904
T H E JOURNAL OF INDUSTRIAL AND ENGINEERING CHEMISTRY
There is b u t little change in the specific gravity of t h e fruit held in storage during the first 60 t o 7 0 days, and even u p t o t h e end of t h e experiment t h e increase is slight a n d may be accounted for b y a normal drying-out or withering of t h e fruit. But with t h e fruit t h a t remained on t h e tree there is a t first a very marked decrease in specific gravity and t h e n apparently a gradual increase. It was noticed in cutting this orchard fruit t h a t during t h e latter half of t h e observation period t h e lemons appeared t o be filling out a little. S UJ?hf A R Y
I n so far as a preliminary experiment of this kind can be considered as giving definite results, t h e following conclusions may be drawn: I-The lemons t h a t remained on t h e trees after being frozen retained less juice and considerably less acid t h a n t h e fruit picked immediately after being frozen and stored a t a temperature of 45 t o 50' F. 11-The frozen fruit left on t h e trees decreased rapidly in specific gravity due t o t h e loss of juice a n d acid and t h e formation of thick, puffy skins, dried-up cells and hollow centers. 111-Lemons picked immediately after being frozen and stored under ordinary packing house conditions retained somewhat less juice t h a n the unfrozen fruit, b u t t h e composition of t h e juice was nearly the same, and in weight and appearance t h e slowly thawed lemons compared well with normal fruit. We wish t o acknowledge our indebtedness t o Mr. G. 'A7. Hosford of t h e San Dimas Lemon Association for his valuable suggestions and hearty cooperation, and t o t h e association for so generously supplying us with t h e fruit used in this investigation. BUREAUO F
CHEMISTRY, WASHINGTON
AN IMPROVED M E T H O D F O R THE DETECTION O F ARACHIDIC ACID By ROBERT H. KERR
Vol. 8, No.
IO
METHOD
Weigh out 20 g. of t h e oil t o be tested in a 300 cc. Erlenmeyer flask, pour in 2 0 0 cc. of 95 per cent ethyl alcohol, and heat t o boiling on t h e steam bath. When t h e alcohol is boiling add I O cc. of t h e potassium hydroxide solution. Saponification begins immediately and is soon complete. After t h e saponification has been completed add a few drops of phenolphthalein and neutralize t h e excess alkali with t h e alcoholic solution of acetic acid. Next add jo cc. of t h e 5 per cent magnesium acetate solution and heat t h e whole mixture t o boiling. Allow t o cool t o room temperature with occasional shaking and then place in a refrigerator a t a temperature of I O t o I j o C. and leave until next day. Filter off t h e solution, wash t h e precipitate twice with j o per cent alcohol a n d three times with distilled water, and return t o t h e flask in which precipitation took place. Pour I O O cc. of hot distilled mater into the flask and add sufficient dilute sulfuric acid t o decompose the magnesium salts. Heat until t h e separated acids form a clear layer. Cool t h e flask, pour off t h e acid solution, add I O O cc. of hot water. When the f a t t y acids have melted and solidified, pour off water as before. Free t h e cake of acids of water as far as possible b y draining; dissolve in I O O cc. of 90 per cent alcohol and separate t h e arachidic acid by crystallization, according t o t h e present provisional method of the Association of Official Agricultural Chemists as given in Bulletin 107, Revised, Bureau of Chemistry, p. 146. The method as outlined above has been used on a number of samples of peanut oil and mixtures of peanut and other vegetable oils. T h e results obtained are qualitative only, no attempt having been made t o apply t h e method for quantitative purposes. It has been found t o be capable of detecting j per cent of peanut oil in olive oil, cottonseed oil, soy bean oil and corn oil. These results are quite as good as t h e best which have ever been obtained with Renard's method. RCREAU O F ANIMAL ISDUSTRY,
WASHINGTON
Received May 16, 1916
T h e method described below for t h e detection of arachidic acid in peanut oil and mixtures of oils containing peanut oil has been found t o offer certain advantages over t h e Renard method adopted b y t h e Association of Official Agricultural Chemists. These advantages consist in greater convenience, lessening of t h e number of operations, reducing t h e amount of attention required, and avoidance of t h e use of ether. REAGENTS
Potassium Hydroxide Solutioiz-Dissolve I O O g. of stick potassium hydroxide in I O O cc. of water. Magnesium Acetate Solution-Dissolve I O g. of magnesium acetate in a mixture of I O O cc. distilled water and I O O cc. of 95 per cent alcohol. Acetic Acid Solution-Mix 50 cc. glacial acetic acid with 150 cc. of 95 per cent ethyl alcohol. jo cc. concentrated Suljuric Acid Solution-Mix sulfuric acid with 150 cc. of distilled water. 90 per cent E t h y l Alcohol (by volume).
THE HYDROLYSIS OF ETHYL-SULFURIC ACID AND THE ASSAY O F AROMATIC SULFURIC ACID, U. S. P. By W. B. D. P E N X I M A N ,W.W. RAKDALL, c.0. M I L L E R Received September 1, 1916
AND
1,. H. ENSLOW
Aromatic sulfuric acid is a mixture of sulfuric acid, ethyl-sulfuric acid, certain aromatics and alcohol. I t is prepared b y bringing together, according t o t h e U. S. Pharmacopoeia, 111 cc. of concentrated sulfuric acid and 7 0 0 cc. alcohol, and, after t h e mixture has cooled, adding jo cc. of tincture of ginger, I cc. of oil of cinnamon, and sufficient alcohol t o bring t h e total volume up t o one liter. The proportion of ethyl-sulfuric acid present depends upon the strength of t h e sulfuric acid and alcohol used and upon the length of time t h e mixture remains hot, and probably increases t h e longer the liquid stands, even when cold. The Pharmacopoeia (Eighth Revision) is not concerned with t h e ethyl-sulfuric acid: its only demand is t h a t t h e mixture shall contain not less t h a n 2 0
Oct., 1916
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
per cent, b y weight, of sulfuric acid, HzS04,after t h e ethyl-sulfuric acid has been completely hydrolyzed by t h e action of water. T h e method of assay made s t a n d a r d b y t h e P h a r m a copoeia has varied in some of its details with each of t h e recent editions. I n t h e Eighth Revision (September, 1905) t h e analyst was directed t o mix I O g. ’ of aromatic sulfuric acid with 30 cc. of water a n d t o boil this mixture “for several minutes,” after which t h e total acid present (assumed now t o be sulfuric) was t o be determined b y means of titration with a s t a n d a r d solution of alkali. I n t h e Supplementary Revision of 1907 t h e only change made was t h a t t h e words “for four hours” were substituted for “for several minutes.” I n t h e Ninth Revision, about t o appear, t h e requirement will be “free sulfuric acid a n d ethylsulfuric acid together equivalent t o not less t h a n 19 per cent nor more t h a n 2 1 per cent of HzS04 ( 9 8 . 0 9 ) ~ ” T h e method of assay is also changed a n d directs t h a t a known weight (which will be about 9 grams) of aromatic sulfuric acid shall be mixed with six times its volume of water, boiled for six hours in a flask fitted with a reflux condenser, a n d later titrated with standa r d solution of alkali. I n a n article published four years ago, L. A . Brown1 called attention t o the fact t h a t t h e methods of assay given in t h e U. S. Pharmacopoeia (8th Revision, 1905, a n d Supplementary Revision, 1907) fail t o determine all t h e sulfuric acid present. Accordingly, Brown advocated a method in which all free acid present (sulfuric a n d ethyl-sulfuric) is first determined by titration; b y t h e action of hydrochloric acid a n d barium chloride t h e alkali sulfate a n d ethyl-sulfate t h u s formed are converted into barium sulfate a n d t h e yield of t h e latter salt determined b y weighing. It is t h u s possible t o determine t h e total acid present, calculated as sulfuric, a n d also t h e proportion of ethylsulfuric acid. I n trying out this method, Brown analyzed a “standard sample of aromatic sulfuric acid,’’ b u t does not inform us what its acid strength actually was. B y t h e U. S. Pharmacopoeia method t h e percentage found was 18.40 in each of two determinations; b y his method t h e percentages found were 20.18, 19.73, a n d 19.80, respectively. Again, he records t h e results obtained on analyzing b y both methods a series of commercial samples, b u t as we have no means of knowing t h e actual strength of a n y of these, t h e only safe conclusion we can draw is t h a t t h e U. S. Pharmacopoeia method is faulty a n d t h a t Brown’s method is more nearly accurate; we are not in a position t o say t h a t a satisfactory method has been found. Only where a series of assays upon a sample of known strength yield closely agreeing results are we t o claim t h a t t h e goal has been attained. Brown concludes from his work t h a t diethyl sulfate is one of t h e constituents of aromatic sulfuric acid, a t a n y rate after long- standing. If, as seems t o t h e present writers, this conclusion has f o r i t s basis t h e fact t h a t his analytic method failed t o show t h e presence of all t h e sulfuric acid originally used, i t rests 1
THISJOURNAL, 4 (1912), 512.
905
upon a doubtful foundation. T h e work of Claessonl a n d t h a t of Villiers2 seem t o prove t h a t , in order t h a t diethyl sulfate shall be formed, absolute alcohol a n d very concentrated sulfuric acid must be employed, a n d t h a t diethyl sulfate decomposes comparatively readily when heated with either water or alcohol. T h e fact t h a t i t yields alcohol a n d ethyl-sulfuric acid when heated in t h e presence of water, would appear t o disprove Brown’s assumption t h a t such part of t h e sulfuric acid as could not be recovered as such, was locked u p i n t h e form of diethyl sulfate at t h e end of t h e boiling process. It fell t o t h e chemists of this bureau this summer t o determine t h e strengths of a large number of samples of aromatic sulfuric acid purchased from retail druggists. Making use of t h e U. S. Pharmacopoeia (1907 Revision) method, it was soon found t h a t few of t h e samples would show results which could be considered as satisfying t h e demands of t h e Pharmacopoeia. Table I gives t h e percentage figures for a series of samples of various strengths as found b y this method and, below t h e m , t h e figures obtained b y t h e method soon t o be described. TABLEI-PERCENTAGES 11297 11300 2 3 . 6 8 17.13 Maryland 2 5 . 5 8 1 9 . 0 8 Diff, 1.90 1 . 9 5
u.;.Y;j
OF TOTAL ACIDASSUMEDTO B E SULFURIC, B Y T W O METHODS 11.188 11391 11393 11394 11395 11396 11397 11398 16.77 11.86 15.92 1 4 . 3 0 1 5 . 0 0 1 5 . 7 6 1 2 . 9 6 17.34 1 9 . 7 6 14.24 2 1 . 7 4 1 7 . 7 6 1 8 . 0 9 19.91 16.79 2 0 . 2 9 2.99 2.38 5.82 3.46 3 . 0 9 4.15 3 . 8 3 2 . 9 5
It will be seen t h a t , if judged b y t h e U. S. Pharmacopoeia method, only one out of t h e t e n samples is satisfactory, a n d t h a t t h e other nine give figures from 2.66 to 8 . 1 4 per cent too low. On t h e other hand, judged b y t h e improved method, three samples are satisfactory, two approximate 2 0 per cent, a n d t h e worst is j . 76 per cent short of t h e required amount. Our attention having been called t o t h e revised method t o become standard with t h e publication of t h e Ninth Revision of t h e Pharmacopoeia, a series of assays upon samples of unknown strength was carried out, t h e amount of added water being increased t o 6 volumes a n d t h e time of boiling with reflux condenser lengthened t o 6 hrs. Under “ N i n t h Revision Method” in Table I1 are given t h e results of duplicate assays a n d t h e differences between them, while another column contains t h e figures found b y our revised method: OF TOTAL ACID, ASSUMEDTO BE SULFURIC NINTHRET , MARYLAND IISION u. s.P . METHOD Diff. A B METHOD 1.10 11.49 12.47
TABLP: 11-PERCENTAGE
Lab. No.
20.70 18.45 17.65 23.00 14.50 18.75 26.90 18.40 24.30 31.80 14.60 17.25 14.75 23.00 27.80
4.81 1.97 1.83 0.18 0.84 0.85 0.71 0.80 0.22 0.88 0.36 0.51 2.90 0.10 0.78
23.22 20.41 18.56 23.97 15.24 20.59 27.57 19.84 25.53 32.72 17.01 19.40 16.16 25.26 28.52
It will be noted t h a t parallel determinations b y t h e new U. S. Pharmacopoeia method rarely showed
,
prakt. C h e m , , 12119, 257; Bull. sac. chim., 34, 26.
J ,
2
13, 1699.
906
T H E J O U R N A L O F I N D U S T R I . 4 L AATD ENGINEERIAVG C H E M I S T R Y
satisfactory agreement, and t h a t , judged b y our method, all these results are too low. For a thorough s t u d y of t h e two standard (U. S. P.) methods of assay, there was prepared a standard aromatic sulfuric acid, in accordance with t h e Pharmacopoeia. A careful specific gravity determination was made in t h e case of the acid, of t h e alcohol and of the final product; and a weighed amount of t h e sulfuric acid used was titrated against standard alkali solution. F r o m these d a t a the percentage of total acid, assumed t o be sulfuric, in the product was calculated t o be 20.14. With this standard material t h e three series of experiments indicated in Table I11 were conducted.
5'01. 8, NO. I O
copoeia method will always give low results, t h e actual figure obtained being apparently dependent largely upon t h e rate of boiling and the e$ciency of t h e condenser. These same conclusions might be drawn as t h e result of t h e following considerations: The reaction between sulfuric acid and alcohol is expressed b y the equation H2S04 CzHb.0H = CzHb.HS04 H20 (a) T h e acid must be a t least fairly strong and t h e alcohol must not contain much water. Again, ethylsulfuric acid is decomposed, t o some extent perhaps in the cold, t o a large extent when heated, in contact with water, with the formation of sulfuric acid and TABLE 111-EFFECT OB VARIOUSTREATMEKTS alcohol, Results in Percentages Total Acid, A4sumed t o he Sulfuric (1) ROILWJ U N D E R CzHb.HS04 HzO = C2Hb.OH HZSO? 13) BOILEDU N D E R (b) REFLUXCONUEKSER (2) $Low EVAPORATION KEFLTJXCONDEKSER The reaction is reversible, and should be expressed Water Added IN THP, COLD Cc Water No. Hrs. 3 Vols 6 1'01s. h-0. Hrs. Results KO Added Results as follows: 17.23 17.23 1 24 17.23 10 19.05 17.56 17.67 2 48 17.23 20 19,33 CzH6.OH HzS04 CzHj.HS04 HzO 2 17.67 17.83 3 (c) 72 25 18.85 17.23 4 18,32 18.27 4 144 19.64 30 19.36 Reaction (a)begins upon mixing t h e acid with alco6 18 81 18.70 40 18.93 12 19 41 19.63 50 18.96 hol, and continues. T h e accumulation of water, 18 19.63 19,85 7 60 19.53 24 19.79 19.90 8 70 19.41 however, soon starts reaction ( b ) , which develops I t will be seen t h a t in Series I all t h e sulfuric acid until, in a given period of time, as much ethyl-sulfuric was not recovered even after 24 hours' boiling. While, acid is being decomposed as is being formed. After on t h e whole, better results were obtained when a this point has been reached, t h e composition of t h e larger proportion of mater Kas added t o decompose mixture remains constant and equilibrium is estabt h e ethyl-sulfuric acid, this outcome may be charge- lished among 'the four reacting substances. The relative amounts of sulfuric and ethyl-sulfuric acid in able t o some other cause. In Series 2 , weighed amounts of t h e standard aro- t h e resulting mixture will therefore depend, physical matic sulfuric acid (to each of which 6 volumes of water conditions being the same, upon t h e amount of water had been added) were placed, cold, in a vacuum originally present in t h e sulfuric acid and in t h e alcodesiccator over sulfuric acid and left for different hol. I t is a well-known fact t h a t fuming sulfuric periods of time before titration. The results seem t o acid and absolute alcohol yield a high proportion of show t h a t t h e decomposition of the ethyl-sulfuric ethyl-sulfuric acid a n d t h a t dilute sulfuric acid has acid begins only after a certain concentration has been little or no effect upon a1cohol.l Could we remove reached, and then proceeds slowly towards comple- t h e water as fast as it is formed in accordance with reaction ( a ) , we should be able t o secure a I O O per tion. This will be referred t o again. Series 3 had t o do with t h e effect of varying propor- cent yield of ethyl-sulfuric acid. Similarly, were m7e tions of water t o acid, time of heating remaining con- able t o remove the alcohol formed in reaction ( b ) s t a n t . I n each case 5 g. of aromatic sulfuric acid as fast as it is produced, we should secure a complete were boiled for 6 hrs. in a flask under a reflux condenser, decomposition of t h e ethyl-sulfuric acid present. after t h e addition of the quantities of water indicated. S o w this is just t h e problem before us. The U. S. T h e results fail t o show t h a t any appreciable advan- Pharmacopoeia assay method is not successful, betage is t o be secured b y t h e addition of 6 volumes of cause i t fails t o bring about t h e removal of t h e alcohol already present as such in t h e aromatic sulfuric acid, water in place of 3. Finally, two j-gram quantities of t h e standard or t h a t is being formed through hydrolysis of ethylaromatic sulfuric acid were each mixed with I j cc. sulfuric acid. The reflux condenser, in so far as it is of water: one was titrated a t once, t h e other after efficient, prevents t h e escape of t h e very reagent standing in t h e cold for 48 hrs. I n each case t h e per- whose presence renders impossible t h e completion of centage of total acid, assumed t o be sulfuric, was the reaction. Even t h e best of our water-jacketed condensers 16.96. As a result of these various experiments, t h e follow- probably allows t h e escape of slight quantities of alcoing conclusions appear t o be justified: hol-vapor, a n d hence in process of time the hydrolysis 1-The ethyl sulfuric acid in aromatic sulfuric may be complete. The experiments reported in acid is not appreciably hydrolyzed on standing mixed Table I V were undertaken t o see if evidence of a s t a t e with cold water. of equilibrium among t h e four reacting substances a-This ethyl-sulfuric acid is slowly hydrolyzed, could be secured. Five-gram quantities of aromatic after dilution with water, as a result of prolonged 1 T h u s in one of our experiments dilute sulfuric acid (1 of acid t o 36 boiling under a reflux condenser, t h e decomposition of water, by volume) was boiled continuously f o r 61/2 hrs. with a mixture of alcohol and extract of ginger; samples were removed from time t o time never, however, becoming complete, no matter how and titrated. h'ot the slightest change in t h e strength of the acid could be much water may have been added. Hence the Pharma- detected.
+
+
+
+
+
Jr
+
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
Oct., 1916
sulfuric acid were heated with 3 volumes of water i n sealed glass tubes, in a b a t h of boiling water, for different periods of time, after which t h e tubes were cooled a n d opened a n d their contents titrated. (The percentage of acid in this preparation, after complete hydrolysis, was 2 4 . 9 4 . ) REQUIRED FOR EQUILIBRIUM 1 2 3 4 5 6 7 8 0 4 6 8 10 12 14 16 18.57 21.66 2 1 . 7 7 22.60 24.08 2 4 . 1 0 24.08 24.10 74.46 8 6 . 8 5 87.29 9 0 . 6 1 9 6 . 5 5 96.63 9 6 . 5 5 9 6 . 6 3
TABLE IV-TIME Hrs. heated yo Acid found % of Total Acid recoverable]
Apparently equilibrium i n t h e system-sulfuric acid, ethyl-sulfuric acid, alcohol, water-for t h e concentrations peculiar t o these experiments a n d at t h e temperature of boiling water, was attained after t e n hours' heating. From this time on about 3 . 4 per cent of t h e sulfuric acid present remained in t h e form of ethyl-sulfuric acid, in spite of t h e presence of a relatively very large volume of water. T h e problem is, therefore, after all very simple. We have only t o heat a weighed q u a n t i t y of aromatic sulfuric acid, after dilution with water, in such fashion t h a t t h e alcohol vapors may escape, a n d for such a time t h a t all ethyl-sulfuric acid shall have been decomposed. I n Table V are given t h e results of a series of experiments in which some of t h e ideas expressed above were tested. I n each case j g. of t h e standard aromatic sulfuric acid ( 2 0 . 1 4 per cent) were heated with 30 cc. of water, t h e period of heating ranging from z t o 8 hrs. TABLEV-COMPARISON OF RBSIJLTS USING REFLUX CONDENSRRS OR WATCHGLASSCOVERS ON FLASKS, A N D HEATING BY FREEFLAMR OR Heated Hrs. 2 ...... 4 ...... 6 . . ,. 8
.. ......
ON WATERBATH REFLUX CONDENSERS Free Flame Water Bath 17.53 17.58 1 8 . 7 4 18.73 17.88 17.86 18.97 19.18 17.86 18.01 19.46 19.34 19.61 15.44 18.50 19.14
W.4TCH
was reduced in each flask t o not more t h a n about I O cc., t h e figures obtained were 2 0 . 1 2 per cent in each case. These, as well as many other results obtained later, show, as was found b y Kastle a n d Fraser' a n d b y Brown,' t h a t t h e hydrolysis of ethyl-sulfuric acid proceeds more rapidly when t h e solution is concent r a t e d t h a n when i t is dilute. These experiments indicated t h a t satisfactory results might be looked for if t h e heating were so conducted t h a t all free alcohol present might quickly evaporate a n d the aqueous solution of ethyl-sulfuric acid left be kept concentrated a n d hot, until hydrolysis is complete. A large excess of water serves no useful purpose, as t h e following experiments (Table V I ) suggest: Three portions of an aromatic sulfuric acid, which contained 2 4 . 9 5 per cent of total acid calculated as sulfuric, were diluted with large quantities of water a n d distilled rapidly. This process removed t h e free alcohol, of course, b u t failed t o break down t h e ethyl-sulfuric acid satisfactorily. TABLEVI Aromatic sulfuric acid No. Grams 1 10 2 10 3 5
Water added Cc. 160 115 160
I t is clear t h a t much better results are obtained b y t h e watch glass method, t h a n where a condenser is used; also t h a t continuous heating for mor; t h a n two hours was necessary, i n this method, t o bring about t h e hydrolysis of all t h e ethyl-sulfuric acid. A weighed a m o u n t of t h e s t a n d a r d aromatic sulfuric acid was mixed with 3 volumes of water a n d slowly boiled for 3 hrs. in a flask fitted with a Hempel t u b e a n d condenser. T h e alcohol was t h u s slowly distilled off a n d t h e water vapor, condensed in t h e Hempel t u b e , returned t o t h e flask. Titration of t h e residual liquid gave for t h e total acid percentage, calculated as sulfuric, 1 9 . 9 4 , instead of 2 0 . 1 4 , actually present. T w o flasks, each fitted with a wide t u b e about 15 in. long, which served as a condenser, a n d each containing a weighed amount of t h e s t a n d a r d aromatic sulfuric acid diluted with 6 volumes of water, were each heated over a small flame in a Kjeldahl rack. T h e alcohol vapors slowly distilled off, while t h e water vapor was for t h e most p a r t condensed in t h e glass tubes a n d ran back. On titrating, t h e liquid whose volume had been reduced t o about 15 cc. gave 2 0 . 0 6 per cent; t h e other, reduced t o about 2 5 cc., gave 19.88 per cent, acid calculated as sulfuric. Repeating t h e experiment in such fashion t h a t t h e liquid volume
Tine Min. 30 25 45
Acid and Ratio acid Water Vol. t o T o t a l acid Residue Water in Found Calc. Cc. Residue as H2SO4 70 1:6. 19.43 25 1:l.S 19.71 100 15 1:2 70.31 150
Distillate Cc. 100
T h e least satisfactory result was obtained where t h e largest proportion of water remained a t t h e end of t h e experiment. Although t h e concentration was ultimately greater i n ( 2 ) t h a n in ( 3 ) , yet t h e last result was t h e best, probably because much more t i m e h a d been given for hydrolysis t o t a k e place. METHOD A D O P T E D
GLASSCOVERS
Water Bath 18.76 18.79 20.14 19.25 20.16 20.13 19.77 20.05
907
Eight 5-gram portions of our s t a n d a r d aromatic sulfuric acid ( 2 0 . 1 4 per cent) were diluted each with 6 volumes of water and heated in open beakers upon a strongly heated water b a t h for different periods of time. T h e results appear in Table V I I . Evidently, as soon a s No. Time hrs. T oCalc, t a i acid as HzSOl
TABLE VI1 2 3 4 5 6 7 8 '/E 1 I'/a 2 21/a 3 31/n 4 17.23 17.23 17.23 17.45 20.14 20.14 20.14 20.14 I
]
t h e proper concentration of acids a n d water is secured, t h e hydrolysis of t h e ethyl-sulfuric acid is fairly rapid a n d soon becomes complete, provided t h e water in t h e b a t h is kept vigorously boiling. If t h e beakers are covered with watch glasses, naturally more time will be required for completing t h e reaction t h a n if t h e escape of vapor is unimpeded. I t m a y as well be pointed out here t h a t , although ethyl-sulfuric acid may be quantitatively converted, in t h e presence of water, into sulfuric acid a n d alcohol, a similar statement may not be made with respect t o its alkali salts. Aqueous solutions of t h e l a t t e r are b u t slightly, if a t all, affected b y boiling. Even when heated in t h e presence of sulfuric acid a n d water, t h e decomposition of alkali ethyl-sulfates into sulfuric acid, sulfate a n d alcohol, is very slow. Ostwald pointed out many years ago2 t h a t ethyl-sulfuric is a far stronger acid t h a n sulfuric; a n d our work, in so far as it may be 1
THISJ O U R N A L ,
2
J . prakt. C h e m , 121 28, 449, 29, 3 8 5 .
4 (19121, 512
908
T H E J O U R N A L O F I N D C ‘ S T R I A L AiVD E N G I N E E R I N G C H E M I S T R Y
said t o furnish evidence on this point, bears out this statement. One of the methods of assay which we attempted t o employ was this: A known weight of aromatic sulfuric acid, diluted with water, was titrated while cold; an amount of sulfuric acid exactly equivalent t o t h e alkali used in t h e titration was then added, a n d t h e liquid heated in a n open beaker on t h e water bath. A second titration, it was hoped, mould now show t h e total acid present t o be sulfuric. a n d a comparison of t h e two titrations would tell us what amount of ethyl-sulfuric acid was originally in t h e mixture. B u t in this we were disappointed. The original diluted aromatic sulfuric acid contained 5 , I 2 per cent total acid calculated as free sulfuric. T h e first titration showed 4 . 3 7 per cent; after heating 2 1 / 2 hrs., t h e second titration showed 4 . jo per cent; after 31/2 hrs., 4 . 9 4 per cent; and after 41,f2 hrs., 4 . 9 4 per cent. T h e presence of alkali ethyl-sulfate thus greatly retarded, if i t did not altogether prevent. t h e conversion of all the acid present into sulfuric. It is difficult t o say how long a given sample of aromatic sulfuric acid must be heated in t h e presence of added water in order t h a t t h e hydrolysis of t h e ethylsulfuric acid may be assumed t o be complete. I n addition t o the physical conditions of the experiment, we must naturally take into account the proportion of ethyl-sulfuric acid present in the mixture. Certainly in most cases four hours’ heating in a n open beaker on a water b a t h , in which t h e boiling is vigorous, will be found ample. I n Table VI11 are some results obtained with samples of unknown strength, where the periods of heating were not t h e same. The figures represent t h e percentage of total acid, assumed t o be sulfuric, as found b y titration. TABLEVI11 Lab. No. 11038 11047 1105 1 11085 11092 11190 11 195 11261 11563
U.s. P. (1916)
METHOD Time Results 6 hrs 22.20 22.16 10.39 20.90 20.70 23.00 27.80 28.58 35.28
F L A S K WITH AIR CONDEN- OPEN BEAKEROH SER OVER FREE FLAME WATERBATH
Time
Results 24.54 24.57 11.83 22.25 22.22 24.95 28.37 32.49 35.81
Time 4 hrs. 4 3 ‘/2 4 4 4 4 4‘./2 4
Results 24.83 24.63 12.47 21.96 23.22 25,26 28.52 32.53 35.77
hours’ It will be noted t h a t , speaking generally, boiling in a flask was not sufficient. I n t h e case of KO. I I O j I , t h e boiling was longer continued b u t was so slow t h a t t h e volume a t t h e end of 3l/2 hours’ heating was only slightly reduced and, hence, t h e reaction was incomplete. The U. s. P. method gave low figures in every case. I t seemed t o us desirable t o study t h e hydrolysis of ethyl-sulfuric acid in t h e case of samples of aromatic sulfuric acid which had not been recently prepared. Accordingly a series of samples, p u t a t our disposal b y an important drug manufacturing house, were assayed, with t h e results given in Table I X . Apparently the age of t h e sample affects very slightly, if a t all, t h e hydrolysis of t h e ethyl-sulfuric acid present. It would also appear t h a t , in order t o meet t h e U.S. P. assay requirement, more sulfuric acid had been used in t h e manufacture of these preparations t h a n corresponds t o 20 per cent by weight.
Vol. 8, NO. I O
TABLEIX-ANALYSES
OF A SERIES OF MANUFACTURERS’ SAMPLES -4-Percentages obtained b y t h e chemists of t h e manufacturing firm a t t h e time of the preparation of t h e samples in question, t h e U. S. P. method having presumably been used. B-Figures obtained by us by t h e use of the same method. C a n d D-Results obtained with open beakers on t h e water bath, according, respectively, a s 3 o r 6 volumes of water were added for hy. drolysis. E-Results of direct titrations, made after dilution with water b u t without previous heating. F-Percentages of t h e total sulfuric acid which have been converted into ethyl-sulfuric acid in t h e respective samples. hT0.
..,,.,. 2 . . . .. . . . 3 . . .. . . .. 4. . . . . . . . 5 , . .. . . . . 6 . . .. . . , , 7... ,.. , . 8....., . . 9. .,.., . 10.. ., . . . . 11 . . . . . , . . 1.
I
Ace 41’/2 mos. 37l/z 35 33’/2 27 25 22 16 13 8 lliz
i 19.90 19.00 19.60 19.70 20.10, 20.40 21 .oo 19.80 19.50 21.00 20.00
Z
B
C
25.40 2 7 , 5 6 2 5 . 8 5 27.49 27.30 28.38 26.51 27.69 23.89 26.02 27.25 28.49 27.41 28.86 26.32 2 7 , 8 6 26.24 27.07 25,67 26.71 23.78 25.05
D 27.56 27.43 28.65 27.69 25.95 28.65 28.86 27.90 26.95 26.65 25.00
E 19.11 19.93 21.34 20.05 18.67 20.85 20.95 20,00 19.53 19.41 18.21
F 15.3 13.7 12.6 13.8 14.0 13.5 13.7 14. I 13.8 13.6 13.6
I n conclusion, we have thought it well t o p u t on record the results of a progressive distillation of aromatic sulfuric acid which had been diluted with over three times its volume of water. Our object was t o show t h a t hydrolysis hardly begins until after t h e removal of t h e alcohol present as such in t h e mixture, and t h a t thereafter t h e increase in t h e proportion of free sulfuric acid keeps pace with t h e removal of alcohol by distillation. As t h e apparatus used possibly exhibits some points of novelty, a short description of it may not be out of place. T h e boiling liquid was contained in a roundbottomed flask through t h e stopper of which were fitted: (I)-A thermometer whose bulb was in the boiling liquid. (2)-A tube through which an aliquot of t h e liquid could be withdrawn b y means of a pipette, and (3)-The distilling t u b e shown in t h e figure. T h e inner t u b e B (about 2 7 X 2 cm.), closed a t t h e bottom and provided with a reflux condenser, E , a t the top, has an outside diameter about 4-5 mm. less t h a n the bore of t h e outer tube A ; t h e latter is provided a t A’ with three “dimples” which hold B in place, and a side-tube, C , which contains a thermometer and connects with a condenser, D. T h e t u b e B is E partly filled with t h e same liquid which it is proposed t o distil off from t h e flaskin this case alcohol. The mixed vapors of alcohol and water rise into t h e annular space between A and B , where t h e water is condensed on 4 coming in contact with B and flows back; as soon as t h e alcohol in B is heated t o boiling t h e alcohol vapor from the flask passes into C and so t o t h e condenser D. T o secure a constant boiling temperature for t h e condensing t u b e B , a bundle of
Oct., 1916
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
capillary tubes closed a t t h e t o p a n d projecting above t h e liquid surface are placed in B . I n order t o give a n idea of t h e efficiency of this apparatus, t h e following may be s t a t e d : Redistilled alcohol was p u t in B a n d a mixture of I O cc. alcohol a n d 240 cc. water was boiled i n t h e flask; when I O cc. of distillate h a d been collected, t h e operation was stopped. T h e distillate proved t o be 95.6per cent alcohol a n d there was no alcohol in t h e residual liquid i n t h e flask. A mixture of 180 cc. aromatic sulfuric acid a n d 580 cc. water was placed in t h e flask a n d boiled. Ordinary alcohol was placed in B . At t h e e n d of each halfhour t h e thermometer readings a n d t h e volume of t h e distillate were noted, after which 5 cc. of t h e mixt u r e were withdrawn from t h e flask, cooled, diluted a n d titrated, while t h e distillation was resumed. I n Table X are given t h e results. T h e respective weights TABLEX Time Hrs. 0.0 0.5 1 .o 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5
Distil- Thermom- Reading late eter in in boiling Cc. vapor liquid 789 0.0 89.10’ 78 43.50 91.40 78 80.00 93.50 78 109.20 95.90 78 129.60 97.90 78 141.80 99.30 78 148.70 99.80 78 152.20 100.10 74 154.30 100.30 69 155.80 100.50 60 in0.70 156.30 60 156.90 100.85 50 157.20 101 . O O 48 157.50 101.15 48 101.30 157.70 i57.8n 48 101.45 158.40 48 101.70 158.80 48 101.85 101.90 159.00 48 102,05 159.00 48 41 102.10 159.00 40 102.10 159.00
Ratio of Weights of HzS04 Free to Total in 2 cc. of Liquid HnSO, Free Combined P e r cent 0.0488 0.0343 79.35 0.0532 0,0328 79.51 0.0593 0.0314 82.70 0.0652 0.0304 84.10 0.0721 0.0255 86.93 0,0758 0.0231 ’ 88.34 0.0821 n.0181 91.44 0.0870 0.0152 92.56 0.0888 0.0147 92.89 0.0118 0.0917 94.31 n.. 0093 95 0.0939 ~ . . . .. 49 .. 0.0956 0,0088 95.77 0.0964 0.0073 96.46 0.0991 0.0050 97.64 0.1003 0.0034 98.34 0.1005 0.0029 98.58 0,1010 0.0019 99.05 0.1020 0.0015 99.52 0.1033 0.0003 99.86 0.1040 0,0000 100.00 0.0000 0.1040 100.00 0.0000 100.00 0.1040
of “free H2S04”a n d “combined H2S04”were obtained in this way: t h e j cc. of liquid withdrawn from t h e flask were made u p t o 2 5 cc. with water; of this dilute solution I O cc. were titrated a t once a n d I O cc. were completely hydrolyzed in a n open beaker on t h e water b a t h a n d t h e n titrated. F r o m t h e d a t a t h u s secured t h e amount of free sulfuric acid a n d t h a t of t h e ethylsulfuric acid present in each 2 cc. of t h e liquid i n t h e flask are calculated. It will be noted t h a t throughout t h e distillation t h e temperature of t h e vapor in t h e side-tube C never rose above 7 8 ” ; after t h e bulk of t h e alcohol h a d been removed, t h e reading of this thermometer slowly fell, since t h e q u a n t i t y of vapor passing through C became less a n d less a n d t h e loss of heat through radiation from t h e thermometer bulb was not compensated for. At t h e same time t h e temperature of t h e boiling liquid slowly rose as alcohol was distilled off. T h e s u m of t h e “free” a n d t h e “combined” acid also slowly increases, for t h e percentage of total acid in t h e boiling liquid rises as t h e alcohol is removed. Finally, we may note t h a t under t h e conditions of t h e experiment over 9 hrs.’ boiling was needed for t h e complete hydrolysis of t h e ethyl-sulfuric acid present. L A B O R A T O R Y O F TMARYLANDSTATE HB DEPARTMENT O F HEALTH, BALTIMORE
RENNIN-A
909
NOTE AND A CORRECTION By HOWARDT. GRABER Received May 27, 1916
Rennin is not official in t h e sense t h a t i t is recognized b y either t h e United States Pharmacopoeia or t h e National Formulary, hence there is a wide variation in t h e article as found upon t h e market, as well as i n t h e methods used for its standardization. I t is universally recogniFed as a milk coagulant a n d all methods use milk as a medium for standardization. I n 1911 I published an article’ entitled “Some Observations upon t h e Assay of Digestive Ferments.” I n this article I included t h e assay used b y me in t h e standardization of rennin. There was a n error in this script which has since been corrected. This error t o which I want t o call especial attention was i n t h e amount of milk specified t o be used for t h e test. I t was just twice t h e volume intended, so t h a t t h e results b y this error were just half what t h e y should be, or in other words t h e rennin seemed t o be just half its real strength. I have tried other methods proposed, b u t I recommend t h e method as published b y me both for its simplicity of operation a n d t h e reliability of t h e results obtained when one uses a standard of known value as control. T h e enzyme rennin, like all other enzymes, is distinctly specific in its reaction a n d it shows its selective characteristics as t o t h e nature of t h e proteid, t e m perature a n d reaction of t h e media as well as t h e presence or absence of organic a n d inorganic salts, especially those of calcium a n d t h e phosphates. Results often vary when samples of milk are used from different cows, as well as different lots from t h e s a m e cow, a n d t h e best results are obtained when using a mixt u r e from t h e milking of many cows. There also seems t o be a seasonal variation in t h e ability of t h e milk t o coagulate. T h e following table gives t h e results of many of t h e tests: DATE 4/ 1 to 4/ 15/ 14 8/ 7/14 8
10 15 9/ 1/14 1/24/15 21 2/16/15 22 3 / 8/15 31 5 / 5/15 13 17 27 29 30 6/ !/I5 1
12 17 22 22 29 7 / 2/15 7 9
IO 21 22
MONTHLY ASSAY AVERAGE 1 : 30,000 in Min. Sec. Min. 7.5 10
io
7
7 9 16 11 8
30 30 30 30 30 30
10 10 11
8 11 11 10 10 10 9 11 8 10 12 12 11 8 8 8 8 12 10
9 9 13.75 9.25 11
30 10
7 10.3
45 45 10 5 30 15
10.6
30 45 45
DATE 11/ 5/15 12/ 3/15 22 23 24 27 28 29 30 31 1/ 3/16 4 5 6
9.3
8 2/14/16 3/ 6/16 7 8 9 10 11 12 14 23 24 28 29 30 31
MONTHLY ASSAY AVERAGE 1 : 30,000 in Min. Sec. Min. 10 30 10 30 11 30 8
16 13 7 11 11 11 10 11 10 11 11 10 11 10 9
7
7 5 9 9 8 1;
30 30
10.5
30 30
10.5
11 45 30 30 30 30 30
7.5
15
4
6 4 7
Several years ago I selected a rennin t o be used as a standard in t h e assay of this enzyme, a n d I have 1
THISJOURNAL,3 (1911), 919.
