ISOLATION AND CHARACTERIZATION OF DOUGLAS-FIR ORGANOSOLV LIGNIN BY HERN J. CHO B. ENG. (Chemical Engineering) Seoul National University, Korea, 1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Faculty of Forestry We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1981 @ Hern J. Cho, 1981 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Forestry The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date April 27, 1981 /Ten i ABSTRACT Granular water-insoluble l i g n i n s were is o l a t e d from a series of aqueous organic solvent (organosols) cooks designed for pulping/sac-c h a r i f i c a t i o n of Douglas-fir sawdust. Among the factors a f f e c t i n g y i e l d and c h a r a c t e r i s t i c s of the i s o l a t e d organosolv l i g n i n s , only cooking time (5-20 minutes) and concentration of acid c a t a l y s t (0-0. IN HC1) were investigated as cooking variables. Cooking temperature (200°C) and s o l -vent composition (acetone/water=60:40) were held constant. It was learned that the a c i d i f i e d organosolv cooking system i s f a r more e f f i c i e n t i n d e l i g n i f i c a t i o n and s a c c h a r i f i c a t i o n than a-queous acid hydrolysis under i d e n t i c a l conditions. In organosolv cook-ing, simultaneous d i s s o l u t i o n of l i g n i n and sugars occurs i n the cook-ing l i q u o r , allowing continued and t o t a l d i s s o l u t i o n of the wood cons-t i t u e n t s . In the present study, only the water-insoluble l i g n i n f r a c -t i o n was i s o l a t e d and analyzed. An almost quantitative recovery of the p r e c i p i t a b l e l i g n i n was accomplished by evaporation of the organic solvent from the spent liquor, followed by removal of sugars dissolved i n the aqueous s o l u t i o n and r e p r e c i p i t a t i o n of the crude l i g n i n i n t o water. To eliminate the interference from hydrogen bonding and unconjugated carbonyl group i n the i s o l a t e d organosolv l i g n i n s , a c e t y l a t i o n or reduction was' c a r r i e d out before the l i g n i n samples were characterized. The r e s u l t i n g l i g n i n samples were found to be completely free of cabohydrate contaminants. Both cooking time and acid concentration were found to have a profound e f f e c t on the y i e l d of l i g n i n f r a c t i o n s , and chemical and macromolecular properties of the l i g n i n molecules due to two competing i i reactions, h y d r o l y t i c depolymerization and recondensation. These re-actions take place simultaneously i n the cooking l i q u o r during organo-solv cooking. The balance between these two reactions i s believed to be responsible f o r not only the content of functional groups, as revealed by nuclear magnetic resonance, infrared and u l t r a v i o l e t s p e c t r a l ana-lyses, but also the si z e of l i g n i n molecules, as measured by gel per-meation chromatographic and scanning electron microscopic analyses of the i s o l a t e d organosolv l i g n i n s . The f u n c t i o n a l group contents, determined by elemental and s p e c t r a l analyses, were found to be 0.86-0.97 methoxyl, 0.20-0.49 aro-matic hydroxyl and 0.68-0.99 a l i p h a t i c hydroxyl groups per C^-unit of the organosolv l i g n i n molecules. I t was also noted that 63-68% of aro-matic n u c l e i have condensed forms with carbon-carbon linkages, having only two hydrogens on each guaiacyl nucleus. The organosolv l i g n i n s were found to have much lower molecu-l a r weights than those of pr o t o l i g n i r i i n wood;, Ty p i c a l values of the number average molecular weight of the i s o l a t e d l i g n i n s ranged from 823 to 1,144. The low molecular weight values are due to degradation reac-tions during the cooking by cleavage of a r y l - a l k y l linkages of l i g n i n molecules. The p a r t i c l e size of the sph e r i c a l p r e c i p i t a t e d l i g n i n s ranged from 25 to 500 nm. i i i TABLE OF CONTENTS PAGE ABSTRACT i TABLE OF CONTENTS ,. i i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENT i x 1. INTRODUCTION 1 2. LITERATURE REVIEW 4 2. 1 D e f i n i t i o n s 4 2. 2 D i s t r i b u t i o n of Lignin 5 2.3 Formation and Chemical Structure of Lignin... 7 2.4 I s o l a t i o n of Lig n i n 14 2. 4.1 Native l i g n i n 14 2.4.2 Lignins from i n d u s t r i a l pulping processes 16 2.4.3 Lignins from organosolv pulping 18 2.5 Characterization of Lignin...... 21 2.5.1 Degradation of l i g n i n 21 2.5.1.1 Strong oxidation 21 2.5.1.2 Mild oxidation. 22 2.5.1.3 Ethanolysis 23 2.5.1.4 Hydrogenolysis 26 2.5.2 Spectroscopic studies on functional groups 26 2.5.2.1 Benzyl alcohol and benzyl ether groups 27 2. 5: 2. 2 Phenolic hydroxyl group 27 2. 5. 2. 3 Methoxyl group 28 2.5.2.4 Carbonyl group 29 i v 2.5.3 Macromolecular properties of l i g n i n 29 2.5.3.1 Molecular weight d i s t r i b u t i o n of l i g n i n . . . . 29 2.5.3.2 Shape and s i z e of l i g n i n molecules 31 3. MATERIALS AND METHODS 33 3. 1 Materials 33 3.1.1 Selection of s t a r t i n g material 33 3.1.2 Preparation of e x t r a c t i v e - f r e e sawdust samples 36 3.1.3 Preparation of organosolv l i g n i n samples 36 3.1.4 Preparation of acetylated l i g n i n samples 37 3.1.5 Preparation of reduced l i g n i n samples 38 3.2 Methods 38 3.2.1 Analysis of l i g n i n f r a c t i o n s 38 3.2.1.1 Klason l i g n i n 38 3.2.1.2 Acid-soluble l i g n i n 39 3.2.1.3 Residual l i g n i n 39 3.2.1.4 Water-soluble l i g n i n and degradation products 40 3.2.2 Chemical analyses of is o l a t e d organosolv l i g n i n s . . . . 41 3.2.2.1 Elemental analysis and methoxyl content determination 42 3. 2. 2. 2 U l t r a v i o l e t spectra 43 3.2.2.3 Infrared spectra 43 3.2.2.4 Nuclear magnetic resonance spectra 44 3.2.3 Macromolecular analyses of i s o l a t e d organosolv l i g n i n s 45 3.2.3.1 Gel permeation chromatography 45 3.2.3.2 Scanning electron microscopy 48 4. RESULTS, 49 V 5;. .DISCUSSION 52 5.1 Chemical Composition of Ex t r a c t i v e - f r e e Douglas-Eir Sawdust.. 52 5^ 2 I s o l a t i o n of Organosolv Lignins 55 5.3 E f f e c t of Cooking Conditions on Yiel d s of Fiber Residue and L i g n i n Fractions 58 5.3.1 E f f e c t of cooking time 59 5.3.2 E f f e c t of acid c a t a l y s t concentration 65 5.4 Water-soluble Li g n i n F r a c t i o n 69 5.5 Thin-Layer Chromatographic Analysis of Water-soluble Lig n i n F r a c t i o n from Spent Cooking Liquor. 70 5.6 Microanalysis of Isolated Organosolv Lignins........ 75 5.7 Spectroscopic Analyses of Isolated Organosolv Lignins........ 78 5.7.1 Nuclear magnetic resonance spectra...... 78 5.7.2 Infrared spectra,. 95 5. 7. 3 U l t r a v i o l e t spectra. 101 5.8 Macromolecular Analyses of Isolated Organosolv Lignins 105 5.8.1 Gel permeation chromatographs .105 5.8.1.1 E f f e c t of cooking time on molecular weights..105 5.8.1.2 E f f e c t of acid concentration on molecular weights 110 5.8.2 Scanning el e c t r o n photomicrographs 113 6. RECOMMENDATIONS 119 7. CONCLUSION 120 LITERATURE CITED 122 v i LIST OF TABLE TABLE PAGE 1. Sieve analysis of Douglas-fir sawdust 35 2. Chemical composition of e x t r a c t i v e - f r e e Douglas-fir sawdust.... 52 3. E f f e c t of cooking conditions on y i e l d s of pulp and l i g n i n f r a c t i o n s from e x t r a c t i v e - f r e e Douglas-fir sawdust............. 60 4. E f f e c t of cooking time on y i e l d s of f i b e r and water-insoluble l i g n i n from various wood species, 62 5. pH Values of acid-catalyzed cooking l i q u o r before and a f t e r 20-min cooking 68 6. R.£ Values and c h a r a c t e r i s t i c colors of s e l e c t i v e l i g n i n deg-radation products and phenolic extractives 72 7. Thin-layer chromatography of water-soluble f r a c t i o n from organosolv spent l i q u o r 73 8. Elementary composition of acetylated Douglas-fir l i g n i n samples 77 9. Assignments of signals i n NMR spectra of acetylated Douglas-f i r l i g n i n samples.. 81 10. Relative i n t e n s i t y of various proton types i n NMR spectra of acetylated l i g n i n samples , 89 11. Empirical formulae and functional group contents of i s o l a t e d l i g n i n samples 90 12. Assignments of absorption bands i n IR spectra of reduced Douglas-fir l i g n i n samples. • • • • - * ^8 13. Spectrophotometry determination of phenolic hydroxyl groups of reduced Douglas-fir l i g n i n samples 103 14. Molecular weight averages and p o l y d i s p e r s i t y indices of acetylated Douglas-fir l i g n i n samples ,. .....106 15. Frequency of p a r t i c l e size of acetylated l i g n i n samples 116 v i i LIST OF FIGURES FIGURE PAGE 1. Pathways for the conversion of glucose to lignin-monomers i n plant 9 2. Dehydrogenation of c o n i f e r y l alcohol 10 3. "End-wise" polymerization 11 4. Freudenberg's formulation of softwood l i g n i n 13 5. Prominent structures i n spruce l i g n i n 13 6. Formation of Hibbert's ketones 24 7. F r a c t i o n a t i o n of l i g n i n from organosolv cooking 34 8. GPC c a l i b r a t i o n curve 47 9. E f f e c t of cooking time on y i e l d s of pulp and i s o l a t e d l i g n i n (FRACTION II) from e x t r a c t i v e - f r e e Douglas-fir sawdust. 61 10. Cooking-bomb temperature vs. cooking time..... 64 11. E f f e c t of acid concentration on y i e l d s of pulp and i s o l a t e d l i g n i n (FRACTION II) from e x t r a c t i v e - f r e e Douglas-fir sawdust.. 66 12. T y p i c a l NMR spectrum of acetylated. Douglas-fir l i g n i n 79 13. NMR spectrum of sample PC-11 82 14. NMR spectrum of sample PC-12 83 15. NMR spectrum of sample PC-13 84 16. NMR spectrum of sample PC-14 85 17. NMR spectrum of sample PC-15 86 18. NMR spectrum of sample PC-9 87 19. NMR spectrum of sample PC-19 88 20. D i f f e r e n t i a l NMR-spectra of cooking time and acid concent-r a t i o n s e r i e s 91 21. E f f e c t of cooking time on IR spectra of reduced Douglas-fir l i g n i n samples 96 22. E f f e c t of acid concentration on IR spectra of reduced Douglas-f i r l i g n i n samples 97 23. Difference UV spectra of reduced Douglas-fir l i g n i n samples.... 102 v i i i FIGURE PAGE 24. E f f e c t of cooking conditions on molecular weight averages of acetylated l i g n i n samples 107 25. Change i n molecular weight d i s t r i b u t i o n with cooking time.... 108 26. Change i n molecular weight d i s t r i b u t i o n with acid concen-t r a t i o n I l l 27. Scanning electron photomicrographs of p a r t i c l e size v a r i -a t i o n with cooking time 114 28. Scanning el e c t r o n photomicrographs of p a r t i c l e size v a r i -ation with acid concentration 115 29. E f f e c t of cooking time on p a r t i c l e size frequency d i s t r i b u -t i o n 117 30. E f f e c t of acid c a t a l y s t concentration on p a r t i c l e s i z e d i s -t r i b u t i o n 118 ix ACKNOWLEDGEMENT The author wishes to express his deepest gratitude to his major professor, Dr. L. Paszner, Faculty of Forestry, for h i s d i r e c t i o n , i n s p i r a t i o n and invaluable suggestions throughout the course of t h i s work. Grateful acknowledgement i s also made to Dr. S.T. Chiu and Mr.T.Kaneko, Western D i v i s i o n of Borden Chemical Co.,Vancouver, f o r numerous discussions on gel permeation chromatography and for making f a c i l i t i e s a v a i l a b l e ; to Dr.B. A. Bohm, Department of Botany, for help-f u l suggestions and c r i t i c i s m on nuclear magnetic resonance spectros-copy; to Dr. J.A.F.Gardner, Faculty of Forestry, for valuable sugges-tions on th i n - l a y e r chromatography; to Mr.G.M. Barton, Forintek Canada Corp., Vancouver, f o r providing various model compounds f or thi n - l a y e r chromatographic an a l y s i s ; to Mr. A.Lacis, Department of M e t a l l u r g i c a l Engineering, for preparation of scanning electron photomicrographs; to Mr. G.Bohnenkamp, Faculty of Forestry, for various help during the ex-perimental phase of t h i s work. In addition, the author wishes to thank to fellow students, Messrs.P.-C.Chang and N.Charleson for various suggestions and discus-sions. The help of Mr. G. Muir and Miss C. Ng, undergraduate students, for t h e i r laboratory work during the summer session i s also appreci-ated. F i n a n c i a l support by the Faculty of Forestry and the Nat i -onal Science and Engineering Research Council of Canada has been great-l y appreciated. F i n a l l y , s p e c i a l thank i s due to my wife, Sharon, f o r her patience and devotion throughout these memorable years. 1 1. INTRODUCTION The r i s i n g cost and expected shortage of crude o i l and natural gas, which are the main raw material sources f o r the organic chemical industry, have stimulated search f o r alternate chemical feed-stocks. L i g n o c e l l u l o s i c materials, such as wood and straws which represent the largest renewable bio-resources on the earth, are consi-dered to be one of the most important near-term substitutes f o r o i l and natural gas (74,75,78,79,154). During the past several years, considerable attention has been given to chemical u t i l i z a t i o n of wastes from the chemical pulping industry, and the l i t e r a t u r e i s abundant with suggestions f o r the re-covery of by-products from spent li q u o r s (74,118,129,131,133). Spent cooking and wash liquors, recovered from chemical pulping processes, contain p r a c t i c a l l y a l l the n o n - c e l l u l o s i c wood constituents such as l i g n i n , hemicelluloses and minor constituents of wood. Based on data of the primary production of wood and other annual and perennial land plants, i t can be calculated that the world-wide annual production of l i g n i n s , which represent about a quarter of l i g n o c e l l u l o s i c materials, i s about 20 b i l l i o n tons (154). According to FAO information (154), the t o t a l amount of l i g n i n obtained e i t h e r as a l k a l i l i g n i n or as l i g n o s u l f o n i c acid i s approximately 40 m i l l i o n tons annually. About 70 to 80% of t h i s amount i s burt f o r heat re-covery i n the pulp m i l l s (131). 2 Although t h i s transformation of l i g n i n into heat i s an econo-mic method of disposal of the waste l i q u o r as f a r as the pulping proces-ses are concerned, i t seems l i k e a wasteful manner of t r e a t i n g such a valuable chemical raw material. With marketable l i g n i n preparations, the chemical value of l i g n i n i s greater than i t s f u e l value,and l i g n i n becomes an important revenue generating by-product of pulping (118,133, 154). At the present time, l i g n i n i s not only used as s t a r t i n g or intermediate raw material for monomeric organic chemicals, but i s also widely used i n adhesives, binders, dispersants, and as extenders for resins and rubbers, emulsion s t a b i l i z e r , grinding aid, b o i l e r water i n -gredient and ion exchange resins (74,78,132). The features of l i g n i n important for chemical u t i l i z a t i o n are i t s aromatic character and co-valent carbon-carbon bonding (78). Although biomass processing holds promise for generation of chemical feedstocks for both chemical and processing i n d u s t r i e s , only few economic processes are known to produce organic chemicals from l i g -nin today. D i f f i c u l t i e s arise mainly due to the condensed and contami-nated state of i n d u s t r i a l l i g n i n s (154). Pulping processes, which allow separation of the main components of l i g n o c e l l u l o s i c materials i n such a manner that l i g n i n can be obtained free of contaminants ( e s p e c i a l l y without sulfur-substituents and sugar residues) and i n a less condensed state, may o f f e r better p o t e n t i a l f or i t s u t i l i z a t i o n as a raw material for the chemical industry. With increasing provisions by law and public concern over the environmental impact of pulp m i l l e f f l u e n t s , development of new non- or l e s s - p o l l u t i n g pulping processes i s also one of the most important prob-3 lems to be solved f o r the pulping industry (112). A new organosolv pulping process, which may provide some of the answers to these problems, have been worked out i n the pulp and paper laboratory of the Faculty of Forestry over the l a s t few years. The present study i s concerned with i s o l a t i o n of l i g n i n from the organosolv cooking and ch a r a c t e r i z a t i o n of the is o l a t e d l i g n i n . In e a r l i e r studies, Chang and Paszner (36,37) concentrated on describing the processes which lead to maximum sugar y i e l d s and the t o t a l d i s s o l u t i o n of aspen and Douglas-fir woods i n organosolv cooking l i q u o r s , but no attention was paid to the q u a l i t y of the d i s -solved l i g n i n . Thus, the objectives of t h i s study were: 1) to i s o l a t e the organosolv l i g n i n from Douglas-fir sawr dust which comprises a substantial portion of the wood raw material supply to many pulp m i l l s i n the P a c i f i c Northwest, 2) to investigate the e f f e c t of various cooking conditions on chemical and macromolecular properties of the i s o l a t e d l i g n i n , and 3) to draw inferences from these data as to the s e n s i t i v i t y of organosolv l i g n i n to degradation, d i s s o l u t i o n and recondensation as well as molecular condensation with other sugars or t h e i r d e r i v a t i v e s formed during the high temperature cooking process. 4 ( 2. LITERATURE REVIEW 2.1 D e f i n i t i o n s L i g n i n : L i g n i n has never been s p e c i f i c a l l y defined because i t i s not a d e f i n i t e chemical e n t i t y , and i t s polymeric chemical s t r u c -ture has not been f u l l y ; e l u c i d a t e d . L i g n i n , however, i s generally considered to be a system of a thermoplastic tridimensional polymer i n which C g-phenylpropane u n i t s ( I ) , linked together by C-O-C and C-C linkages appear to be the basic units (26,68,131). The concept of l i g n i n derived from an enzyme-initiated dehydrogenation polymerization of a mixture of three primary precursors, namely c o n i f e r y l alcohol ( I I ) , s i n a p y l alcohol ( I I I ) and p-coumaryl a l -cohol (IV), i s now well established (65,66,67). (I) (II) (III) (IV) 5 Organosolv Lign i n : Lignins obtained by procedures of ex-t r a c t i o n by means of organic solvents, usually i n the presence of a ca t a l y s t , have been c a l l e d "organosolv l i g n i n s " (131). These orga-nosolv l i g n i n s are soluble i n organic solvents employed as well as other solvents generally known as l i g n i n solvents. 2.2 D i s t r i b u t i o n of Lig n i n U n t i l early i n the 19th century, wood was considered to be a single chemical e n t i t y . This b e l i e f was held u n t i l Payen (130), i n a paper published i n 1838, showed that wood i s composed of several com-ponents including a fibrous material, c e l l u l o s e and an "encrusting ma-t e r i a l " which was l a t e r termed as " l i g n i n " . In plants, the f i r s t i n -d i c a t i o n of l i g n i f i c a t i o n can be seen at the time of the onset of the wall thickening phase (163,164). In 1965, Wardrop (164) showed that the f i r s t deposition was at the c e l l corners within or just inside the primary wall. Following t h i s i n i t i a l deposition at the c e l l corners, l i g n i f i c a t i o n then extends along the middle lamella and into the secon-dary wall (63,162). L i g n i n d i s t r i b u t i o n i n wood has been of considerable i n t e r -est f o r both t h e o r e t i c a l and p r a c t i c a l reasons. According to R i t t e r (140), l i g n i n e x i s t s i n wood i n two forms, namely, the 'middle lamella l i g n i n ' , and the ' c e l l wall l i g n i n ' , implying differences not only i n lo c a t i o n but a c c e s s i b i l i t y , composition and possible a s s o c i a t i o n with other c e l l w all components, mainly hemicelluloses. 6 The r e s u l t s of most recent workers i n the f i e l d were reviewed by Sarkanen and Hergert i n 1971 (148), and Cote i n 1977 (43). In 1965, Berlyn and Mark (19) showed that l e s s than 40% of the t o t a l l i g n i n i n softwood i s i n the middle lamella, most of i t being found i n the secondary wall of coniferous tracheids. This new p o s i t i o n has been supported by more recent evidences (18,44,45,69,128). Using u l t r a v i o l e t and fluorescence o p t i c s , Frey-Wyssling (69) demonstrated the uniformity - of d i s t r i b u t i o n of l i g n i n across the secondary c e l l w all as w e l l as across the middle lamella. Sacks et a l . (142) suggested that a greater portion of l i g n i n i s concentrated i n the compound middle lamella of maple, and that the l i g n i n network i n the secondary w a l l appears le s s dense than i n softwoods. The same conclu-sion has been drawn from u l t r a v i o l e t i n v e s t i g a t i o n on hardwood tissues (101). More recently, i n 1978 G r a t z l and his co-workers (143) deve-loped a new method to determine l i g n i n d i s t r i b u t i o n by using energy-dispersive X-ray analysis of brominated wood sections coupled with scanning electron microscopy. The data from the corresponding peaks of brominated wood samples show that the l i g n i n concentration i s very high i n the middle lamella region, decreases toward middle part of the c e l l w a l l , and s l i g h t l y increases again near the lumen. The o v e r a l l l i g n i n d i s t r i b u t i o n i s i n agreement with the r e s u l t s of e a r l i e r microscopic studies (128,142) on l i g n i n skeletons created by the removal of carbo-hydrates with hydro f l u o r i c acid. L i g n i n d i s t r i b u t i o n across the c e l l wall has important implications i n d e l i g n i f i c a t i o n and f i b e r separation from l i g n o c e l l u l o s i c s a f f e c t i n g both f i b e r y i e l d and c e l l u l o s e purity of pulps. 7 2.3 Formation'and Chemical Structure of L i g n i n The plant l i g n i n s of i n t e r e s t can be divided into three classes, which are commonly c a l l e d (i) gymnosperm or softwood l i g n i n s , ( i i ) angiosperm or hardwood l i g n i n s and ( i i i ) monocotyledonous angiosperm or grass l i g n i n s (131). According to several e a r l i e r investigations (65,158,161), i t was known that the most p r i m i t i v e land plants, as well as softwoods, have l i g n i n s i n which g u a i a c y l n u c l e i or c o n i f e r y l alcohol (II) predominates whereas i n hardwood l i g n i n s , both c o n i f e r y l alcohol (II) and s y r i n g y l n u c l e i or sinapyl alcohol (III) are present even though there are some exceptions to t h i s generalization (54,55,73). Grass or annual plant l i g n i n s generally are polymers of synapyl alcohol (III) and p-hydroxylphenyl propane (I) u n i t s . In common with a l l other organic plant constituents, l i g n i n must be derived u l t i m a t e l y from carbon dioxide. Although the complete scheme of biogenesis of l i g n i n i n the tree i s s t i l l f a r from t o t a l l y known, there appears to be l i t t l e doubt that l i g n i n o r i g i n a t e s from the carbohydrates which are formed from atmospheric carbon dioxide by the process of photosynthesis (87,131). Thus, the f i r s t phase of l i g n i n biogenesis involves the conversion by l i v i n g plant c e l l s of non-aromatic precusors such as carbohydrates into compounds containing benzenoid type rings which becomes a part of the basic structure of l i g n i n . As the f i r s t clues to t h i s conversion, around 1955, Davis, Sprinson, and t h e i r co-workers (47,103,157) demonstrated that radiation-induced 8 mutants of the bacterium Escherichia c o l i , which lacked enzymes necessary for aromatic r i n g formation, accumulated i n growth-medium compounds that have proved to be obligatory intermediates i n the conversion of sugars to benzenoid compounds. The so-called Davis-Sprinson pathway (102,131), as understood at present time on the basis of more recent findings (28,29,50,71,152), for the biosynthesis of the aromatic precursors of l i g n i n i s pictured i n F i g . 1. D-Erythrose-4-phosphate (V) and 2-phosphoenolpyruvic acid (VI) , both formed from glucose combined to form an intermediate phosphate (VII) , which then forms the c y c l i c 5-dehydroquinic acid (VIII). The biosynthesis then proceeds through the obligatory intermediates, 5-dehydroshikimic acid (IX) and shikimic acid (X). On the basis of tracer and enzyme studies, Brown (28,29) proposed the pathways from shikimic acid (X) to the three lignin-monomers ( I I , I I I , I V ) . He pointed out the fact that not a l l reactions i n t h i s pathway occur i n a l l species. The scheme indicates that a l l l i g n i f i e d plants possess the enzymes necessary to carry out the reactions i n the sequence. It should be emphasized that l i g n i f i c a t i o n pathways other than those shown i n F i g . 1 may also e x i s t . The second phase of l i g n i n biosynthesis involves the dimerization of the monomer precursors (II,III,IV) and the continued growth of molecule by the oxidative polymerization. The e f f o r t s to c l a r i f y the structures of the d i f f e r e n t types of l i g n i n have resulted i n a d e t a i l e d p i c t u r e of the various modes i n which the phenylpropane units 9 GLUCOSE n ~ <[H2 ® O H 2 C - C - i - C H O • C-COOH 1 1 o© OH OH (v) (VI) > - \ C C O H \ O H / HONl / O H ( V I I I ) H H OH ® O H 2 c - c - i — t — OH OH H C H , (VII) - C — C O O H > o HO \ \ / H2COH n CH OCHj OH OH ( I I I ) ( I D t Ofv) Figure 1. Pathways for the conversion of glucose to lignin-monomers i n plant (131). 10 (I) are l i n k e d together i n the polymer. This problem has been inv e s t i g a t e d by two general methods, degradation and synthesis of l i g n i n . As early as 1933, Erdtman was succe s s f u l i n dehydrogenating a number of monomer model compounds to dimeric products (51,52). He suggested that l i g n i n i s formed i n nature by an oxidative polymerization of phenolic precursors. Freudenberg and co-workers (67,68) showed that enzymes with laccase and peroxidase a c t i v i t i e s are probably responsible f o r dehydrogenation. Freudenberg formulated the following mechanism for i n i t i a l r e actions of the dehydrogenation polymerization of c o n i f e r y l a l c ohol (II) as shown i n F i g . 2 (2). H2COH H2COH H2COH h^COH HjCOH H2COH CH CH II - H II CH » CH " ' OH OCH3 (II) (II-a) ( H -b) (I I - c ) (II-d) (II-e) Figure 2. Dehydrogenation of c o n i f e r y l a l c ohol ( I I ) . 11 The enzymatic dehydrogenation is a one-electron transfer resulting i n the formation of a resonance-stabilized phenoxy radical, dehydrogenated from coniferyl alcohol (II). Stabilization of the radical occurs by coupling to another radical in any of the positions of the unpaired electron given i n resonance structures (Il-a,II-b,II-c, II-d,II-e). These mesomeric radicals then intercombine. The continued growth of the molecule w i l l predominantly take place by what has been called "end-wise" polymerization (145). The process i s il l u s t r a t e d by an example in Fig. 3 where a coniferyl alcohol radical in i t s resonance form (Il-b) i s attached by /^ -O-4 coupling to an end group radical (Il-a). The result of this coupling w i l l be a quinonemethide (XI) which w i l l react further by addition of a molecule of water to give the ether structure (XII). OH (II-b) Figure 3. (H-a) (XI) "End-wise" polymerization (2). (XII) 12 The formation of dimers i s followed by further poly-merization to tetrameric and high molecular weight aggregates. A great many formulae f o r l i g n i n polymers have been proposed over the years (3,24,51,52,64,65,67). In 1965, Freudenberg (65) proposed a s t r u c t u r a l formulation of F i g . 4 as a c o n s t i t u t i o n a l model for softwood l i g n i n based on enzymatic dehydrogenation: experiments. The r e s u l t i n g formulation containing 18'Cg-units are i n t e r l i n k e d i n a fashion corresponding to the biochemical growth of the n a t u r a l l y occurring l i g n i n molecule. It represents only a f r a c t i o n of a l i g n i n molecule. More recently the prominent substructures of spruce l i g n i n were c o l l e c t e d i n a scheme (Fig. 5) comprising 16 Cg-units (2,53). In 1974, Glasser and Glasser (73) developed a mathematical simulation of reactions with softwood l i g n i n b u i l d i n g units by computer. The simulated structure of softwood l i g n i n molecule, which consists of 81 Ccj-units, involves rather large globular configurations that are d i f f i c u l t to represent on a two-dimensional scale. The s t r u c t u r a l sketch depicted by them shows that 15% of the C^-units are derived from p-coumaryl alcohol (IV), 79% from c o n i f e r y l alcohol (II) and 6% from sinapyl alcohol ( I I I ) . The proportions of the three monomers (II,III,IV) involved i n the copolymerization process vary i n d i f f e r e n t woods (53) and even i n d i f f e r e n t morphological parts of the wood, thus giving r i s e to the d i f f e r e n t l i g n i n s (2). These studies point out the poten-t i a l d i f f i c u l t i e s i n obtaining uniformly depolymerized l i g n i n s . Figure h. Fre'udenberg' s formulation of softwood l i g n i n (65) Figure 5. Prominent structures i n Spruce l i g n i n (2). 14 2.4 I s o l a t i o n of Lignin No method has yet been developed f o r the i s o l a t i o n of the p r o t o l i g n i n i n i t s e n t i r e t y o r i g i n a l l y present i n the wood. Many common methods of i s o l a t i o n cause fundamental changes i n the l i g n i n structure and the l i g n i n s obtained are d i f f e r e n t i n many physi c a l and chemical properties from the native l i g n i n i n wood (131). In order to i s o l a t e l i g n i n from l i g n i f i e d substances, Brauns (26) has noted that the extraneous materials of the s t a r t i n g wood must be pre-extracted as completely as possib l e , because they might not only be i s o l a t e d as an inseparable part of the l i g n i n but also might form condensation products with the l i g n i n during the i s o l a t i o n procedure. It should be noted, however, that the s t a r t i n g wood has never been pre-extracted i n some cases, such as studies on the chemistry of l i g n i n s i s o l a t e d from the spent pulping l i q u o r s . 2.4.1 Native l i g n i n s In 1939, Brauns (25) reported that a few per cent of the l i g n i n of black spruce i s found among the extractives obtained by ext r a c t i o n with aqueous ethanol and can be p u r i f i e d by series of p r e c i p i t a t i o n s with water and ether. The r e s u l t i n g cream-colored powder was found to possess a l l of the chemical properties associated with the t o t a l l i g n i n and thus was termed Brauns Native L i g n i n (BNL). On account of i t s low 15 y i e l d , however, i t may be doubled whether BNL can be considered as re-presentative f o r the bulk of the l i g n i n i n a l l respects (21). In 1951, Nord and Schubert (123) t r i e d to set the l i g n i n s of hardwood and softwood free for extraction with neutral solvents by removal of carbohydrates by biochemical decomposition. They u t i l i z e d the "brown-rotting fungi", one of two main types of fungi which decom-pose the components of wood, to digest polysaccharides leaving l i g n i n more accessible to solvent extraction. Enzymatically liberated l i g n i n and BNL are outstandingly s i m i l a r , as proven by extensive studies by Nord and his co-workers (122,123,152). A few years l a t e r , Bjorkman (21) reported investigations on milled-wood l i g n i n (MLW), is o l a t e d from spruce by using a v i b r a t i n g b a l l m i l l i n the presence of a non-swelling solvent, such as toluene. Bjorkman's method i s based on the finding that about 30% of the l i g n i n becomes extractable with dioxane-water, i f wood i s suspended i n toluene and f i n e l y disintegrated i n a vibr a t o r y b a l l m i l l (21). A conventional rotary b a l l m i l l was introduced by Brownell (31) to overcome some disad-vantages, such as poor y i e l d s and length of time required f o r Bjorkman's procedure. According to his method, the milled wood i s completely s o l -uble i n an aqeuous s o l u t i o n of sodium thiocyanate, and the l i g n i n i s liberated by various treatments, such as tr a n s f e r i n t o the organic phase by l i q u i d - l i q u i d p a r t i t i o n i n g . ( 3 1 ) . In 1979, Wegener and Fengel (167) used u l t r a s o n i c s to speed up the dioxane extraction of b a l l - m i l l e d wood i n t h e i r e l ectron microscopic studies of lignin-polysaccharide complexes. By using a modified Bjorkman's 16 procedure with shaking and u l t r a s o n i c extraction, t h e i r l i g n i n i s o l a t i o n method supplied highly reproducible y i e l d s of well defined l i g n i n s i n a reasonably short time. However, the best l i g n i n preparation now ava i l a b l e i s probably the c e l l u l o l y t i c enzyme l i g n i n , developed by Chang et_ a l . (35). They treated wood meal which had been m i l l e d under toluene with an enzyme , preparation possessing high c e l l u l o l y t i c and h e m i c e l l u l o l y t i c a c t i v i t i e s , and the l i g n i n was i s o l a t e d by extracting the digested material successively with aqueous dioxane. 2.4.2 Lignins from i n d u s t r i a l pulping processes Lignins obtained from i n d u s t r i a l pulping processes are always heterogeneous i n nature. In a l l pulping processes the l i g n i n i s obtained i n aqueous s o l u t i o n along with spent cooking chemicals and other materials dissolved from the wood. These l i g n i n s are usually not sui t a b l e f o r fundamental studies because of the presence of extractives i n the o r i g i n a l wood chips. Ready a v a i l a b i l i t y of the l i g n i n s from i n d u s t r i a l pulping, however, caused these l i g n i n s to be used widely as experimental l i g n i n s , even without any p u r i f i c a t i o n (131). A c i d i f i c a t i o n of any of the commercial black l i q u o r s from the a l k a l i n e pulping, both k r a f t and soda processes, y i e l d s an a l k a l i l i g n i n . I s o l a t i o n of a l k a l i l i g n i n s , e s p e c i a l l y k r a f t l i g n i n s , was thoroughly reviewed by P e a r l and h i s co-workers i n a se r i e s of annual reviews (132). 1 7 In 1962, Merewether (117) investigated the p r e c i p i t a t i o n of l i g n i n from commercial Eucalyptus k r a f t black li q u o r s with acids and reported on the optimum conditions necessary f o r s a t i s f a c t o r y i s o l a t i o n He also studied k r a f t black l i q u o r s prepared i n the laboratory from e x t r a c t i v e - f r e e wood (117,118). The kra f t l i g n i n s d i f f e r from lignosulfonates i n that they are soluble only i n a l k a l i n e s o l u t i o n above a pH of approximately 9. The i n s o l u b i l i t y of k r a f t l i g n i n s i n a c i d i c s o l u t i o n has been overcome by Westvaco Corporation at Charleston, South Carolina (88) and a commercial l i g n i n "Indulin" has been produced i n three grades: I) Indulin C (crude sodium s a l t of l i g n i n ) , i i ) Indulin B ( p u r i f i e d sodium s a l t of l i g n i n ) and I i i ) Indulin AT ( a c i d i f i e d - l i g n i n ) . In 1980,Lundquist and Kirk (109) reported a simple p u r i f i c a t i o n procedure of an i n d u s t r i a l k r a f t l i g n i n , Indulin ATR-C by f r a c t i o n a t i o n through a serie s of l i q u i d - l i q u i d extractions. A f r a c t i o n which i s water-insoluble, chloroform-soluble and ether-soluble i s considered to be the p u r i f i e d k r a f t l i g n i n and i t accounts for more than 60% of the s t a r t i n g Indulin ATR-C. The spent l i q u o r s from s u l f i t e pulping processes contain more than 50% l i g n i n i n the form of l i g n o s u l f o n i c acids, which are mixed with sugars and other carbohydrate decomposition products, wood extractives, and pulping chemicals (131). Lignosulfonates have been i s o l a t e d from spent s u l f i t e l i q u o r s by a v a r i e t y of means. Most of the procedures f a l l w ithin a few general classes, including p r e c i p i t a t i o n as an insoluble basic lignosulfonate, s a l t i n g out with acids or s a l t s , p r e c i p i t a t i o n with alco-hols and ion exchange ( 1 3 2 , 1 3 3 ) . 18 The most important way of i s o l a t i n g and p u r i f y i n g lignosulfonates i s the Howard process inaugurated by Marathon Corporation at Rothschild, Wisconsin (132). After removal of most of the s u l f i t e and s u l f a t e by lime addition to pH 10.5, more lime i s added to the f i l t r a t e to give a basic calcium lignosulfonate which p r e c i p i t a t e s i n the pH i n t e r v a l of 10.5 - 12.2. More than ha l f the lignosulfonates quantity can be recovered by t h i s process (84). 2.4.3 Lignins from organosolv pulping Organosolv cooking of wood i n an aqueous organic solvent system with a proper ca t a l y s t at an elevated temperature provides an excellent procedure f o r simultaneous d i s s o l u t i o n and almost quantitative recovery of both sugar and l i g n i n f r a c t i o n s of wood. Organosolv pulping may be the only procedure which y i e l d s l i g n i n as a by-product of pulping process i n a le s s condensed state and free of inorganic contaminants(154). Since Klason used 5% HCl-ethanol to extract l i g n i n from spruce sawdust i n 1893, many investigators have reported on a wide v a r i e t y of organosolv lignins.-. In 1978 Paszner (129) reviewed a/large, number of papers" on organosolv pulping. Among the organic.solvents most frequently used are lower a l i p h a t i c alcohols, such as ethanol and butanol, ethylene g l y c o l , g l y c e r o l , dimethyl sulphoxide and dioxane. In an e a r l i e r study of various organic solvents that exhibit a c e r t a i n degree of solvent action on the i s o l a t e d l i g n i n s , Schuerch (97,153) showed that the a b i l i t y of solvents to dissol v e or swell the 19 i s o l a t e d l i g n i n s increases as the hydrogen-bonding c a p a c i t i e s of solvents increase and as t h e i r solubility-parameters ( £ ) approach a value of around 11. Although very powerful l i g n i n - s o l v e n t s , ketones, such as acetone ( S ~ 10), have not been used as often as lower a l i p h a t i c alcohols (129). A's early as 1931, K l e i n e r t and Tayenthal (96) introduced aqueous ethanol s o l u t i o n with hydrochloric acid as a cat a l y s t to cook wood above 150°C and obtained good y i e l d of c e l l u l o s e of low l i g n i n content. In 1936, Aronovsky and Gortner (10) c a r r i e d out a serie s of cooking on aspen sawdust and chips at constant pressure and i n the temperature range of 160° to 185°C with aqueous solution (1:1 r a t i o ) of various organic solvents such as methanol, ethanol, propanols (n-, iso-), butanols (n-, i s o - , t e r t - ), amyl alcohols (n-, i s o - , t e r t - ), dioxane and ethylene g l y c o l as cooking agents. They found that the normal primary alcohols were better pulping agents than the secondary or t e r t i a r y alcohols, and n-butanol yielded better pulp than was obtained with other solvents. However, when the same procedure was applied to pine, the r e s u l t of d e l i g n i f i c a t i o n was poor and no pulp was produced (149). It was l a t e r found that d e l i g n i f i c a t i o n of hardwoods was about twice as fast as that of the softwoods when cooks of. spruce and poplar sawdust were com-pared (94). In a serie s of studies (93,94,95), K l e i n e r t investigated the k i n e t i c s of bulk d e l i g n i f i c a t i o n that apply generally to organosolv pul-ping using aqueous ethanol solutions. I t was found that d e l i g n i f i c a t i o n proceeded i n two stages, an i n i t i a l f a s t bulk d e l i g n i f i c a t i o n followed by a slow removal of the remaining l i g n i n . K l e i n e r t also demonstrated 20 that aqueous solutions of ethanol were better d e l i g n i f y i n g agents than ethanol alone ( 9 3 , 9 4 , 9 5 ) . . The preferred pulping agents were mixture of ethanol and water i n the range between 20 to 75% ethanol by weight. K l e i n e r t also studied the influence of pH changes on organosolv pulping and reported that organic acids l i b e r a t e d i n the pulping process had an acc e l e r a t i n g e f f e c t upon d e l i g n i f i c a t i o n (94). In 1973, Kosfkova and P o l c i n reported on the influence of varying concentrations of added acid c a t a l y s t and water content i n the cooking l i q u o r on the y i e l d of extracted l i g n i n by aqueous solutions of dioxane (97). It was found that pure dioxane was able to dissolv e only very small amounts of l i g n i n from wood. Addition of acid c a t a l y s t such as HC1 increased the rate of d e l i g n i f i c a t i o n s i g n i f i c a n t l y (97, 153). The i s o l a t i o n of l i g n i n with dioxane i s b a s i c a l l y an a c i d o l y t i c s p l i t t i n g of the lignin,macromolecule and a lignin-carbohydrate complex into i n d i v i d u a l components which are soluble i n dioxane. In previous inv e s t i g a t i o n s on wood a c i d o l y s i s , great importance was attached to the presence of a polar-solvent, mainly water, which s u b s t a n t i a l l y improved y i e l d s of the i s o l a t e d l i g n i n ( 1 2 1 , 1 3 5 , 1 5 3 ) . The p o t e n t i a l of recovering l i g n i n s from large scale organo-solv pulping process i s very promising because of i t s many desirable properties. Among these, i t s high, s o l u b i l i t y i n the usual l i g n i n solvents such as ethanol, methanol, pyridine, chloroform, THF and acetone i s most important. The i s o l a t e d organosolv l i g n i n retains -it's good s o l u b i l i t y i n l i g n i n solvents-even a f t e r repeated p r e c i p i t a t i o n and'isO-l a t i o n from the spent l i q u o r ( 3 7 , 3 9 ) . 21 2.5 Characterization of L i g n i n ,: . 2.5.1 Degradation of l i g n i n D irect proofs of structure of native l i g n i n have been very lew because the polymeric l i g n i n involves many complex linkages. Nevertheless, the c h a r a c t e r i z a t i o n of l i g n i n . has evolved from degradation and synthetic i n v e s t i g a t i o n s . .2,5.1.1 Streng oxidation Strong oxidative degradation of methylated spruce l i g n i n with permanganate (68) y i e l d s methoxyl-substituted aromatic acids, v e r a t r i c acid (XIII), isohemipinic a c i d (XIV) and dehydro-diveratric acid (XV). The formation of isohemipinic a c i d (XIV) seemed to support the occurrence of o<-5 or ^-5 condensed structures and that of v e r a t r i c a c i d (XIII) i n d i c a t e d that noncyclic ether bridge between a side chain hydroxyl group and phenolic hydroxyl group of the adjacent unit were also important. OH OH OH OH i • • (xiii) (xrv) (xv) 22 Though the oxidation of l i g n i n with permanganate a f t e r methylation l e d to many i n t e r e s t i n g suggestions about the possible s t r u c t u r e of l i g n i n , the oxidation products f a i l e d to provide information concerning arrangement of side chains (144). 2.5.1.2 Mild oxidation Mild o x i d a t i v e degradation method such as nitrobenzene i n the presence of hot a l k a l i produced s u b s t a n t i a l y i e l d s of aromatic aldehydes (20,136,159-); Spruce wood gave about 25% v a n i l l i n (XVI) based on the Klason l i g n i n content, whereas mixtures of v a n i l l i n and syringaldehyde (XVII) were obtained from hardwoods. In a d d i t i o n to these two aldehydes, grasses afforded p-hydroxybenzaldehyde (XVIII). These degradation reactions g i v i n g three major aldehydes became one of the most important t o o l s f o r the i n v e s t i g a t i o n of l i g n i n m aterials. Later, small amounts of p-hydroxybenzaldehyde (XVIII) were also found i n the oxidation mixtures from both softwoods and hardwoods (2).' o o o OH OH OH (XVI) (XVII) (XVIII) 23 In 1951, Stone and Blundell (159) published a simple procedure f o r the rapid microdetermination of aldehydes found i n the nitrobenzene oxidation of l i g n i f i e d materials. This method involved separating the aldehydes chromatographically on a paper s t r i p and thereby has become a valuable t o o l i n d i f f e r e n t i a t i n g between l i g n i f i e d and n o n - l i g n i f i e d materials. 2.5.1.3 Ethanolysis Solvolysis methods applied to l i g n i n y i e l d derivatives of phenylpropane (I). Acid catalyzed ethanolysis of coniferous wood l i g n i n produces Hibbert's ketones (7,46,119,134) and i s considered to be one of the mildest methods to i s o l a t e arylpropane monomers from l i g n i n (100). A great many investigators applied t h i s ethanolysis technique to a variety of materials as quantitative and q u a l i t a t i v e a n a l y t i c a l methods. Hibbert and his co-workers (27,46,119) succeeded i n i s o l a t i n g several monomeric phenylpropane u n i t s , so-called Hibbert's ketones (XIX, XX, XXI, XXII), from spruce wood by refluxing with 2-3% ethanolic hydrochloric acid. (XIX) (XX) (XXI) (XXII) 24 The importance of g u a i a c y l g l y c e r o l - ^ - a r y l ether (XXIII), from which the phenolic Hibbert's monomers ori g i n a t e d during ethanolysis, has been recognized by numerous researchers. ( X X I I I ) In 1952, Alder and h i s co-workers (5) synthesized dimeric phenylpropane compounds of j j - a r y l ether which was considered to be incorporated i n the l i g n i n macromolecule e i t h e r as end group or as an e a s i l y hydrolysable u n i t . They showed that Hibbert's ketones were formed through s p l i t t i n g of benzyl ether and jS-aryl ether bonds of l i g n i n . According to Gardner (70), Hibbert's ketones (XIX, XX, XXI, XXII) were derived from ethanolysis of a k e t o l , 3-hydroxy-l-(4-hydroxy-3-methoxyl)-2-propanone (XXIV) v i a i t s enol (XXV). The side-chain structures of these Hibbert's ketones c e r t a i n l y had to be regarded as modifications of the o r i g i n a l structures caused by the a c i d during ethanolysis (2). Thus, the exact 7 nature of side-chains i n l i g n i n i s s t i l l an open question. H I H - C - O H I c=o H - C - H O C H , H I H-C-OH C - O H H - C O H OCH, OCH-, O H O H (XXIV) (XXV) O C H 3 H I H - C - H I H - C = 0 I H - C - 0 - C 2 H 5 O C H , O H O C H - , O C H O H OH (XIX) (XX) (XXI) (XXII) gure 6. Formation of Hibbert's ketones (70). 26 2.5.1.4 Hydrogenolysis Together, mild hydrolysis and c a t a l y t i c hydrogenolysis products from l i g n i n represent almost a l l the linkage patterns which e x i s t i n the enzymatic dehydrogenation products of c o n i f e r y l alcohol. C a t a l y t i c hydrogenolysis mainly cleaves ether linkages and reduces i n part the hydroxyls on side-chains (1,9,14,83,85,125,155). In 1938, Harris and h i s co-workers (85) hydrogenated aspen methanol l i g n i n i n dioxane under high temperature and high pressure of hydrogen over a copper-chromatic c a t a l y s t obtaining f a i r y i e l d s of monomeric propylcyclohexane d e r i v a t i v e s and i t was established that l i g n i n might be b u i l t up from C^(C^ - C^) u n i t s . This experiment also constituted the f i r s t p o s i t i v e proof that l i g n i n was predominantly aromatic i n character. Recently, dimers and trimers were i s o l a t e d from hydrogenolysis products of p r o t o l i g n i n and t h e i r structures were i d e n t i f i e d by means of u l t r a v i o l e t (UV) spectroscopy, i n f r a r e d (IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy and mass spectroscopy (114,115,116, 126). 2.5.2 Spectroscopic studies on functional groups Most of the e a r l i e r studies on UV spectra, IRu-speetra and NMR spectra of l i g n i n , for determination of f u n c t i o n a l groups and linkages i n l i g n i n preparations, have been reviewed i n d e t a i l by Goldschmid (76), Hergert (86) and Ludwig (105), r e s p e c t i v e l y . 27 2.5.2.1 Benzyl alcohol and benzyl ether groups Since Holmber, i n the middle of 1930's, made the important suggestion that c h a r a c t e r i s t i c reactions of l i g n i n were reactions of benzyl alcohol or benzyl ether, numerous inv e s t i g a t i o n s by UV spectra (5,22), I R spectra (56,67) and NMR spectra (120) have lent support t h i s suggestion. The t o t a l amount of benzyl alcohol and benzyl ether per 100 Cg-units of spruce l i g n i n was estimated 24 groups (2,67). Benzyl alcohol and benzyl ether groups are known to be highly unstable under acid or a l k a l i n e conditions and therefore, they are almost l i k e l y absent i n most pulp l i g n i n s (120). 2.5.2.2 Phenolic hydroxyl group In the middle of 1950's Aulin-Erdtman (11,12) and Goldschmid (77,111) c a r r i e d out ser i e s of studies independently to determine phenolic hydroxyl content of l i g n i n . I R spectral analyses by Alder and h i s co-workers (2,4) showed that phenolic hydroxyl groups of the l i g n i n u nits are l a r g e l y e t h e r i f i e d ( ot-aryl ether or |3 - a r y l ether structure) and determination of the amount of free phenolic hydroxyl groups should give a measure of the number of ether groups present. They reported that the amount ofC^-units with free phenolic hydroxyl group i n unaltered spruce l i g n i n ,1s ..less than. 20 per 100 28 Cg-units which means i n a great majority of the guaiacylpropane units (II) the phenolic hydroxyl group i s e t h e r i f i e d (4). 2.5.2.3 Methoxyl group In 1967, Chang et a l . (147) showed that the 280. nm UV absorption maximum of reduced softwood and hardwood l i g n i n s c orrelates w e l l with the values of methoxyl groups vs. carbon r a t i o . They also reported that the c a l i b r a t i o n curves obtained from the r a t i o of the IR absorbance of the i n d i v i d u a l maxima from 1,600 cm ^ to 1,045 cm ^ and that of the maximum at 1,500 cm ^ can be used to determine the corresponding methoxyl group per Cg-unit of the l i g n i n . More recently Faix and Schweer (57) determined the methoxyl content per Cg-unit from the integrated NMR spectra of the acetylated l i g n i n polymer models. Their r e s u l t s showed that the c a l c u l a t i o n by NMR spectra gives a l i t t l e higher values than those obtained by the conventional methods. 2.5.2.4 Carbonyl group Studies on the IR spectra of various l i g n i n s indicated the presence of minor amounts of conjugated as well as non-conjugated carbonyl groups (6,35). T o t a l number of carbonyl group i s known to be 20 per 100 C q-units, of which ha l f was found to be conjugated•carbonyl groups (2,6). 29 2.5.3 Macromolecular properties of l i g n i n 2.5.3.1 Molecular weight d i s t r i b u t i o n of l i g n i n The molecular weight of l i g n i n and i t s d i s t r i b u t i o n i s one of the most fundamental c h a r a c t e r i s t i c s of l i g n i n . The determination of molecular weight of l i g n i n macromolecules has been reviewed i n . . d e t a i l by Brauns (26) and Goring (81). In a serie s of studies (15,16,17) Benko characterized lignosulfonates by the d i f f u s i o n c o e f f i c i e n t method. He reported molecular weights of f r a c t i o n s obtained from a v a r i e t y of lignosulfonates and calculated a molecular weight d i s t r i b u t i o n curve from o p t i c a l density readings of the d i f f u s a t e . He also found that v i s c o s i t y measurements on i d e n t i c a l l i g n i n samples i n d i f f e r e n t solvents showed changes i n molecular weight values due to i n t e r a c t i o n of the dissolved l i g n o s u l -fonates with the solvent (15). Marton and Marton (113), using a vapor pressure osmometer, obtained highly consistent number average molecular weights (Mn) of several k r a f t l i g n i n s . The Mn values they obtained, however, ranged from 900 to 2,500 and gave only a one-sided picture of the p o l y d i s p e r s i t y of l i g n i n . Therefore, i t s use f o r macromolecular ch a r a c t e r i z a t i o n of l i g n i n i s of l i m i t e d importance. In 1970,Brownell (30) measured the i n t r i n s i c v i s c o s i t i e s and . Mn values of fractionated m i l l e d wood l i g n i n . The r e s u l t s obtained suggested that the degree of branching was greater i n high molecular 30 weight (5,000 - 19,000) than i n low molecular weight (ca. 3,500) l i g n i n f r a c t i o n s . Because of the non-linear structure of l i g n i n , the i n t r i n s i c v i s c o s i t y depends not only on the molecular weight but also on the degree of c r o s s - l i n k i n g and the i n t e r a c t i o n of e l e c t r o s t a t i c charges on molecular chains (82,139). V i s c o s i t y measurements were, therefore, not very valuable i n molecular weight measurements of l i g n i n solutions. Goring and h i s co-workers (110), using the u l t r a c e n t r i f u g e method, determined weight average molecular weight (Mw) of kr a f t l i g n i n s prepared from spruce sawdust. They obtained Mw ranging from 1,800 to 51,000. The disadvantage of the u l t r a c e n t r i f u g e method i s that the p o l y d i s p e r s i t y of l i g n i n s o l u t i o n a f f e c t s the sedimentation speed and thus the molecular weight r e s u l t s . Another d i f f i c u l t y i s the intense c o l o r of l i g n i n solutions, because the concentration gradients developed i n the u l t r a c e n t r i f u g e c e l l are usually detected o p t i c a l l y (81). Currently, the most r a p i d l y developing method i s gel permeation chromatography (GPC). Since i t s discovery i n 1959, GPC has gained r a p i d l y i n success because the molecular weight d i s t r i b u t i o n (MWD) can be determined quickly and e a s i l y . Depending on t h e i r s i z e , the l i g n i n macromolecules can d i f f u s e i n varying proportions into the porous volume of the column. Thus the e l u t i o n volume of any p a r t i c u l a r f r a c t i o n i s a function of the dimension of l i g n i n macromolecules and the siz e of the pores i n the gel (81). 31 A great many GPC investigations on l i g n i n have used dextran gels (Sephadex) (41,42,110,124,151,166,168) or agarose gels (Sepharose) (89,90) as the stationary phase for GPC to determine molecular weights and MWD of l i g n i n . Both types of gels (Sephadex gels and Sepharose gels) have c e r t a i n disadvantages. The Sephadex gels can be used only up to molecular weight of 100,000 (89) 6 and Sepharose gels up to 4 x 1 0 ( 9 0 ) . T h e l a t t e r contains charged groups which may i n t e r f e r e with the l i g n i n (89). Although the cross linked copolymer of styrene and divinylbenzene beads (Styragel) i s the most . commonly used column packing gel f o r high polymers (127), no a p p l i c a t i o n of t h i s g e l for l i g n i n macromolecules has been reported yet. 2.5.3.2 Shape and size of l i g n i n molecules There are only a few papers describing l i g n i n investigations by electron microscopy, mostly connected with degradation of the c e l l wall or with the i n v e s t i g a t i o n of polysaccharides containing a c e r t a i n amount of l i g n i n (57,58,60,61,62,99). In 1963 Rezanowich e_t a l . (139) reported that the molecules of dioxane l i g n i n had s p h e r i c a l configuration i n s o l u t i o n . The sp h e r i c a l shape was also supported by agreement between the sedimentation equilibrium molecular weights and values obtained by s u b s t i t u t i o n of i n t r i n s i c v i s c o s i t y and d i f f u s i o n constant into an equation derived from the E i n s t e i n v i s c o s i t y r e l a t i o n s h i p for s p h e r i c a l p a r t i c l e s . The low i n t r i n s i c v i s c o s i t y of l i g n i n solutions suggested that l i g n i n molecules behave l i k e E i n s t e i n spheres i n s o l u t i o n (81). 32 Further evidence for the sphe r i c a l shape of l i g n i n macromolecules.. was provided by • electron micrographs of high molecular weight f r a c t i o n s of sodium lignosulphonates (138). A l k a l i l i g n i n s and organosolv l i g n i n s were found to behave inore l i k e E i n s t e i n spheres than the lignosulfonates (81). In 1978 Kosikova et a l . (99) confirmed that BNL and methanol l i g n i n from beech wood and MWL from spruce wood showed c h a r a c t e r i s t i c s p h e r i c a l aggregates of lignin-macromolecules. In their/electron: microscopic investigations on the above l i g n i n samples, they found that a l l these l i g n i n s . had c h a r a c t e r i s t i c structures of small sphe r i c a l p a r t i c l e s of about 100 to 400 nm. . A s t a t i s t i c a l p a r t i c l e s i z e d i s t r i b u t i o n was reported for Bjorkman l i g n i n and BNL by Fengel (59). Further, his studies indicated l i t t l e , i f any, e f f e c t of the i s o l a t i o n method on granular shape and siz e d i s t r i b u t i o n of the above l i g n i n s . In summary, i t i s evident that most i s o l a t e d l i g n i n s e x i s t as high molecular weight f r a c t i o n s and show behavior i n s o l u t i o n cha-r a c t e r i s t i c of E i n s t e i n spheres of microscopic to macroscopic size. The inside structure of such spheres has not been investigated yet. 3 3 3. MATERIALS AND METHODS The present i n v e s t i g a t i o n involved the i s o l a t i o n and limited chemical and physical c h a r a c t e r i z a t i o n of the organosolv l i g n i n s from ex-t r a c t i v e - f r e e Douglas-fir sawdust. Dissolved sugars were not analyzed i n t h i s study. Nor were the pulps analyzed beyond t h e i r y i e l d and residual l i g n i n content. Though Douglas-fir was e x c l u s i v e l y used as the s t a r t i n g ma-t e r i a l for t h i s thesis, other species were also investigated during the preliminary cooking experiments for comparative purpose and included sp-ruce, aspen, birch, sugar cane and wheat straw ( 3 9 ) . The f r a c t i o n a t i o n of the l i g n i n s from organosolv cooking was done according to the scheme summarized i n Fig. 7 . D i f f i c u l t i e s were experienced with quantitative i s o l a t i o n of the water-soluble f r a c t i o n . For c h a r a c t e r i z a t i o n of the i s o l a t e d l i g n i n s , some of the most powerful tools available f or l i g n i n i n vestigations, such as high-speed GPC, scanning electron microscopy (SEM) as well as UV, IR, and NMR spectrometries were employed. 3.1 Materials 3.1.1 S e l e c t i o n of s t a r t i n g material Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco,) sawdust served as s t a r t i n g material for t h i s study to i s o l a t e and characterize the l i g n i n s from organosolv cooking. Douglas-fir sawdust comprises a a s u b s t a n t i a l portion of the raw material supply for many pulp m i l l s i n 34 Douglas-fir sawdust 1) Cook at 200° C. 2) F i l t e r 3) Wash with acetone F i l t r a t e s + Washings Residue -Evaporate acetone (Fiber) Aqueous s o l u t i o n S o l i d p r e c i p i t a t e s Residual l i g n i n (FRACTION I) 1) Dilute with water 2) F i l t e r / c e n t r i f u g e 3) L i q u i d - l i q u i d ex-t r a c t i o n 1) Re-dissolve i n acetone 2) P r e c i p i t a t e from water 3) F i l t e r 4) Wash with water 5 ) Dry Water-insoluble l i g n i n (FRACTION II) Aqueous layer Organic layer Sugars,etc. -Evaporate organic solvent Water-soluble l i g n i n (FRACTION III) Figure 7. Frac t i o n a t i o n of l i g n i n from organosolv cooking. 3 5 i n the P a c i f i c Northwest (34) and i s r e a d i l y available i n large quanti-t i e s . The fresh sawdust was obtained from the production l i n e of L & K Lumber, Ltd., North Vancouver, B r i t i s h Columbia. The Douglas-fir trees ware about 80 years old and originated from the P a c i f i c coastal region. The p a r t i c l e size of the sawdust (sp. gr.=0.42) selected for t h i s study covered a wide range. The r e s u l t s of a sieve analysis on p a r t i c l e s i z e d i s t r i b u t i o n of the a i r - d r y Douglas-fir sawdust sam-ple are shown i n Table 1. Table 1. Sieve analysis of Douglas-fir sawdust. Sieve size(mesh) <10 10-20 20-40 40-60 60-80 >80 Total Frequency(%) 30,9 36. 7 21.9 6.3 1.6 2. 6 100 The f r a c t i o n accepted for t h i s study was that passed through a 10-mesh sieve and retained on a 40-mesh sieve, which constituted about 60% (based on a i r - d r y weight) of the f r e s h sawdust c o l l e c t e d . 3.1.2 Preparation of e x t r a c t i v e - f r e e sawdust samples In order to obtain e x t r a c t i v e - f r e e sawdust as cooking material, the a i r - d r y sawdust sample was extracted with a mixture of 95% ethanol and benzene (1:2 by volume) i n a large Soxhlet extractor for 8 hours followed by extraction with 95% ethanol for 40 hours (a modified procedure of TAPPI Standard T12m-59). The content of alcohol-benzene extractives was found 36 to be 3,9%, of oven-dry un-extracted sawdust. After proper washing with ether and a i r - d r y i n g , the extrac-t i v e - f r e e sawdust sample was stored i n the CTH room (21°C/50%'RH) before moisture content was determined. The moisture content of the ex t r a c t i v e -free sawdust was 9.44%. Analysis of chemical composition of the extrac-ted sawdust was also c a r r i e d out, and the r e s u l t s are given i n Table 2. 3.1.3 Preparation of organosolv l i g n i n samples A 5 g ("oven-dry. basis) portion of the e x t r a c t i v e - f r e e saw-dust sample was placed i n a 65 ml capacity s t a i n l e s s s t e e l bomb-diges-ter along with 50 g of cooking l i q u o r (wood/liquor ratio=l:10). The cooking l i q u o r consisted of acetone and water (60:40 by volume), with various concentrations of hydrochloric acid as the c a t a l y s t . Cooking was carried out i n a glycerine-bath equipped with Universal Relay (Type R-10), PTR-Electronic Controller (Type R-20/2) and P-120 E l e c t r o n i c Programmer, for the desired periods of time at 200°C. Each cook was duplicated to obtain r e p l i c a t e y i e l d s of pulp and l i g n i n f r a c t i o n s . After cooking, the undissolved l i g n o c e l l u l o s i c residue was separated from the spent l i q u o r by vacuum f i l t r a t i o n and washed with acetone (ca. 100 ml). The residue was slushed with acetone (ca. 300 ml) i n a blender at a low speed for further d i s i n t e g r a t i o n to remove the trapped l i g n i n . The s l u r r y was f i l t e r e d and washed with fresh acetone (ca. 200 ml). The r e s i d u a l f i b e r s were then dried i n an oven at 105+3°C and pulp y i e l d and the r e s i d u a l l i g n i n (FRACTION I ) were determined. 37 The combined s o l u t i o n of f i l t r a t e s and washings was evaporated on a f l a s h evaporator at 50°C to obtain dark brown mass (a quasi-molten •phase) of crude l i g n i n and a cl e a r yellowish aqueous s o l u t i o n which contained sugars and water-soluble l i g n i n (FRACTION I I I ) . The dark brown l i g n i n mass was redissolved i n a minimum amount of acetone (ca. 20 ml) and pre c i p i t a t e d into an excess amount of d i s t i l l e d water (1,000 ml) with vigorous s t i r r i n g . The water-in-soluble l i g n i n p r e c i p i t a t e s were c o l l e c t e d by vacuum f i l t r a t i o n and washed thoroughly with warm (40-45°C) water. This powdered water-insoluble organosolv l i g n i n (FRACTION II) was dried over phosphoric anhydride i n a desiccator placed i n an oven at 50°C. 3.1.4 Preparation of acetylated l i g n i n samples Acetylation of the is o l a t e d organosolv l i g n i n samples was done by the method used by DeStevens and Nord (48). The l i g n i n sample (0.4 g) was dissolved i n pyridine (6 ml), and acetic anhydride (5 ml) was added to the s o l u t i o n with s t i r r i n g . The mixture was then allowed to stand for 48 hours at room temperature and centrifuged at a rotor-speed of 12,000 rpm for 15 minutes. The cle a r s o l u t i o n portion was separated from the fine p r e c i p i t a t e s and poured into ice-water (200 g) to p r e c i p i t a t e the acetylatedr l i g n i n . The p r e c i p i t a t e s were c o l l e c t e d by vacuum f i l t r a t i o n through a M i l l i -pore f i l t e r (pore size:0.2 um) and washed with 0.1 N hydrochloric acid (100 ml) to neut r a l i z e any remaining pyridine. The acetylated l i g n i n was then washed with d i s t i l l e d water several times u n t i l the f i l t r a t e was no more a c i d i c and dried over phosphoric anhydride at 50°C as men-tioned above. 38 3.1.5 Preparation of reduced l i g n i n samples Borohydride-reduced l i g n i n samples were prepared by a modi-f i e d method of the procedures adapted by Alder ejt al. (4) and Gierer e_t a l . (72). The i s o l a t e d l i g n i n (0.12 g) was dissolved i n a mixture of ethanol (8 ml) and 0.1 N sodium hydroxide (2 ml) under nitrogen atmos-phere. Sodium borohydride (0.04 g) and a d d i t i o n a l water (4 ml) were added to the mixture with s t i r r i n g . The reaction mixture was allowed to stand overnight and a c i d i f i e d to pH 2 with d i l u t e hydrochloric acid. The p r e c i p i t a t e s formed were co l l e c t e d by centrifuging and washed with water several times. The reduced l i g n i n was then dried over phosphoric anhydride at 50°C. 3.2 Methods 3.2.1 Analysis of l i g n i n f r a c t i o n s 3.2.1.1 Klason l i g n i n To determine acid-insoluble Klason l i g n i n content of the e x t r a c t i v e - f r e e sawdust, the procedure described i n TAPPI Standards . T13 os-54 was followed. A modified secondary hydrolysis with 3% s u l -f u r i c acid was used by t r e a t i n g the reaction mixture i n an autoclave under pressure of 20 psig steam pressure and 127.5°C f o r 1 hour. The insoluble residue (Klason l i g n i n ) was c o l l e c t e d by vacuum f i l t r a t i o n on a medium porosity glass c r u c i b l e , dried at 105 +3°C and weighed. The f i l t r a t e from the f i l t r a t i o n was saved for the 39 determination of acid-soluble l i g n i n content. 3.2.1.2 Acid-soluble l i g n i n The acid-soluble l i g n i n was determined according to TAPPI Useful Method 250. The acid solution, which contained the acid-soluble l i g n i n , was obtained from the Klason l i g n i n determination. The maximum UV absorbance of the acid s o l u t i o n was measured around 205 nm and used for c a l c u l a t i o n of acid-soluble l i g n i n content by using the following equation (TAPPI UM 250). A Unicam SP 800 Spec-trophotometer was used to obtain the UV spectrum. T . . „ B x V x 100 Lignin, /o = —• 1000 x W where V = t o t a l volume of s o l u t i o n (ml) W = oven-dry weight of wood (g) B = l i g n i n content (g/lOOOml) and B can be calculated by: B - A X D 110 where A = UV absobance D = d i l u t i o n factor 3.2.1.3 Residual l i g n i n The r e s i d u a l l i g n i n content (FRACTION I) i n the f i b e r residue was determined by the micro Kappa number method described i n TAPPI Useful 40 Method 246. Sample preparation was done according to TAPPI Stan-dards T 236 m-76. The r e s i d u a l l i g n i n i n the pulp was computed from the following equation (32): Residual l i g n i n , % = Kappa number x 0.15 3.2.1.4 Water-soluble l i g n i n and degradation products The aqueous portion, following evaporation of the organic solvent of the cooking liquor, was separated and d i l u t e d to 100 ml with d i s t i l l e d water. In order to separate the water-soluble l i g n i n from the dissolved sugars, the yellowish aqueous sol u t i o n was extracted with chloroform i n a s p e c i a l l y designed l i q u i d - l i q u i d extractor. The organic layer from the e x t r a c t i o n was concentrated to about 10 ml on a rotary evaporator at 50 + 5°C and a t h i n - l a y e r chromatography (TLC) sample was taken from the concentrated s o l u t i o n at t h i s stage. The evaporation of the organic layer was continued u n t i l a highly v i s -cous syrup-like residue (FRACTION III) was obtained. A small portion (0.1-0.5 g) of the water-soluble l i g n i n (FRACTION I I I ) , which was not r e a d i l y soluble i n neutral water or met-hanol, was re-dissolved i n acetone (15 ml) and d i l u t e d to 50 ml with methanol. The c l e a r s o l u t i o n was concentrated on a rotary evaporator at a low temperature (40°C) to remove the acetone. To remove any re-maining acetone, a large amount (caP 50 ml) df methanol was -added to the sample and concentrated again.. This procedure was repeated several times u n t i l no detectable acetone by UV absorption remained. The acetone-free methanol sol u t i o n was then d i l u t e d to the desired concentration with methanol and UV spectrum was taken on a Uni-41 cam SP 800 Spectrophotometer. The c a l c u l a t i o n for the water-soluble l i g n i n content was e s s e n t i a l l y the same as that suggested by TAPPI Useful Method 250, but methanol was used as reference instead of 3% s u l f u r i c acid. Q u a l i t a t i v e TLC analysis was conducted by the methods described by K r a t z l and Paszner (100) and Barton (13) with minor modifications, using TLC glass plates (20 cm x 20 cm) coated with 0.25 mm thick S i l i c a Gel G. Benzene-acetone (60:40), methanol-chloroform (30:70), benzene-ethanM (150:22) and benzene-chloroform-methanol (70:28:2) were used as developing systems. The plates were f i r s t examined under UV l i g h t and the spots were i d e n t i f i e d by t h e i r R^ values and colors developed upon spraying with the various reagents. The spraying reagents used were Folin-Denis reagent (100) and diazotized s u l p h a n i l i c acid (13). To detect the presence of carbonyl groups and Hibbert's ketones, 2,4-dinitophenyl hydrazine (80) and f e r r i c chloride-potassium f e r -ricyanide reagents (70) were also used. 3.2.2 Chemical analyses of i s o l a t e d organosolv l i g n i n s Some equipment, such as elemental analyzer, was not d i r e c -t l y accessible f or the present study. Due to the limited funds a v a i l -able for r e n t a l , a minimum number of acetylated or reduced organosolv l i g n i n samples, representing series of two cooking variables (cooking time and acid c a t a l y s t concentration), were selected for the extensive chemical c h a r a c t e r i z a t i o n of the is o l a t e d organosolv l i g n i n s . For cooking time series, selected samples PC-11 (5 min), 42 PC-12 (9 min), PC-13 (12 min), PC-14 (17 min) and PC-15 (20 min), a l l prepared with the same concentration of aeid c a t a l y s t (0.05 N H G 1 ) , -and for acid concentration series, PC-9 (0.025 N HC1), PC-14 (0.05 N HC1) and PC-19 (0.1 N HC1), a l l of the same cooking period (17 min), were selected for elemental and s p e c t r a l analyses. These selected samples were part of the complete experi-mental scheme for both cooking time and acid concentration series, which w i l l be presented l a t e r (Table 3) i n connection with discussion of the e f f e c t of various cooking conditions on l i g n i n y i e l d . 3.2.2.1 Elemental analysis and methoxyl content determination The elementary composition of the acetylated l i g n i n was determined by standard methods of organic combustion analysis for per cent carbon and hydrogen contents based on the freeze-dry l i g n i n samples. A small amount(0.7 mg) of the freeze-dried acetylated l i g -n i n sample was weighed i n a t i n container loaded into the sample hol-der and injected i n t o a combustion reactor at 1,010°C. The combus-t i o n gases were carried by a constant flow of helium through to the c a t a l y t i c section of the reactor for complete oxidation to CO^, H^ O, N„ and N 0 . The gas mixture flowed into a second reactor kept at 2 x y 644°C which was f i l l e d with copper for reduction of the nitrogen oxides. The gas mixture was then directed into a chromatographic column for N^, CO^, H^ O separation. The gas components were q u a n t i t a t i v e l y ana-lyzed by a thermal conductivity detector. The machine used was an 43 Elemental Analyzer-Model 1106 equipped with Model CSI 38-Digital Integrator. For the determination of the methoxyl content of the i s o l a t e d l i g n i n and e x t r a c t i v e - f r e e sawdust samples, TAPPI Standards T209 su-72 was followed with a few modifications. The t e s t specimens (0.1 g for l i g n i n ; 0.3 g for sawdust) were reacted with 56.6% hydroiodic a c i d (6 ml) and propionic acid (2 ml) at 150°C for 40 minutes. The resultant methyl iodide was removed from the reaction f l a s k by a current of nitrogen and oxidized i n an a c i d i c s o l u t i o n of potassium acetate containing bromine to give i o d i c a c i d . The i o d i c a c i d was determined by t i t r a t i o n with 0.1 N sodium t h i o s u l f a t e . The methoxyl content was then calculated by the following equation (TAPPI T 209 su-72). v i v 0.0517 (A - B) Methoxyl, % = — w where, A = volume of 0.1 N N a2^2^3 so-'-ut:'-on required for specimen (ml) B = volume of 0.1 N Na^S^O^ so l u t i o n required for blank (ml) W = moisture-free weight of the specimen (g). 3.2.2.2 U l t r a v i o l e t spectra The UV spectra for the i s o l a t e d l i g n i n and the reduced l i g n i n samples were recorded with a Unicam SP 800 Spectrophotomer. The 4 4 procedure selected for the determination of the phenolic hydroxyl group was the method of Goldschmid (77). The i s o l a t e d l i g n i n sample (0.2 g) was dissolved with gentle heating (50-60°C) i n pH 12 buffer s o l u t i o n (100 ml) which was made of 6.2 g of boric acid i n 1,000 ml of 0.1 N sodium hydroxide. A portion of t h i s l i g n i n s o l u t i o n (2 ml) was d i l u t e d to 50 ml with pH 12 buffer s o l u t i o n ( a l k a l i n e s o l u t i o n ) , and another portion (2 ml) was neutralized with 0.1 N s u l f u r i c acid (2 ml) and d i l u t e d to 50 ml with pH 6 buffer s o l u t i o n (neutralized s o l u t i o n ) . The d i f f e r e n t i a l spectrum was determined by measuring the absorbance of the a l k a l i n e s o l u t i o n r e l a t i v e to that of the neutralized s o l u t i o n which was placed i n the reference c e l l of the spectrophotometer as the blank. Phenolic hydroxyl groups were estimated by using the following equation (77). Phenolic hydroxyl, % = A x 17/41 a, max where A = a b s o r p t i v i t y d i f f e r e n c e at maximum peak a,max (1/g.cm). 3.2.2.3 Infrared spectra IR spectra of the borohydride-reduced l i g n i n samples were obtained with a Perkin-Elmer 521 Spectrophotometer with. an. extended 45 range interchange which can eliminate the environmental problems, such as s e n s i t i v i t y to moisture and temperature. The frequency range was 4,000 cm ^ to 250 cm \ with accuracy of + 0.5 cm ^ and r e p r o d u c i b i l i t y of 0.25 cm \ The procedure followed was s i m i l a r to the method adapted by Naveau (121). The KBr p e l l e t s were made by mixing the reduced l i g n i n (4 mg) and potassium bromide (200 mg) and pressing under a pressure of 12,000 p s i into a 1 cm diameter p e l l e t . 3.2.2.4 Nuclear magnetic resonance spectra NMR spectra of the acetylated l i g n i n samples were obtained on a Varian EM-390 90 mHz NMR Spectrometer. The acetylated l i g n i n (ca. 10 mg) was dissolved i n deutero-chloroform (300 jul ) and f i l t e r e d through glass wool into a 5 mm thin-wall sample tube. Tetramethylsilane (TMS) was added as an i n t e r n a l reference standard. Sweep width was 10 ppm and sweep time was 2 minutes. Spectrum amplitude varied from 5000 to 6000. The integ-r a t i o n of the spectrum was "recorded to obtain the r e l a t i v e peak areas. 3 .2.3 Macromolecular analyses of i s o l a t e d organosolv l i g n i n s 3 . 2.3 . 2 Gel permeation chromatography GPC r e s u l t s were obtained on a high-speed GPC, Water Associates Model ALC/GPC-201 equipped with a d i f f e r e n t i a l r e f r a c t i v e index detector. . 46 The acetylated l i g n i n sample (25 mg) was dissolved i n tetrahydrofuran (5 ml) to make about 0.5% s o l u t i o n . To minimize the p o s s i b i l i t y of viscous f i n g e r i n g , the s o l u t i o n was f i l t e r e d through two M i l l i p o r e f i l t e r s (pore s i z e : 0.45 Jll m). The i n j e c t i o n of the sample (250 jul ) was done with the aid of a Model U6K u n i v e r s a l i n j e c t o r . The separation of f r a c t i o n s with d i f f e r e n t molecular weights was accomplished through a seri e s of four columns packed with d i f f e r e n t sizes of highly porous gel p a r t i c l e s (^t-Styragel). The columns used were 10 A , 10 A , 500A and 100A for molecular weights of 10,000-200,000, 1,000-20,000, 50-10,000 and 0-700, re s p e c t i v e l y . The pressure of the flowing solvent system was 1,000 p s i and the flow rate was 1 ml per minute. The d i f f e r e n t i a l refractometer detected a change i n r e f r a c t i v e index as small as 10 7 Rl units which corresponds to a concentration change of 1 ppm of l i g n i n sample. An X-Y recorder converted the d i f f e r e n t i a l refractometer s i g n a l to a continuous trace on the chart. The time required for a complete run was about 50 mi-nutes. To construct a c a l i b r a t i o n curve, the detector count number was p l o t t e d on the X-axis against the corresponding value of a known molecular weight on the logarithmic Y-axis on semi-log paper (Fig. 8). To c a l c u l a t e the weight average molecular weight (Mw) and the number average molecular weight (Mn), peak height of each count number was measured and the p o l y d i s p e r s i t y was expressed by the r a t i o of Mw/Mn. ,r Mw and Mn were calculated by the following equations ,(8). ; 47 10 I I I : ! 1 I : : : I i I ! ! I : ; : i I ! I .^ 4+++: I: : i. 1 R - ^ 4 f - U - + - -72 78 84 90 96 102 108 114 120 126 Counter•Number Figure 8. GPC c a l i b r a t i o n curve. 48 Mn= Z w /EHh where, H = height of peak of each count number M = molecular weight converted from count number (from c a l i b r a t i o n curve, F i g . 8) Two selected sample series, PC-11, 12, 13, 14 and 15 for cooking time and PC-9, 14 and 19 for acid concentration s e r i e s , were investigated for GPC analysis of the iso l a t e d organosolv l i g n i n s . 3.2.3.2 Scanning electron microscopy An ETEC Autoscan SEM was used to investigate the nature of the p a r t i c l e s of the acetylated l i g n i n samples. The primary beam voltage applied was 20 KV. The SEM specimen was prepared by d i s s o l v i n g a small amount (0.01 g) of the acetylated l i g n i n sample i n acetone (1 ml) and the sol u t i o n was added drop by drop to d i s t i l l e d water (ca. 8 ml) with vigorous s t i r r i n g and d i l u t e d to 10 ml with d i s t i l l e d water. One drop of the suspension was put on a M i l l i p o r e f i l t e r and a i r - d r i e d . In order to prevent e l e c t r o s t a t i c charging, a t h i n gold f i l m was deposited on the surface of the specimen. The specimens thus prepared were observed by SEM and photographed with a Polaroid 545 Camera at 20,000 magnification. The samples used for the SEM i n v e s t i g a t i o n were exactly the same as those used i n the GPC analysis. 49 4. RESULTS The chemical composition of the e x t r a c t i v e - f r e e Douglas-f i r sawdust i s presented i n Table 2. The contents of »<-cellulose, hemicellulose, Klason l i g n i n and acid-soluble l i g n i n were not cor-rected f o r ash content which i s about 0.2% for Douglas-fir wood (141). Table 2 also includes methoxyl content of the e x t r a c t i v e - f r e e sawdust. The y i e l d s of pulp and l i g n i n f r a c t i o n s are tabulated i n Table 3 . The y i e l d s of pulp and the i s o l a t e d organosolv l i g n i n (FRAC-TION II) are further plotted against cooking time and acid concentra-t i o n i n Figs. 9 and 11, respectively. F i g . 10 shows temperature r i s e i n s i d e the bomb-digester, which was measured by copper-constantan thermocouple, during a prolonged period ( 3 0 min) of cooking. The re-s u l t s of preliminary cooking experiments with various wood species are presented i n Table 4. Changes i n pH value of the cooking li q u o r s with various concentrations of acid c a t a l y s t , before and a f t e r cooking for 20 minutes, are compared i n Table 5. Table 6 indicates c h a r a c t e r i s t i c TLC colors and R^ values of the known degradation compounds and phenolic model substances which are most l i k e l y present i n the spent cooking liquor. TLC r e s u l t s , ob-tained from PC-15 (0.05 N HC1;20 min. cook) a f t e r removing the sugars from the spent liquor, are presented i n Table 7. Table 8 presents the elementary compositions of some se-l e c t e d acetylated l i g n i n samples representing several cooking time and acid concentration series. Methoxyl contents of the selected acetylated l i g n i n samples and t h e i r parent l i g n i n s are also given i n the same table. 50 F i g . 12 demonstrates a t y p i c a l NMR spectum of the a c e t y l - . ated organosolv l i g n i n from e x t r a c t i v e - f r e e Douglas-fir sawdust with d e s c r i p t i o n of each region of the spectrum. Assignments of the s i g n a l regions are l i s t e d i n Table 9. Figs. 13-19 reproduce the NMR spectra of the selectedr acetylated l i g n i n samples. Comparison of these NMR spectra i s i l l u s t r a t e d i n Fig. 20. From each NMR spectrum, the r e l a -t i v e i n t e n s i t i e s of various proton types were obtained and the r e s u l t s are computed i n Table 10. From these values, contents of the various fu n c t i o n a l groups i n l i g n i n molecules were estimated and the r e s u l t s are expressed as the number of fu n c t i o n a l groups per C^-unit of l i g n i n molecule i n Table 11. E f f e c t s of cooking time and acid concentration on the IR spectra of the selected reduced organosolv l i g n i n samples are compared i n Figs. 21 and 22, respectively. Assignments of absorption bands i n the IR spectra are tabulated i n Table 12. Fig. 23 presents the difference curve of UV absorption of the selected reduced l i g n i n samples. From maximum peaks of the d i f -ference curve, the molar a b s o r p t i v i t y was measured for each sample and the r e s u l t s along with the calculated phenolic contents are given i n Table 13. The values of Mw and Mn as well as the p o l y d i s p e r s i t y i n -dices obtained from GPC analysis are presented i n Table 14. E f f e c t of cooking conditions on Mw and Mn of the selected, acetylated l i g n i n samples are summarized i n four diagrams i n F i g . 24. Comparative mole-cular weight d i s t r i b u t i o n s , as affected by cooking time and acid cata-l y s t concentration,are i l l u s t r a t e d i n Figs. 25 and 26, respectively. 51 Scanning electron photomicrographs depicting p a r t i c l e size v a r i a t i o n of the uniformly p r e c i p i t a t e d l i g n i n s as affected by cooking time and acid concentration are shown i n Figs. 27 and 28, respectively. Changes i n p a r t i c l e s i z e due to varying cooking time and concentration of acid c a t a l y s t are shown i n Table 15 by tabulating p a r t i c l e size f r e -o quency d i s t r i b u t i o n s of the acetylated l i g n i n samples. From the data, p a r t i c l e s i z e d i s t r i b u t i o n diagrams for 'the cooking time and acid con-centration series were constructed and are presented i n Figs. 29 and 30, respectively. 52 5. DISCUSSION In the present study, the main objectives were to i s o l a t e the p r e c i p i t a b l e organosolv l i g n i n from Douglas-fir sawdust and to characterize i t s chemical and macromolecular properties. For t h i s reason, only b r i e f treatment was given to other f r a c t i o n s , such as water-soluble l i g n i n or degradation products of l i g n i n , without mak-ing a serious e f f o r t i n completing the picture. No analysis was conducted to investigate the dissolved carbohydrates (hemicelluloses and glucose). Such sugar ana-l y s i s was^ carried out e a r l i e r and the r e s u l t s were published (36, 37), and thus was not considered as part of the present study. 5.1 Chemical Composition of E x t r a c t i v e - f r e e Douglas-fir Sawdust As shown i n Table 2, the average l i g n i n content of the e x t r a c t i v e - f r e e Douglas-fir sawdust was found to be 31.81 7», based on the oven-dry e x t r a c t i v e - f r e e sawdust, or 30.66 %, based on the oven-dry unextracted sawdust. While most of the recorded l i g n i n contents of Douglas-fir l i e between 27-29 %, a substantial v a r i a -t i o n (24.5-33.5 7») i n the content has also been reported (148). Such a v a r i a t i o n e x i s t s not only between members of a single spe-c i e s grown under d i f f e r e n t environmental conditions or from d i f -ferent seed source, but also within incremental growth zones of a single tree (170). Since the sawdust used i n the present study was obtained from an i n d u s t r i a l lumber production l i n e , i t was not 53 Table 2. Chemical composition of e x t r a c t i v e - f r e e Douglas-fir sawdust. Wood component Amount ( % ) a Test method used Holocellulose 68.19 b (65. 74) C Acid c h l o r i t e method o<-cellulose 43.04(41.49) TAPPI T203 os-61 Hemicellulose 25.15(24.25) Li g n i n 31.61 (30.66) Klason l i g n i n 31.50(30.37) TAPPI T13 os-54 Acid-soluble l i g n i n 0.31(0.29) TAPPI UM 250 Weight l o s s ( E x t r a c t i v e s ) - (3.60) Tot a l 100(100) Methoxyl content 5.19(5.00) TAPPI T209 su-72 Not corrected f o r ash content. 'Percentage values based on oven-dry e x t r a c t i v e - f r e e sawdust. Percentage values in-parentheses based on oven-dry unextracted sawdust. 54 possible to trace the natural o r i g i n of the r e l a t i v e l y high l i g n i n con-tent. Klason l i g n i n , which was 31.50 % of the e x t r a c t i v e - f r e e oven-dry sawdust, accounted for 99.06 % of t o t a l l i g n i n , whereas the acid-soluble l i g n i n f r a c t i o n was less than 1 % of the t o t a l l i g n i n content. The contents of <<-cellulose and hemicellulose of the extrac-t i v e - f r e e sawdust were found to be 43.04 % and 25.15 %, re s p e c t i v e l y (Table 2). The c e l l u l o s e content of softwoods was reported to be be-tween 41-45 % (141, 160) and i t i s also well known that higher or low-er values indicate the presence of reaction wood (160). Kennedy and Jaworsky (92) reported that no f u l l y s a t i s f a c t o r y explanation for a v a r i a t i o n i n c e l l u l o s e content of Douglas-fir (70 to 85 years old) could be found i n spite of thorough analyses on crown cl a s s , s i t e , ra-d i a l p o s i t i o n , growth rate and per cent summerwood. However, i t was suggested that most of the v a r i a t i o n i n c e l l u l o s e content could be at-tributed to inherent genetic c h a r a c t e r i s t i c s of i n d i v i d u a l trees. Con-sid e r i n g the fact that the c o l l e c t e d sawdust was a mixture of a l l pos-s i b l e cases, the r e s u l t of <<-cellulose content (43.04 % ) , which i s very close to the reported average value, i s quite normal. The weight loss of o r i g i n a l sawdust due to alcohol-benzene extraction, which can be considered to be the content of extraneous substances extracted was found to be 3. 60 7» of the unextracted sawdust. This value i s s l i g h t l y lower than the recorded value of 4.4 7» (141). The reason f o r t h i s may be explained by the fact that extractive depo-s i t s i n s i d e lumens can not be completely extracted even with prolonged alcohol-benzene e x t r a c t i o n (91). 55 The methoxyl content of the e x t r a c t i v e - f r e e sawdust was found to be 5.0%, which i s well within the reported range of 4.97-5.67%, for Douglas-fir wood (148,170). 5.2 I s o l a t i o n of Organosolv Lignins As mentioned e a r l i e r , no method by which p r o t o l i g n i n can be i s o l a t e d i n an unchanged state has yet.been developed. Isolated l i g -nins from organosolv pulping or s a c c h a r i f i c a t i o n processes seem to be the only l i g n i n s which can be generated on a large scale, i n a less-con-densed state and free of organic or inorganic impurities (154). It i s known that i s o l a t i o n of l i g n i n from wood by acid-catalyzed organosolv cooking i s e s s e n t i a l l y an acid hydrolysis of polymeric l i g n i n molecules and of the lignin-carbohydrate matrix (97, 121,169). The a c i d i f i e d organosolv cooking system i s far more e f f i -c ient i n d e l i g n i f i c a t i o n and sugar hydrolysis than aqueous acid hydrolysis due to superior penetration power of the organic solvent and simultaneous d i s s o l u t i o n of a l l hydrolysed products, including l i g n i n . In order to find the optimum composition of the aqueous organic cooking liquor, not only the y i e l d of l i g n i n s , but also so-l u b i l i t y of the i s o l a t e d l i g n i n s i n various solvent systems were com-pared. The optimum composition of the cooking l i q u o r was found to be an acetone-water system with a r a t i o of 3:2 by volume. Acetone was chosen as the organic component:of the aqueous organic solvent system because of i t s excellent solvent power for the polymeric l i g n i n fragments. It i s reported that the a b i l i t y of s o l -vents to dissolve an a c i d o l y t i c a l l y attacked l i g n i n macromolecule or lignin-carbohydrate complex increases as t h e i r s o l u b i l i t y parameter 56 ( 6 ) approaches the value of around 11 (153). Acetone has a value of ^"=10, i . e . i t i s very close to the optimum value for l i g n i n s o l u b i l i -ty. For the simultaneous removal of liberated l i g n i n f r a c t i o n s during acid hydrolysis, a solvent having such high s o l u b i l i t y parameter must be chosen (97,129,153). Aft e r cooking, there are several choices available for i s o -l a t i o n of the dissolved l i g n i n s from organosolv spent cooking liquor. While perhaps d i r e c t p r e c i p i t a t i o n of the spent l i q u o r i n t o an excess (8 to 15 volumes) of d i s t i l l e d water i s the easiest way, the recovered l i g n i n f r a c t i o n s a f t e r such a procedure do not provide good y i e l d s for l i g n i n mass balance because of the large amounts l o s t due to p a r t i a l d i s s o l u t i o n of low molecular weight l i g n i n f r a c t i o n s i n water. Quantitative recovery of the water-insoluble f r a c t i o n of l i g -nin i s best accomplished by evaporation of the organic solvent from the spent l i q u o r on a f l a s h evaporator at low temperature. This obtains a mass of crude l i g n i n and s l i g h t l y yellowish c l e a r aqueous s o l u t i o n which contains dissolved sugars and the water-soluble l i g n i n f r a c t i o n . The concentration of dissolved sugars i n the aqueous s o l u t i o n did not exceed 21% even from the prolonged cooks (20 min), and i t was noted that a l l the dissolved sugars were present as monomers i n the spent l i -quor (37). Water-soluble l i g n i n (FRACTION III) w i l l be discussed l a t e r i n connection with TLC analysis. A f t e r removing the yellowish aqueous solution, the mass (a quasi-molten phase) of crude l i g n i n was redissolved i n a minimum amount of acetone to r e p r e c i p i t a t e i t i n a large excess of d i s t i l l e d water. "Yield of the i s o l a t e d water-insoluble organosolv l i g n i n s (FRACTION II) varies between 8.65-31.15% (based on oven-dry e x t r a c t i v e - f r e e sawdust), depend-57 ing on the cooking conditions as shown i n Table 3. More detai l e d d i s -cussion on y i e l d and purity as well as methoxyl content of the i s o l a t e d l i g n i n s w i l l be presented i n the following-sections. There are several methods av a i l a b l e to p u r i f y the p r e c i p i -tated organosolv l i g n i n . However, no p u r i f i c a t i o n was c a r r i e d out be-cause such methods involve d i s s o l u t i o n of the crude l i g n i n i n c e r t a i n organic solvents for r e p r e c i p i t a t i o n . This may a f f e c t the structure of l i g n i n molecules due to solvent e f f e c t s . Most of the tests for the pre-sent study were conducted on e i t h e r acetylated or reduced organosolv l i g n i n s which can be considered to be p u r i f i e d (freed from sugars) dur-ing the reactions and work-up processes.. There i s a p o s s i b i l i t y that the crude l i g n i n s from the spent l i q u o r might have been i s o l a t e d together with hemicellulose f r a c t i o n s . The sugars could have been covalently bonded to l i g n i n molecules or trap-ped i n the tridimensional l i g n i n matrix (26,97,98,141). This possi-b i l i t y w i l l be discussed l a t e r i n conjunction with microanalysis of the i s o l a t e d organosolv l i g n i n s . From a preliminary experiment on moisture hysteresis of the i s o l a t e d organosolv l i g n i n samples, i t was learned that they picked up about 12-16% moisture when the samples were exposed to saturated a i r con-d i t i o n i n an Amineo cabinet (31°C/96'98% RH). At GTH room condition (21° C/50% RH), the moisture content of these l i g n i n samples was found to be 4. 43-4. 81%, which'-is about h a l f of that of Douglas-fir sawdust (9.44%) under 1'identical conditions. In order to prevent any p o t e n t i a l moisture e f f e c t , the-isolated organosolv l i g n i n samples were kept dry i n a phos-phoric anhydride desiccator at 50°C. Underi such drying conditions, less than 1% moisture content was obtained within 12 hours. 58 5.3. E f f e c t of Cooking Conditions on Yield s of Fiber Residue and L i g n i n Fractions Among the factors a f f e c t i n g the out-come of organosolv cooking, only cooking time and concentration of acid c a t a l y s t were chosen as cooking var i a b l e s . Other important cooking conditions, such as temperature, cooking l i q u o r composition and wood/liquor r a t i o , were kept constant because optimum conditions for these va-r i a b l e s were worked out i n previous studies (36,37,129) under simi-l a r or i d e n t i c a l conditions as used i n the present study. I t was found that the cooking "temperature had a profound e f f e c t on the rate of hydrolysis of Douglas-fir sawdust. -For example, the f i b e r residue y i e l d a f t e r 20-min cooking at 160°C was 63. 787o, whereas the values obtained at 180° and 200°C a f t e r the same period of cooking were markedly reduced to 41.88% and 6.50%, r e s p e c t i v e l y (37). In order to obtain high l i g n i n y i e l d s , 200°C was chosen as constant cooking temperature for this study. The e f f e c t s of temperature and concentration of acid c a t a l y s t on the rate of hydrolysis were found to be interchangable to some extent, i.e. a low acid concentration can be o f f s e t by r a i s i n g the cooking temperature and vice versa (37). Acetone/water r a t i o of the organosolv cooking l i q u o r sys-tem and wood/liquor r a t i o were 3:2 by volume and 1:10 by weight, res-pectively, as mentioned before. E a r l i e r , a s i g n i f i c a n t e f f e c t of p a r t i c l e size of Douglas-f i r sawdust on pulp y i e l d and Kappa number was reported (34). To e l i -minate the extremely fin e andicoarse . p a r t i c l e s , only'-+the 10-40 mesh f r a c -t i o n of the sawdust c o l l e c t e d was selected as cooking material. Since moisture content i s one of the most important factors 59 a f f e c t i n g the rate of liqu o r penetration (37), the e x t r a c t i v e - f r e e sawdust samples were kept i n the CTH room to maintain a constant moisture content (9.44%) before cooking. 5.3.1 E f f e c t of cooking time As Tables 3 and 4 show, cooking time seems to be the most -significant single parameter i n regulating the r e s u l t s of the cooks. In general, longer cooking time gives lower f i b e r y i e l d and higher re-covery of p r e c i p i t a b l e l i g n i n (Fig.9). In a preliminary cooking experiment (39), under s i m i l a r cooking conditions as used i n the present study, i t was found that the f i b e r y i e l d for long cooks (20 min) was only one-quarter to one-third of that for short cooks (7 min). On the other hand, l i g n i n recovery from long cooks was about 2 to 5 times of that from short cooks. These trends were found to hold for a l l wood species studied as i l l u s t r a t e d i n Table 4. In Table 3, i t can be seen that the t o t a l l i g n i n content accounted for i n cook nos. 15, 18, 19 and 20 were higher than the po-t e n t i a l l i g n i n content (31.81%). It i s believed that substantial amo-unts of hemicelluloses removed from wood were i s o l a t e d together with the l i g n i n f r a c t i o n (26). Hydrolysis, which aids the d i s s o l u t i o n of l i g n i n into the cooking liquor, also occurs at aryl-glycoside bonds of l i g n i n - s a c c h a r i d i c complex during the acid-catalyzed cooking (98,141). In e a r l i e r studies (36,37), i t was found that about one-t h i r d of the l i g n i n and a large f r a c t i o n (71%,) of hemicelluloses were dissolved i n the f i r s t 5 minutes during the hydrolysis of Douglas-fir Table 3. Ef f e c t of cooking conditions on y i e l d s of f i b e r residue and l i g n i n f r a c t i o n s from extractive-free Douglas-fir sawdust. Cook Cooking Acid(HCl) Yield of Yie l d of Yields of l i g nin fractions ( 7 o ; a No. time concent- f i b e r re- l i g n i n - Residual Isolated Water-soluble Total - . (min) r a t i o n ^ ) s i d u e ^ ) free pulp l i g n i n , l i g n i n , l i g n i n , l i g n i n FRACT'N I FRACT'N II FRACT'N I I I accounted 1 5 _ _ _ _ _ -2 9 - 92.06 - - - - -3 12 - 90. 16 - - - - -4 17 - 86.09 - - - - -5 20 - 83.14 - - - - -6 5 0.025 69.03 50.99 18. 04 8. 65 1. 20 27. 89 7 9 0.025 33. 74 27.70 6.04 14.95 1.94 22.93 8 12 0.025 12.94 11. 17 1.77 18.45 2.66 '22. 88 9 17 0. 025 11. 71 10.13 1. 58 25.00 2. 27 28. 85 10 20 0.025 9.97 8. 67 1.30 26. 20 2.55 30.05 11 5 0.05 67.02 49. 81 17. 21 9.12 1.10 27.43 12 9 0.05 29.37 24.18 5.19 17.80 2.04 25.03 13 12 0.05 13. 75 11. 82 1.93 23.65 2.25 27. 83 14 17 0.05 6.36 5.55 0.81 26.95 2.25 30.01 15 20 0.05 3.03 2.59 0.44 29.12 2. 69 32. 29 16 5 0.1 64. 45 48.65 15.88 9.85 1.11 26. 84 17 9 0.1 22.54 19.08 3.46 23.40 2.08 28.92 18 12 0.1 7.11 6.23 0. 88 28. 25 2. 76 31. 89 19 17 0.1 3.88 3.42 0.46 30.30 2.33 33.09 20 20 0.1 2.47 2. 18 0. 29 31.15 2.66 34. 10 (Cooking temperature=200°C; Wood/liquor r a t i o ^ l r l O by weight) Percentage values based on oven-dry extractive-free sawdust. 61 Cooking time (min. ) Figure 9. E f f e c t of cooking time on y i e l d s of pulp and i s o l a t e d l i g n i n (FRACTION II) from e x t r a c t i v e - f r e e Douglas-fir sawdust. 62 Table 4. E f f e c t of cooking time on y i e l d s of f i b e r and water-insoluble Si l i g n i n from various wood species - preliminary cooking experi-m e n t ^ ) . b c Species Cooking time Fiber y i e l d ' Lignin y i e l d (min. ) (%) (%) b. Spruce 20 19. 78 19.94 7 57.97 9.74 Douglas-fir 20 14. 44 24. 78 7 63.53 5.74 Aspen 20 23. 78 16.34 7 57. 88 6.89 Birch 20 12. 68 18.36 7 49. 44 8. 44 Sugar cane 20 6.45 9.37 7 38. 23 2. 61 lWood samples were not pretreated to remove extractives. Percentage values based on oven-dry wood samples. Yields were not corrected for re s i d u a l l i g n i n . 63 sawdust by a c i d i f i e d organosolv cooking under i d e n t i c a l cooking condi-tions as those used i n the present study. In a s i m i l a r organosolv cooking experiment on Eucalyptus v i m i n a l i s , Gomide (80) also reported that about 607, of hemicellulose monomeric units and more than one-th i r d of the l i g n i n were removed i n the i n i t i a l pulping stage. In the present study, i t was found that about 10-13% of l i g n i n , which i s equivalent to about one-third of the t o t a l l i g n i n content (31.81%), was released from the wood during the i n i t i a l 5-min period of the d e l i g n i -f i c a t i o n process (Table 3). The temperature at this stage was only 175°C (Fig. 10). The amount of hemicellulose dissolved within t h i s pe-riod was about 21-22.5%, which i s equivalent to about 83.5-89.57, of the t o t a l p o t e n t i a l hemicellulose content of 25. 157o (Table 2). Even though the bulk of l i g n i n and hemicelluloses were removed "during 5-9 minutes of cooking, samplesiof cook hos.6,7,11,12,16 and 17 se-em to be:incompletely d e l i g n i f i e d . Residual l i g n i n content of these cooks ranged between 3. 46-18.047,, based on the oven-dry e x t r a c t i v e r f r e e sawdust (Table 3).. The high:*residual l i g n i n content i n short cooks might have been caused by. experimental errors i n Kappa number determination because, the re-s i d u a l f i b e r s could not be blended before the addition of 0.IN potassium permanganate solution. As a result,the standard reaction time (10 min) might have been too" short to complete^ the reaction, r e s u l t i n g i n lower consumption of 0.IN potassium permanganate solution, and thus giving er-roneous Kappa numbers. Another possible explanation for the high content of the res i d u a l l i g n i n may be the fact that the decomposed sugars conden-sed with l i g n i n and pre c i p i t a t e d on the f i b e r residue during the organo-solv cooking. However, no v i s u a l evidence of t h i s was found. It i s also known that c e l l u l o s e degradation i s time depen-dent (3). After 20-min cooking, cook no.15 (20 min;0.05N HC1) and cook 64 10 15 20 25 Cooking. time(min. ) Figure 10. Cooking-bomb temperature vs. cooking time. 65 no. 20 (20 min;0. IN HC1) gave almost t o t a l d i s s o l u t i o n of the wood cons-t i t u e n t s leaving less than 3% of the s t a r t i n g material as f i b e r residue (Table 3 and Fig.9). When the cooking temperature reached 200°C,-after about 11-12 minutes (Fig.10), the maximum pressure registered about 320 psig and was s t a b i l i z e d . Maximum pressure i n the organosolv cooking system was obtained f a s t e r (6-7 min) than the maximum temperature. 5.3.2 E f f e c t of acid c a t a l y s t concentration It was found that the increase of acid c a t a l y s t concentra-t i o n generally increased the rate of d e l i g n i f i c a t i o n i n organosolv pul-ping (36,153). This i s attributed to the f a s t e r a c i d o l y t i c s p l i t t i n g of the lignin-carbohydrate complex into fragments small enough to be soluble i n the aqueous organic cooking system (97). Cooking r e s u l t s presented i n Table 3 show that f i b e r re-sidue y i e l d decreased and the i s o l a t e d l i g n i n (FRACTION II) y i e l d i n c -reased as the acid concentration increased from 0.025 to 0.1 N HC1. This general observation can be observed i n Fig, 11. The e f f e c t of acid concentration on y i e l d s of f i b e r residue and the organosolv l i g n i n , however, i s not as s i g n i f i c a n t as that of cooking time ( F i g . 9). The increased c a t a l y s t l e v e l seems to cause rapid d i s s o l u t i o n of polysac-charides as well as rapid d e l i g n i f i c a t i o n because both phenomena must be regarded e s s e n t i a l l y as the h y d r o l y t i c s o l v b l y s i s process of wood (153). At a high acid concentration (0.IN), extensive d i s s o l u t i o n of the wood constituents, beyond the amount represented by hemicellu-loses and l i g n i n , occurred. As mentioned before, cook nos,18. (0.1 >N HC1;12 min), 19 (0,1 N HC1;17 min) and 20 (0.1 N HC1;20 min) produced more l i g n i n than the p o t e n t i a l l i g n i n content (31,81%) possibly due 66 A — _ . A Lignin Pulp A 5 min. cook ^ 9 min. cook • 12 min.cook • 17 min.cook O 20 min. cook —O 0.025 0.05 0.075 Acid-catalyst concentration (N,HC1) 0.1 gure 11. Effect of acid concentration on yields of pulp and isolated lignin (FRACTION II). from extractive-free Douglas-fir sawdust. 67 to contamination by hemicelluloses (26). Cook no. 15, having been made at an intermediate acid concentration of 0.05 N, gave s i m i l a r r e s u l t s due to the long cooking time (20 min), leading to nearly t o t a l d i s s o l u -t i o n of wood and thus r e s u l t i n g i n a mere 2.59% f i b e r residue y i e l d and nearly quantitative recovery of the p r e c i p i t a b l e l i g n i n . The pH value of pure acetone-water cooking l i q u o r was about 6 and those of 0.025, 0.05 and 0.1 N hydrochloric acid solutions were found to be 2,5, 1.5 and 1.2, respectively. Table 5 shows the change of pH values a f t e r 20-min cooking. This change seems to be due to an accumulation of organic acids, such as a c e t i c acid and formic acid, l i -berated from the wood during cooking (36). Though such organic acids can a f f e c t the rate of d e l i g n i f i c a t i o n (94), t h e i r e f f e c t was found to be i n s i g n i f i c a n t . In the presence of a strong mineral acid such as hydrochloric acid, t h e i r e f f e c t was completely unnoticed and unimportant i n spite of the fact that s u b s t a n t i a l decreases i n the f i n a l pH of the cooking l i q u o r was noticed (Table 5). In the absence of acid catalyst, the rate of h y d r o l y t i c d i s s o l u t i o n was obviously very slow, even though the pH of the cooking l i q u o r had been lowered to 3.2 at the end of a 20-min cook from the near neutral s t a r t i n g pH as shown i n Table 5. The amount of l i g n i n ex-tracted fromr t h i s series (cook nos.1-5) was too small to i s o l a t e , therefore no further analysis was attempted. By a simple extraction with organic s o l v e n t s , r i t was found that only about 1% of the o r i g i n a l l i g -nin can be extracted (137). This series (cook nos.1-5;without c a t a l y s t ) proves that the d e l i g n i f i c a t i o n process i s not just a s o l v o l y s i s process, but requires s u f f i c i e n t strength of c a t a l y s t for the hydrolysis reaction, i f s u b stantial amount of l i g n i n are to be removed from the wood i n the course of high temperature organosolv cooking. 68 Table 5. pH values of acid-catalyzed cooking l i q u o r before and a f t e r 20-min cooking. HC1 concentration(N) before cooking a f t e r cooking 0 6.0 3. 2 0.025 2.5 2.2 0.050 1.5 1. 2 0. 100 1.2 1.1 Cooking liquor:acetone/water=60/40, by volume. Cooking with e x t r a c t i v e - f r e e Douglas-fir sawdust(wood/cooking l i q u o r =1/10) 69 5.4 Water-soluble L i g n i n F r a c t i o n It was not the i n t e n t i o n of the present study to discuss i n d e t a i l the water-soluble l i g n i n (FRACTION I I I ) , but i t must be men-tioned that some important observations were made with regard to t h i s f r a c t i o n . As mentioned before, repeated attempts to i s o l a t e the water-soluble l i g n i n f r a c t i o n i n the s o l i d state had f a i l e d . Even quantitative separation of t h i s f r a c t i o n from aqueous f i l t r a t e s of the spent l i q u o r by l i q u i d - l i q u i d e x t raction was found to be somewhat a problem since the s o l u b i l i t y of t h i s f r a c t i o n i n water was not very much lower than that of chloroform or that of other water-immiscible or-ganic solvents. The semi-quantitative determination of the water-soluble l i g n i n by UV spectrophotometry also provided somewhat ques-tionable r e s u l t s (Table 3). In any cook, less than 370 of the e x t r a c t i v e - f r e e sawdust was detected as water-soluble l i g n i n . However, i t was found that as cooking time increased, the y i e l d of the water-soluble l i g n i n f r a c t i o n seemed to increase. For example, increasing theucooking time from 5 to 20 minutes almost doubled the y i e l d of water-soluble l i g n i n f r a c t i o n s i n a l l cases. P e c u l i a r l y , maximum y i e l d of these f r a c t i o n s occurred for the 12-min cooks with only a s l i g h t f a l l - o f f f o r 20-min cooks, i n d i c a t i n g a f a i r degree of thermal s t a b i l i t y of water-soluble l i g n i n s . E f f e c t of i n -creasing concentration of acid c a t a l y s t on y i e l d of water-soluble l i g -n i n seems to be i n s i g n i f i c a n t . For the lack of an ,adequate i solation-method, an accurate o v e r a l l l i g n i n mass-balance-could not be obtained. .. The quantitative determination of t h i s f r a c t i o n , however, w i l l be discussed i n connec-70 t i o n with TLC analysis i n the following section. 5.5 Thin-Layer Chromatographic Analysis of Water-soluble L i g n i n F r a c t i o n from Spent Cooking Liquor Regarding the l i g n i n mass-balance, i t i s evident that con-siderable amounts of l i g n i n and lignin-degradation products were pre-sent as water-soluble substances i n the spent cooking liquor. Reac-tions that convert p r o t o l i g n i n into water-soiuble d e r i v a t i v e s have been the subject of numerous inv e s t i g a t i o n s . Hibbert and his co-workers (27,46,119) were the f i r s t group to investigate these water-soluble materials from the ethanolysis of l i g n i n . A number of water-soluble compounds which were found to be monomeric phenylpropane C^-units were is o l a t e d , i d e n t i f i e d and assumed to be lighin-degr.adation products(26, 131). The organosolv cooking of e x t r a c t i v e - f r e e sawdust i n acid medium i s e s s e n t i a l l y an acid hydrolysis. It was reported that aque-ous hydrolysis l i q u o r s of softwood contain low molecular weight aroma-t i c materials such as c o n i f e r y l alcohol ( I I ) , v a n i l l i c acid (XIII), v a n i l l i n (XVI), acetyl v a n i l l o y l (XXI) and guaiacyl acetone (XXII) as well as some Hibbert's ketones (26,97,131). Those compounds which are l i k e l y present i n the aqueous f r a c t i o n of the spent cooking liqu o r of the present study are l i s t e d i n Table 6 along with the known R^ values and c h a r a c t e r i s t i c colors on s i l i c a gel TLC (13,70,100). Table 7 presents TLC r e s u l t s of the aqueous f r a c t i o n from cook no.15 (20 min;0.05 N HC1) a f t e r removing the sugars. Among the compounds i d e n t i f i e d were Hibbert's ketones such as a c e t y l v a n i l l o y l (XXI), guaiacyl acetone (XXII), 1.38 175.12 2.37 1.35 0.39 3. 79 0.99 9.61 0.97 17.17 PC-12 C9 HS.O5°O.68 ( O C H3 )0.95 ( O H )1.31 175;65 2.35 1.34 0.44 4. 26 0.87 8.42 0.95 16.77 PC-13 SH4.94°0.80. -of p a r t i c l e s ^ (nm) \ PC-11 PC-12 PC-13 PC-14 PC-15 PC-9 PC-19 < 25 5.2 3.5 4.2 3.4 8.1 2.3 2 5 — 50 21.5 12.8 10.4 10.3 26.8 11.1 -5 0 — 75 34.8 27.9 16.2 1.6.2 30.9 46.9 2.6 75 — 1 0 0 32.6 33.3 26.2 20.5 12.1 33.4 7.9 100—125 4.1 10.9 24.2 27.4 3.4 3.6 28.3 125 — 1 5 0 • 1.1 3.8 11.2 15.0 2.0 2.0 31.6 150 — 175 - 4.5 1.9 3.4 1.3 - 9.2 175 — 200 0.3 1.9 3.1 0.9 - 0.7 4.6 200 — 225 0.3 - - 0.4 2.0 - 6.6 225 — 250 - 0.3 0.8 1.3 0.7 - 5.3 2 5 0 — 275 - 0.3 0.8 - 1.3 - 0.7 275 — 300 - - • - 0.4 - - 2.0 3 0 0 — 325 0.3 - 0.4 0.4 0.7 - -325 — 350 - 0.6 0.8 - 4.7 - -350 — 375 - - - 0.4 0.7 - 0.7 375 — 4 0 0 - - - - 0.7 - • -4 0 0 — 425 - - - - - -4 2 5 — 450 - - - 2.0 - 0.7 4 5 0 — 475 - - - • - - - -4 7 5 — 500 - - - - 1.3 - -117 Too 300 460 500" Diameter of particle(nm) 'Figure 29. Effect of cooking time on particle size frequency distribution. 50 40 30 20 10 0 PC-9 (0.025N HC1) i i i -i 1 1 r-* 1 r £-5 >, CJ C cu XT 0) U P n 30, 20 10 40 1 30 20 4 10 0 PC-14 (0.05N HC1) i ' i i i i PC-19 (0. IN HC1) ' 1 100 200 300 i-j r- r 400 500 Diameter of particle(nm) Figure 30. E f f e c t of acid c a t a l y s t concentration on p a r t i c l e s i z e frequency d i s t r i b u t i o n ^ 119 6. RECOMMENDATIONS The information derived i n th i s study on the i s o l a t i o n and c h a r a t e r i z a t i o n of Douglas-fir organosolv l i g n i n from the acid-catalyzed cooking allows recommendations for further research on organosolv l i g n i n . 1. An improvement of the i s o l a t i o n technique for the water-soluble l i g n i n fraction,which seems to have very low molecu-l a r weight and high content of phenolic hydroxyl group, i s required. Such water-soluble l i g n i n s , i s o l a t e d i n large amounts, could have commercial importance. 2. Simple and e f f e c t i v e separation techniques for d i s -solved sugars'from ithe'-spent .liquor-should be developed. Determina-t i o n o f these sugars could give" invaluable information on pulp and the i s o l a t e d organosolv l i g n i n . 3. Detailed studies should be i n i t i a t e d regarding to competition between the l i g n i n degradation reaction (hydrolysis) and recondensation reaction since the l a t t e r l i m i t s l i g n i n fragmen-t a t i o n to low molecular weight products during the cooking. 4. Influence of various organic solvents (both polar and non-polar) used as organic component of the cooking l i q u o r on eontent of functional groups o f the i s o l a t e d l i g n i n s should be stu-died i n more d e t a i l . 5. Further studies on l i g n i n macromolecular properties by GPC and SEM are required i n order to find firm evidence i n sup-port of a close r e l a t i o n s h i p between MWD of l i g n i n molecules and PSD of l i g n i n p a r t i c l e s . 120 7. CONCLUSION The following general conclusions can be drawn from t h i s study. 1. At a constant reaction temperature (200°C), both cooking time and concentration of acid c a t a l y s t were found to have a profound e f f e c t on the rate of hydrolysis of wood c o n s t i -tuents during organosolv cooking. These parameters provide the means to maximize y i e l d s of l i g n i n and sugars. 2. Cooking time seems to be a more important parameter than acid-concentration i n regulating the quantity and q u a l i t y of the i s o l a t e d organosolv. lignins.. In general, lignin-.yield increased with prolonged cooking. Cooking time was f®und to a f f e c t the content of functional groups and molecular weight.of the organosolv l i g n i n . 3. The molecular weights of the i s o l a t e d organosolv l i g n i n seem to be much lower than those of the p r o t o l i g n i n i n wood. The probable reason for t h i s i s thought to be s c i s s i o n of ether linkages of p r o t o l i g n i n molecules through^acid, hydro-l y s i s during cooking. However, prolonged cooking seems to i n -crease the molecular weights due to autocondensation of l i g n i n molecules? • . 4. Analyses of NMR, IR, and UV spectra of the i s o -lated l i g n i n s provide good evidence of acid hydrolysis of a r y l -a l k y l ether linkages and the e f f e c t of autocondensation reaction during organosolv cooking. The balance of degradation and re? condensation reactions was found to be the deciding 7 f a c t o r f o r 121 the size of molecular weight and p o l y d i s p e r s i t y of the i s o l a t e d or-ganosolv l i g n i n s . 5. SEM photographs show that p r e c i p i t a t i o n of the acety-lated organosolv l i g n i n s from Douglas-fir sawdust occurs i n shape of sp h e r i c a l p a r t i c l e s with wide v a r i e t y of size. The p a r t i c l e diame-ters ranges from somewhat smaller than 25 nm t o . about 500 nm. PSD obtained from SEM and MWD curves from GPC were found to have s i -milar d i s t r i b u t i o n -patterns. 122 LITERATURE CITED 1. Adkins, H. , Frank, R.L. and E.S. Bloom. 1941. The products of the hydrogenation of l i g n i n . J. Am. Chem. Soc. 63:549-555. 2. Alder, E. 1977. Li g n i n chemistry - past, present and future. Wood Sc i . Technol. 11:169-218. 3. . 1957. St r u c t u r a l elements of l i g n i n . Ind. Eng. Chem. 49: 1377-1383. 4. , Hernestam, S. and I. Wallden. 1958, Estimation of phe-n o l i c hydroxyl groups i n l i g n i n . Svensk Papperstid. 61:641-647. 5. , Lindgren, B. 0. and U. Saeden. 1952. The beta-guaiacyl ether of alpha-veratryl g l y c e r o l as a l i g n i n model. Svensk Papperstid. 55:245-253. 6. and J. Marton. 1959. Zur Kenntnis der Carbonylgruppen im Lignin. I. Acta Chem. Scand. 13:75-96. 7. and S. Y l l n e r , 1952, Some reactions of v e r a t r y l g l y c e r o l related to l i g n i n chemistry. Svensk Papperstid. 55:238-244. 8. Anon. 1977. C a l i b r a t i o n of GPC Systems. Waters Associates, Inc. , Mi l f o r d , Massachusetts. 8 pp. 9. A r l t , H.G., Gross, S.K. and C. Schuerch. 1958. A study of the l i g n i n f r a c t i o n obtained from the a l k a l i n e hydrogenation of maple wood. Tappi 41:64-70. 10. Aronovsky, S. I. and R. A. Gortner. 1936. The cooking process. IX. Pulping wood with alcohols and other organic reagents. Ind. Eng. Chem. .28:1270-1276. 11. Aulin-Erdtman, G. 1958. Studies on u l t r v i o l e t absortion changes caused by modification of chromophores, with s p e c i a l reference to l i g n i n chemistry. Svensk Kern. Tidskr. 70:145-156. 12. and L. Hegbom. 1958. Spectrographs contributions to l i g n i n chemistry. VIII. A£-Studies on Brauns' native l i g -nins from coniferous woods. Svensk Papperstid. 61:187-210. 13. Barton, G.M. 1967. Thin-layer chromatography of guaiacylpropanone monomers, selected l i g n i n s and phenolic wood extractives. J. Chromatolog. _26:320-322. 123 14. Benigni, J.D.^ and I. S, Goldstein. 1971. Neutral hydrolysis of a l k a l i l i g n i n to monomeric phenols. J. Polymer Sci.(Part C) 36:467-475. 15. Benko, J. 1964. The measurement of molecular weight of ligno-s u l f onic acids and related materials by d i f f u s i o n . Tappi 47:508-514. 16. . 1961. Measurement of the r e l a t i v e molecular weight of lignosulfonate by d i f f u s i o n . I. Methods for molecular weight determination and for separation of i n t e r f e r i n g substances by ion exclusion or by exhaustive d i a l y s i s . Tappi 44:766-771. 17. . 1961. Measurement of the r e l a t i v e molecular weight of lignosulfonates by d i f f u s i o n . I I . Determination of r e l a t i v e molecular weights on various lignosulfonates. Tappi 44:771-775. 18. Bentum, A.L.K. , Cote, (Jr.) W.A. , Day, A. C. and T.E. Tim e l l . 1969. D i s t r i b u t i o n of l i g n i n i n normal and tension wood. Wood S c i . Technol. 3:218-231. 19. Berlyn, G.P. and R.E. Mark. 1965. L i g n i n d i s t r i b u t i o n i n wood c e l l walls. Forest Prod. J. 15:140-141. 20. Bicho, J.B., Zavarin, E. and D.L. Brink. 1966. Oxidative degradation of wood. II. Products of a l k a l i n e nitroben-zene oxidation by a methylation gas chromatography tech-nique. Tappi 49:218-226. 21. Bjorkman, A. 1956. F i n e l y divided wood. I. Extraction of l i g -n i n with neutral solvents. Svensk Papperstid. 59:477-485. 22. and B. Person. 1957. Studies on f i n e l y divided wood. II. The properties of l i g n i n s extracted with neutral solvents from softwoods and hardwoods. Svensk Papperstid. 60:158-169. 23. Bolker, H. I. and N.G. Somerville;" 1963. Infrared spectroscopy of l i g n i n s . Part II:Lignins i n unbleached pulps. Pulp paper Mag. Can. 64:T187-193. 24. Brauns, F.E. 19,62. Soluble native l i g n i n , m i l l e d wood l i g n i n , synthetic l i g n i n and structure of l i g n i n . Holzforschung 16:97-102. 25. 1939. Native l i g n i n . I. Its i s o l a t i o n and methyl-ation. J. Am. Chem. Soc. 61:2120-2127. 26. and D.A. Brauns. 1960. The Chemistry of Lignin, Sup-plement Volume. Academic Press, New York. pp.91-92,157-167, 119-222. 124 27. Brickman, W. , Brickman, L. and H. Hibbert. 1940. Studies on l i g n i n and related compounds. XLVIII. I d e n t i f i c a t i o n of v a n i l l i n and v a n i l l o y l methyl ketone as ethanolysis pro-ducts from spruce wood. J. Am. Chem. Soc. 62:2149-2154. 28. Brown, S.A. 1964. L i g n i n and Tannin Biosynthesis. In:Bio-chemistry of Phenolic Compounds. J.B. Harbone (ed.). Academic Press, London, pp.361-387. 29. . 1961. Chemistry of l i g n i f i c a t i o n . Science 134: 305-313. 30. Brownell, H. H. 1970. Spruce milled wood l i g n i n and l i g n i n -carbohydrate complex:Molecular siz e and shape and f r e -quency of lignin-carbohydrate linkage. Tappi 53:1278-1281. 31. . 1968. L i q u i d - l i q u i d p a r t i t i o n of b a l l m i l l e d wood. Tappi 51:359-363. 32. Browning, B.L. 1967. Methods of Wood Chemistry. Vol. II. Interscience Publishers, New York. pp.717-746. 33. Bublitz, W.J. and T. Y. Meng. 1974. The e f f e c t of c e r t a i n f l a -vonoids on the bleaching of Douglas f i r r e f i n e r ground-wood. Pulp Paper Mag. Can. 75:T91-96. 34. and T.Y. Yang. 1975. Pulping c h a r a c t e r i s t i c s of Douglas f i r sawdust. Tappi 58:95-99. 35. Chang, H, -m. , Cowling, E.B., Brown, W. , Alder, E. and G. Mik-sche. 1975. Comparative studies on c e l l u l o l y t i c enzyme l i g n i n and m i l l e d wood l i g n i n of sweetgum and spruce. Holzforschung 29:153-159. 36. Chang, P.-C., and L. Paszner. 1976. Recovery and GC analyr. s i s of wood sugars from organosolv s a c c h a r i f i c a t i o n of Douglas-fir heartwood. Paper, Canadian Wood Chemistry Symposium, Mont Gabriel (Sept. 1-3). 15 pp. 37. and G. Bohnenkamp. 1977. Compa-ra t i v e d i s s o l u t i o n rates of carbohydrates and l i g n i n durr ing a c i d i f i e d aqueous organosolv (AAOS) s a c c h a r i f i c a t i o n of alcohol-benzene extracted Douglas-fir and aspen woods. Paper, Tappi Forest Biology/Wood Chemistry Symposium, Madison (June 20-22). 29 pp. 38. Charbonnier, H.Y. 1942. L i g n i n i s o l a t e d i n the presence of butanol. Paper, Tappi Annual Meeting, New York. Feb. 16-19). pp.31-36. 39. Cho, H.J. , Muir, G. and C. Ng. 1979. Biomass processing:Iso-l a t i o n , c h a r a c t e r i z a t i o n and u t i l i z a t i o n of l i g n i n . Re-port, Fac. of Forestry, Univ. of B.C. 26 pp. 125 40. Clermont, L. P. 1962. Charaterization of a l i g n i n I s olated by-action of sulphur dioxide i n dimethylsulphoxide on spruce wood. Pulp Paper Mag. Can. 63:402-404. 41. Connors, W.J. 1978. Gel chromatography of l i g n i n s , l i g n i n mo-del compounds and polystyrenes using Sephadex LH-60. Holz-forschung 32:145-147. 42. , Lorenz, L.F. and T.K. Kirk. 1978. Chromato-graphic separation of l i g n i n models by molecular weight using Sephadex LH-20. Holzforschung 32:106-108. 43. Cote, J r . , W.A. 1977. Wood Ul t r a s t r u c t u r e i n Relation to Chem-i c a l Composition. In: Recent Advances i n Phytochemistry, Vol. 11. F.A. Loewus and V.C. Runeckles (eds.). Plenum Press, New York. pp.1-44. 44. , Day, A. C. and T.E. Ti m e l l . 1968. D i s t r i b u -bution of l i g n i n i n normal and compression wood of Tama-rack. Wood S c i . Technol. _2:1 3 - 3 7. 45. , T i m e l l , T.E. and R.A. Zabel. 1966. D i s t r i -bution of l i g n i n i n compression wood of red spruce, Holz a l s Roh-Werkstoff. J24: 432-438. 46. Cramer, A.B. , Hunter, M.J. and H. Hibbert. 1939. Studies on l i g n i n and related compounds. XXXV. The ethanolysis of spruce wood. J. Am. Chem. Soc. 61:509-515. 47. Davis, B. 1955. Advances i n enzymology and related subjects of biochemistry. Advan. Enzymol. 16:247-312. 48. DeStevens, G. and F.F. Nord. 1953. Investigations on l i g n i n and l i g n i f i c a t i o n XI. S t r u c t u r a l studies on Bagasse Na-t i v e L i g n i n . J. Am. Chem. Soc. 75:305-309. 49. Dimmel, D.R., Shepard, D., Brown T.A. and R.D. Mckelvey. 1979. Molecular weight changes i n l i g n i n during anthraquinone/ a l k a l i pulping. Paper, Canadian Wood Chemistry Symposium, Harrison Hot Springs, B.C.(Sept. 19-21). pp.37-39. 50. Eberhardt, G. and W.J. Schubert. 1956. Investigations on l i g -n i n and l i g n i f i c a t i o n . XVII. Evidence for the mediation of shikimic acid i n the biogenesis of l i g n i n b u i l d i n g stones. J. Am. Chem. Soc. 78:2835-2837. 51. Erdtman, H. 1949. The chemical nature of l i g n i n . Tappi 32:71-74. 52. . 1933. Dehydrierungen i n der C o n i f e r y l r e i h e . I. De-hydrodi-eugenol und Dehydroduesoeugenol. Biochem. Z. Ber-l i n . 258:172-180. 126 53. Erickson, M., Larsson, S. and G.E. Miksche. 1973. Gaschromatogra-phische Analyse von Ligninoxyldations-Producten. VIII. Zur Struktur des Lignins der Fichte. Acta Chem. Scand. 27:903-914. 54. and G.E. Miksche. 1974. On the occurrence of l i g n i n or polyphenols i n some mosses and liverworts. Phytochem. 13: 2295-2299. 55. . 1974. Two dibenzofurans obtained on oxidative degradation of the moss Polytrichum commune Hedw. Acta Chem, Scand. 28:109-113. 56. Faix, 0. and W. Schweers. 1974. Vergleichende Untersuchungen an Polymermodellen des Lignins (DHP's) verschiedener Zusammen-setzungen. 3. Mitt. IR-spektroskopische Untersuchungen;, Holz-forschung 28:50-54. 57. . 1974. Vergleichende Untersuchungen an Polymermodellen des Lignins (DHP's) verschiedener Zusammen-setzungen. 5. Mitt. 1 H-NMR-spektroskopische Untersuchungen, Holzforschung .28:179-185. 58. Fengel, D. 1976. Studies on the supermolecular structure of c e l l w a ll components. Part 5. Ul t r a s t r u c t u r e of the f r a c t i o n s from i o n exchange chromatography and of i s o l a t e d l i g n i n . Svensk Papperstid. 79:24-28. 59. . 1976. Chemische und elektronenmikroskopische Unter-suchungen an schonend i s o l i e r t e m Nadelholzlignin. Holzfor-schung,, 30:1-6. 60. . 1975. Untersuchung von Lignin-Kohlenhydrat-Komplexen mit H i l f e der hochauflOsenden Elektronenmikroskopie. Holz-forschung 29:160-164. 61. . 1970. U l t r a s t r u c t u r a l behavior of c e l l wall poly-saccharides. Tappi 53:497-503. 62. , S t o l l , M. and G. Wegener. 1978. Studies on milled wood l i g n i n from spruce. Part I I . Electron microscopic ob-servations on the milled wood. Wood S c i . Technol. 12:141-148. 63. Fergus, B.J. , Procter, A.R., Scott, J, A.N. and D.A.I. Goring. 1969. The d i s t r i b u t i o n of l i g n i n i n sprucewood as deter-mined by u l t r a v i o l e t microscopy. Wood S c i . Technol. ^:117-139 64. Forss, K. and K.-E. Fremer. 1965. The repeating unit i n spruce l i g n i n . Paperi puu. 47:443-454. 127 65. Freudenberg, K. 1965. L i g n i n ; Its c o n s t i t u t i o n and formation from p-hydroxycinnamyl alcohols. Science 148:595-600. 66. . 1964. Entwurf eines Konstitutionsschemas fUr das L i g n i n der Fichte. Holzforschung 18:3-9. 67. and J.M. Harkin. 1964. Ergaenzung des Konsti-tutionsschemes fUr das L i g n i n der Fichte. Holzforschung 18:166-168. 68. and A. C. Neish. 1968. Constitution and Bio-synthesis of Lignin. Springer-Verlag B e r l i n , Heidelberg, pp. 1-9,60-77,82-85. 69. Frey-Wyssling, A. 1964. U l t r a v i o l e t and Fluorescence Optics of L i g n i f i e d C e l l Walls. In: The Formation of Wood i n Forest Trees. M. H. Zimmermann(ed.). Academic Press, New York. pp.153-167. 70. Gardner, J. A.F. 1954. The ethanolysis of 3-hydroxy-l-pro-panone i n r e l a t i o n to l i g n i n chemistry. Can. J. Chem, 32:532-537. 71. Gibson, F. and L.M. Jackman. 1963. Structure of chorismic acid, a new intermediate i n aromatic biosynthesis. Nature,, 198:388-389. 72. Gierer, J. and S. SOderberg. 1962. tfber die wirkung einer Borohydridbehandlung auf die Sulfonierbarkeit von L i g -ninprHparaten. Acta Chem. Scand. 16:629-639. 73. Glasser, W.G. and H.R. Glasser. 1974. Simulation of reactions with l i g n i n by computer (Simrel). I I . A model f or s o f t -wood l i g n i n . Holzforschung 28:5-11. 74. Goheen, D.W. 1978. Chemicals from l i g n i n . Paper, the Eighth World Forestry Congress. Jakarta (Oct. 16-28). 13 pp. 75. . 1971. Low Molecular Weight Chemicals. In;Lignins. K.V. Sarkanen and C. H. Ludwig (eds.). Wiley-Interscience, New York. pp. 797-831. 76. Goldschmid, 0. 1971. U l t r a v i o l e t Spectra. In:Lignins. K.V. Sarkanen and C.H. Ludwig (eds.). Wiley-Interscience, New York. pp.241-266. 77. . 1954. Determination of phenolic hydroxyl con-tent of l i g n i n preparations by u l t r a v i o l e t spectrophoto-metry. Analyt. Chem. 26:1421-1423. 128 78. Goldstein, I.S. 1978. Forestry f o r I n d u s t r i a l Development. Chemicals from Wood. Paper, the Eighth World Forestry Congress. Jakarta (Oct. 16-28). 15 pp. 79. . 1975. Perspectives on production of phenols and phenolic acids from l i g n i n and bark. Applied Polymer Symposium. No.28. pp.259-267. 80. Gomide, J.L. 1978. Ethanol pulping of Eucalyptus V i m i n a l i s wood and chemical c h a r a c t e r i z a t i o n of the pulping spent l i q u o r . Ph.D. Thesis, North Carolina State Univ., Raleigh. 113 pp. 81. Goring, D.A.I. 1971. Polymer Properties of Lignin.and L i g n i n Derivatives. JjirLignins. K.V. Sarkanen and C. H. Ludwig (eds.). Wiley-Interscience, New York. pp.695-768. 82. Gupta, P.R. and D.A.I. Goring. 1960, Physiochemical studies of a l k a l i l i g n i n s . I. Preparation and properties of reac-tions. Can. J. Chem. 38:248-258. 83. Hachihama, Y. and A. Jodai. 1941. Studies on c a t a l y t i c hyd-rogenation of l i g n i n . Kygyo Kagaku Zasshi 44:773-775. 84. H a l l , L. 1965. S u l f i t a v l u t e n s tekniska anvandning Del. I. Fraktionerad u t f a l l n i n g med slacks Kalk. Svensk Pap-p e r s t i d . 59:716-720. 85. Harris, E. E. , D'lanni, J. and H. Adkins. 1938. Reaction of hardwood l i g n i n with hydrogen. J. Am. Chem. Soc. 60:1467-1472. 86. Hergert, H.L. 1971. Infrared Spectra. Ln:Lignins. K.V. Sar-kanen and C. H. Ludwig (eds.). Wiley-Interscience, New York. pp.267-297. 87. Higuchi, T. , Shimada, T. , Nakatsubo, F. and M. Tanahashi. 1977. Differences i n biosynthesis of guaiacyl and s y r i n g y l l i g -n i n i n woods. Wood S c i . Technol. 11:153-167. 88. Hoyt, C. H. and D.W, Goheen. 1971. Polymeric Products. In:Lig-nins. K.V. Sarkanen and C.H. Ludwig (eds.). Wiley-Inter-science, New York. pp. 833-865, 89. Huttermann, A. 1978. Gel permeation chromatography of water-insoluble l i g n i n s on con t r o l l e d pore glass and Sepharose CL-6B. Holzforschung 32:108-111. 90. . 1977, Gelchromatographie von' :Na-Ligninsulfo-naten an Sepharose CL-6B, Holzforschung 31:45-50, 91. Kennedy, R.W. 1970. An outlook f o r basic wood anatomy re-search. Wood and Fiber 2:182-187. 129 92. Kennedy, R.W. . and J.M. Jaworsky. 1960. V a r i a t i o n i n c e l l u l o s e content of Douglas-fir. Tappi 43:25-27. 93* K l e i n e r t , T.N, 1975. Ethanol-water d e l i g n i f i c a t i o n of wood. Rate constants and a c t i v a t i o n energy. Tappi 58:170-171. 94. . 1974. Organosolv pulping with aqueous alcohol. Tappi 57:99-102. 95. . 1967. Thermal wood deformation i n alcohol-water mixture. Holzforsch. Holzverwert. 19:60-65. 96. and K. Z. Tayenthal; 1931. Process of decompos-ing vegetable fibrous matter for the purpose of the simul-taneous recovery both of the c e l l u l o s e and of the incrus-t i n g ingredients. US Patent No. 1856567. 97. Kosikova, B. and J. Po l c i n . 1973. I s o l a t i o n of l i g n i n from spruce by a c i d o l y s i s i n dioxane. Wood S c i . Technol. 7_: 308-316. 98. and D. Joniak. 1977. NMR studies on the l i g n i n - s a c c h a r i d i c complexes. II . Hydrolysis of the lignin-saccharide complex i s o l a t e d from acetylated beech-wood. Holzforschung 31:191-193. 99. , Zakutna, L. and D. Joniak. 1978. Investigation of the l i g n i n - s a c c h a r i d i c complex by electron microscopy. Holzforschung 32:15-18. 100. K r a t z l , K. and L. Paszner. 1970. The aqueous hydrolysis, ethanolysis and oxidation of /8-arylether bonded l i g n i n b u i l d i n g units and t h e i r o t-sulfonic acids. C e l l . Chem. Technol. 4:293-303. 101. Lange, P.W. 1954. The d i s t r i b u t i o n of l i g n i n i n the c e l l w all of normal and reaction wood from spruce and a few hard-woods. Svensk Papperstid. 57:525-533. 102. Lehninger, A. L. 1976. The Biosynthesis of Amino Acids and some Derivatives. In:Biochemistry. Worth Publishers, Inc., New York. p.708. 103. Levin, J.G. and D.B. Sprinson. 1960. The formation of 3-enol-pyruvyl shikimate 5-phosphate i n extracts of Escherichia C o l i . Biochem. Biophys. Res. Commun. 3:157-163. 104. Lora, J.H. and M. Wayman. 1978. D e l i g n i f i c a t i o n of hardwoods by autohydrolysis and extraction. Tappi 61:47-50. 105. Ludwig,, C.H. 1971. Magnetic Resonance Spectra. In:Lignins. K-.V. Sarkanen and C.H. Ludwig (eds.). Wiley-Interscience, New York. pp.299-344. 130 106. Ludwig, C. H. , Nist, B.J. and J.L. McCarthy. 1963. Lig n i n XII. The high r e s o l u t i o n nuclear magnetic resonance spectros-copy of protons i n compounds related to l i g n i n . J. Am, Chem, Soc. .86:1186-1196. 107. . 1963. L i g n i n XIII. The high r e s o l u t i o n nuclear magnetic resonance spectros-copy of protons i n acetylated l i g n i n s . J. Am. Chem. Soc. 16:1196-1202. 108. Lundquist, K. 1979. NMR studies of l i g n i n s . II. Interpreta-t i o n of the lH NMR spectrum of acetylated b i r c h l i g n i n . Acta Chem, Scand. 33:27-30. 109. and T.K. Kirk. 1980. F r a c t i o n a t i o n - p u r i f i c a t i o n of an i n d u s t r i a l k r a f t l i g n i n . Tappi 63:80-82. 110. McNaughton, J.G., Yean, W.Q. and D.A. I. Goring. 1967. Macro-molecular properties of k r a f t l i g n i n s from spruce made soluble by a continuous flow process. Tappi 50:548-553. 111. Maranville, L.F. and 0. Goldschmid. 1954. U l t r a v i o l e t absorp-t i o n spectra as a measure of phenolic hydroxyl group con-tent i n pQlyphenolic tanninlike materials, Analyt. Chem, 26:1423-1427. 112. Marks, R.E; 1974. Environmental c o n t r o l and i t s r e l a t i o n to pulping by-product recovery processes. Tappi 57:63-65. 113. Marton, J. and T. Marton. 1964. Molecular weight of Kraft l i g n i n . Tappi 47:471-476. 114. Matsukura, M. and A; Sakakibara, 1973. Hydrogenolysis of pro-t o l i g n i n , V. I s o l a t i o n of some dimeric compounds with carbon to carbon linkage, Mokuzai Gakkaishi 19:131-135. 115. . 1973. Hydrogenolysis of pro-t o l i g n i n . VI. I s o l a t i o n of a new t r i m e r i c compound with carbon to carbon linkage. J. Jap. Wood Research Soc. 19:137-140. 116. . 1969. Hydrgenolysis of pro-t o l i g n i n . IV. I s o l a t i o n of a dimeric material of conden-sed type. Mokuzai Gakkaishi 15:279-302. 117. Merewether, J.W.T. 1962. Lignin . XV. The coagulation of l i g -n i n s a l t from a c i d i f i e d k r a f t black l i q u o r . Holzforschung 16:26-29. 118. . 1961. Lignin . XIV. The p r e c i p i t a t i o n of l i g n i n from k r a f t black l i q u o r . Holzforschung 15:168-177. 131 119. M i t c h e l l , L. and H. Hibbert. 1944. Studies on l i g n i n and related compounds. LXXX. The ethanolysis of 1-acetoxy-l-(4-acetoxy-3-methoxyphenyl)-2-propanone and i t s r e l a -t i o n to l i g n i n structure. J. Am, Chem, Soc. 66;602-604. 120. Mogharab, I. and W.G. Glasser. 1976. The structure of l i g n i n s i n pulps . A new i s o l a t i o n method with t h i o g l y c o l i c acid. Tappi 59:110-113. 121. Naveau, H. P. 1968. Degradation of wood constituents during self-agglomeration of an oak sawdust. I I . Infared study of two l i g n i n s . Can. J. Chem. 46:1893-1902. 122. Nord, F.F. and W.J. Schubert. 1957. On the mechanism of l i g -n i f i c a t i o n . Tappi 40:285-294. 123. . 1951. Enzymatic studies on c e l l u l o s e , l i g n i n and the mechanism of l i g n i f i c a t i o n . Holzforschung J5:1-9. 124. Obiaga, T.I. 1972. L i g n i n molecular weight and molecular weight d i s t r i b u t i o n during a l k a l i n e pulping of wood. Ph. D. Thesis, Univ. of Toronto, Toronto. 164 pp. 125. Ohta, M. and A.Sakakibara. 1969. Hydrogenolysis of proto-l i g n i n . I I I . I s o l a t i o n of diguaiacylpropanol and chro-matographic detection of some monomers. Mokuzai Gak-kaish 15:247-250. 126. Omori, S. and A. Sakakibara. 1974. I s o l a t i o n of new dimeric and t r i m e r i c compounds with l i g n i n type linkages from hydrolysis products of hardwood l i g n i n . Mokuzai Gak-kaish _20: 48-49. 127. Ouano, A;C., B a r r a l l , E.M. and J.F. Johnson. 1975. Gel Per-meation Chromatography. JCn:Polymer Molecular Weights. Part I I . P.E. Slade, J r . (ed.). Marcel Dekker, Inc., New York. pp.287-378. 128. Parham, R.A, and W.A. Cote, J r . 1971. D i s t r i b u t i o n of l i g n i n i n normal and compression wood of Pinus taeda L. Wood Sc i . Technol. 5:49-62. 129. Paszner, L; and P.-C. Chang. 1978. COPE:Catalysed Organosolv Pulp Extraction. A v e r s a t i l e and economical process f o r small scale biomass conversion i n t o pulp and chemical feedstocks. Paper, the Eighth World Forestry Congress, Jakarta (Oct. 16-28). 11 pp. 130. Payen, A. 1838. Memoire sur l a composition du t i s s u propre plantes et du ligneux. Comp. Rend. Tj,1052-1056. 132 131. Pearl, I.A. 1969. The Chemistry of Lignin. Marcel Dekker, Inc., New York. pp. 2-47,65-123,136-147, 238-261. 132. . 1965, 1964, 1963, 1962. Annual review of l i g n i n chemistry. Forest Prod. J. 15:379-399, 14:435-445, 13:373-385, 12:141-150. 133. . 1957. Present status of chemical u t i l i z a t i o n of l i g n i n . Forest Prod. J. 2:427-432. 134. and S.F. Darling. 1968. Mass spectrometry as an aid for determining structures of natural glucosides. Phyto-chem. 2=831-837. 135. Pepper, J.M. , B a y l i s , P.E.T. and E. Alder. 1959. The i s o l a t i o n of l i g n i n s obtained by the a c i d o l y s i s of spruce and aspen woods i n dioxane-water medium. Can. J. Chem. 37:1241-1248. 136. Pew, J.C. 1954. Nitrobenzene oxidation of l i g n i n model com-pounds, spruce wood and spruce "native l i g n i n " . J. Am. Chem. Soc. 77:2831-2833. 137. Polcm, J. and B. Bezuch. 1978. Enzymic i s o l a t i o n of l i g n i n from wood and pulps. Wood Sc i . Technol. 12.: 149-158. 138. Rezanowich, A., Yean, W. Q. and D.A.I. Goring. 1964. High re-so l u t i o n electron microscopy of sodium l i g n i n sulfonate. J. Appl. Polymer S c i . 8:1801-1812. 139. . 1963. The mole-cular properties of milled wood and dioxane l i g n i n s : S e d i -mentation, d i f f u s i o n , v i s c o s i t y , r e f r a c t i v e index incre-ment and u l t r a v i o l e t absorption. Svensk Papperstid. 66: 141-149. 140. R i t t e r , G.J. 1934. Structure of the c e l l wall of wood f i b e r s . Paper Ind. 16:178-183. 141. Rydholm, S.A. 1965. Pulping Processes. Interscience Publishers, London, pp.90-93,200-204. 142. Sacks, I.B., Clark, I.T. and J.C. Pew. 1963. Investigation of l i g n i n d i s t r i b u t i o n i n the c e l l wall of c e r t a i n woods. J. Polymer S c i . (Part C.) 2:203-212. 143. Saka, S., Thomas, R.J. and J.S. G r a t z l . 1978. L i g n i n d i s t r i b u -t i o n : Determination by energy-dispersive analysis of X-rays. Tappi 61:73-76. 133 144. Sakakibara, A. 1977. Degradation Products of P r o t o l i g n i n and the Structure of Lignin. JEnrRecent Advances i n Phyto-chemistry. Vol. 11. F.A. Loewus and V. C. Runeckles (eds.). Plenum Press, New York. pp.117-139. 145. Sarkanen, K-.V. 1971. Precursors and t h e i r Polymerization. _In: Lignins. K.V. Sarkanen and C. H. Ludwig (eds.). Wiley-Interscience, Inc., New York. pp.93-163. 146. ., Chang, H.-m. and G.G. Allan. 1967. Species v a r i a t i o n i n l i g n i n . I I . Conifer l i g n i n s . Tappi 50:583-587. 147. . 1967. Species v a r i a t i o n i n l i g n i n . III. Hardwood l i g n i n s . Tappi 50:587-590. 148. and H. L. Hergert. 1971. Lignins i n the Plant King-dom. C l a s s i f i c a t i o n and D i s t r i b u t i o n . _In:Lignins. K.V. Sar-kanen and C.H. Ludwig (eds.). Wiley-Interscience, Inc., New York. pp.43-94. 149. , Johanson, L. and G. Meshitsuka. 19 78. Catalyzed organosolv pulping of hardwood species and wheat straw. Annual Report. College of Forest Resources, Univ. of Was-hington. P. 9. 150. Sarkanen, S., S e l i n , J.F., Hope, N.R., Woerner, D. and J.L. Mc-Carthy. 1979. Molecular weight d i s t r i b u t i o n s of organosolv and a l k a l i l i g n i n s . Annual Report. College of Forest Re-sources, Univ. of Washington, pp.70-97C. 151. , T e l l e r , D. C. , H a l l , J. and J.L. McCarthy. 1979. Associative e f f e c t s among organosolv l i g n i n components. Annual Report. College of Forest Resources, Univ. of Was-hington, pp. 98-138. 152. Schubert, W.J. 1965. Aromatization i n Microorganisms. In:Lignin Biochemistry. Academic Press, New York. pp.39-42. 153. Schuerch, C. 1952. The solvent properties of l i q u i d s and t h e i r r e l a t i o n s to the s o l u b i l i t y , swelling and f r a c t i o n a t i o n of l i g n i n . J. Am. Chem. Soc. 74:5061-5067. 154. Schweers, W. 1978. U t i l i z a t i o n of l i g n i n s i s o l a t e d under mild conditions from wood or wood waste for the production of useful chemical products. Paper, the Eighth World Forestry Congress. Jakarta (Oct. 16-28). 7 pp. 155. . 1969. Uber die Hydrogenolyse von Lignin. 2. Mitt. UbeT die Hydrierung verschiedener Lignine mit Komplexver-bindungen der Ubergangsmetalle Eisen, Kobalt und Nickel a l s Katalysatoren. Holzforschung 22:5-9. 134 156. Smith, D~C.C. 1955. Ester groups i n l i g n i n . Nature. 176:267-268. 157. Sprinivasan, P.R. and D. B. Sprinson. 1959. 2-Keto-3-deoxy-D-arabo-heptonic acid 7-phosphate synthetase. J. B i o l . Chem. 234:717-722. 158. Srivastava, L.M. 1966. Histochemical studies on l i g n i n . Tappi 49:173-183. 159. Stone, J.E. and M. J. Blundell. 1951. Rapid micromethod for a l -kali n e nitrobenzene oxidation of l i g n i n and determination of aldehydes. Analyt. Chem. 23:771-774. 160. Timell, T.E. 1965. Wood and Bark Polysaccharides. I n : C e l l u l a r U l t r a s t r u c t u r e of Woody Plants. W.A. Cote, Jr. (ed.). Syracuse Univ. Press, Syracuse, pp.127-156. 161. Towers, G. H.N. and R.D. Gibbs. 1953. Lign i n chemistry and taxonomy of high plants. Nature 172:25-26. 162. Wardrop, A. B. 1965. C e l l u l a r D i f f e r e n t i a t i o n i n Xylem. In: C e l l u l a r U l t r a s t r u c t u r e of Woody Plants. W.A. Cote, Jr. (ed.). Syracuse Univ. Press, Syracuse, pp.61-124. 163. . 1964. The Structure and Formation of the C e l l Wall i n Xylem. _In:The Formation of Wood i n Forest Trees. M. H. Zimmermann (ed.). Academic Press, New York. pp.87-137. 164. . 1957. The phase of l i g n i f i c a t i o n i n the d i f f e r -e n t i a t i o n of wood f i b e r s . Tappi 40:225-243. 165. Wayman, M. and J. H. Lora. 1978. Aspen autohydrolysis. The e f f e c t s of 2-naphthol and other aromatic compounds. Tappi 61:55-57. 166. and T.I. Obiaga. 1974. Molecular weight of m i l l e d -wood l i g n i n s . Tappi 57:123-126. 167. Wegener, G. and D. Fengel. 1979. Rapid u l t r a s o n i c i s o l a t i o n of m i l l e d wood l i g n i n . Tappi 62:97-105. 168. . 1977. Studies on mi l l e d wood l i g -nins from spruce. Part I. Composition and molecular pro-pe r t i e s . Wood Sc i . Technol. 11:133-145. 169. Wenzl, H.F. J. 1970. The Chemical Technology of Wood, Academic Press. New york. pp.157-173,229-237,240-243. 170. Wilson, J.W. and R.W. Wellwood. 1965. Intra-Increment Chemical Properties of Certain Western Canadian Coniferous Species. JEn:Cellular U l t r a s t r u c t u r e of Woody Plants. W.A. Cote, J r . (ed.). Syracuse Univ. Press, Syracuse, pp. 551-559.