VOL. 8, NO. 4
INDUSTRIAL AND ENGINEERING CHEMISTRY
218
Finally, use of the modifications for nitrate nitrogen in addition to total nitrogen gives quantitative estimation of both forms, as will be noted in Table IV. The authors have found no evidence that the organic matter present is able to reduce the nitrate during the process of digestion, and reduction with nascent hydrogen must be resorted to. TABLEIV. NITRATE NITROQEN DETERMINATION -Nitrogen FoundNo. of Nitrogen Present ReSamples Organica Nitrate Total Nitrate covery Mg. Mg. MQ. Me. % Pure NaNOs 6 0.45 0.45 *0.02 0.45 *0.02 100 2125 0.45 2.69 *0.06 0.44 *0.06 97 Sap NaNOs NaNos 2.25 0.20 2.45 * 0.03 0.20 * 0 03 100 a Determined on sap before addition of nitrate. Material
‘5
+
Literature Cited (1) Assoc. Official Agr. Chem., Official and Tentative Methods, 3rd ed., 1930. (2) Dsvisson and Parsons, J. IND.ENG.CHIM., 11, 306 $:919). (3) Fisher, “Statistical Methods for Research Workers, London, Oliver and Boyd, 1930. (4) Folin, J. Bid. Chem., 97, 141 (1932). (5) Meeker and Wagner, IND. ENG.CHEM.,Anal. Ed., 5, 396 (1933). (6) Parnas and Wagner, Biochem. Z., 125, 253 (1921). (7)Ranker, E.R . , Ann. Missouri Botan. Gardens, 13, 391 (1926). (S) Sessions and Shive, Plant Physiol., 3, 501 (1928). (9) Smgth and Wilson, Biochem. Z.,282, 1 (1935). (10) Umbreit and Wilson, to be published. (11) Van Slyke and Cullen, J. Biol. Chem., 19, 218 (1914). R E C E I V ~April D 23, 1936. Herman Frasch Foundation in Agricultural Chemistry, Paper No. 117: Contribution from the Departments of Agricultural Bacteriology and Agricultural Chemistry, University of Wisconsin.
Determination of Hydroxyl Groups in Organic Compounds M. FREED AND A. M. WYNNE, Department of Biochemistry, University of Toronto, Toronto, Canada
D
URING an investigation of the enzymic synthesis of
glycerides it was desirable to have information as to the degree of esterification accomplished at various stages of the reaction. For this purpose a method for the determination of the hydroxyl content of the product, based upon that of Verley and Bolsing {2), was devised. The method of these workers, as originally described, required more material than could be conveniently obtained in the synthetic experiments and, in addition, was not sufficiently rapid. The use of the reagent of Verley and Bolsing-namely, acetic anhydride in pyridine solution-has been applied by other workers to the analysis of small amounts of material [of. Peterson and West ( I ) , and West, Hoagland, and Curtis (S)],but it seemed possible that some of the precautionary measures, such as the use of condensers and ground-glass stoppers, adopted by these workers to prevent possible losses of reactants might
not be necessary in the analysis of many nonvolatile compounds, especially in view of the fact that the original authors found such measures unnecessary, Moreover, the modified methods were found to be still somewhat time-consuming. Because of the observation that no loss of titratable acid occurs even on fairly vigorous boiling of the acetic acidpyridine reagent in open vessels without condensers, it has been possible to simplify and shorten the analytical procedure very considerably without loss of accuracy. Before using the method for the purpose for which it was designed it was tested on a number of representative compounds, with the results recorded in Table I.
Experimental Methods REAQENT.This was a solution of acetic anhydride (either 12 or 20 per cent) in dry pyridine, which was prepared by redis-
TABLEI. HYDROXYL CONTENT OF ORQANICCOMPOUNDB
Compound 1-Naphthol Hydroxyiscbutyric acid Salicylic acid Catechol
Phloroglucinol Arabinose
Xyloee
-OH -OH EqmvaEquivalents lents per Mole, & e? Aver- Theoreti- Error ’ Founb age cal Per CeAt 20 Per Cent Reagent 1.000 1,000 0.996 0.998 0.995 0.997 0.098 0.909 0.998 2.05 1.96 1.93 1.92 2.12 2.99 3.03 3.88 3.98 4.14 3.94 3.90 4.00 4.00 4.18 3.82 4.01 4.13 3.89 3.94 3.94
-OH
Source of Compound
Equivalents per Mole Compound Founh Glucose
0.998
1,000
-0.2
Eastman
0.997
1,000
-0.3
Kahlbaum
Mannitol
0.999
1,000
-0.1
Merck
Sorbitol
2.00
2.00
0.0
Eastman
3.01
3.00
f0.33
Eastman
Dulcitol
Trichloro - tertbutyl alcohol 0 . 0 0 Monobutvrin Triolein
3.97
4.00
-0.75
Pfanstiehl Cholesterol Oestrone Pregnandiol
3.99
4.00
-0.25
Pfanstiehl
5.00 4.99 5.00 5.10 5.90 5.83 5.83 5 89 A RA -.I5.84 5.85
2.06 1.97 0.35 0.44 0.41 1.018 1.004
-OH Equivalents Der hlole, AverTheoreti- Error, age ad Per Cent 20 Per Cent Reagent (Cont’d)
Source of Compound British Drug House8
5.02
5.00
+0.40
5.85
6.00
-2.5
British Drug Houses
5.86
6.00
-2.4
Pfanstiehl
5.84
6.00
-2.4
Pfanstiehl
. ..
..
1.00 12 Per Cent Reagent 2.02 2.00 1.0
+
0.40
0.00 1.00 1.00
*.
+1.8 4-0.4
Eastman Schuchardt Kahlbaum British Drug Houses Isolated from pregnant mares’ urine Iaolated from pregnant humun urine
1.67a 1.44 2.00 -28.0 1.30a 1.96b 1.97 2.00 -1.5 1.99b Determinations made by the standard method. b The reaction mixture, after the preliminary heatinq, was allowed to stand for 13 hours at room temperature before the final titration. @
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. In 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 anhydrideis 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-
