Despite significant differences in their densities, lignin and carbohydrates associate so strongly, even in physical mixtures, that no separation takes place in liquid density gradient columns. Ball-milled cellulose, lignin, and wood exhibit much higher densities in salt gradients than in organic liquid gradients indicating penetration of the saline solutions into voids or "free volume" in the polymers that are inaccessible to organic liquids

Harkin, John M.

Obst, John R.

English

Wood and Fiber Science (E-Journal)

INTERACTIONS BETWEEN LIGNIN AND CELLULOSE IN ORGANIC LIQUID AND SALINE DENSITY GRADIENT COLUMNS1 John  M .  Harkin2 and John  R. Obst Forest Product Laboratory,:' Forest Service, U.S. Department of Agriculture ( Received 1 October 1973 ) ABSTRACT Jlespite significant differences in their densities, lignin and carbohydrates associate so strongly, even in phys cal mixtures, that no separation takes place in liquid density gradient columns. Ball-milled cellulose, lignin, and wood exhibit much higher densities in salt gradients than in organic liquid gradients indicating penetration of the saline solntions into voids or "free volume;" in the polymers that are inaccessible to organic liquids. Additional keywords: Popz~lus trenzuloides, Picca sitchensis, ball-milled wood, enzyme lignin, cotton, Avicell. INTRODUC rION Wood is a composite I laterial made up of the structural po1ysacch;~rides cellulose and hemicellulose and the r imorphous phenyl- propanoid polymer lignin. These polymers are so closely interrelate11 in wood that none of them can be completely extracted, even if powerful solvents or reagents are used that dissolve the isolated substances. I t  is not known whether this behavior merely reflects an intimate phj sical intermingling of the polymers in l ~ o o d  (incrustation theory) or whether the) are interlinked by occasioi~al covaleilt cher ~ i c a l  bonds to form a true graft polymer in i he so-called lignin- carbollydrate complex ( ior :I reccnt review, see Grushnikov and Sh ~rygina 1970). Little or no ligniil call be extracted with organic solvents from jawdust or Wiley- inilled wood. However, when wood is sub- jected to vibratory ball-inilling, some lignin of low carbohydrate co~ltcilt (milled wood ' We thank Dr. Daniel ?. Caulfield for mea- snrelnents of the X-ray diffiaction of our cellulose samples. The use of trade, firm, or corporation ligniil, MWL) can be extracted, for example with dioxane-water mixtures ( Bjbrkman 1956). The amount of lignin extractable from aspen (Populus tremuloides Michx.) and the carbohydrate content of the extract increase with milling time (see Table 1). The distribution of lignin across wood cell walls is not uniform; the narrow com- pound middle lamella between cells and the interstices at cell corners contain much higher concentrations of lignin than the broad secondary cell-wall thickenings (Fergus and Goring 1970). Vibratory ball- milling produces particles smaller than wood cell-wall cross sections (Bjdrkman 1956). If ligilin and the polysaccharides are merely physically intermixed in wood, it call be conjectured that MWL of low carbo- hydrate content is derived from the lignin- rich fragments of middle lamellae that have become exposed to solvent by extensive mechanical breakdown of the cell wall during milling. The carbohydrate-rich frac- tions are derived from secondary cell-wall particles ( Sarkanen and Ludwig 1971 ) . Published values for the densitv of Urr -  " names in this publication is {or the information and nin-about 1.34 S/cc (stamm 1969)-are convenience of the reader. Such use does not constiti~te official endorsemc nt or approval by the inuch lower than those for cellulose. It  U.S. Department of Agricultilre. therefore seems that physical separation of ' J.  &I .  Ifarkin, currently P ssociate Professor, Soil iniddle lanlella lignin from lig~lin-~arbo- Science, University of Wisc~nsin; J. R. Obst, Re- hydrate complexes of secondary cell-wall warch Chemist, Forest Prolucts Laboratory. Maintained at Madison, Wisconsin, in origin should occur in liquid density gradi- ation with the University t f  Wisconsin. ent columns (Oster and Yamamoto 1963) \\'OOD AND FIBEH 343 WINTER 1974, V. 5 ( 4 )  344 JOHN hI. EIAHKlN AND JOHN R .  013ST TABLE I .  A W L O ~ L I L ~ S  and carbohydrate contents of lignin" from "bone-dry" ball-milled aspen Milling tim& Total lignin Klason lignin Carbohydrate extracted content o f  content of extract extract H r  - PC t - PC t - PC t -4 27.8 6 9 . 1  8.4 a~xtractable with dioxane-water ( 9 6 : 4  v/v) .  b~illed in air. and perhaps lead to isolation of purer lignin fractions. RESULTS AND DISCUSSION Organic liquid density gradients A suspension of vibratory ball-milled aspen was dispersed ultrasonically in a mix- ture of dried, degassed toluene/carbon tet- rachloride (d = 1.300) and added cautiously to a density gradient column made from the same liquids (density range 1.300- 1.590). A sharp band formed at density 1.440; no particles floated at the levels for lignin or cellulose densities. An enzyme lignin was isolated from Sitka spruce (Picea sitchen~is) by the method of Pew and Weyna ( 1962) ; this had a residual carbohydrate content of 6%. A dispersion of this lignin formed a narrow band at d = 1.340 on a toluene/CCl4 colunin. A dis- persed sample of ball-milled cotton linters was added to the same column. As the cellulose particles sedimented through the lignin layer, they picked up varying amounts of lignin, so that a distribution of agglom- erates resulted between the density of the lignin (1.340) and that of pure cellulose ( 1.510). Similarly, when milled cellulose sedimented through a layer of milled aspen wood in another toluene/CC11 column, a distribution of agglomerates was obtained between d = 1.440 and 1.510. This indicates a vcry pronounced physical attraction be- tween the cellulose and lignin particles. This lignin-carbohydrate interaction was further demonstrated: Homogeneous mix- tures of 1 g of spruce lignin ( d  = 1.340) with either 4 g of sorbitol ( d  = 1.467 i11 tol- uene/CC14 columns) or 4 g of milled linters ( d  = 1.510) were made by prolonged spatu- lation in toluene/CC14 or by milling for 5 to 30 min, both followed by ultrasonic dis- persion. When these mixtures were added to colurnns, only single sharp bands were formed at d = 1.432 (sorbitol/lignin) and 1.465 ( cellulose/lignin) . The sorbitol could be extracted quantitatively from the initial mixture with water, and the MWL fraction from the cellulose/lignin mixture with di- oxane-water (9: 1 v/v) . Thus, although the lignin and carbohy- drates were not chemically bonded and physical incrustation was not involved, no separation according to density occurred. Salt density gradients The interaction of the lignin and carbo- hydrates to yield particles of homogeneous consistency might be ascribed to hydrogen bonding or to electrostatic charges acquired during milling. Although such forces were not overcome in the nonpolar organic sol- vents, they ought to be dissipated in strong electrolyte solutions. Columns were therefore prepared from saturated potassium iodide and water (den- sity range 1.300-1.720) and saturated ce- sium chloride and water (density range 1.250-1.850). I11 the KI columns, ball- milled wood again formed only a single narrow band. However, this band occurred at a much higher density, viz. 1.675. Milled cotton cellulose sank to the bottom of KI columns, indicating a density of over 1.720. Enzyme lignin showed a density of 1.450. A 4 : l  mixture of milled cellulose and en- zyme lignin gave only a single band at  d = 1.680. Similar results were observed in CsCl columns. The densities of enzyme lignin, milled wood, and milled cotton cellulose in CsCl columns were 1.449, 1.673, and 1.724. A 4 : l  cellulose/lignin mixture gave only a single sharp band at 1.680. Thus, the physical forces between lignin LIGNIN .4NI) CELLULOSE IN DENSITY GRADIENT COLUh'lNS 345 TABLE 2. Densl~itie~~ (g tin" of wood, lignin, und cellt~lose in gradient coltimns - - Column Enzyme lignin Wood Ball-milled cotton Avicell Toluene/carbon tetrachloriiie 1.340 1.440 1.510 1.549 PoLassium iodide Cesium chloride - - %ensity values are expressed in terms of the density of the liquid at the height of the column where the particles come to rest. and cellulose are too strong to be neutral- ized even in concentrated salt solutions. Even though vibratory ball-milling may liberate some carbohydrate-free (i.e., chem- ically unbonded) lignin From wood-as in- dicated by its solubilit) in dioxane-water ( Table 1 )-the physical association of this lignin with the wood polysaccharides pre- cludes its separation from lignin-carbohy- drate complex by flotation in density gradi- ents. Anomalies in densities The values for the densities of lignin, cellulose, and wood observed in saline columns (Table 2 )  were much higher than those found in organic liquid columns. A sample of microcrystalli~le Cellulose I (Avi- cell) had a density of 1.648 in a CsCl column and 1.650 in a KI column. Ball- milling destroys the crjstallinity of cellu- lose, but upon treatment with water it partially recrystallizes as Cellulose I1 ( Caul- field and Steffes 1969) However, X-ray diffraction showed that material treated with KI or CsCl solutiori did not recrystal- lize. It  therefore appears that amorphous cellulose has a higher density (ca. 1.724) than crystalline cellulose (ca. 1.650) in saline systems. The density values for cellulose in saline columns were higher than any in the liter- ature (Table 3 ) .  The density of cellulose in organic liquid columns has been related to its degree of crystallinity (Caulfield and Steffes 1969); higher densities are asso- ciated with greater crystallinity. Because the density of Avicell measured in the salt gradients is slightly higher than the maxi- mum density calculated from X-ray data, some residual content of amorphous cellu- lose in this preparation seems indicated. An antiparallel relation between the density of cellulose in organic liquids and its higher "apparent" density in water as a buoyancy medium (Table 3)  has been discussed by EIermans (1949). The den- sity of a solid measured by liquid displace- ment is unique only when there is no inter- action to change the internal strl~cture and dimensions of the solid or its components. \Vater penetrates into portions of the cellu- lose matrix inaccessiblc to nonswelling organic liquids. Depending on the extent of swelling, an infinite number of displaced volumes are possible and computed density ' ~ABI ,E  3. Densities (g lc~ t~"  of ccdltdose ~rleastrred hy oariotls ~netlzo(Z,s (Ruck 1967) Method X-ray diffrac ion Water displacement Helium displacement Organic liquid displacement Cellulose I 1.591-1.6311 1.604-1.612 1.567-1.588 1.516-1.553 Cellulose I1 1.580-1.6611 1.601-1.615 1.531-1.550 1.512-1.536 346 JOIIN hf. IIAHKIN ANI) JOHN R. OHST \~alaes have less meaning. The high den- sities of lignin, cellulose, and wood now fount1 in saline solntions (Table 2 )  are ascriljable to even better swelling and liquid peljetration into the free volume (Tobolsky 1960) in the polymers. This enhanced swelling rnay l ~ e  due to the large amount of tmstructured water present in solutions of high ionic strength (Dobbins 1970). Ailom- alol~sly high derlsity values would also be o1)tained using conventional pycnometric procedures (Kellogg and Wangaard 1969), as deeper penetration and even solubiliza- tion would result in a reduced displace- meilt VO~IIIIIE. SUMMARY Attempts to separate physical mixtures of celll~lose and lignin in organic liquid and saline solution density gradient columns failed. In  spite of appreciable differences in density, such mixtures showed little ten- dency to fractionate according to their den- sities, sl~ggestir~g a strong physical attraction 1)etween the two polymers. Densities of the individual components measured in cohnnns of nonswelling liquids are normal. Densities measured in columns capable of swelling the components are ai~omalously high 1)ecaase of the penetration of the liquid into portions of the free volume of the polymers. HEFEIIENCES BJKRK~IAN, A. 1956. Studies on finely divided wood. Part 1. Extraction of lignin with neutral solvents. Sven. Papperstidn. 59:477-485. CAULFIELD, D. F., AND R. A. STEFFES. 1969. Water-induced recrystallization of cellulose. Tappi 52: 1361-1366. DOBBINS, R.  J. 1970. The role of water in cellulose- solute interactions. Tappi 53:2284-2290. FERGUS, H. J., ASD D. A. I. CORING. 1970. Distri- bution of lignin in birchwood as determined 11y ultraviolet microscopy. Holzforschung 24: 118-124. (;RUSHYTKOY, 0. P., . ~ N I )  N. N. SIIORYGINA. 1970. The present state of the problem of lignin- carbohydrate bonds in plant tissues. Russ. Chem. Rev. 39( 8 )  :684-694. IIERMANS, P. H. 1949. Physics and chemistry of cellulose fibers. Elsevier, New York. Pp. 197-213. KELLOGC:, R. hl.,  AND F. F. WANGAARD. 1969. Variation in the cell-wall density of wood. Wood Fiber 1 ( 3 ) : 180-204. OSTER, C;., AND hl .  YAMAKIOTO. 1963. Density gradient techniques. Chen~. Rev. 63:257-268. PEW, J. C., AND P. WEYNA. 1962. Fine grinding, enzyme digestion, and the lignin-cellulose bond in wood. Tappi 45:247-256. RUCK, H. 1967. Zur Dichtebestimmung der Cellu- lose. Das Papier 21 : 697-713. SARICANEN, K. V., ASD C. H. LUDWIG. 1971. Lignins; occurrence, formation, structure and reactions. Wiley-Interscience, New York. S~hnrxi, A. J. 1969. Correlation of structural vari- ations of lignins with their specific gravities. Tappi 52: 1498-1502. TOBOI.SKY, A. V. 1960. Properties and structures of polymers. Wiley, New York. Pp. 61-65. 

Interactions Between Lignin and Cellulose in Organic Liquid and Saline Density Gradient Columns

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Interactions Between Lignin and Cellulose in Organic Liquid and Saline Density Gradient Columns

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