b b When this new approach was applied to the subdivision principle b Spruce wood suspended in a nonswelling solvent and fed to this mill in small charges released for extraction 50% of its lignin
I
ANDERS BJORKMAN Billeruds AB, Saffle, Sweden
Lignin and Lignin-Carbohydrate Complexes Extraction from
Pmmw
Wood Meal with N e u t r a l Solvents
THE MOST annoying feature of lignin has been the difficulty in isolation without severe change. By applying several methods, a set of different lignins can be obtained from one piece of wood. Certain types are soluble in cold neutral solvents but the same solvents dissolve from common wood meal only a small amount of lignin material which has been known for 18 years (6) as Brauns’ native lignin (BNL). There are three conceivable explanations for the difficulty in extracting lignin from wood-first, strong covalent bonds exist between lignin and carbohydrates; second, lignin has a high molecular weight and possibly forms a three-dimensional network; and third, hydrogen bonds and physical phenomena such as solid solution may be involved in retention of lignin in the wood-fiber wall. Evidence for the first and second explanation is only indirect and mostly doubtful, whereas the third possibility has been overlooked almost entirely. However, it is impossible to exclude any of these possibilities for the present; perhaps all three contribute to the status of lignin in wood. The work reported here was based on the assumption that thoroughgoing subdivision of wood might facilitate extraction of lignin. This is not a new ap-
proach but earlier attempts have been unsuccessful. A considerable part of the lignin can be extracted from finely ground wood meal with neutral solvents at room temperature if in grinding (3) an efficient mill is used and the wood is dispersed in a nonswelling liquid. The wood charge to the mill must be small. A vibrational ball mill (8) originally of German design (Schwingmlihle) was used. The grinding vessel is oscillated by a n eccentric weight rotating behind the vessel with a frequency of 1410 r.p.m.; moreover, the vessel turns slowly. The wood was milled in toluene suspension and subsequently treated with methyl Cellosolve in room temperature. The amount of lignin obtained in the extract was estimated by measuring the extinction at 280 mp and also it was determined by weighing after purification. Table I.
Lignin Extracted from Spruce Wood (Picea abies milled 48 hr.) Extracted Lignin. % ’ of Klason Lignin
Wood Charge, Gr.
Estd. from A , at 280 mp
Weighed aftler purification
25
9
5
44
5 33
1
92
54
The weighed amount of lignin was much lower than shown by the light absorption; this is caused mainly by extraction of some lignin-carbohydrate complexes together with lignin (Table I). Wood charge has a great influence on the yield of lignin. The peak of original particle-size distribution is lowered and practically disappears while another peak grows at successively smaller particle sizes, leaving a tail of larger particles (Figure 1). Finally, a kind of dynamic equilibrium is reached (5) characterized by an essentially unchanged distribution. The ultimate action of the mill would be a reversible agglomeration and separation of already existing fragments of the wood, whereas no further subdivision of the wood structure would be obtained. When the amount of wood in the mill is reduced, this equilibrium, or a t least its peak, is displaced to smaller particle sizes; thus, more lignin is set free for subsequent extraction. Choice of solvent for the extraction step is not so critical as grinding conditions for lignin yield. Physicochemical factors may be involved--e.g., two samples of wood were ground under identical conditions and extracted, one with aqueous dioxane and the other with a mixture of ethylene chloride and ethyl alcohol. After the latter extraction had ceased to yield more lignin, the lignins VOL. 49,
NO.
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SEPTEMBER 1957
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Table II. Relative Amounts of p-Hydroxybenzyl Alcohol Groups in Lignin Preparations from Norway Spruce BNL M WL After standard purification After several repurifications Parallel sample purified with dioxane instead of acetic acid Dioxane lignin, according to Freudenberg (21) Ethyl alcohol lignin (37, HCl) Lignin in wood meal
100
44 51 40 9 6 21
Table 111. Phenolic Hydroxyl Groups in Various Softwood Lignins ( 7 ) Lignin
MWL Norway spruce Scots pine Western hemlock Black spruce BNL, Norway spruce
Phenolic O H per CHaO 0.30 0.27 0.29 0.28
0.46
obtained from both extractions were purified. The yields were 275 and 47 mg., respectively. When the second sample was re-extracted with aqueous dioxane, an additional 306 mg. was obtained which, although easily soluble in ethylene chloride-ethyl alcohol mixture, could not be extracted by the same solvent. For practical work, it is convenient to use dioxane for extracting lignin from milled wood. The dioxane must contain a few per cent of water because the lignin obtained is not soluble in pure dioxane. The extracted lignin is called milledwood lignin (MWL) but although convenient, this is not quite adequate. The extraction is fairly rapid at first but
complete extraction requires months. The highest yields of MWL, obtained by grinding and extracting spruce wood are around 50% of the Klason lignin. However, to obtain a substantial amount of lignin per day it was preferred to use conditions which give a total yield of about 357,. I n the method used, wood is reduced to pass 20-mesh by conventional methods, extracted io remove resins and BNL, and finally dried over phosphorus pentoxide. Twelve grams of wood are given a 48-hour premilling in a LampCn mill with the use of toluene. Portions of 6 grams suspended in toluene are then milled further for 48 hours in the vibrational ball mill. The wood is separated by centrifugation and extracted at room temperature with dioxane containing a few per cent of water. After evaporation to dryness under vacuum, the lignin is dissolved in acetic acid and precipitated into water. .4fter centrifugation, the precipitate is dried as a thin layer with air, redissolved more or less entirely in a mixture of ethylene chloride and ethyl alcohol, and precipitated into ethyl ether. The MWL is obtained as a slightly creamcolored, ash-free powder. This method allows production of more than 100 grams of lignin a year. The over-all temperature increase during milling is about 10' to 15' C. but higher local temperatures, though of short duration, are reached at collision surfaces. If oxygen is not excluded, some oxidation may occur. I t is necessary to find out if, during milling, the protolignin suffers chemical alteration in addition to possible depolymerization. A corresponding treatment of cellulose, made at the National Bureau of Standards (8), gave depolymerization followed by small chemical changes, but lignin may be more sensitive to this kind of treatment. MWL is lighter in color than any other lignin preparation, including BNL. This in itself may be considered an indication of mild isolation. Low-sul-
.. particle diameter Figure 1. Change of particle-size distribution curve during milling
1 396
250
fonated lignosulfonic acids, isolated according to Kullgren, may be considered structurally similar to protolignin. Figure 2 shows a comparison of such a lignosulfonic acid and MWL as revealed by curves for A6 (difference of the extinction coefficients obtained in alkaline and neutral solutions) according to AulinErdtman (2). Both preparations are from Norway spruce (Picea abies). Uifferences between the curves are rather insignificant. Incidentally, hlWL of Scots pine (Pinus siluestris), black spruce (Picea marianu), and western red cedar (Thuja plicata) give curves almost identical to those of Korway spruce, whereas the lignin of western hemlock (Tsugu heterophylla) has somewhat higher maxima. Regarding action of acids and elevated temperatures, ;b-hydroxybenzyl alcohol groups are probably the most sensitive structural elements in lignin. The relative amounts of such groups, determined with the indophenol method of Gierer (70) in various lignin preparations, are shown in Table 11. The value for BNL is double that for MWL-this agrees with the fact that BNL contains more phenolic hydroxyls. Repeated contact with acetic acid does not seem to affect the p-hydroxybenzyl alcohol groups, but action of hydrochloric acid a t room temperature as used in preparing dioxane lignin, or of boiling ethanolic hydrochloric acid destroys or blocks most of them. The low value for wood meal lignin is quite conceivable if we assume that part of the p-hydroxybenzyl alcohol groups are bound to the lignin in position 5 and therefore do not yield the soluble indophenol dyestuff. It can be assumed that MWL has retained the p-hydroxybenzyl alcohol groups of protolignin, at least the main part, during its isolation. The content of phenolic hydroxyl groups in MWL of spruce has been determined with different methods by Adler (7), Freudenberg ( 9 ) , and Enkvist. (7) and their results agree very well.
300
350
mP Figure 2. Ae curves of MWL and a low-sulfonated lignosulfonic acid using methyl Cellosolve as the solvent
INDUSTRIAL AND ENGINEERING CHEMISTRY
MWL.
--- Lignosulfonk
acid
LIGNIN
2 50
200 Figure 3. Ultracentrifugation of MWL in methyl Cellosolve
~
%
150
After; 1, 90 rnin.; 2 , 1 5 0 m i n . ~ and 3, 240 min.
100
hydroxybenzyl alcohol groups are much the same for all three fractions. I t may be satisfactory to extract about half the lignin of spruce wood with neutral solvents but the question arises: Why is the remaining lignin not extracted? Subsequent extraction with dimethylformamide or dimethyl sulfoxide, after dioxane, yielded additional material called lignin-carbohydrate complexes (LCC) which contained a large amount of carbohydrate together with lignin. Purification of this material was somewhat difficult because dimethylformamide holds dispersed wood particles very strongly and their separation is difficult. A procedure has been worked outafter the dimethylformamide solution
50 I
I
600
650 em.
BNL contains more phenolic hydroxyl groups than MWL, whereas the various softwood lignins are much alike (Table 111). Values for lignin derivatives like lignosulfonic acids and thioglycolic acid lignin are of the same order; this indicates that M W L and protolignin carry similar amounts of phenolic hydroxyls. I t is too early for definite conclusions about the chemical identity or possible differences between MWL and protolignin, but M W L may be a useful material for the lignin chemist. However, what happens during milling and extraction must still be explained. These problems are being studied from several angles, one of which is the determination of molecular weights, carried out a t the University of Uppsala. Figure 3 demonstrates the course of an ultracentrifugation of a MWL from
I
I
,
I
Norway spruce. The derivate of the concentration gradient is plotted against the distance from the center of rotation, The broadening of the peak indicates that M W L is polymolecular. Three fractions of M W L as defined by the ease of extraction can be distinguished-one fraction dissolves rapidly, another slowly, and the third is in between. All three fractions have nearly the same average molecular weight, around 11,000, corresponding to about 60 phenylpropane units. They were also similar in specific viscosity (Figure 4); the curves indicate a normal viscosity behavior. The molecules are not much elongated but it cannot be said what their actual form is. It must be concluded that the slow dissolution of lignin from milled wood is not caused by a difference in molecular weight. Incidentally, the contents of phenalic hydroxyls as well as of p-
r
10
Table IV.
Carbohydrate Composition of LCC (From Norway spruce and Scots pine) HemiLCC, LCC, cellulose Spruce Pine Spruce5 Relative amounts, %
200
10 17
Galactan Glucan Mannan Araban Xylan
15 25 32 13 15
50
4 19
8 18 46 4 24
Uronic acidsb,
% 7-10 8.4 Calculated for 42% glucan in wood as cellulose (4). Calculated as glucuronic acid.
Table V. Composition of LCC and Hemicellulose from Birch Hemicellulose”, LCC, % % Ligninb Uronic acidsC Galactan Glucan Mannan Araban Xylan
r7 13
3 2 86
...
14.3 4 7 7 7
75 glucan in wood as
Calculated for cellulose (4). As Klason Iignin, obtained hydrolysis. As glucuronic acid.
f i *
during
Table VI. Composition of Carbohydrate Fraction of MWL from Spruce Relative Amounts, % HemiPolysaccharide MWL cellulose
Figure 4. The ratio specific viscosity to volume fraction vs. volume fraction for different fractions of MWL in methyl Cellosolve
Galactan Glucan Mannan Araban Xylan
VOL. 49, NO. 9
13 18 31 10 28
0
SEPTEMBER 1957
8 18 46
4 24
1397
Yields of MWL from Norway Spruce under Different Milling Conditions Yield, Relative Amounts CH~O Conditions Estimated Weighed in First Milling Second Millingfrom A , at after MWL, Liquid Days Liquid Days 280 mp purification yo
Table VII.
_ _ I _ -
Toluene Toluene Ethyl alcohol, 99% Toluene Toluene Ethyl alcohol, 99% Isoamyl alcohol
3 2 1 2 2 3 3
...
Ethyl alcohol, 99% Toluene Ethyl alcohol, 95% Dioxane
... e . .
is evaporated to dryness under vacuum, the residue is dissolved in a mixture of equal amounts of acetic acid and water and the wood particles are removed by centrifugation. The wood residue is washed and the combined solutions are evaporated to dryness. The residue is then dissolved in dimethylformamide and precipitated in a mixture of ethylene chloride and ethyl alcohol to remove ligninlike materials which remain in solution. The precipitate is washed with ethylene chloride-ethyl alcohol and ether, dried, and redissolved in aqueous acetic acid. The final precipitation is made into acetone with several washings of ether. The LCC are obtained as slightly grayish white powders. Molecular weight of LCC from Norway spruce was determined as 8000. Analytical figures on the carbohydrate composition of LCC from spruce and pine are given in Table IV. The lignin contents in various samples of LCC from spruce were from 16 to 34y0 but the carbohydrate composition was always the same. The LCC may well be a mixture of true lignin-carbohydrate compounds and pure carbohydrate. The polysaccharide component of LCC contains the same sugars as hemicellulose and in about the same proportions. Uronic acids are also present in LCC. For pine, no values for comparison with wood are available, but glucan content in the LCC is also 10%~. The carbohydrate composition of LCC from wood of white birch (Betula ueirucosu) is shown in Table V. Results for birch indicate that its LCC, too, contain hemicellulose. Extraction of MiVL with dioxane is almost complete after 1 or 2 months,
Table VIII.
Methoxyl Content in MWL
from Various Species of Wood Norway spruce Scots pine Black spruce Western hemlock Thuja plicata White birch Common beech Aspen
1 398
CHDO,% 15.8 15.7 15.4 15.7 16.1 21.5 21.4 21.5
..
100 105
1 2 1
100 125
15.6 15.6
217
14.9
35
15.3
15 .I
1
.. ..
135 166 31 30
but extraction of LCC requires considerably longer. It is difficult to understand why the extractions are slow; if it is from a slow chemical reaction releasing MWL and LCC, these materials ought to be extractable in a similar way fr,om ordinary wood meal. This, however, is not the case. The yield of lignin extracted as LCC seems to be of the same magnitude as that of
MWL. The isolation of products which contain lignin and hemicellulose indicates that a large part of the lignin in wood is attached to carbohydrate but not to cellulose. The latter is probabk because cellulose forms strings which are partly crystalline. I t might be possible Lcith the help of these soluble LCC's to elucidate the nature of the bonds between lignin and carbohydrate, MWL has not been obtained completely free of carbohydrate. The first fraction obtained from wood of h-orway spruce may contain 15.8 to 15.9YC methoxyl, whereas subsequent fractions give somewhat lower figures. A lignin preparation from spruce \vith 3 5.8Yc methoxyl contained 1.9% polysaccharides which had the susar composition shown in Table V1. It is apparently close to hemicellulose. If all lignin molecules in this preparation, with the molecular weight 11,000, were bound to sugars, there would be an average of only one sugar monomer left for each lignin molecule. This is not a probable arrangement; therefore, it is better assumed that the MWL contained a small amount of LCC. At present, it is difficult to judge if in wood there is any lignin at all which is completely free from carbohydrate. To get a more complete picture it is necessary to fractionate both MWL and LCC. Now, the materials are defined to some extent, only by their preparation methods. Nevertheless, it is probable that half the lignin in spruce is free and the other half combined with hemicellulose. The covalent bonds between them may not be numerous and may resemble spot-welding, whereas hydrogen-bonding and physical phenomena are more important than is generally assumed. The importance of the liquid used for
INDUSTRIAL AND ENGINEERING CHEMISTRY
dispersion during milling is demonstrated by Table VII, which shows relative yields of lignin as compared with .the yield obtained after milling in toluene. Milling with ethyl alcohol after toluene seems to increase the yield of MWL somewhat, whereas milling in the opposite order-ethyl alcohol before toluene-gives a low yield. Ethyl alcohol alone as well as isoamyl alcohol, give moderate yields. If milling after toluene is continued with 95% ethyl alcohol or dioxane, the yield is increased considerably. However, as seen from the methoxyl content of purified lignin in the case of dioxane, the carbohydrate impurities tend to increase at these high yields. When MTVL contains carbohydrate impurities: it is not correct to compare analytical figures on lignins from different woods. A first fraction obtained during extraction of MWL from Korway spruce contained only a small amount of sugars. The methoxyl content \vas 15.8 to 15.9yoand the calculated value for pure spruce lignin was 16.1yo. If it is assumed that the first fractions from different kinds of wood contain about the same small amount of carbohydrate, then the methoxyl contents can be compared (Table VIII). Values are the same within each botanical order but vary between them. Experiments with sulfite cooking of MTVL from spruce have been made. The lignin "melts" and dissolves rapidly in an ordinary sulfite cook if efficient stirring is provided, but even at pH 7, nearly all lignin is dissolved after 2 hours at 135' C. This is a somewhat unexpected result which may necessitate some revision of our present views regarding the mechanism of the sulfite cook. literature Cited Adler, E., Hernestam, S., Acta Chem, Scand. 9, 319 (1955); Saensk Kern. Tid. 67, 37 (1955). Aulin-Erdtman, G., SLvnsk Papperstidn. 55, 745 (1952); 56, 287 (1953); 57, 745 (1954). Biorkman. A.. Ibid.. 59. 477 11956). Bjorkqvist, k. J.; Jorgensen, L., Gustafsson, S.:Ibid., 56, 734 (1953). Bradshaw. B. C., J . Chem. Phys. 19, I057 (1951). Brauns, F. E., J . Am. Chem. SOC.61, 2120 (1939). Enkvist, T., Paperija Puu 38, 1 (1956). Forziato, Florence H., Stone, W. K., Rowen, S. Mi., Appel? W. D., J . Research Natl. Bur. Standards 45, 209 11950).
RECEIVED for review December 3, 1956 ACCEPTED July 1 1957 Division of Cellulose Chemistry, Symposium on Lignin, 130th Meeting. XCS, Atlantic City X. J., September 1956.
