SEPTEMBER 15, 1936
ANALYTICAL EDITION
of the apparatus: a pair of straight-end tongs to remove and replace the metal cup, feed funnel, and fat flask; a straight funnel tube to add ether; an ether bottle with constricted tip for a tin container with twenty places for the feed funnels, of such size as to fit a high-form 20.4-cm. (8-
389
inch) desiccator; and a tin container with twenty places for the fat flasks, of such size as to fit a 20.4-cm. (8-inch) desiccator. RECEIVED May 21, 1936. Published with the approval of the Director, West Virginia Agricultural Experiment Station, as Scientific Paper 167
Lignin in Douglas Fir The Pentosan Content of the Middle Lamella A. J. BAILEY,' College of Forestry, University of Washington, Seattle, Wash.
I
N AN EARLIER communication (1) a method of isolating the middle lamella of wood was described. The isolated material was subsequently analyzed for lignin by a micromethod (2) developed for the purpose. The present report describes the analysis of another sample of middle lamella, which was isolated by the same method, in order to establish further the composition of the lamella by the identification of a t least a portion of the unknown 29 per cent. As was pointed out earlier (I), several investigators produced evidence indicative of the presence of hemicelluloses in the middle lamella.
Methods of Hemicellulose Analysis An inquiry into the prior work on hemicelluloses brought to light such an overwhelming mass of incomplete, contradictory, and apparently inaccurate information that there is little wonder that the nomenclature is so unsatisfactory. A brief but reasonably complete bibliography (3, 9-11, 17, 20-23, 27, 34) on hemicelluloses, including recent excellent reviews, offers a summary of present knowledge; hence i t is unnecessary to epitomize the experimental work or to take up in detail the viewpoints of the various investigators. Inasmuch as the estimation of the hemicelluloses-rather than their constitution-was the primary consideration, only the analytical methods need discussion here. Unfortunately, there is no method of making a complete analysis of hemicelluloses in a single sample. It is even impossible to define hemicelluloses in a way that has analytical significance. Supposedly, they are anhydrides of hexose and pentose sugars which are soluble in dilute alkalies and which are less resistant to hydrolysis by dilute acids than the remainder of the plant tissue. It is well known that these properties do not furnish a means of sharp distinction. There is no known method of determining the total hemicelluloses or the total hexoses in a single sample. Pentoses are usually determined by conversion to furfural, which may be estimated by oxidation or precipitation methods. Hexoses are usually determined by oxidation of the sugar or by conversion to the phenyl hydrazone or osazone (S, 18,19, 30, 3s-36). While 2 pure carbohydrates can be characterized fairly easily, their identification or quantitative separation from mixtures is exceedingly difficult. An attempt to determine the total hemicelluloses was made by extracting a sample of wood with hot water, and then subjecting the residual wood to hydrolysis. The hot-water extractive of oven-dry Douglas fir (Pseudotsuga tazijoZiolia) powder which had been put through a 100-mesh screen was 13.68 per cent; the subsequent loss in this sample, based on the original oven-dry weight of the wood, due to hydrolysis by 2 per cent sulfuric acid was 15.99 per cent; the loss in unextracted wood when refluxed with 2 per cent sulfuric acid was 27.15 per cent. On the basis of Schorger's definition, 1
Present address,lInstitute of Paper Chemistry, Appleton, Wis.
that hemicelluloses are insoluble in hot water but are hydrolyzed by heating with dilute acids at atmospheric pressure (.%), i t would seem that the total hemicelluloses in Douglas fir constitute 16 per cent of the wood. That this is not accurate is shown by the fact that the extractive plus the loss by hydrolysis is greater than the loss by hydrolysis of the unextracted wood. In other words, the hot water alone either extracts some hemicelluloses or changes some constituents so that they are soluble in the dilute acid. Further evidence that hemicelluloses are removed by hot water alone was found by an examination of the hot-water extractive. Treatment with phenylhydrazine and acetic acid in the cold yielded no mannose phenylhydrazone. When the hot water extract, however, was made up to a concentration of 5 per cent with hydrochloric acid, hydrolyzed, neutralized, and treated with phenylhydrazine and acetic acid in the cold, a small quantity of mannose phenylhydrazone was formed, together with a large quantity of an as yet unidentified compound with a melting point of 235" C. Hence, it may be concluded that mannan in Douglas fir is soluble in hot water but is not hydrolyzed by it, The total mannan by Schorger's method (18, 29) was found to be 5.33 per cent (Schorger found 6.65 per cent). After filtering off the mannose phenylhydrazone and other material from the hydrolyzate, heating in the presence of an excess of phenylhydrazine yielded the brownish yellow oily drops of the osazone of arabinose, phenylglucosazone, and another crystalline solid. When the hot-water extract, with no prior treatment, was made up to a concentration of 12 per cent with hydrochloric acid and distilled, the addition of thiobarbituric acid solution to the distillate produced furfuralmalonylthiourea. This fact extends earlier knowledge of the resistance of pentosans to hydrolysis to include not only those that occur in purified cellulose or those that are readily hydrolyzed by dilute acids, but also to those which are removed by hot water. In other words, hot-water extraction alone removes a t least three hemicelluloses and other carbohydrates. Likewise, there seems to be no way of determining the hexose hemicelluloses in a group in a single sample. Pentose hemicelluloses, however, can be distinguished easily from hexoses. Since the existing knowledge of hemicellulose chemistry was insufficient to permit an even reasonably complete determination, it was necessary to attempt to ascertain the hemicellulose most likely to occur in the middle lamella. Prior work pointed the way: Ritter and Fleck (16) found that the pentosan content of springwood was greater than that of summerwood. It was suspected that the higher volumetric fraction of lamella in the springwood was responsible. Other speculations (16), many of a teleological nature, however, had been voiced, showing the mechanism of chemical metamorphosis from pentoses to lignin, hence the possibility of a close relation between pentosans and the middle lamella.
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VOL. 8, NO. 5
INDUSTRIAL AND ENGINEERING CHEMISTRY
Since it was found earlier that the lignin content was higher in the springwood than in the total wood and even higher in the wood rays ( I ) , check analyses were carried out, by a method described below, on ray tissue isolated by the micromanipulator to learn if this same pentosan-lignin association obtained. The pentosan content of the rays was found to be about 6.6 per cent, as compared to about 5.5 per cent for the total wood, or in linear relation to the lignin content. Accordingly, it appeared probable that the pentose hemicelluloses should constitute at least an important fraction of the total hemicelluloses and that their close and constant association with tissue which had both a high lignin content and a high volumetric fraction of middle lamella should assure their presence in the middle lamella in significant quantities.
Determination of Pentosans The determination of pentosans, for practical purposes, may be said to depend upon their conversion to furfural or methylfurfural, as the case may be, by distilling with a mineral acid 'of proper concentration and subsequent determination of the furfural by oxidation, precipitation by various reagents, by colorimetric means, or by other methods (23, 26). While the common method of determining pentosans consists of a strictty standard distillation and precipitation with phloroglucinol, many objectionable features are apparent. The distillation itself is not quantitative (7), while some furfural is destroyed by the high acid concentration during the latter part of the distillation (8). Furthermore, precipitation by phloroglucinol is not quantitative (8), necessitating a correction factor, while hydroxymethylfurfural, which is of hexose origin, is precipitated in small yield (24). The phloroglucide of furfural is not a compound of definite composition, varying with the temperature a t which it is precipitated (Sa). Virtually all objections may be overcome by proper modifications. By using the exceedingly accurate method of distillation with steam, first proposed by Jolles (16) and later modified by Pervier and Gortner (2S), quantitative yields have been obtained from pure pentose materials (23). The accuracy seems to be due to the furfural's being swept out by the steam and to the constancy of acid concentration preventing destruction of the furfural. While many precipitants other than phloroglucinol have been used, only two merit attention. Barbituric acid was found to give nearly quantitative yields as a precipitant for furfural without precipitating hydroxymethylfurfural (7, $4, S I ) . Dox and Plaisance (4) found that thiobarbituric acid gave quantitative or slightly better yields, that only a slight excess of the precipitant was necessary thus avoiding the danger of inclusion, that it was very sensitive to exceedingly small amounts of furfural, and that the precipitate was a compound of definite and uniform composition-namely, furfuralmalonylthiourea. Other investigators found that thiobarbituric acid precipitates both furfural and methylfurfural quantitatively and that while it reacts with hydroxymethylfurfural, the resulting product is soluble, coloring the solution, but not interfering with the determination of pentoses (6, S I ) . The superiority of thiobarbituric acid over phloroglucinol has been verified by the researches of other workers ($4, 26). It appeared, therefore, that the most promising method for the determination of pentoses in microquantities was to reduce the method of steam distillation of Pervier and Gortner and to use thiobarbituric acid as the precipitant. PROCEDURE. On a macrobasis high precision was obtained on samples of 100-mesh (rasped and passed through a 100mesh screen) oven-dry mood ranging in size from 0.1 to 3.0 grams. One modification of the Pervier and Gortner method increased the precision and shortened the time-viz., the
substitution of a 125-ml. flask for the 75Gml. distilling flask and the addition of 50 ml. of 12 per cent hydrochloric acid instead of 200 ml. The increase in precision was presumably due to better contact of steam and furfural. In distilling, a moderate stream of steam was passed in a t a constant rate throughout the distillation and the temperature, as measured by a thermometer in the vapor in the neck of the flask, was maintained between 103" and 105" C. by boosting the distilling flask with a burner. Distillation was continued until a sma.11 sample of the distillate in thiobarbituric acid solution gave no precipitate or turbidity after standing 5 minutes. The value of aniline acetate paper (or hydrochloride) as an indicator, while entirely satisfactory on pure pentose material, was found to be worthless on wood, since the hydroxymethylfurfural from the cellulose, etc. ( I S , I 4 ) , reacted with the thiobarbituric acid as Unger and Jiiger pointed out ( S I ) , giving a colored compound which was also found to be the case with both the papers and solutions of aniline salts. In thiobarbituric acid solution, however, the difference between a color and turbidity could be easily detected, thus providing a simple and accurate means for determining the end of the distillation. Pervier and Gortner found their method of distillation to give theoretical yields of all pure pentose materials. Thiobarbituric acid was obtained from the Eastman Kodak Company and was prepared by the method of Gabriel and Coleman (6). Both gave satisfactory results. The furfural was precipitated from the distillate by adding a slight excess of thiobarbituric acid in 12 per cent hydrochloric acid a t room temperature and allowing to stand overnight. The lemonyellow compound formed was filtered on a tared Gooch crucible, dried a t 105", and weighed as furfuralmalonylthiourea : CH-CH I
CO-NH
I
I
which is uniform and constant in composition (4). Values obtained in the presence of methylfurfural are only very slightly in error, because the molecular weight of the residue is between furfural and methylfurfural. Calculations were based upon the following relations:
+
Pentosan ---t furfural 2 H20 Weight of furfural = furfural 2 H20 furfuralmalonylthiourea
+
~
59.6
Microdetermination of Pentosans
A microprocedure was developed which is as follows: A 6-ml. flask was blown from tubing having an inside diameter of 9 mm., and a long side tube was sealed on the neck of the flask.
A tube larger than the side tube was fitted with two side tubes to form the outer condenser shell, and slipped sleevewise over the long side tube of the flask, which formed the inner condenser shell. The ends of the outer tube were then made tight with rubber tubing, thus providing a means of condensation in the side tube of the flask and eliminating all joints from the system. Another flask of the same size was blown for the steam generator and connected to the distilling flask by small-bore tubing. A microthermometer was made by blowing a bulb on a capillary tube, filling it with an alcoholic solution of safranin, and sealing. It was calibrated in a thermostat at 103" and 105' C. The distillation was conducted in the same manner as in the macroprocedure, samples in each case being introduced into the flask in small glass boats and the flask being filled half full (50 ml. in the macro- and 3 ml. in the microprocedure) of 12 per cent hydrochloric acid. In the microprocedure the boat was first dried at 105", cooled in a desiccator, then placed in a weighing bottle kept in the balance case, and weighed. This process was repeated with a sample in the boat in order t o obtain identical anhydrous surface conditions on the glass boat without error due to hygroscopicity. The same technic was used in weighing the
SEPTEMBER 15, 1936
ANALYTICAL EDITION
micro-Gooch crucible. Samples of approximately 3 mg. of wood were used in the microprocedure.
Results of Pentosan Analyses Table I summarizes the results of analyses by macro- and microprocedures. The analysis of a single sample of the middle lamella weighing 1.127 mg. showed a pentosan (including methylpentosan) content of 14.21 per cent. Subsequent filtration and chlorination of the residue showed a cellulose content of 4.1 per cent. TABLEI. PENTOSAN CONTENT O F DonaLAs FIR Aa
4.53 4.69 4.53 4.61 4.58 4.65 A.v. 4.60 a
h c
d
Bh 5.38 5.53 5.43 5.41 5.49 5.46 5.45
C C
5.46 R . 62 8.54 5.35 5.41 5.50 5.48
Dd 6.61 6.45 6.78 6.71 6.49 6.58 6.60
A total wood sawdust b macroprocedure.
B’100-mesh total wood
macroprocedure. C: 100-mesh total wood by microprocedure. D, ray tissue by microprocedure.
Significance of Pentosan Analyses The difference between sawdust and 100-mesh wood is typical of many wood analyses, the results depending upon the degree of subdivision of the sample. The precision of the micromethod shown in C and D, Table I, probably obtained in the analysis of the middle lamella, as the quantity of furfuralmalonylthiourea obtained was approximately the same in each case. Ritter and Fleck (25) showed both the lignin and pentosan content of springwood to be about 10 per cent greater than in the summerwood. It has been shown above that both the lignin and pentosan content of the rays were about 20 per cent greater than in the total wood, and by analysis of the middle lamella that the lignin and pentosan content in the latter tissue was 215 and 260 per cent greater, respectively, than in the total wood. While this clear-cut relation between lignin and pentosan content and their constant association may be entirely accidental, it is equally possible that these data point to the origin of lignin. Schorger ( l a , 29) and Ritter and Fleck (8.56 found ) the pentosan content of Douglas fir to be nearly double that found above for the total wood. While their results are in good agreement with each other, the phloroglucinol method which was employed to obtain these results has been shown to be greatly in error by Gierisch (7, 8 ) , Heuser and Stockigt ( I d ) , Pervier and GortPeter, Thaler, and Taufel (24), Unger and Jager ner (W), ( S I ) , and VotoEek (Si?). Summary of Middle Lamella Composition The small quantity of cellulose present in the isolated sample of middle lamella affords an accurate index of the extraneous material which was unavoidably included in the isolation. The composition of the middle lamella may be summarized as follows: lignin 71 per cent, pentosans 14 per cent, unknown 15 per cent. The 4 per cent of cellulose in the isolated sample probably should be regarded as the error in isolation. If this be done, the values for lignin and pentosan are slightly low. There is no reason to believe that the composition of the middle lamella is uniform in different species and genera or even in the same species. The association of lignin and pentosans in nearly direct relation suggests, but offers little conclusive evidence, that pentoses antecede lignin. Such association is difficult to interpret on the basis of present knowledge of wood chemistry. The fact that pentosans may be present in the middle lamella necessitates modifications in present opinions of both chemical and botani-
391
cal investigators as to the cause and significance of solubility and staining reactions of the middle lamella. It appears that the major contribution of these findings, at the moment, has fundamental rather than practical importance.
Summary This paper describes a continuation of the analysis of the middle lamella which was isolated mechanically by a micromanipulator in order to determine the pentosan content of the middle lamella. Methods of hemicellulose analysis are discussed and data on hemicelluloses in Douglas fir reported. An accurate gravimetric method of determining pentosans by steam distillation and precipitation of the furfural by thiobarbituric acid is suggested. The procedures for the determination of pentosans on both macro- and microsamples are outlined and the precision of each is indicated. An isolated sample of middle lamella of Douglas fir showed a pentosan content of 14.21 per cent (the first paper describing this work indicated a lignin content in the middle lamella of 7 1 per cent). A direct and nearly constant relation between lignin and pentosan content was found in different wood elements. This suggests that pentoses take part in the formation of lignin, although the indicia are circumstantial. The importance of the findings is discussed.
Literature Cited Bailey, A. J., IND.ENG.CKEM.,Anal. Ed., 8, 52 (1936). Bailey, A. J., Mikrochemie, 19, 98 (1936). Dische, X., Ibid., 10, 128 (1931). Dox, A. W., and Plaisance, G . P., J . Am. Chem. Soc., 38, 2156 (1916). Ekenstein, W. A. van, and Blanksma, J. J., Ber., 43, 2355 (1910). Gabriel, S.,and Coleman, J., Ibid., 37, 3657 (1904). Gierisch, W., CeZZulosechem., 6, 61 (1925). Ibid., 6 , 81 (1925). Hiigglund, E., “Holachemie,” Leipaig, Akademische Verlagsgesellschaft, 1928. Hawley, L. J., and Norman, A. G., IND.ENG.CHEM.,24, 1190 (1932). Hawley, L. F., and Wise, L. E., “Chemistry of Wood,” New York, Chemical Catalog Co., 1926. Heuser, E., and Dammel, W., CeZZuZosechem., 5, 45 (1924). Heuser, E., and Schott, W., Ibid., 4, 85 (1923). Heuser, E., and Stockigt, F., Ibid., 3,61 (1922). Jolles, Sitz. Akad. Wiss. Wien, Aht. 11, 114, 1191 (1905). Klason, P., Ber., 63, 1548 (1930). Kriiger, D., 2eZlsto.f u. Papier, 14, 89 (1934). Morris, V. H., J . Am. Chem. Soc., 54,2843 (1932). Mulliken, S. P., “Methods of Identifying Pure Organic Compounds,” Vol. 1,pp. 29 ff., New York, John Wiley & Sons, 1904. Nanji, D. R., Paton, F. J., and Ling, A. R., J . SOC.Chem. I n d . , 44, 253T (1925). Norman, A. G., Science Progress, 28, 229 (1933). O’Dwyer, M. H., Chemistry & Industry, 10, 968 (1932). Pervier, N. C., and Gortner, R. A , , IND.ENG.CHEM.,15, 1167, 1255 (1923). Peter, B., Thaler, H . , and TRufel, K., 2. Untersuch. Lebensm., 66, 143-57 (1933). Ritter, G. J., and Fleck, L. C., IND. ENG.CHEM.,18, 608 (1926). Schmidt-Nielsen, S.,and Hammer, L., Kgl. Norske Videnskab. Selskabs, Forh., 5 , 84-7 (1932). Schorger, A. W., IND. ENG.CHEM.,16, 141 (1924); “Chemistry of Cellulose and Wood,” New York, McGraw-Hill Book Co., 1926. Ibid., p. 141. Schorger, A. W., J. IND.ENG.CHEY.,9, 748 (1917). Schorger, A. W., and Smith, D. F., Ibid., 8, 494 (1916). Unger, E., and Jnger, R., Ber., 36, 1222 (1903). Votodek, E., Ibid., 30, 1195 (1897). Wagenaar, H., Pharm. WeekbZad, 71, 229 (1934). Wasitzky, A., Mikrochemie, 16, 87 (1934). Webster, R . W., “Diagnostic Methods,” pp. 275 ff., Philadelphia, P. Blakiston’s Son &z Co., 1913. RECEIVED May 18, 1936. Presented before the Division of Cellulose Chernistry a t the 92nd Meeting of the American Chemical Society, Pittaburgh, Pa.. September 7 t o 11, 1936.
