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Isolation and characterization of Douglas-fir organosolv lignin Cho, Hern J. 1981

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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), <K-hydroxypropiovanlllone (XXVII) and 7 1 O C H , OCH, H C = 0 O C H , O C H , O C H 3 OCH, H I H-C-OH I C-H II H-C O C H 3 O C H 3 (XXVII) (XXVIII) (XXIX) H I H - C - O H (XIII) (XXIV) OH (XXIII) Table 6. Rf values and characteristic colors of selective lignin degradation products and extractives(31,70,100). R.f x 100 Color on s i l i c a gel Compound B-C-M3 B-Eb B-AC M-Cd Folin-Denis Reagent Diazotized sul-f a n i l i c acid Acetyl vanilloyl(XXI) 53 47 52 83 dark brown yellow Guaiacyl acetone(XXII) 49 44 60 93 mustard brown Vanillin(XVI) 48 40 59 93 orange-red red Coniferyl aldehyde(XXVI) 42 47 55 93 wine-red red-brown <<-Hydroxypropiovani Hone ( XXVII) 36 30 37 77 yellow brown JJ-Hydroxypropiovanillone(XXVIII) 31 20 32 77 brown brown Coniferyl alcbhol(II) 28 37 40 76 blue red-brown 0<-Hydroxyguaiacyl acetone(XXIX) - - - - - -V a n i l l i c acid(XIII) 25 23 17 42 pale yellow yellow-orange w-Hydroxyguaiacyl acetone(XXIV) - 32 43 70 - pink Guaiacylglycerol-^-guaiacyl ether (XXIII) 17 - - - yellow -aBenzene-chloroform-methanol(70:28:2) bBenzene-ethanol(150:22) "Benzene-acetone(3:2) ^Methanol-chloroform(3:7) 73 Table 7. Thin-layer chromatography of water-soluble fraction from organdsolv spent liquor(after removal of carbohydrates). Cpd. Tentative Rf x 100 No. identification B-C-M* B-Eb B-AC M-Cd 1. Acetyl vanilloyl(XXI) 53 49 52 89 6 2. Guaiacyl acetone(XXII) 50 43 62 94 e 3. Vanillin(XVI) 47 41 58 92 e 4. Coniferyl aldehyde(XXVI) 44 45 54 90 e 5. «<-Hydroxypropiovanillone(XXVII) 36 29 32 79 6. |* - Hyd roxypropiovani Hone ( XXVIII) 31 19 27 74 7. Coniferyl alcohol(II) 1 37 39 1 8. V a n i l l i c acid(XIII) 25 ? ? 47 9. w-Hydroxyguaiacyl acetone(XXIV) ? 1 ? 66 10. 11. Guaiacylglycerol-fl-guaicyl ether (XXIII) Undeveloped starting material & Furfural(XXX) 17 2 ? 3 7 2 32 11 tiBenzene-chlor6form-methanol(70: 28: 2) bBenzene-ethanol(150:22) cBenzene-acetone(3:2) dMethanol-chloroform(3:7) Partially overlapped. 74 j8-hydroxypropiovanillone (XXVIII). The presence of these ketones i s a good i n d i c a t i o n of lignin-degradation by cleavage of ether bonds during the acid-catalyzed organosolv cooking. From the data presented i n Table 7, i t can be seen that be-side the Hibbert's ketones, l i g n i n molecules further proportioned into degradation products such as v a n i l l i n (XVI) and c o n i f e r y l aldehyde (XXVI), even though the amounts of these f r a c t i o n s seems to be small. K r a t z l and Paszner (100) reported that simple aqueous hydrolysis of wood at 100°C f o r 2-4 hours also y i e l d s these compounds i n somewhat larger pro-portions. TLC r e s u l t s of cook n o s . l F (5 min;0.05 N HC1) and 13 (12 min; 0.05 N HC1) were found to be s i m i l a r to those of cook no.15 (20 min;0.05 N HC1), i n d i c a t i n g that the degradation of l i g n i n molecules takes place to about the same extent during the i n i t i a l d e l i g n i f i c a t i o n period of organo-solv cooking as mentioned before. This observation i s i n good agreement with the r e s u l t s obtained from NMR spectra which w i l l be discussed l a t e r . The presence of c o n i f e r y l alcohol ( I I ) , v a n i l l i c acid (XIII) orco-hydroxyguaiacyl actone (XXIV) could not be confirmed due to the absen-ce of the corresponding spots i n the developing systems used (Table 7). For the same reason, guaiacylglycerol-jj-guaiacyl ether (XXIII), which i s pr i m a r i l y responsible for formation of Hibbert's ketones, could not be i d e n t i f i e d . Notable missing compounds are benzyl alcohols, presumably because of t h e i r i n s t a b i l i t y under a c i d i c cooking conditions. Their self-condensation i n the acid medium i s well known (141). Inasmuch as a l l of the compounds mentioned above are phenolic i n nature, i t appeared that hydrolysis of the non-carbohydrate portion of wood reduced part of the l i g n i n molecule to low molecular degradation 75 products (131). The formation of phenolic hydroxyl groups as r e s u l t of s p l i t t i n g of ^ - a r y l ether bonds of l i g n i n molecules i s another im-portant f a c t o r f o r the increased s o l u b i l i t y of lignin-degradation pro-ducts i n aqueous solution. Not s u r p r i s i n g l y , traces of f u r f u r a l (XXX), which i s a sugar degradation product (36), were also detected. There were some spots which could not be e a s i l y i d e n t i f i e d . These spots might have o r i g i -nated from some extract i v e s , such as dihydroquercetin (XXXI), because some e x t r a c t i v e deposits i n lumens were reported to r e s i s t a l c o h o l -benzene e x t r a c t i o n (33,91). OH O -(XXX) (XXXI) Since water-soluble l i g n i n or l i g n i n - l i k e compounds were not the prime target of the present study and i n s u f f i c i e n t amounts of the water-soluble f r a c t i o n were obtained, no further a n a l y s i s was car-r i e d out as mentioned before. 5.6 Microanalysis of Isolated Organosolv Lignins L i g n i n contains only carbon, hydrogen and oxygen, and the elementary compositions reported i n the l i t e r a t u r e show considerable v a r i a t i o n because of the v a r i e t y of sources and methods i n l i g n i n pre-paration. 76 The carbon content of softwood l i g n i n s are i n a range of 60. 2-67.5% and the corresponding hydrogen content ranges 4.5-6.4% (32). As shown i n Table 8, carbon content of the acetylated Douglas-fir l i g n i n samples ranged from 62.99 to 67.66%, while hydrogen contents were 4.80 to 6.14%. Average elementary composition of Douglas-f i r MWL from normal wood are reported to be 63.37% carbon, 6.07% hydrogen and 30.56% oxygen (148). In Table 8, i t can be seen that methoxyl group content of the acetylated l i g n i n generally decreased as both cooking time and acid concentration increased. The reason for t h i s observation may be ex-plained by the as s o c i a t i o n of the i s o l a t e d l i g n i n (FRACTION II) with hemicellulose contaminants (26). As the cooking time or acid concen-t r a t i o n increased, the i s o l a t e d l i g n i n f r a c t i o n seemed to contain i n -creasingly larger amounts of carbohydrate fragments due to secondary condensation between s o l u b i l i z e d l i g n i n and carbohydrate fragments. As a r e s u l t the methoxyl content decreases s i g n i f i c a n t l y . However, there are strong points against the secondary con-densation between l i g n i n and carbohydrates. As an evidence for t h i s argument, NMR spectra of the same acetylated l i g n i n samples show no signals for hemicellulose contaminants, which w i l l be discussed l a t e r . E a r l i e r an attempt to condense l i g n i n and l i g n i n model substances with f u r f u r a l gave negative r e s u l t s (169). From t h i s i t may be concluded that l i g n i n which has been i s o l a t e d from wood by hydrolysis has under-gone c h i e f l y an autocondensation i n which the functional groups of the side-chain, the phenolic hydroxyl groups, and the reactive hydrogen atoms of the aromatic rings are involved (169). There i s an a l t e r n a t i v e explanation for the v a r i a t i o n of methoxyl content with cooking period. P r o t o l i g n i n i s non-homogeneous 77 Table 8, Elementary composition of acetylated Douglas-fir l i g n i n samples. :(%) b a Sample No. Carbon Hydrogen Oxygen OCHg PC- 11 65.62 5.01 29.37 12.90(14. 84) PC- 12 64. 25 5. 29 30.46 12.42(14.37) PC- 13 64. 50 5. 22 30. 28 12.11(14. 18) PC- 14 65. 14 4.91 29.95 11.36(13.24) PC- 15 .62. 64 5.48 31. 88 11.18(13.20) PC-9 65.08 5.11 29. 81 12.33(13.54) PC- 19 66. 61 5.07 28.32 11.07(12. 80) Values i n parentheses are for parent i s o l a t e d l i g n i n . Sample number code 'PC' was used to distinguished the treated (acetylated or reduced) samples from t h e i r parent l i g n i n sam-ples (FRACTION I I ) . 78 i n terms of methoxyl content (135), and the l i g n i n f r a c t i o n s having higher methoxyl contents are most r e a d i l y l i b e r a t e d from wood during the organosolv cooking. Thus, short cooks, such as cook nos.11 (5 min) and 12 (9 min) produced l i g n i n s with higher methoxyl contents (14.17-14.6470), while long cooks, such as cook nos.9 (17 min), 14 (17 min), 19 (17 min) and 20 (20 min) gave l i g n i n s with much lower methoxyl con-tents (11. 20-13. 547o). 5.7 Spectroscopic Analyses of Isolated Organosolv Lignins 5.7.1 Nuclear magnetic resonance spectra The l i b e r a t i o n of l i g n i n from wood by acid-catalyzed hyd-r o l y s i s at high temperature y i e l d s l i g n i n with changed chemical struc-ture, even when mild reaction conditions are used (107,137). NMR ana-l y s i s i s one of the best techniques to examine the chemical changes of the l i g n i n molecules caused by the various cooking conditions. Though NMR s p e c t r a l analysis was done on the acetate derivatives of the i s o -lated (parent) organosolv l i g n i n samples, the discussion of the func-t i o n a l group contents ref e r s to the parent organosolv l i g n i n samples. A t y p i c a l NMR spectrum of the acetylated Douglas-fir l i g -n i n i s shown i n F i g . 12. To determine quantitative estimations of the functional groups, several selected ranges of {{-value were constructed according to the method used by e a r l i e r investigators (57,98,105,107). The NMR spectra of protons i n organic compounds can usually be integ-rated e l e c t r o n i c a l l y with a high degree of preci s i o n . However, the complexity of l i g n i n spectra made i t necessary to use the method men-0 ppm(«rj Figure 12. Typical NMR spectrum of acetylated Douglas-fir l i g n i n . 80 tioned above to determine the percentages of t o t a l s i g n a l strength found within the selected ranges (107). Table 9 gives the assign-ments of signals . for the selected ranges of the NMR spectrum of the acetylated l i g n i n sample, shown i n Fig, 12. Range A (5 7.20-6,15) gives signals for aromatic and <x-vinyl protons. However, since the number of unsaturated struc-tures of the v i n y l type i n l i g n i n has been shown to be very small, interference from oc-vinyl protons i s not appreciable. F i g . 12 c l e a r l y shows that the guaiacyl unit around & 6.95 predominates over that of s y r i n g y l unit around £ 6.60. Ranges B ( S 6.15-5.75), C ( J 5.75-5.15), D (S 5.15-4.45), E (<T 4.45-4.05) and G ($ 3.40-2.50) represent signals f o r ^ - v i n y l pro-tons (and some oi-protons), o<-protons, ^-protons, Jf-protons and ^-pro-tons, respectively. Range F ($ 4.05-3.40) shows signals from aromatic methoxyl protons. Ranges H (£ 2.50-2.15) and J (S 2.15-1.50) represent signals for aromatic acetoxyl protons and a l i p h a t i c protons, respectively. Table 10 shows the percentage estimations of the various proton types occurring within the assigned ranges of the integrated spectra (Figs. 13-19). From these values and the r e s u l t s from the mic-roanalysis (Table 8), contents of various functional groups of the pa-rent l i g n i n samples were estimated and the res u l t s are presented i n Table 11. Due to the lack of microanalysis data on parent l i g n i n s , accurate empirical formulae could not be calculated. However, s t r i c -t l y from the NMR spectra and elemental analysis of acetylated l i g n i n s , approximate empirical formulae could be constructed (Table 11). 81 Table 9. Assignments of signals i n NMR spectrum of acetylated Douglas-fir lignin., sample. Range Chemical s h i f t s -value (ppm) Assignment(types of proton) Symbol </" -value (ppm) A 7.20-6.15 6.95 aromatic(also some e C-vinylic) B 6.15-5.75 6.05 /8-vinylic c<-proton of side chain(^-0-4 linkage) C 5.75-5.15 5.40-5.80 c<-proton of side chain(^8-5 linkage or benzyl aryl; ether) D 5.15-4. 45 4. 55 /S-proton of side chain(£-0-4 linkage) E 4. 45-4.05 4. 25 Jf-proton of side chain F 4. 05-3.40 3. 75 (3.81) methoxyl G 3.40-2.50 2. 60-3. 40 /8-proton of side chain(y8-y5 linkage) H 2.50-2.15 2. 25 aromatic acetoxyl J 2. 15-1.50 2.00 a l i p h a t i c acetoxyl 82 85 Figure 17. NMR spectrum of sample PC-15. Figure 19. NMR spectrum of sample PC-19. CO CO 89 Table 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. (70) Symbol of range (T-value (ppm) PC-11 PC-12 PC-13 PC-14 PC-15 A 7.20-6.15 20.32 19.87 20.64 20.19 21.57 B 6.15-5. 75 2.58 1.30 2.62 2.88 3.85 C 5. 75-5.15 0.32 0. 65 0. 29 0.96 0.49 D 5.15-4.45 2.58 2.93 4.07 5. 77 2.94 E 4. 45-4.05 8.39 9.77 7.85 9.13 5.88 F 4.05-3.40 24. 84 24.10 25.00 23.03 24.02 G 3.40-2.50 5.48 8.14 8.43 9. 62 9.31 H 2. 50-2. 15 10. 00 11.07 13.03 (29.337 (31.85) J 2.15-1.50 25.48 22. 15 18.08 Combined Ranges H and J together due to overlap. Table 11. Empirical formulae and functional group contecnts of isolated,lignin samples. Sample Empirical formula Apparent Aromatic H Aromatic OH Aliphatic OH 0CH3 No. Weight of -C q-unit per C 9 7. per C 9 % per C 9 % per C9 7. . PC-11 C9H4.63°0.56<0CH3)0.97<OH>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<OCH3)0.94(OH)1.17 174.77 2.35 1.34 0. 49 4.76 0.68 6.61 0.94 16.67 PC-14 C9 H5.34°1.12< O C H3 )0.88 ( O H )1.12 174.86 2.32 1.32 a a a a 0. 88 15.60 PC-15 C9 H4.74°0.95 ( O C H3 )0.86< O H )1.14 173.69 2.32 1.34 a a a a 0.86 15.35 aThese contents were not determined due to poor resolution(overla P of signals for aromatic and aliphatic hydroxyl groups). 91 Signal assignments, 6~-Value (ppm) A: 5 = 2.0, a l i p h a t i c acetoxyl protons B: 6= 2.3, aromatic acetoxyl protons C: 6 = 3. 75, 3.80, methoxyl protons D: «S = 4.1-6.2, a l i p h a t i c protons E: <5= 6.9-7.1, aromatic protons A c B I Figure 20. D i f f e r e n t i a l NMR spectra of cooking time and acid concentration series. 92 Fig. 20 shows the comparison of NMR spectra f o r the two series of samples, i . e . cooking time series (PC-11 through PC-15) and acid concentration series (PC-9, PC-14 and PC-19). Signal broadening, which i s obvious from the appearance of the spectra i s thought to be due to a tendency toward r i g i d i t y caused by cross l i n k i n g i n the mole-cular structure of l i g n i n (107). L i g n i n obtained from the short cook (PC-11) gave more sharply defined spectrum than those from long cooks (PC-12 through PC-15 and PC-19). The reason for t h i s i s the fact that sample PC-11 has r e l a t i v e l y low molecular weight, which makes for gre-ater mobility of the molecules i n solution. The v a r i a t i o n of molecular weight of those two series of l i g n i n samples w i l l be extensively d i s -cussed l a t e r i n connection with the GPC analysis. The spectra shown i n F i g . 20 also demonstrate that the size of a l i p h a t i c acetoxyl signals at S 2.00 decreased markedly as cooking time increased. The i n t e n s i t y of the si g n a l for the a l i p h a t i c acetoxyl protons of the acetylated l i g n i n should correspond to three times of that of., a l i p h a t i c hydroxyl protons of the parent l i g n i n . The s i g n i f i -cant collapse of a l i p h a t i c hydroxyl groups i n longer cooks can be -.ex_-plained by the fact that these hydroxyl groups were used f o r carbon-carbon linkages to form self-condensation products (104). It was noted that high a l i p h a t i c hydroxy!,group content i n the parent l i g n i n was as-sociated with the. low molecular weights of the acetylated l i g n i n . This observation w i l l be discussed i n more d e t a i l l a t e r . There was no noticeable signal around 5 6,1, i n d i c a t i n g the absence of benzylic hydroxyl groups i n the parent l i g n i n samples, d i s p i t e the fact that benzyl ether content i n MWL was reported to be.more than 20 per ,100 C^-units.(2,67). Benzyl alcohol i n a c i d i c medium i s so un-stable (120) that almost complete recondensation reaction involving ben-9 3 z y l alcohol seems to take place (108) during the organosolv cooking and as a result, there were no longer free benzylic hydroxyl groups to be acetylated. Table 11 shows a much higher content of aromatic hydroxyl group i n the parent l i g n i n s than the reported values, which range 20-25 per 100 C -untis. This can be explained by the release of phenolic hyd-9 roxyl group due to almost complete s p l i t t i n g of the a l k y l - a r y l ether bonds of l i g n i n during acid-catalyzed cooking (98), Another reason for this i s the fact that the functional group contents determined by NMR spectra are usually higher than those determined by other methods be-cause of the overlap of signals from d i f f e r e n t types of protons. This comparison w i l l be discussed l a t e r with the r e s u l t s of phenolic hydroxyl determination by the spectrophotometric method. The estimated methoxyl contents range 86-97. per 100 C -units. Table 11 and Fig, 20 show that methoxyl group content of the i s o l a t e d . parent l i g n i n s decreases s i g n i f i c a n t l y as cooking time increases from 5 to 20 minutes, or as acid concentration increases from 0.025 to 0.1 N HC1. The l a t t e r confirms the dependency of methoxyl content on a c i -d i t y of cooking medium (38). In general, the methoxyl contents deter-mined by NMR spectra were higher than those determined by the TAPPI method (Table 8), as to the case of phenolic hydroxyl content, and t h i s general trend i s i n good agreement with previous r e s u l t s (57), Although the change seems to be very small, the content of aromatic protons was found to decrease s l i g h t l y as cooking time increased, as indicated i n Table 1L This fi n d i n g has a very s i g -n i f i c a n t meaning. Table 11 shows that the content of aromatic pro-tons for short cook (PC-11) i s 2,37 per Cg-unit, far smaller value than 3 protons per guaiacyl nucleus. This means, i f there were l i t -94 t i e or no s y r i n g y l units i n the Douglas-fir organosolv l i g n i n mole-cules, about 63% of the aromatic rings i n the l i g n i n molecules must be somewhat condensed form and have only two aromatic protons per Cg-unit, while about 37% of aromatic rings are non-condensed and have three hydrogen atoms on each aromatic ring of the l i g n i n mole-cule; The content of aromatic proton for long cook (PC-15) was found to be 2.32 per C^-unit (Table 11), i n d i c a t i n g 68% of aromatic rings are condensed and 32% are non-condensed. The re s u l t s s i g -n i f y the f i n d i n g that more condensed, l i g n i n s were i s o l a t e d as cook-ing time increased. A l i p h a t i c protons attached to the side-chains of l i g n i n molecules were d i f f i c u l t to estimate due to overlapping and i n t e r -ference from the strong methoxyl proton signal as mentioned before. As a r e s u l t , accurate account for the a l i p h a t i c protons was not pos-s i b l e . However, r e l a t i v e l y Tow content of these protons was be-lieved to be due to an elimination reaction of <=<- and p-protons i n the c y c l i c <=<-aryl ethers by acid hydrolysis (98,106). Methylene protons, terminal methyl protons and possibly some highly shielded a l i p h a t i c protons, which are not attached d i r e c -t l y to oxygen functions, were excluded from the cal c u l a t i o n s because they gave i n s i g n i f i c a n t signals and could hardly be distinguished from the baseline noise. There was no evidence for the presence of any contamination from carbohydrates i n the acetylated organosolv l i g n i n samples used for NMR spectra. None of the l i g n i n spectra shows any d e t e c t i b l e signals i n the S'8-11 range, i n d i c a t i n g none or very low content, i f any, of carboxylic or aldehyde protons. 95 5.7.2 Infrared spectra Infrared spectra obtained from two shortened ser i e s of reduced l i g n i n samples, cooking time seri e s (Fig. 21) and acid con-centration series (Fig. 22) were analyzed, and the r e s u l t s were com-pared with those obtained from NMR spectra of the same samples. The test was designed to confirm the s t r u c t u r a l changes of l i g n i n molecules which may have occurred as the r e s u l t s of the various cooking conditions. Since a general s i m i l a r i t y of^IR'Jspectra i n Figs. 21 and 22 i s evident f o r . a l l the samples, only those bands which varied markedly areodiscusse'd. The absorption band assignments presented i n Table 12, based on ^ e a r l i e r ..investigationsi (23; 86,121,146,147) give-a considerable degree of confidence i n most cases except i n the region 1400-1000 cm \ where various aromatic r i n g v i b r a t i o n modes and C-0 stretching modes occur (86). As with many other polymers, the complexity of l i g n i n molecules causes band overlapping , d i f f u s e bands, aridecross-linking may dampen v i b r a t i o n s (23). Nevertheless, as evident i n Figs. 21 and 22, s t r u e t u a l f .changes ; i n the l i g n i n molecules due to the d i f f e r e n t cook-ing conditions can be observed. In general, samples c o l l e c t e d a f t e r short cooking gave sharper absorption bands i n t h e i r IR spectra upon reduction. On the hand, an increase i n the acid concentration of the cooking l i q u o r did not change the absorption bands markedly. Broad bands at 3440-3460 cm"1 shown i n Figs. 21 and 22 are due to 0-H stretching v i b r a t i o n and medium or weak bands at 1390 cm 1 seem to be due to 0-H bending v i b r a t i o n . Changes of these bands i n . , I I • I I I I 1 '—X 3600 3400 3200 3000 2800 1800 1600 1400 1200 1000 Wave-numberCcm*1 ) Figure 21. E f f e c t of cooking time on IR spectra of reduced Douglas-fir l i g n i n samples. 97 3600 3400 3200 3000 2800 1800 1600 1400 1200 1000 Wave-numberCcm"1) Figure 22. E f f e c t of acid concentration on IR spectra of reduced Douglas-fir l i g n i n samples. Table 12. Assignments of absorption-bands i n IR spectra of reduced Douglas-fir l i g n i n samples. Wave-number(cm ^) Assignments 3440-3460 0-H stretching v i b r a t i o n 2990, 2940, 2850 C-H stretching v i b r a t i o n 1710, 1690, 1640 C=0 stretching v i b r a t i o n 1560 aromatic s k e l e t a l v i b r a t i o n 1470 C-H deformation v i b r a t i o n 1430 C-H bending vibration/aromatic s k e l e t a l v i b r a t i o n 1390 0-H bending v i b r a t i o n ( ? ) 1300 condenced guaiacyl(?) 1260 uncondencd guaiacyl 1230, 1190 asymmetric stretching v i b r a t i o n of aryl-- a l k y l ether(?) 1100 uncondenced guaiacyl(?) 1010 condenced guaiacyl(?) 900 C-H out-of-plane bending v i b r a t i o n 99 various spectra were found to be i n s i g n i f i c a n t , i n d i c a t i n g that the t o t a l hydroxyl (aromatic and a l i p h a t i c ) content changes very l i t t l e - w i t h d i f -ferent cooking conditions. Bands at 2990-2850 cm ^ are c h a r a c t e r i s t i c of various types of C-H bonds. No s i g n i f i c a n t e f f e c t of cooking conditions on the inten-s i t y of these bands was noted, though sample PC-11 (5 min;0.025 NHC1) shows l i t t l e sharp bands. Since the l i g n i n samples were reduced with sodium borohyd-ride, absorption bands which are usually well defined ones f o r car-bonyl group seem to disappear at 1735 cm * and 1375 cm * (146). The IR spectrum of the sample PC-11 shows a strong peak at 1690 cm ^ while spectra f o r the other samples show only ? trace or very weak peaks at t h i s wave number (Fig. 21). Normally, t h i s i s very d i f f i c u l t to exp-l a i n because both unconjugated ketones and conjugated acid or esters absorb at 1715 cm which can be s h i f t e d to 1690 cm * (146). However, a possible explanation for t h i s observation can be found because of the f a c t that the i n t e n s i t y of the band decreases gradually as cooking time increases. As mentioned e a r l i e r i n NMR spectroscopic analysis, there was no evidence f o r any aldehyde protons or carboxylic protons i n the acetylated l i g n i n samples including PC-11. Therefore, the strong band at 1690 cm ^ i n the IR spectrum of PC-11 i s thought to be o r i g i n a -ted from -^(.conjugated ketone and seems to be a t t r i b u t a b l e to the pre-sence of impurities (26,40,86), which can be affected by cooking time. The most probable impurities with unconjugated ketones are flavones, such as dihydroquercetin (XXXI), which can cause a s h i f t of about 60 cm ^ to the longer wave-length due to 0-hydroxyl group chelated to the carbonyl group (26,86). 100 Dihydroquercetin (XXXI), which has been i d e n t i f i e d as major flavanohe of Douglas-fir (33,141), can not be completely removed by alcohol-benzene extraction as mentioned e a r l i e r , and thus i s believed to be l i b e r a t e d from sawdust together with l i g n i n during organosolv-cooking. Because of low y i e l d (9.12%) of the i s o l a t e d l i g n i n (FRACTION II) from short cook (PC-11;5 min), the r e l a t i v e concentration of t h i s impurity i s high, while the i n t e n s i t y of the peak for t h i s impurity i n the sam-ple from long cook (PC-15;20 min) i s almost unnoticed due to high y i e l d (29.12%) of the i s o l a t e d l i g n i n sample (Table 3), As a r e s u l t , the s t -rong peak at 1690 cm 1 decreases as cooking time increases. Probably, the most s i g n i f i c a n t observation from F i g . 21 i s that the i n t e n s i t i e s of the bands at 1260 cm 1 and 1100 cm 1 f o r uncon-densed guaiacyl nucleus decrease s u b s t a n t i a l l y as cooking time increases, confirming the r e s u l t s obtained from NMR spectra. Analysis of NMR spect-ra indicated that non-condensed aromatic n u c l e i were 37% f o r short cook sample PC-11 (5 min)and those f o r long cook sample PC-15 (20 min) were 32% (Table 11). This observation i s the most important evidence f o r high-er ^ molecular, weights of samples from longer cooks.. This w i l l be discussed l a t e r in. connection with GPC analysis., A somewhat smaller decrease i n longer cooks was also noted f o r the band at 1230 cm 1 ( F i g . 21), which has been assigned to asym-metric C-0 v i b r a t i o n s of a r y l - a l k y l ethers. The recondensation reaction, which r e s u l t s i n high molecular weights of l i g n i n samples from longer cooks, must have followed the i n i t i a l s c i s s i o n of a r y l -a l k y l ether linkages due to the prolonged cooking. This evidence was also confirmed by NMR analysis. 101 Methyl and methylene groups, and ethylenic double bond were not analyzed due to the lack of supporting evidences i n NMR spectra. Estimation of methoxyl content by IR spectra (80,147) was not attempted because of poor r e s o l u t i o n between wave-number 1200-1470 cm \ and broadness and overlapping of the absorption bands as mentioned e a r l i e r . The IR spectra of unreduced l i g n i n samples may be more informative as to the o r i g i n a l conditions However, unreduced sam-ples were observed to be less stable since a continued color change was evident on standing at room temperature. 5.7.3 U l t r a v i o l e t spectra A s e r i e s of reduced l i g n i n samples were"analyzed: using UV spectrophotometetr t o examine t h e - e f f e c t of cooking time on the con-tent of'? phenolic—hydroxyl group. F i g , 23 shows that a l l f i v e sam-ples have somewhat ^similar UV spectra. The maxima appeared around 294 to 302 nm of the difference curve which i s c h a r a c t e r i s t i c f or only the phenolic hydroxyl group (11,12,77,131). The method used i n the present study i s based upon the c h a r a t e r i s t i c UV absorption of phenols i n a l k a l i n e solution. According to Aulin-Erdtman (11,12), the bathochromic s h i f t of the c h a r a c t e r i s t i c 280 nm absorption maximum of l i g n i n i n a l k a l i n e s o l u t i o n takes place due to the i o n i -zation of the phenolic group. In the present method, UV dif f e r e n c e curves were obtained d i r e c t l y by scanning the a l k a l i n e vs. the neutral l i g n i n solutions, r e s p e c t i v e l y placed i n the sample and reference c e l l s of'the spectro-photometer (77). \ The r e s u l t s shown i n Table 13 indicate that the 102 0 J — I 1 1 1 i i • . I 270 280 290 300 310 320 330 340 350 Wavelength(nm) Figure 23. Difference UV spectra of reduced l i g n i n samples. Table 13. Spectrophotometric determination of phenolic hydroxyl group i n Douglas-fir l i g n i n . Sample No. A max. Absorbance /Aa,max. Phenolic OH (nm) (1/g-cm) (%) PC-11 294 0.38 4. 75 1.97 PC-12 296 0.44 5.50 2. 28 PC-13 294 0.46 5.75 2.39 PC-14 299 0. 55 6.88 2.86 PC-15 302 0.47 5.88 2.44 104 phenolic hydroxyl group content increased as cooking time increased (up to 17 min) and then s l i g h t l y decreased, confirming the s i m i l a r r e s u l t s of NMR spectra analysis. The calculated phenolic hydroxyl content of t h i s series ranged from 1.97 to 2.86% (based on t o t a l weight of l i g n i n ) according to the equation provided by Goldschmid. (77). This content based on l i g n i n weight i s equivalent to 0.20 to 0.29 hydroxyl groups per Cg-unit, i f converted by using the apparent unit weights l i s t e d i n Table 11. This content i s s l i g h t l y lower than the recorded phenolic hydroxyl contents (0.27-0.29/C -unit) of several softwood MWL (148). y Considering these r e s u l t s i n r e l a t i o n to those obtained from NMR spectra, i t -.seems'likely"that-mainly'the active aromatic hydrogens, rather than phenolic hydroxyl groups, are involved i n the-recondensation reaction of.lrgnrris"to form"C-C linkages because the contents of aromatic hydrogen was found to decrease as the cooking time increased, whereas the phenolic hydroxyl group content a c t u a l l y increased.-.t It i s s i g n i f i -cant that, the net hydroxy! aontent increased gradually due "to a possible cleavage -of the a r y l - a l k y l ether linkages as cooking time increased, even though some of the phenolic hydroxyl groups might have been consumed i n the recondensation.reaction. As mentioned above-*., the phenolic hydroxyl content, deter-mined by the spectrophotometry method, was lower than that obtained from NMR spectra (Table 11). There i s one serious l i m i t a t i o n i n the method used f o r t h i s test. Because of the assumption that every phenyl propane unit of l i g n i n c a r r i e s one methoxyl group, t h i s method can not be applicable to those l i g n i n samples which contain s y r i n g y l n u c l e i (77). 105 5. 8 Macromolecular Analysis of Isolated Organosolv L i g n i n 5. 8.1 Gel permeation chromatographs 5.8.1.1 E f f e c t of cooking time on molecular weight According to Goring (81), during the chemical d e l i g n i f i -c a tion process, penetration by cooking l i q u o r into the secondary wall occurs i n i t i a l l y and the low molecular weight l i g n i n of the secondary c e l l w all i s the f i r s t to be extracted. As cooking time increased, penetration by the cooking l i q u o r into the middle lamella occurred.and l i g n i n of high molecular weight was extracted. Thereby, both Mw and Mn changed as cooking time increased as shown i n Table 14. Mw and Mn f i r s t increased up to about"12 minutes and then decreased as cooking period further increased up to 20 minutes ( F i g . 24). Lora and Wayman (104) suggested e a r l i e r that there are two reactions involved during the d e l i g n i f i c a t i o n under a c i d i c condi-tions. The f a s t e r f i r s t reaction, p r i m a r i l y occurring by breaking of lignin-carbohydrate bonds, produces soluble l i g n i n f r a c t i o n s and the slow second reaction, which i s e s s e n t i a l l y a condensation, r e s u l t s i n insoluble l i g n i n f r a c t i o n s i n the presence of the organic acids ( a c e t i c acid and formic acid) formed during the autohydrolysis. The r e s u l t s shown i n Table 14 c l e a r l y demonstrate that at the beginning of cooking, the rate of d e l i g n i f i c a t i o n was fast due to rapid increase of the tem-perature of the cooking mixture (94,96), and*resulted i n low molecular weight l i g n i n f r a c t i o n s . However, as cooking time increased, a slow recondensation s t a r t s to produce hl"g-He'rX.moi6ouiar.:,wtaight' -lign i n -frae-106 Table 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. Sample No. Cooking time(min. ) Acid(HCl) con-c e n t r a t i o n ^ ) Mw Mn Mw/Mn PC-9 17 0.025 7,675 996 7.71 PC-11 5 0.050 7,984 1,092 7.31 PC-12 9 0.050 9, 821 1,127 8. 73 PC-13 12 0.050 10,122 1,144 8. 85 PC-14 17 0.050 9, 293 1,002 9. 27 PC-15 20 0.050 8, 762 823 10.65 PC-19 17 0. 100 10,186 1,005 10. 13 0.025 0.05 0.075 0.1 1,200 1,100 PC-11 PC-12 P C - 1 3 ^ - ^ £ 1,000 PC-14\ 900 800 • PC-15 •' 5 10 15 Cooking.time (min.) 20 0.025 0.05 0.075 0.1 HC1 concentration (N) Figure 24. Effect of cooking conditions on molecular weight averages of acetylated l i g n i n samples. 108 M.W.2,500 130 120 110 100 90 80 70 60 Counter No. 100 1,000 10,000 100,000 Molecular weight Figure 25. Change i n molecular weight d i s t r i b u t i o n with cooking time. 109 tions (104), By the time (around 12 min) the temperature reaches 200°C, however, the extracted l i g n i n i n the cooking l i q u o r i s subject to degradation due to a c i d i c condition (0.05 N HC1) as cooking time i s prolonged. This r e s u l t s i n a decrease of Mw and Mn as shown i n Table 14 and F i g , 24. F i g . 25 suggests that there i s no substantial change i n the predominant f r a c t i o n (main peak around molecular weight 2,500) of the ;acetylated lignin'samples, even i f cooking time i s chan-ged to 20 minutes. A short cook (PC-11;5 min) gives a narrow main peak while longer cooks (PC-12 through PC-15;9-20 min) give broader peaks which are more or less symmetrical i n shape. Similar d i s t r i -butions were also reported i n e a r l i e r studies (35,89). In the MWD curve (Fig. 25) of the longest cook (PC-15;20 min), i t can be seen that a very d i s t i n c t f r a c t i o n ranged between 10,000 and 50,000. This observation m a y be explained by the s e l f -condensation of l i g n i n through C-C linkages due to carbonium ions formed i n d i f f e r e n t positions on the side-chain of the phenyl pro-pane units under a c i d i c conditions during prolonged periods of cook-ing time (35,165). Somewhat-similar high molecular weight f r a c t i o n s were also found i n the other samples (Fig.25). The unusually low contents of hydrogen and oxygen i n these l i g n i n samples, as mentioned i n NMR s p e c t r a l analysis, may be the d i r e c t r e s u l t of the formation of new C-C linkages. NMR spectra i n F i g . 20 c l e a r l y i l l u s t r a t e that the sharp s i n g l e t s i g n a l f or a l i p h a t i c hydroxyl groups (£ 2.0) c o l -lapses s i g n i f i c a n t l y as cooking time increases. There i s another explanation f o r the high molecular weight l i g n i n f r a c t i o n s . Sarkanen et a l . (151), i n t h e i r study on hardwood l i g n i n s from organosolv cooking (temperature«135-165°C;catalyst«alumi-num chloride;cooking time=l-6 hrs), found that organosolv l i g n i n s have a 110 tendency to form high molecular weight associated complexes even under quite strongly a l k a l i n e aqueous conditions. Even though there i s no evidence f o r t h i s tendency i n the present test, i t i s quite possible that high molecular weight associated f r a c t i o n s might have formed i n the acetylated samples used for GPC analysis. Another s i g n i f i c a n t observation i s the small, but very d i s -t i n c t peaks around molecular weight 96-108 (Fig. 25). E s p e c i a l l y , sam-ple PC-15 c l e a r l y shows three peaks i n i t s MWD curve around molecular weights of 108, 186 and 273, which can be interpreted as monomer, dimer and trimer of the l i g n i n guaiacyl unit, respectively. These peaks seem to represent the degradation products formed during the cooking period. Recently, Sarkanen et al.(150) noted f i v e or s i x peaks i n the lower molecular weight region i n the MWD curve of organosolv l i g n i n from cottonwood by Sephadex G-50. Smaller f r a c t i o n s than tetramer were re-ported., to be i d e n t i f i e d by gas chromatography by Dimmel et al.(49), i n t h e i r study on molecular weight changes i n l i g n i n during anthra-quinone-alkali pulping. Table 24 also shows that p o l y d i s p e r s i t y i n -dex (Mw/Mn) increases as cooking time increases, r i s i n g from 7.31 for a 5-min cook (PC-11) to 10.65 f o r a 20-min cook (PC-15). Such high values of p o l y d i s p e r s i t y , i n d i c a t i n g a wide range of MWD, are not un-usual for these l i g n i n samples because of simultaneous formation of low molecular weight degradation products and high molecular weight recondensation products during cooking as mentioned before. 5.8.1.2 E f f e c t of acid concentration on molecular weights Kosikova and P o l c i n (97) reported that d e l i g n i f i c a t i o n with hydrochloric acid i n aqueous organic solvent i s b a s i c a l l y an I l l o C 0) 3 cr v i-i P M M.W. 94-96 M.W. 1,850 f A PC-9 A PC-14 A PC-19 M.W. 20,000 130 120 110 100 90 80 70 60 Counter No. 100 1,000 10,000 100,000 Molecular weight Figure 26. Change i n molecular weight d i s t r i b u t i o n with acid concentration. 112 a c i d o l y t i c s p l i t t i n g of the l i g n i n macromolecules and of the l i g n i n -carbohydrate complex in t o i n d i v i d u a l components soluble i n the cook-ing l i q u o r . As indicated i n Table 14, and Fig. 24, Mw shows a large increase while Mn shows a moderate increase as acid concentration increased from 0.025 to 0.1 N. The MWD curve f o r sample PC-19 (0.1 N HC1;17 min) shows a new peak around molecular weight 20,000. This high molecular weight f r a c t i o n may be the products of the l i g n i n recondensation re-action or associated complexes, as mentioned before. The balance between decomposition and recondensation i s evidently one of the deciding fac-tors f o r MWD of low and high f r a c t i o n s of the i s o l a t e d organosolv l i g -nin, and the two may be taken as r e s u l t s of two competing reactions. One of the most i n t e r e s t i n g observations i n F i g . 25 i s the s h i f t of the predominant peak toward the high molecular weight end of the MWD curve as acid concentration increased. The main peaks of samples PC-9 (0.025 N HC1), PC-14 (0.05 N HC1) and PC-19 (0.1 N HC1) appear at molecular weights of 1,850, 2,500 and 3,400, respec-t i v e l y . The s h i f t of the main peak, which represents the predominant f r a c t i o n of the i s o l a t e d l i g n i n , toward the highermolecular weight end seems to be the main reason for the increase of Mw and Mn as acid s con-centration increased. The increase of the p o l y d i s p e r s i t y index from 7.71 to 10.13 i s also observed as acid concentration increased from 0.025 to 0.1 N (Table 14). Preliminary cooking experiment with the same a c i d i c condition (0.025 N HC1) i n methanol-water (7:3 by volume) as cooking liquor, resulted i n a much lower "polydispersity index (3.68), i n d i c a t i n g that not only acid concentration, but also solvent power a f f e c t s the p o l y d i s p e r s i t y of molecular weight of the i s o l a t e d organo-113 solv l i g n i n s . These r e s u l t s prove that acetone-water i s a better s o l -vent system.for rapid d e l i g n i f i c a t i o n i n organosolv pulping. 5.8.2 Scanning electron photomicrographs The wide range of l i g n i n p a r t i c l e s i z e can be seen i n the scanning e l e c t r o n photomicrographs (Figs. 27 and 28). For example, the s p h e r i c a l p a r t i c l e s of sample PC-15, having the highest value of p o l y d i s p e r s i t y index, includes-.a wide*range of p a r t i c l e sizes from smaller than 25 nm up to 500 nm (Figs 27 and 28, and'Table 15). The larger p a r t i c l e s which seem to be aggregates of the smaller granules (137,138) may be the high molecular weight f r a c t i o n s shown i n the MWD curves ob-tained by GPC. To c o r r e l a t e MWD curve and p a r t i c l e s i z e d i s t r i b u t i o n (PSD) of the same l i g n i n samples, 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 d i a -grams based • on:,i cooking time ( F i g . 29) and acid concentration (Fig. 30) were constructed. Similar patterns of d i s t r i b u t i o n of MWD curve and PSD were found for both the cooking time series (Figs. 25 and 29) and the acid concentration s e r i e s (Figs. 26 and 30). There i s no hard evidence, however, as to whether there i s a d i r e c t r e l a t i o n s h i p between the MWD curves obtained from GPC and PSD of the same l i g n i n samples. In a l l SEM photos, there i s no evidence of carbohydrate con-tamination of the acetylated organosolv l i g n i n samples, thus confirming the r e s u l t s of NMR spectra analysis on the same l i g n i n samples. 114 PC-13 (12 min) PC-15 (20 min) Figure 27. Scanning e l e c t r o n photomicrographs of p a r t i c l e s i z e v a r i a t i o n with cooking time. PC-9 (0.025N HC1) Figure 28. Scanning electron PC-14 (0.05N HC1) lotomicrographs of p a r t i c l e size PC-19 (0.IN HC1) i a t i o n with acid concentation. Table 15. Frequency of p a r t i c l e sizes of aeetyla-ted; l i g n i n samples. \Sample No. Diameter>. -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. 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