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An Experimental Study of the Significance of “Lignin” Color Reactions ‘,2 By Ernest C. Crocker RESEARCH LABORATORY OF APPLIEDCHEMISTRY, MASSACHUSETT~ INSTITUTE O F TECHNOLOGY, CAMBRIDGG, MASS.
There are certain color reactions3 given by wood and lignified fibers, and not by thoroughly cooked pulp, which are commonly considered by paper-mill chemists and others who work with these substances as more or less specific tests for lignin. I n connection with an investigation carried out in this laboratory for the Mead Research Co., of Dayton, Ohio, with regard to some coloring matters in wood, it was felt desirable to have information concerning the substance or substances in wood responsible for these color reactions. The literature4 upon this subject, while quite voluminous, was found to be contradictory and incomplete in many places, so that it became necessary to make an experimental study of the problem. While this involved some duplication of previous work, it is believed that the results may be of interest, since purer reagents were used than were available to some of the early investigators, and the method of attack of some phases of the problem was different. The Mead Company has kindly given its permission for the publication of the results obtained. The experimental work, here briefly summarized, was carried only far enough to arrive a t an answer to certain practical questions. The field, however, is broad enough for more work, some of which should be of interest to the paper and allied industries. The color reactions investigated most thoroughly were those of wood with phloroglucinol, aniline, p-nitroaniline, and pyrrole, the Maule reaction, and the ferricferricyanide reacti~n.~ These tests were varied in such ways as to aid in determining the essential components both of wood and ’reagent responsible for the tests.
only, or no coloration, were obtained with phenol, guaiacol, thymol, eucalyptol, a- and @-naphthols, p-nitrophenol, m-nitrophenol, picric acid, hydroquinol, pyrocatechol, gallic acid, and tannic acid. Certain phenols which had two hydroxyls meta to each other produced strong red to blue colors. These were, in the decreasing order of rapidity of color formation: phlororesorcinol + pyrogallol. glucinol + orcinol
*
THEACTIONOF VARIOUS AMINESow WOOD Aniline and p-nitroaniline solutions in dilute hydrochloric acid were found to give yellow and orange stains, respectively. The acid strength was increased and decreased, and the rapidity of staining noted. I n every case where there was excess free acid, the stain was readily produced. Other amines were then tried, all in 2 N hydrochloric acid, with the results listed below : Yellow or orange-yellow colors were given by aniline, monomethylaniline [faint], a-naphthylamine, m-aminophenol, anthranilic acid, sulfanilic acid, 0- and p-toluidines, phenylhydrazine, and dimethyl-p-phenylenediamine. Orange colors were given by p-nitroaniline and diphenylamine, and a deep red color by pyrrole. No color was given by formamide, acetamide, urea, acetanilide, dimethylaniline, carbazole, or pyridine. (The diphenylamine was purified by recrystallization from 70 per cent alcohol, and by treatment, in the molten state, with dilute hydrochloric acid, to remove any traces of primary amines.) These experiments indicate that most, if not all, aromatic primary amines, and a t least diphenylamine among the secondary amines, react with a component of wood to form THE ACTIONOF PHLOROGLUCINOL AND OTHER PHENOLS colored compounds. Acid amides do not react, and no ON W O O D reactive tertiary amine has been found. Only a small Phloroglucinol solutions were made up in the customary concentration of free acid is needed for the reaction. way, by dissolving 1 to 2 per cent of the material in 6 N WHICHGIVE COLORS WITH PHENOLS aqueous hydrochloric acid, and in 6 N hydrochloric acid in STUDYOF COMPOUNDS AND AMINES 50 per cent alcohol. Some of the above reagents were applied to several kinds Phloroglucinol in 6 N hydrochloric acid and aniline in 2 N of deciduous and coniferous woods, and to the woody fiber (free) acid were used as reagents to add to small amounts of of the achlorophyllous plant, Monotropa uniflora (Indian chemicals on a porcelain spot plate, and to alcoholic solutions pipe). I n each case, a strong red color was produced in containing one-tenth of their volume of concentrated hydroa few moments. chloric acid. (It was noted that in any case where a color The reagent containing alcohol did not give such strong or was formed on the spot plate, it was also formed in alcoholic clean-cut tests as the one containing only water, as the alcohol solutions and vice versa.) caused spreading of the stain. The colors given with these reagents are shown in the Other solutions were made up with sulfuric acid of \-a- accompanying table. rious strengths. It was noted that the stronger the acid the COLOR WITH COLORNITH quicker the reaction. From 20 per cent to 70 per cent SUBSTANCE PHLOROGLUCINOL AKILINE Wood Strong cerise Strong yellow sulfuric acid were found to be effective as a reagent, but in no Eugenol Strong cerise Strong yellow way better than strong hydrochloric acid. (oil of cloves) Eugenol methyl ether Strong cerise Strong yellow As many phenols other than phloroglucinol are mentioned Safrol Strong cerise Strong yellow (oil of sassafras) in the literature as being effective as reagents, a considerSalicyl aldehyde Strong scarlet Strong yellow able number were tried out, in 6 N hydrochloric acid, in Vanillin Scarlet Yellow Pink Faint yellow Piperonal 10 N hydrochloric acid, and in 50 per cent sulfuric acid. Strong orange Yellow Cinnamic aldehyde Making the tests on various kinds of wood, strong red, Cinnamic alcohol Strong orange Yellow violet, or blue colors were obtained in the cases of orcinol, Anisaldehyde Furfural Orange+olive+black Orange Strongyellow Faint yellow resorcinol, and pyrogallol. Pale greenish or brownish colors Citral 1 Presented before the Section of Cellulose Chemistry a t t h e 61st Meeting of theAmerican Chemical Society, Rochester, N. Y.,April 26 t o 29, 1921. 2 Published as Contribution No 32 from the Research Laboratory of Applied Chemistry, Massachusetts Institute of Technology. 3 A. Klein, Papier-Zlg , 34 (1909), 267; Paper, 24 (1919), 351. 4 V. Grafe, Monatsh., 25 (1904), 987; F. Czapek, “Biochemie der Pflanzen,” Jena, 1913. R Haller, C A , 10 (1916), 530
Citronellal
Benzaldehyde Anethole Heptaldebyde
Orange Orange+pink+colorless Buff Paie orange Pale orange
Faint Faint yellow yellow None None None
No color appeared with either phloroglucinol or aniline in cases of formaldehyde, acetaldehyde, duodecylaldehyde, anisole, phenol, resorcinol, hydroquinol, pyrogallol, pyro-
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catechol, thymol, eucalyptol, glucose, cinnamic acid, and benzoic acid. Among the compounds conceivably present in wood which gave no colors with either phloroglucinol or aniline were many simple phenols, polyphenols, alcohols, acids, and the modified aldehyde, glucose. All the substances which gave the color tests strongly were pure aldehydes, except for a few materials derived from vegetable sources, where there was opportunity for contamination with any materials present in the plant from which they were obtained. In order to determine whether the colors given by these compounds might be due to traces of aldehydic impurities, the eugenol, safrol, eugenol methyl ether, and cinnamic alcohol were each shaken up with saturated sodium bisulfite solution for some time and then separated. This treatment was repeated five or six times, and the essentially aldehyde-free materials were again tested. In each case, the color-forming power was very greatly reduced, and in the case of eugenol methyl ether, practically eliminated. The aldehyde-reduced eugenol was tested when freshly treated, and again after four months had elapsed. In each case, the amount of color produced was about 5 per cent of that given by the untreated eugenol, indicating that airoxidation was not responsible for the aldehyde in eugenol which gave the color with ‘(lignin” reagents. Eugenol was treated with phloroglucinol plus hydrochloric acid, and steam-distilled from this mixture. It gave no coloration whatever with phloroglucinol and no yellow with aniline. After steam-distillation from aniline and hydrochloric acid, eugenol gave a faint orange color with phloroglucinol, which, on standing, became greenish: (This is an evidence of a trace of furfural in the eugenol, which was not held by the aniline.) Many of the references in the literature to color reactions with phloroglucinol, on the one hand, and eugenol, safrol, cinnamyl alcohol, anethole, and anisole, on the other, were doubtless due to the presence of aldehydic impurities in the reagents used. (Anethole and anisole, as tested in this laboratory, gave no color whatever.) No case is known wliere materials conceivably present in mood, other than aldehydes, can react with phloroglucinol reagent, in the cold, to produce a red color. The colors formed with aniline and other amines, if caused by aldehydes, as is so apparent from the phloroglucinol results, are “aniles” (Schiff’s bases) SPECTROSCOPIC STUDYOF COLORSPRODUCED BY “LIGNIN” REAGENTS Some of the aldehyde in wood, which gives the “lignin” tests with phloroglucinol, pyrrole, and aniline, was obtained in benzene solution by the method of Czapek.2 This method consists in boiling finely ground wood with stannous chloride solution, and extracting the wet mass with benzene for several days. To some of this solution, diluted with alcohol, phloroglucinol, pyrrole, and p-nitroaniline reagents were added, and the characteristic cerise, dark red, and orange colors obtained were tested in a spectroscope. Solutions of (unpurified) eugenol and safrol, and pure cinnamic aldehyde, piperonal, vanillin, salicylaldehyde, anisaldehyde, and furfural were likewise treated with reagents and examined spectrometrically. (The compound with furfural changed color so rapidly it could not be measured.) The phloroglucinol compounds of the wood-aldehyde, unpurified eugenol, unpurified safrol, vanillin, and salicylaldehyde, and the pyrrole compounds of the first three, gave more or less clear-cut bands of easy light transmission in the red and orange. In all the p-nitroaniline compounds, and the phloroglucinol and pyrrole compounds of the other aldehydes, the spectra were indefinite, and contained no well-marked 1
L. Zechmeister, “Kenntnis Cellulose uiid Lignins,” Zurich, 1913.
* 2. physto2. Chem , 27 (18991, 141.
Vol. 13, No. 7
bands, either of absorption or transmission. Using Formanekl units, the bands of highest transmission (minima of absorption) with phloroglucinol were at about 11.30 for wood, 11.30 for eugenol, 11.30 for safrol, 13.50 for vanillin, and 15.75 for salicylaldehyde. With pyrrole,the minimum absorption for wood aldehyde was a t about 9.60 units; for eugenol and for safrol, a t about 9.90 units. (In these three cases, t h e minima were not sharp, and might have been identical.) The spectroscopic study indicates that oils of cloves and sassafras contain traces of a color-forming material very similar to, if not identical with, that in wood, with its probable origin in the woods from which these oils were distilled. Czapek’s work and this spectroscopic work indicate that not more than a small fraction of the color-forming aldehyde of wood-Grafe2 notwithstanding-can be vanillin,3 although i t might be similar to or decomposable into vanillin.4 The tinctorial power of phloroglucinol compounds is high, SO that very slight traces of aldehyde suffice to produce strong colors. The amount in wood, as well as in oils of cloves and sassafras, is probably of the order of hundredths of a per cent. COMBINATION OF ANILIXEAND PHLOROGLUCIKOL TESTS Vnpurified eugenol was diluted with 20 volumes of alcohol to serve as a reagent. This reagent was treated with aniline in hydrochloric acid, with immediate formation of a strong yellow color. When the eugenol reagent was treated with phloroglucinol in strong hydrochloric acid, a deep red color formed in less than 5 see. When, now, some aniline solution was added to the eugenol reagent, followed by phloroglucinol, the red color did not appear strongly for nearly 60 see. Pine wood was treated with aniline plus hydrochloric acid, and the yellow-stained material dried. This treated wood and some plain wood were then treated with phloroglucino1 solution in hydrochloric acid. The plain wood developed a red color in a few seconds, but the treated wood developed the color only after a minute. Reversal of the test was next tried with eugenol. Phloroglucinol was first added, and when the color was a t a maximum (about 15 min.), some aniline hydrochloride was added. A slight yellowing of the solution was noticeable on direct comparison with some of the solution to which the aniline salt had not been added. These experiments and the one involving steam-distillation of eugenol from acid plus phloroglucinolmixtureindicate that the material which causes the aniline test is also responsible for that with phloroglucinol. The aniline compound is formed a t once, whereas that with phloroglucinol appears only after an appreciable lapse of time. Apparently the phloroglucinol and aniline divide the aldehyde between themselves in an equilibrium mixture, with the larger part going to the phloroglucinol. The very excellent work by Czapek,6 published in 1899, wherein he arrives a t the conclusion that an aldehyde is present in wood which gives the color tests with phloroglucinol and the amines. is well borne out by these experiments. Czapek named the aldehyde “hadromal.” He succeeded in concentrating it, but could not get it pure. Subsequent work by Grafee seemed to show that hadromal was a mixture of vanillin, methyl furfural, and pyrocatechol. Czapek,7 again, in turn, showed that no mixture of these three could duplicate the tests given by hadromal. Very recently, KlasonS has come to the conclusion that the “Spektral Analytische Nachweis,” Berlin, 1900. Monalsh., 26 (1904), 987. 8 Wichelhaus and Lange, Ber , 4 9 (1916), 2001. 4 M. Singer, Monalsh., 3 (ISSZ), 395. 5 Z . physzo2. Chem , 27 (1899). 141. 6 Monalsh., 25 (1904), 987. 7 “Biochemie der Pflanzen,” Jena, 1913. SSoensk Pappers-Tzd , 2 S (1920). 70, C. A , , 15 (1921), 526. 1
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T H E JOURhTAL OF I N D U S T R I A L A N D ENGINEERING CHEiWISTRY
principal color-forming aldehyde of wood is coniferyl aldehyde. The work in this laboratory, particularly the spectral analysis, points strongly to the soundness of Czapek’s original work, and is entirely consistent with that of Klason. U p to the present, little success has attended the attempts of this laboratory to prepare coniferyl aldehyde from coniferin, so that it has not been possible to confirm Klason’s work definitely by a comparison of the absorption spectra of the color tests.
STUDYOF
THE ~ ~ A U LREACTION E
I n 1900, Maulel published the results of some experiments on wood, wherein he found that a bright red color often results when wood is treated, in succession, with neutral potassium permanganate solution, dilute hydrochloric acid, and finally ammonia. It has been noted in references to the Maule reaction that pine wood gives a brown rather than red color, but so far as known, only one2 clear-cut statement has been made regarding which kinds of woods do, and which do not, give the red color. This matter was, therefore, experimented upon, and some woods of uncommon botanical families, such as gingko, were tested. Some eighteen species of deciduous woods, including the interesting balsa wood, eighteen species of coniferous lvoods, and gingko wood, were tested with Maule reagent. All the woods of deciduous angiosperms gave distinct red colors. All those of coniferous gymnosperms, and of the deciduous gymnosperm, Gingko biloba, gave only indefinite yellow or pale brown colors. The test is thus a convenient one for differentiating between the woods of angiosperms and those of gymnosperms. If gingko wood is excepted, the test distinguishes between deciduous and coniferous woods. KOwoody fiber of cycads or other possible ancestors of the angiosperms was available for study. It would be very interesting, indeed, to determine if this reaction takes place with these now rare woods. If it does, the reaction would be a valuable tool in the hands of the botanist. The reagents used were varied to determine the essential conditions and chemicals needed to produce the red color with “hard” woods. The results may be summarized as follows. 1-Potassium permanganate deposits manganese dioxide O h the wood. This reacts with the hydrochloric acid t o produce chlorine, which forms a compound which turns red with alkalis, in this case ammonia. 2-ChIorine water will replace permanganate and hydrochloric acid 3-Bromine can replace chlorine, b u t the color in this case is a somewhat brownish red. Iodine wilt not produce the reaction. 4-Other oxidizing reagents, such as permanganate plus sulfuric acid (which deposits no manganese dioxide), do not give the red color. 5-Any alkali or organic base can replace ammonia, b u t usually not with advantage. Strong alkalis dissolve the color from the wood and thus weaken the test. Organic bases, and strong inorganic alkalis, give yellowish shades. Sodium bicarbonate can replace ammonia with the saJlle deep red color, however. 6-Heat does not destroy the test at any part of the process, but reduces the sensitiveness at the last stage b y dissolving color from the wood.
The Maule test was applied to wood which had been extracted with various organic solvents, refluxed with acids, alkalis, and formaldehyde. The red color was given strongly. Jute, which was bleached nearly white with sodium per1 Funfsfzlcks, Beitr IViss. bot., 4 (1900), 166; also Habilitats Schrift, S t u t t g a r t , 1901. 2 A W. Schorger, THIS J O U R N A L ,9 (1917), 561.
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oxide, still gave a strong red color. Sulfate or soda-cooked, unbleached papers, however, gave no red color. It is not improbable that the red color often obtained when chlorine-treated wood is treated with alkaline sodium sulfite, as in the Cross and Bevan method for cellulose determination, is a hlaule test. The color-making material produced by chlorination is similar in nature to phenolphthalein, in that its acid form is light colored, and its salts deep red. STUDYOF THE FERRICFERRICYANIDE TEST^ If a ferric chloride solution is added to a freshly prepared solution of potassium ferricyanide, a dark brown solution is obtained. This sorution, when applied to wood, produces a deep green-blue color. Such a color is not given when the reagent is applied to filter paper. In this case, a diffuse pale blue is produced. Some experiments have shown that this reaction is given by many other materials than wood. Any phenol or any amine gives the test. Inorganic reducing agents, such as stannous chloride, sulfurous acid, or hydrogen peroxide in acid solution also produce the green-blue color. (Certain aldehydes, as benzaldehyde and formaldehyde, do not give the test.) The fact that so many materials give the test makes its qualitative value as a lignin reagent very small.
CONCLUSIONS 1-Phloroglucinol, p-nitroaniline, and the other singlereagent color tests are not to be thought of as indicators of any appreciable part of the composition of lignin, but rather of traces of aldehyde which usually2--perhaps always-accompanies lignin. 2--All experiments, and particularly the spectroscopic work, are entirely consistent with the presence of one single color-producing aldehyde in wood, or, a t least, the strong predominance of one aldehyde. 3-This aldehyde is not improbably coniferyl aldehyde, H
H
I 1
0
//
c HoO = C-c-H
CH3
as suggested very recently by the work of KIason. 4-Vanillin or furfural, if present, constitutes only a small fraction of the aldehyde content of the wood. 5-This work is in essential agreement with that of Czapek, who called the aldehyde of wood “hadromal.” 6-Several of the nonaldehydic materials mentioned in the literature as giving color reactions similar to those of “lignin” have been shown to contain traces of aldehydes which are responsible for the color formation. In the cases of oils of cloves (eugenol) and sassafras (safrol) the aldehyde seems to be identical with that of wood, and is probably distilled from the wood along with the essential oils. 7-The Maule reaction was found to consist essentially of a chlorination in acid solution of some compound in the wood, followed by alkaline treatment. A strong red color is developed in general in the case of deciduous woods, and only a weak indefinite brown color in the case of coniferous woods. 8-The Maule reaction may prove valuable as a means of distinguishing between deciduous and coniferous woods, in mixtures of chips. It may, further, prove of interest to botanists, in tracing the ancestors of the present angiospermous trees, for the production of the reacting chemical is quite possibly inherited. 9-The ferric ferricyanide test (acting as it does in the presence of almost any reducing agent) is too general to be considered as a “lignin” reaction. Haller, Fbrbev-Zlg., 26 (1915), 167, C. A , , 10 (1916), 630. Cross, Bevan and Syndall, “Wood Pulp and Its Uses,” I,ondon,
1R.
1911.
