JULY 15, 1936
-4NALYTICAL EDITION
tilling p ridine which had been dried by refluxing over barium oxide. %he 20 per cent reagent is preferred for sugars, whereas either reagent may be used with the other compounds analyzed in the present work. PROCEDURE. Exactly 2 cc. of reagent are pipetted into a clean dry test tube containing an accurately weighed portion of the substance under analysis, care being taken to wash down with reagent any particles adhering to the side. The weight of the sample varies with the hydroxyl content of the compound-for example, 50 mg. of glucose are sufficient. The mixture is carefully heated over an open flame until the solution boils and for 1 minute longer. After cooling it to room temperature, the solution is diluted with 5 cc. of carbon dioxide-free water and transferred t o a small flask; three washin 8 of the tube are made, two with 10-cc. portions of water, andone with 10 cc. of ethyl alcohol. The acid is titrated with 0.1 N sodium hydroxide to the cresolphthalein end point. It is necessary, of course, to run blanks for the reagent alone. In the case of substances containing appreciable proportions of fatty acids which may form difficultly decomposable mixed anhydrides with the reagent it is desirable, after the dilution of the reaction mixture with 5 cc. of carbon dioxide-free water, to boil the solution for 1 minute before finally cooling and titrating. Furthermore, in the analysis of lipoidal substances the final titration is carried out in the presence of sufficient ethyl alcohol to provide a concentration of approximately 50 per cent after the addition of the alkali. Since only one-half of the titratable acid is available for reaction as acetic anhydride, it is desirable, in order to ensure an excess of the anhydride, that three-quarters of the original titratable acidity remain after the completion of the acetylation reaction. I n the analysis of the compounds listed in the second part of Table I the aqueous solution of the reactants was boiled, then cooled, and alcohol was added to the flask before titration. Practically theoretical results were obtained for all compounds listed except the sugar alcohols, the results for
279
which were about 2.5 per cent low, and the single tertiary alcohol, which did not react. The results for pregnandiol, obtained by the method described, were low; allowing the reaction mixture, after the preliminary heating, to stand overnight (13 hours) a t room temperature the results obtained were much closer to the theoretical value. The sample of triolein analyzed was obviously impure, having a deep red color. The method of recording the results tends to emphasize slight errors.
Summary 1. The acetylation method of Verley and Bolsing has been modified to make possible the analysis of small samples of organic compounds. 2. The new method is rapid and convenient and does not require the use of condensers or ground-glass stoppers. It has been applied successfully to representative organic compounds. 3. Tertiary alcohols cannot be estimated by this method.
Acknowledgment The writers are indebted to G. F. Marrian of this department for the samples of oestrone and pregnandiol used in the investigation.
Literature Cited (1) Peterson,V. L., and West, E. S., J . Bid. Chem., 74,379 (1927). (2) Verley, A., and Bolsing, F., Bey., 34,3354 (1901). (3) West, E. S., Hoagland, C. L., and Curtis, G. H., J. Biol. Cham., 104, 627 (1934). RECEIWD February 13, 1936.
Estimation and Identification of the Glucoside
Salicin MORRIS B. JACOBS, Department of Health, 125 Worth St., New York, N. Y. NICHOLAS T. FARINACCI, 336 West Twelfth St., New York, N. Y.
A qualitative and quantitative method for the determination of salicin, a glucoside, is presented. The quantitative method depends on the production of a polymerized cleavage product, obtained by acid hydrolysis, which is estimated gravimetrically. A qualitative test which is outlined depends on the solubility of the polymer in a solution of sodium hydroxide with the production of a violet color, and is modified so as to be used as an approximate quantitative colorimetric method. Another qualitative method based on a coupling reaction with p-diazobenzenesulfonic acid anhydride is detailed.
T
HE identification of salicin and its estimation have been studied over a number of years, but the methods obtained by these studies have been long and not easily adapted for accurate work. The importance of glucosides as a class necessitates the development of short and accurate means for
their estimation. The methods in use a t present rely on the hydrolysis of salicin either by enzymatic action (7) or by the action of acids ( I S ) and the subsequent estimation of the glucose part of the molecule. None of the methods available are dependent upon the specific chemical nature of salicin, itself, or of the cleavage product, saligenin. It is well known that when salicin (saligenin-/3-glucoside) is hydrolyzed by enzymes or acids, the products are glucose and saligenin. These reactions are noted in Beilstein (S), where it is also noted that saligenin polymerizes on boiling with dilute sulfuric acid to saliretin. * Much work was done by Piria ( 1 4 , who noted that both salicin and saligenin boiled with dilute hydrochloric acid yielded saliretin. He concluded that saliretin prepared from salicin did not have a definite composition, but prepared from saligenin its formula was GHsO. Kraut (10) took powdered salicin and ten parts of hydrochloric acid (sp. gr. 1.125), warmed until dissolved, heated to 80' C., and noted the yellowish red color of the resultant saliretin. He concluded that it was saligenosaligenin, HOCeH&H*.O.CeHcCH20H. On the other hand, von Gerhardt ( 6 ) and Beilstein and Seelheim (8) found higher polymers. Kraut also noted that saliretin was soluble in alkalies. Voswinkel (16)did not obtain the same product from salicin plus hydrochloric acid as with sulfuric acid. Wischo (17) treated salicin with nitric acid, producing picric acid. He boiled an aqueous solution of salicin with dilute hydrochloric acid, forming saliretin, dissolved the saliretin in potas-
280
INDUSTRIAL AND ENGINEERING CHEMISTRY
sium hydroxide, and added this solution to the picric acid solution, producing picramic acid and a bluish red color. Dott (5) gives the following test to identify salicin: To an aqueous solution of the substance to be investigated add twotenths its volume of hydrochloric acid and warm. The solution develops an aromatic odor and precipitates saliretin. Extract this with ethyl ether and evaporate to dryness. If salicin is present the amorphous residue will be red. Jackson and Dehn (8) present in tabular form various reactions for the identification not only of salicin but of many other glucosides. Ware (16) notes that salicin gives a color when warmed with resorcinol and phosphoric or sulfuric acid.
It occurred to the authors that salicin, which has the nature of a phenolic ether, should under the proper conditions couple ( I , 11, 12) with a diazonium compound. Salicin does couple with p-diazobenzenesulfonic acid anhydride, but only with an alkaline solution prepared from the dry compound.
mean is 0.006. The method may be applied in the presence of small amounts of lactose, sucrose, inulin, maltose, arabic, tragacanth, karaya, agar, Irish moss, locust kernel, ghatti, starch, and the glucoside amygdalin. These substances do not interfere, for they do not produce a precipitate on evaporation with concentrated hydrochloric acid. Diluted normal urine also does not interfere. On the other hand, the glucosides digitonin and saponin do interfere, for they yield an acid-insoluble precipitate when treated as directed. These precipitates do not have a red color and are insoluble in 10 per cent sodium hydroxide solution. B y means of the method given below, salicin may be determined in the presence of these glucosides. TABLE I. CONVERSION OF SALICIN TO SALIRETIN A:id Precipitate
Salicin
Qualitative Tests TEST1. Dissolve 15 mg. of p-diazobenzenesulfonic acid anhydride (4) in 2 cc. of 10 per cent sodium hydroxide solution. Add this to 5 cc. of salicin solution, mix, and place in a warm water bath (approximately 80' C.) for 1 minute. At the end of this time, if salicin is present, a deep red color will develop. Upon the addition of acid the color changes to orange. A very deep red color will be produced by 1 mg. of salicin per cc., and will also develop in the cold, in the case of 10 mg. per cc. at the end of 30 minutes, and in the case of 1mg. per cc. at the end of 2 hours. It is best to run a blank with the diazonium reagent, in order to be sure that the colors are not due to decomposition of the reagent itself.
This method for the identification of salicin is very simple except for the preparation and keeping qualities of the diazonium reagent. However, at best, it can only be indicative of the presence of this glucoside because of the numerous other substances which will couple with this reagent under exactly the same conditions. The color produced by the reaction between salicin and the diazonium reagent is apparently proportional to the concentration of the former, for the depth of color increases with the concentration of salicin. TEST2. Proceed as directed under quantitative colorimetric method, evaporating almost to dryness. A red precipitate soluble in alkali with the production of a violet color, which on dilution becomes salmon-colored, shows the presence of salicin. This color will be produced with as little as 0.2 mg. per cc.
Quantitative Gravimetric Method The regularity of the changes produced in salicin during evaporation with concentrated hydrochloric acid led to the belief that the production of the precipitate was entirely quantitative. Place 25 to 50 cc. of the solution to be analyzed containing 1 to 5 mg. of salicin per cc. in a tall-form 100-cc. beaker and add 25 cc. of concentrated hydrochloric acid. Cover with a watch glass on glass hooks and evaporate very slowly on a hot plate carefully regulated to 80' C. or less, down to a volume of not more than 10 cc. Prepare a Gooch crucible or a porous glass crucible with a thin pad of acid-washed asbestos in the usual manner and dry in a constant-temperature oven to constant weight at 100' C. Cool in a desiccator and weigh. Filter the precipitate obtained from the salicin through the Gooch crucible. Carefully transfer all the precipitate to the crucible by means of a rubber policeman and wash bottle. Wash thoroughly with distilled water, suck dry, place in a constant-temperature oven at 100" C., and dry to constant weight. Multiply the weight of the precipitate by the factor 2.524 to obtain the quantity of salicin present in the original aliquot taken for analysis. Table I gives the results of this method on known quantities of recrystallized salicin. Acid hydrolysis of salicin conducted as outlined yields results, based on an average recovery of saliretin, which are at least 98.5 per cent accurate. The average deviation from the
VOL. 8 , NO. 4
MO./d5-60
39 41 50 50 50 62.5 62.6 75 75 100 100 125 125 250 260
CC.
Ratio of Column 2 to Column 1
Mo. 15.5 16.0 19.0 19.8 20.0 24.2 24.6 29.2 29.3 39.6 39.8 52.9 49.7 101.2 100.6
0.397 0.390 0.380 0.396 0.400 0.387 0.394 0.389 0.391 0.396 0.398 0.423 0.398 0.405 0.402 Mean 0.396
Assuming that salicin splits quantitatively, yielding saligenin and glucose C1aHisO.l
+ Ha0 +CEHI~OE + OHCBH~CH~OH
and that saligenin goes completely to saliretin 2GHsO2 +Cl4H1408
+ HzO
then two moles of salicin are equivalent to one mole of saliretin-that is, 572 grams of salicin are equivalent to 230 grams of saliretin. The theoretical yield, if the above holds true, would be 230/572 or 40.21 per cent of salicin used. Table I demonstrates that we recover, expressing the ratio of columns 2 to 1 in percentage, 39.6 per cent of the salicin used, or 98.5 per cent of the theoretical yield. These results indicate that only one polymer of saligenin is formed when conditions of acid hydrolysis are rigidly controlled. Kraut ( I O ) , it may be noted, felt that the production of saliretin is quantitative. The factor used in the method is obtained from the reciprocal of the average recovery of saliretin and will, of course, yield the quantity of salicin sought.
Quantitative Colorimetric Method Place 5 cc. of a solution of salicin containing ap roximately 1 to 2 mg. per cc. in a 10- to 15-00. beaker with a mar[ at 2 cc., add 5 cc. of concentrated hydrochloric acid, and evaporate slowly on a hot plate kept at 80" C. or less to 2 cc. Allow to cool and filter through a small filter. Wash both beaker and filter well with cold water, and replace the receiver of wash water with a tube having a graduation at 10 cc. or with a 10-cc. volumetric flask. Wash beaker and filter paper with 4 successive I-cc. portions of 10 per cent sodium hydroxide, wash filter once more with 1cc. of 10 per cent sodium hydroxide, and again wash beaker and filter faper with four successive 1-cc. portions of distilled water. Ma e receiver tube or volumetric flask up to volume, mix thoroughly, and read in a colorimeter against standard control treated in exactly the same way. The ratio of fourteen determinations comparing unknown to standard had a minimum of 0.925, a maximum of 1.17, and a mean of 1.02 as the variation from unity.
It is obvious that this method has not been completely developed to yield high accuracy; however, the results are of the correct order and are better than some quoted in the
JULY 15, 1936
ANALYTICAL EDITION
literature (9). Using standard of concentration close to that of unknown would aid in obtaining higher accuracy. This method is far more rapid than others and where interfering substances are present in the gravimetric method, it is better than none. It is a t present being studied for better development. As the hydrolysis of salicin proceeds, the following changes take place: Shortly after the solution reaches a temperature of 80” C., a white turbidity is noticeable. This turbidity changes slowly in an hour or so to a pink precipitate which grows deeper in color as the time of heating and evaporation proceeds. For quantitative conversion, it is essential to evaporate very slowly and a t a temperature never higher than 80” c. The length of time necessary for accurate results indicates that the formation of saliretin proceeds with a definite velocity. The rate of this reaction has never been studied. The concentration of hydrochloric acid is of great importance for, when it falls below a certain minimum, the reaction does not yield quantitative results within a reasonable time. This work was Derformed in the Chemical Laboratorv of the Bureau of Food and Drugs, Department of Health, City of New York.
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Literature Cited Anwers, K., and Michaelis, F., Ber., 47,1275 (1914). Beilstein, F.,and Seelheim, F., Ann., 117,83 (1861). Beilstein, 3rd ed., Vol. 111, p. 608 (1897); Vol. 11, p. 1109 (1896); Vol. 11,supplement, p. 680 (1903). Cumming, W. M., Hopper, I. V., and Wheeler, T. S., “Systematic Organic Chemistry,” 2nd ed., p. 441, New York, D. Van Nostrand Co., 1931. Dott, H. B., Pharm. J.,119, 691 (1927). Gerhardt, von, Ann. Chim., [317,215 (1843). Hudson, C. S., and Paine, H. S., J. Am. Chem. Soc., 31, 1242 (1909). Jackson, K. E., and Dehn, W. M., IXD.ENO.CHEM.,Anal. Ed., 6,382 (1934). Kerstan, G., Planta (Abt. E., 2. wiss. Biol.), 21,657 (1934). Kraut, K., Ann., 156,123 (1870). Meyer, K. H.,Irschick, A., and Schlosser, H., Ber., 47, 1741 (1914). Meyer, K. H., and Lenhardt, S., Ann., 398,66 (1913). Moelwyn-Hughes, E. A., Trans. Faraday SOC.,24,309 (1928). Piria, R.,Ann., 56,35 (1845). Voswinkel, A., Chem. Zentr., (1900) I, 771. Ware, A. H.,Chemist and Druggist, 120,74 (1934). Wischo, P.M. F., Pharm. Post, 28.42 (1895). RECEIVED February 4, 1936.
Battery-Type Stand Assembly for Distilling Equipment *
ROY L. MOBLEY, P. 0. Box 1021, Baton Rouge, La.
T
H E efficiency of distillation has in many ways been lowered by the equipment available. I n the older types of apparatus vapors and liquids were brought in contact with metals, and with cork and rubber joints, stoppers often leaked, and in some instances the units making up the apparatus were inefficient. The advent of all-glass interchangeable-joint apparatus and improved condensers has brought about much greater efficiency and more accurate results. However, the complete change of type and general form of equipment has made older accessories obsolete, and it is extremely time- and accessory-consuming to set up two or more of the newer types a t a time. I n an attempt to overcome these handicaps, the author made the necessary calculations and arrangements, diagrammatic sketches, etc., and developed a working model of a battery-type stand assembly which has proved very satis-
factory during the past 3 years. The photographs show front and diagonal end views of the assembly as built for 500-cc. distilling flasks and indicate arrangement of the parts. The apparatus was built and used in the laboratory of William L. Owen, Baton Rouge, La. RBCEIVED May 14,1936.
CORRECTION. In the article on ILDeterminationof Nitric Oxide in Coke-Oven Gas” [IND. ENG.CHEM.,Anal. Ed., 8, 164 (1936)] an error was made in Figure 2. The glass balls should be of 6or 6-mm. diameter, as the 8-mm. balls specified would be entirely too large for this apparatus. J. A. SHAW
