is formed, while with tilled water, filter if necessary, and dilute to 5 0 0 cc. more acid there is a decided precipitation. A large in a volumetric flask. Withdraw a 2 5 cc. portion excess of acid generally results in solution of the prewith a pipette and place in a 5 0 0 cc. receptacle wiih cipitate. Acid will cause a precipitation in peptonized ground-glass stopper. Add 50 cc. N/IO bromine solu- milk no matter how long the digestion takes place. tion and 50 cc. dist. water. Add 5 cc. conc. HC1, In view of the importance of trypsin in pancreatin shake .and let set about I minute. Dilute with about the writer wishes to propose a milk test which, if z o o cc. water, add 5 cc. 2 0 per cent. K I , shake vigorcarried out exactly in accordance with directions, ously and let set about 5 minutes. Titrate, using will give very accurate results. In testing the activity starch as indicator. Divide number of cc. of N/IO of enzymes, i t is very important to adhere to certain bromine solution consumed by 0.4 (or multiply by conditions such as temperature, time for digestion, 2. j) for correct percentage of resorcinol. etc. So it is in testing trypsin. This test determines A blank should be run along with the determina- the amount of pancreatin necessary to peptonize tion, using the same pipette and draining in the same a given quantity of milk in fifteen minutes. The end manner in order to obtain an exact duplicate titre of reaction is determined by adding a slightly acidified N/IObromine solution. 7-10 cc. of 2 0 per cent. KI solution of rennin t o a portion of the peptonized should be used instead of 5 cc. as in regular deter- milk and noting if precipitation or coagulation takes mination. place. The final end point of the test is reached when BIBLIOGRAPHY. the milk is just sufficiently peptonized so thatpit 1. Volumetric Estimation of Phenol. W. F. Koppeschaar. Zeitschr. will not be coagulated by the rennin. To determine anal. Chem., 1876, 233-245; through (Abst. Jr. Chem. Soc., 1877, 746). this it is necessary t o test the pancreatin first at wide 2 . Reactions of B r with Phenol and the Cresols, A Process of Calcu-
lating the Composition of Mixtures Thereof. Ditz and Cedivoda. Zeitschr. a m l . Chem.. 1899, 873 and 897: through (Abst. Analyst. 1900, 74).
1 Paper read before the Pharmaceutical Division, June meeting, American Chemical Society.
Kov.,
1911
T H E J O C R S A L OF I A - D U S T R I A L AND ELYGI-YEERI~YGC 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.
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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.
