THE CHEMISTRY OF LIGNIN X. LIGNIN FROM OAT STRAW* BY MAX PHILLIPS AND M. J. GOSS (From the Industrial Farm Products Research Division, Bureau of Chemistry and Soils, United States Department of Agriculture, Washington) (Received for publication April 20, 1936) In a previous communication (1) results of an investigation were presented dealing with the chemistry of ligqin from barley straw. A review of the literature was then presented. Inadvert,ently, reference to a paper by Marion (2) dealing with the lignin of oat and wheat straws was then omitted. In this paper results of a study of the lignins from oat straw are presented. Three lignin fractions were isolated from oat straw. The first two fractions were isolated by successive and exhaustive extractions, first with a 2 per cent alcoholic sodium hydroxide solution at room temperature, and then by refluxing with a 4 per cent aqueous sodium hydroxide solution, according to the method previously described (1). The third fraction was isolated from the straw remaining from the extraction operations referred to above with fuming hydrochloric acid. The composition of the first lignin fraction agreed with that represented by the formula C40H48015. On the basis of a compound having a molecular weight represented by this formula, approximately four methoxyl groups and four hydroxyl groups were shown to be present. Of the four hydroxyl groups, two could be methylated with diazomethane, thus indicating that these are more acidic, possibly phenolic or enolic in character. On methylation with dimethyl sulfate and sodium hydroxide solution (9 per cent) incomplete methylation of the four hydroxyl groups resulted. However, by remethylating this preparation with dimethyl sulfate and 40 per cent sodium hydroxide solution, a product was obtained containing five methoxyl groups in addition to those naturally present in this lignin * Industrial Farm Products Research Division Contribution No. 260. 557
558 Chemistry of Lignin. X fraction, thus indicating that this drastic treatment resulted in the formation of one more hydroxyl group which was then methylated by the dimethyl sulfate. When this lignin fraction was subjected to fusion with potassium hydroxide, protocatechuic acid (isolated as its dimethyl ether, veratric acid) in approximately 4 per cent yield was obtained. The analytical results on the second lignin fraction are more in agreement with those represented by the formula CMHUOS In this lignin fraction approximately four methoxyl groups and four hydroxyl groups were shown to be present. Two of the hydroxyl groups could be methylated with diazomethane. The methoxyl content of the third lignin fraction was found to be about the same as that of the other two lignin fractions. This lignin fraction was found to contain a higher percentage of carbon than either of the other two lignin fractions. By means of a method previously described (3), the alkoxyl groups in the three lignin fractions were shown to be methoxyls. The presence of other alkoxyl groups, such as, for example, ethoxyl groups, was definitely excluded. The three lignin fractions when distilled with 12 per cent hydrochloric acid afforded small quantities of formaldehyde. The significance of the isolation of this fission product from lignin from the standpoint of the presence of a methylene dioxide group in this substance has already been discussed in the paper referred to (1). In comparing the lignin fractions isolated from oat straw with the corresponding lignin fractions isolated from barley straw one is led to the conclusion that, if these lignins are not identical, they are certainly closely related. EXPERIMENTAL 1 kilo of ground oat straw (910 gm. of moisture-free material) was subjected to an exhaustive extraction with an alcoholic sodium hydroxide solution according to the procedure described in a previous communication (1). The crude lignin thus obtained, after being dried in a desiccator over sulfuric acid, amounted to 61 gm. (6.7 per cent yield calculated on the weight of dry straw taken). The weight of the straw which had been extracted with 6he alcoholic sodium hydroxide solution was 855
M. Phillips and M. J. Goss gm. (dry weight). The crude lignin was treated with 1 liter of an acetone-alcohol solution (2 volumes of acetone to 1 volume of 95 per cent ethanol), the lignin solution was filtered, and the alcohol and acetone were removed by distillation under reduced pressure. The lignin thus obtained was washed with water and dried at 56 over phosphorus pentoxide in the vacuum Abderhalden drier. The yield of this purified lignin amounted to 30 gm. (3.3 per cent calculated on the weight of dry straw taken). The lignin obtained was a light tan amorphous powder. A carbon and hydrogen determination made upon the lignin gave the following results: Found, C 62.6, 62.5, H 6.2, 6.1. The lignin gave no test for furfural with aniline acetate paper when distilled with 12 per cent hydrochloric acid. The nature of the alkoxyl group present in this lignin fraction was established by the method of Willstatter and Utzinger (4). The crystalline trimethylphenylammonium iodide was identified by its melting point and mixed melting point. In order to prove definitely that no alkoxyl groups other than methoxyl are present in this lignin fraction, the method described by one of us in a previous communication (3) was used. Found, (Zeisel method) OCHI 14.76, 15.04, (Kirpal and Biihn method) 14.84, 14.88. The ratio of the percentage of total carbon to the percentage of carbon present in the form of methoxyl is 10.8:1. The data obtained on the percentages of carbon and hydrogen in this lignin fraction agree with the percentage composition as represented by a compound of the empirical formula C%~H~~OE,. The calculated percentages are C 62.50, H 6.25. The percentage of methoxyl found in this lignin fraction is somewhat low as compared with that calculated for four methoxyl groups in a compound represented by the above formula. The results obtained correspond to 3.7 methoxyl groups in every unit represented by the above formula. Distillation with 19 Per Cent Hydrochloric Acid-2 gm. of lignin were distilled with 12 per cent hydrochloric acid by the procedure recommended by the Association of Official Agricultural Chemists for the determination of pentosans. The distillate was neutralized with sodium bicarbonate, then made slightly acid with acetic acid, and redistilled, and the first 50 cc. of distillate were retained. This distillate was treated with an alcoholic
Chemistry of Lignin. X solution of dimethylcyclohexanedione, as suggested by Weinberger (5). The crystalline precipitate was recrystallized from dilute alcohol, m.p. 191-192, mixed m.p. 191-192. The presence of formaldehyde in the distillate was, therefore, definitely established. Alkali Fusion-To 50 gm. of potassium hydroxide contained in a nickel crucible, 25 cc. of water and 10 gm. of zinc dust were added and the mixture was heated to 100. To these were added portionwise 5 gm. of lignin while the reaction mixture was stirred. The temperature of,the mixture was gradually raised to 250 and maintained at that temperature for 30 minutes. The reaction was completed by heating the mixture at 310 for 15 minutes. The melt was allowed to cool and then dissolved in water, filtered, acidified with dilute sulfuric acid, and distilled in a current of steam until the distillate no longer gave an acid reaction. The total distillate was titrated with 0.1 N sodium hydroxide solution, phenolphthalein being used as the indicator. 181 cc. of the alkali were required. The entire neutralized distillate was evaporated to dryness on the steam bath. The acid p-toluide was prepared according to the method of Mulliken (6). The toluide melted at 148%149, and, when mixed with some pure acet-ptoluide, there was no depression in the melting point. The acid was, therefore, acetic acid. The solution remaining in the distilling flask was filtered and the filtrate was repeatedly extracted with ether. After removal of the ether a dark brown syrup was obtained. To this were added 25 cc. of 10 per cent sodium hydroxide solution, followed by 10 cc. of dimethyl sulfate. The methylation was completed by warming the reaction mixture on the steam bath. The cooled solution was extracted with ether and after removal of the ether a small quantity of syrup was obtained which had the odor of veratrole. The amount obtained was insufficient for identification. The alkaline solution which had been extracted with ether was acidified with hydrochloric acid and again extracted with ether. After removal of the ether a gummy residue was obtained. This was boiled with water, decolorized with norit, filtered, and concentrated. Crystals were obtained which, after three recrystallizations, melted sharply at 180-181 (corrected). When mixed with some pure veratric acid, no depression in the melting point
M. Phillips and M. J. Goss 561 was observed. The yield of pure material amounted to 0.2 gm. = 4 per cent of the weight of lignin. Acetylation-The lignin was acetylated by the method described in a previous investigation (7). The product, light gray in color, was dried at 56 in the Abderhalden drier over phosphorus pentoxide. Andysis- (OCHA / Cadh.On Calculated. CH9aCO 18.3 \ (CO.C&h Found. I 18.1, 18.0 The percentage of acetyl was determined by the method described by one of us (8). Chlorination-The chlorination of this lignin fraction was carried out as described in a previous communication (7). The product was colored lemon-yellow. Analysis- CaoH,O&lw Calculated. Cl 42.98, OCH9 9.40 Found. 43.28, 43.45, 7.07, 7.07 It will be noted that there was a loss of methoxyl brought about by the chlorination. A similar observation has been made in connection with the bromination of lignin from spruce wood (9). Methylation with Diazomethane-2 gm. of lignin were added to an ether solution containing approximately 2 gm. of diazomethane and the mixture allowed to stand at room temperature with occasional stirring for 5 days. The product was filtered off, airdried, and finally dried in vacua at 56 over phosphorus pentoxide. A light tan amorphous product was obtained. Anulysis- (OCHda / C39H3409 Calculated. OCHI 23.36 \ (OCH& Found. 23.51, 23.50 The methylation with diazomethane was repeated but no increase in the methoxyl content was obtained.
Chemistry of Lignin. X Methylation with Dimethyl Suljate-3 gm. of lignin were dissolved in a solution containing 10 gm. of sodium hydroxide in 100 cc. of water, and 24 cc. of dimethyl sulfate were added drop by drop, and the reaction mixture was stirred mechanically. After all the dimethyl sulfate had been added, the reaction mixture was heated on the steam bath for 30 minutes. The product was filtered off, washed with water, and dried over phosphorus pentoxide. A methoxyl determination gave the following results: Found, OCH, 28.37, 28.40. The above product was remethylated with dimethyl sulfate and 40 per cent hydroxide solution. A vigorous reaction took place, and, after it had subsided, the reaction mixture was heated on the steam bath for 20 minutes. The product was washed with water and dried in the Abderhalden drier at 56 over phosphorus pentoxide. Analysis- (OCHJ, / C36H3106 Calculated. OCHI 33.29 \ (OCU Found. 32.77, 32.95 Isolation of Second Lignin Fraction-The straw (855 gm.) which had been extracted exhaustively with alcoholic sodium hydroxide solution, was treated with a sufficient 4 per cent aqueous sodium hydroxide solution to cover it completely and heated under the reflux condenser at 100 for 4 hours. The reaction mixture was filtered and the lignin precipitated by the addition of concentrated hydrochloric acid to the filtrate. The residual straw was again treated with a 4 per cent sodium hydroxide solution, and the operation was repeated until the alkaline extract when acidulated with hydrochloric acid no longer gave a precipitate of lignin. The cellulosic residue obtained amounted to 448 gm. It still contained 4.2 per cent of lignin as determined later by the fuming hydrochloric acid method. The lignin obtained from the several extractions was combined and purified by dissolving it in 500 cc. of 2 per cent aqueous sodium hydroxide solution and adding to it 1 liter of 95 per cent ethanol. The precipitate was filtered off, the filtrate was acidified with hydrochloric acid, and the alcohol was removed by distillation. The lignin was washed with water until the wash water was free of chlorides and dried in vacua at 56
111. Phillips and M. J. Goss 563 over phosphorus pentoxide. Yield, 9 gm. An amorphous brown substance was obtained. Analysis- Found. C 61.8, 61.8, H 5.3, 5.4, OCH, 14.68, 14.84 (ash-free basis) The alkoxyl groups present in this lignin fraction were identified as methoxyls. The method previously referred to for the identification of the alkoxyl groups was used. The percentage composition of a compound represented by the formula C40H4201~ is C 61.68, H 5.43. The calculated percentage of methoxyl in a compound of this empirical formula having three methoxyl groups is 11.95 per cent, and on the basis of four methoxyl groups it is 15.92 per cent. The lignin fraction consists chiefly of a substance containing four methoxyl groups admixed with some material containing fewer methoxyl groups. Acetylation-This was carried out according to the method previously referred to. The percentage of acetyl was determined by the same method used for the analysis of the acetyl derivative of the first lignin fraction. Analysis- CmHas01e(CO.CHs)r. Calculated. CHs. CO 18.1 Found. I 16.68, 16.39 Methylation with Diazomethane-O.5 gm. of lignin was added to an ether solution containing approximately 1.8 gm. of diazomethane and allowed to remain at room temperature for 5 days. The product was filtered off and dried in vacua at 56 over phosphorus pentoxide. Analysis- (OCH& / Cdh0r(OH)~ Calculated. OCHS 23.0 \ (OCHA Found. 22.32, 22.50 Distillation with 12 Per Cent Hydrochloric Acid-The distillation of the lignin with the subsequent cohobation of the distillate was carried out as previously described. Formaldehyde was identified in the distillate by the dimethylcyclohexanedione method already referred to. Isolation of the Residual Lignin-A portion of the residual straw
Chemistry of Lignin. X (112 gm. = 25 per cent of the weight of the straw) which had been ground fine enough to pass through an SO-mesh sieve was boiled for 3 hours under a reflux condenser with 5 liters of distilled water to which sufficient hydrochloric acid had been added to neutralize the sodium hydroxide adhering to the straw. The straw was filtered off and boiled again for 3 hours under a reflux condenser with 5 liters of 2 per cent hydrochloric acid solution, again filtered off, washed with distilled water until free of acid, and dried at 105O. Yield, 73.5 gm. = 65.6 per cent of the weight of the straw. The dry and extracted straw was added portionwise to 1000 cc. of fuming hydrochloric acid (d 1.212 to 1.223 at 15 ) which was maintained at a temperature of +S to +lo. A stream of dry hydrochloric acid gas was passed through the reaction mixture for 2 hours. It was then allowed to stand in an ice box (+8 to + 10 ) for 24 hours. The reaction mixture was diluted with 10 liters of distilled water and boiled under a reflux condenser for 1 hour. It was allowed to cool to room temperature and was then filtered off and washed with distilled water until free of acid. It was dried at 105 and a brown-colored product was obtained. Yield, 4.8 gm. = 4.2 per cent of the original (112 gm.) unextracted straw. Analysis--(Ash-free basis) Found. C 64.7, 64.7, H 5.8, 5.8, OCH3 14.96, 14.86 The character of the alkoxyl group present in this lignin fraction was established by the methods previously described. When this lignin fraction was distilled with 12 per cent hydrochloric acid, some formaldehyde was obtained in the distillate. SUMMARY 1. Three lignin fractions were isolated from oat straw by extracting it successively and exhaustively, first with a 2 per cent alcoholic sodium hydroxide solution at room temperature, then by refluxing it with a 4 per cent aqueous sodium hydroxide solution, and finally subjecting the residue to the action of fuming hydrochloric acid. The composition of the first lignin fraction agreed with that represented by the formula &,H~sO~S. Four methoxyl groups and four hydroxyl groups were found to be present. Of the four hydroxyl groups, two could be methylated with diaeomethane, thus indicating that these are more acidic, possibly
M. Phillips and M. J. Goss phenolic or enolic in character. When fused with potassium hydroxide, protocatechuic acid was obtained. The yield was approximately 4 per cent of the weight of the lignin. The analytical results on the second lignin fraction are more in agreement with that represented by the formula CK,HQZOS In this lignin fraction, approximately four methoxyl groups and four hydroxyl groups were shown to be present. Two of the hydroxyl groups could be methylated with diazomethane. The third lignin fraction was found to have a higher percentage of carbon than either of the other two lignin fractions. The percentage of methoxyl did not differ from that of the other two fractions. 2. The alkoxyl groups present in all the three lignin fractions were proved definitely to be methoxyls. 3. All the three fractions when distilled with 12 per cent hydrochloric acid afforded some formaldehyde. 4. The lignin fractions isolated from oat straw are closely related to the corresponding lignin fractions isolated from barley straw. BIBLIOGRAPHY 1. Phillips, M., and Goss, M. J., J. Am. Chem. Sot., 66,2707 (1934). 2. Marion, L., Canad. J. Research, 6, 521 (1932). 3. Phillips, M., J. Am. Chem. SOL, 62, 793 (1930). 4. Willstatter, R., and Uteinger, M., Ann. Chem., 382, 148 (1911). 5. Weinberger, W., Znd. and. Eng. Chem., Anal. Ed., 3, 365 (1931). 6. Mulliken, S. P., A method for the identification of pure organic compounds, New York, 1, 81 (1965). 7. Phillips, M., J. Am. Chem. Sot., 49, 2037 (1927). 8. Phillips, M., Znd. and. Eng. Chem., Anal. Ed., 6, 321 (1934). 9. Friedrich, A., and Pelikan, E., Biochem. Z., 239,461 (1931).
THE CHEMISTRY OF LIGNIN: X. LIGNIN FROM OAT STRAW Max Phillips and M. J. Goss J. Biol. Chem. 1936, 114:557-565. Access the most updated version of this article at http://www.jbc.org/content/114/2/557.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/114/2/557.citation.full.h tml#ref-list-1