Quantitative Estimation of Acetyl in Carbohydrate Acetates d
ROY L. WHISTLER
AND
ALLENE JEANES, Starch and Dextrose Division, Northern Regional Research Laboratory, Peoria, Ill.
ETHODS for the quantitative determination of carbohydrate acetyl have been developed principally along two separate lines: (1) for the simple sugar acetates which require the use of mild conditions to prevent errors which might arise from the destruction of the labile carbohydrate molecules; (2) for cellulose acetates which, because of the heterogeneous reaction involved, require somewhat more drastic conditions or considerably extended reaction time. Separate, essentially noninterchangeable methods were, therefore, evolved for the two classes of carbohydrate derivatives. Adaptations of the aforementioned methods have been applied to starch acetates, but none appears to have received general acceptance. This paper describes a quantitative method of acetyl analysis which is applicable not only to the polysaccharide acetates of starch and cellulose but, on slight modification, to the simple sugar acetates as well. The method employs the sodium methoxide catalyst of Zemplen (7) to transesterify carbohydrate acetyl in methanol with the production of methyl acetate which is then quantitatively removed and determined b y a modification of the well-known Freudenberg ( 2 ) procedure. Starch and cellulose do not yield volatile acidic decomposition products on contact with alkaline catalyst during the
separation of the methyl acetate by distillation. The lower sugars, being labile, are decomposed on boiling in the presence of the catalyst. Therefore, in these cases, the transesterification is accomplished at room temperature, and the catalyst is neutralized hp the addition of a methanol solution of sulfuric acid before proceeding with the distillation. The transesterification is rapid, requiring only 15 minutes a t room temperature, for acetates of the simple carbohydrates. Even in the case of polysaccharide acetates, ivhich remain insoluble, the reaction is completed in about 25 minutes. In certain instances with dense or somewhat horny acetates the reaction proceeds slowly. I n these cases it is expedient first to swell the acetate by heating in methanol for 0.5 to 1 hour before proceeding with the analysis. The apparatus employed for the analysis is a modification of the Freudenberg ( 2 ) apparatus as shown in Figure 1.
Analytical Procedure REAGEKTS. Anhydrous methanol was prepared by drying methanol with magnesium methoxide (4) and distilling through a fractionating column. Sodium methoxide was prepared by dissolving clean sodium in anhydrous methanol. Th'e sodium ;-as cut und6.r xylene to expose fresh surfaces and weighed. It was then rapidly passed through three separate portions of anhydrous methanol contained in beakers and finally dissolved in anhydrous methanol in a volumetric flask and made up t o volume. Approximately 0.2 N solutions were employed. An approximately 0.2 N solution of sulfuric acid in methanol was prepared by adding a calculated amount of concentrated sulfuric acid to anhydrous methanol and allowing the mixture to stand several days to permit formation of monomethyl sulfate. The solution was then standardized and was found to maintain its normality for a considerable time. PROCEDURE FOR POLYSACCHARIDE ACETATES.A 0 2- to 0.3gram sample of finely divided polysaccharide acetate, previously dried in vacuo over phosphorus pentoxide, is placed in 20 ml. of anhydrous methanol in the reaction flask (C, Figure l),containing a boiling tube to prevent bumping. Approximately 5 to 10 ml. of 0.2 N sodium methoxide (3 to 8 milliequivalents per gram of sample) in anhydrous methanol are added through the dropping funnel, B, which is protected by a small drying tube, A . The temperature of the watw bath, G, is raised until slow distillation into the receiving flask, E, occurs, the rate of distillation being approximately 35 drops per minute. During the distillation, the receiving flask is cooled by immersion in crushed ice. The receiving flask may be protected by a small tube, F , filled with soda lime. When the liquid in the distillation flask has been concentrated to a small volume (approximately 5 ml.) a fresh 20-ml. portion of anhydrous mcthanol is admitted through the dropping funnel and the solution is again distilled to a small volume. Two 10-ml. portions of anhydrous methanol are then added successively and the solution is concentrated to a small volume after the addition of each portion. During the concentration the n ater level in the water bath is maintained just below the level of liquid in the distillation flask in order to prevent baking of material on the flask walls. After the final concentration, the distillation flask is removed or the stopcock on dropping tube B is opened,. and 25 ml. of 0.2 N sodium hydroxide are pipetted into the receiving flask by way of the side neck and the neck closed by a glass stopper. The bath, H , of crushed ice is removed and a hot water bath substituted. The contents of the receiving flask are refluxed 15 minutes, the flask is cooled, 75 ml. of boiled distilled water are added, and the excess sodium hydroxide is titrated with 0.1 N hydrochloric acid to a phenolphthalein end point. The base consumed in the saponification, minus that consumed in a separate blank determination. is eauivalent to the acetyl content of the Carbohydrate acetate. ' Blaxik determinations are made by refluxing 50 ml. of
F
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methanol 15 minutes with 25 ml. of 0.2 N sodium hydroxide and titrating as described above. It is not necessary that the catalyst remain in the reaction mixture throughout the distillation. After the fist concentration of the liquid in the distillation flask the catalyst may be neutralized or the solution slightly acidified by addition of sulfuric acid dissolved in methanol, and the remainder of the distillation carried out as described. This procedure causes no change in the observed acetyl value. Samples not in a state of fine subdivision are refluxed with methanol for 0.5 to 1 hour before introducing the catalyst and proceeding with the analysis. For the reflux, water is circulated through condenser D but at all other times this condenser remains empty. PROCEDURE FOR SIMPLESUQAR ACETATES. A 0.20- to 0.25am sample of sugar acetate is added to the reaction flask and %solved in 20 ml. of anhydrous methanol by gentle heating When complete solution has occurred the mixture is cooled td room temperature, and 5 to 10 ml. of 0.2 N sodium methoxide in anhydrous methanol are added and well mixed with the solution. After 15 minutes, slightly more of a solution of sulfuric acid in methanol is added than is needed to neutralize the catalyst. The methyl acetate is then quantitatively distilled and determined as described in the procedure for polysaccharide acetates. The sample may be left in contact with the catalyst for periods longer than 15 minutes without affecting the results. The equilibrium effected by the catalyst apparently produces methyl acetate to the almost complete exclusion of carbohydrate acetate.
Discussion Since the presence of sodium hydroxide in the solution would tend to prevent quantitative transesterification and distillation of the methyl acetate, it is important that water be carefully excluded from the reaction flask and that dry samples and anhydrous reagents be employed. The amount of catalyst employed need not be precisely controlled. A several-fold variation in the ratio of catalyst to eample may be used without detriment to the analytical results. I n general, 3 to 8 milliequivalents of catalyst per gram of sample were employed. Barium methoxide gives good results with the simple sugar acetates, but is not so effective with the polysaccharide acetates as is sodium methoxide. Methanol is used in preference to ethanol because of the more favorable distillation of methyl acetate from a solution in methanol as compared to ethyl acetate from ethanol. Results are reproducible to about *0.1 per cent acetylfor example, the value reported in Table I for cornstarch acetate I represents the mean value of 18 determinations all values of which were within ~ 0 . per 2 cent of the mean value and 15 results were within *0.1 per cent. The accuracy of the results is good in the case of the lower sugar acetates. Since no method exists for definitely establishing the acetyl content of starch and cellulose acetates, the accuracy of the results in the case of these polysaccharides cannot be precisely determined. For this reason it was of interest to compare the values obtained for the polysaccharide acetates with values obtained by the use of a previously widely used procedure. The methods in the literature, applying principally to the determination of acetyl or combined acetic acid in cellulose acetates, have been comprehensively reviewed and criticized by several workers (3, 6, 6). The method recommended (3,6) as giving the most reliable results is a modification of the method of Eberstadt (I), which consists in saponification of the acetate in standard alkali and subsequent titration of the excess alkali with acid. Values obtained from quadruplicate determinations by this method were compared with values resulting from the sodium methoxide procedure. I n general, the results obtained by the two methods were in good agreement (Table I). In several cases, however, the modified Eberstadt method gave acetyl percentages 0.2 to 0.4 higher than the values obtained by the sodium methoxide procedure. I n these instances, a blank run on the unacetylated starches showed that the samples possessed an acidity equivalent to 0.2 to 0.4 per cent acetyl, Acidic groups may result from the presence
of fats, phosphoric acid residues, or carboxyl groups produced by partial oxidation of the carbohydrate. Correction for these acidities brought the results into agreement with those of the sodium methoxide procedure. Acetyl values which were thus corrected are indicated in Table I. Since cornstarch acetate I11 was a commercial product and none of the unacetylated starch was available, correction of the acetyl value for acidic contaminants could not readily be made. This acetate was impure, as indicated by its brown color. I n contrast to the Eberstadt method, the sodium methoxide procedure is not affected by the presence of nonvolatile acidic contaminants such as fats and phosphoric acid residues or by the existence of free carboxyl groups resulting from oxidation of the polysaccharide. TABLE I. ANALYSISOF CARBOHYDRATE ACETATES Per Cent Acetyl 1 By sodium By Eberstadt Substance methoxide method Theoretioal Cornstarch acetate I 44.36 44.48b 44.78C Cornstarch acetate I1 44.17 44.25d Cornstarch acetate 111 36.94 37.64 White potato starch acetate 44.15 44.20d Waxy cornstarch acetate 43.90 44.046 ..* Wheat starch acetate 44.31 44.10d Cellulose acetate 1’ 40.51 40.21 Cellulose acetate 1 1 8 44 20 43 97 ... _ _ ~. Glucose penta-acetate 55.23 56:i3 Mannose penta-acetate 55.19 55 13 Maltose octa-acetate 50.79 50.74 Gentiobiose octa-acetate 50.77 50.74 a All values are average of a t least tri licste determinations. b No acidity found present in unacetyrated starch. e Calculated for [CsH-Or(CHaCO)rl. d Corrected for acidity in unacetylated starch. e Commercial preparations.
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Attempts to adapt the Freudenberg procedure (2) to polysaccharide acetates were not entirely successful. The slowness of the p-toluenesulfonic acid to cause transesterification required long reflux periods which resulted in abnormally large blank values. However, when the proper blanks were subtracted the acetyl values were in rough agreement with those indicated in Table I.
Summary A method is reported for the quantitative estimation of acetyl in either polysaccharide acetates or simple sugar acetates. The acetates are transesterified by sodium methoxide catalyst in anhydrous methanol to yield methyl acetate, which is quantitatively distilled, and saponified in standard alkali. The method is rapid, gives reproducible results, and possesses the advantage of yielding acetyl values which are not affected by the presence of nonvolatile acidic groups in the sample. Acknowledgment The authors wish to thank P. A. Haeder and N. H. Koenig for performing some of the analytical determinations. Literature Cited (1) Eberstadt, O.,“Uber Acetylcellulose”, dissertation, Heidelberg, 1909; see Knoevenagel, E.,2.angew. Chem., 27, 505-9 (1914); Knoevenagel, E., and Koenig, P., Cellulosechem., 3, 113-21 (1922). (2) Freudenberg, K.,and Harder, M., Ann., 433,230-7 (1923). (3) Genung, L. B., and Mallatt, R. C., IND. ENQ.CHEM.,ANAL.ED., 13, 369-74 (1941). (4) Gilman, H., “Organic Syntheses”, Collective Vol. I, p. 244, John Wilev & Sons. 1932. (5) Kruege”r, D., Furben-Ztg., 35, 2032 (1930); “Zelluloseazetate”, pp. 218-28, Berlin, Theodor Steinkopff, 1933. (6) Murray, T. F., Jr., Staud, C. J., and Gray, H. LeB., IND.ENQ. CHEM.,ANAL.ED., 3,269-73 (1931). (7) ZemplBn, G., Gerecs, A., and HadBcsy, I., Ber., 69, 1827-9 (1936). PRESENTED before the Division of Sugar Chemistry and Technology a t the SOCIETY, Buffalo, S . Y . 104th Meeting of the . ~ V E R I C A X CHEVICAL
