DEGRADATION OF LIGNIN BY OZONE
by
Reginald A.D. Mbachu, B.Sc. (Hons) @ (University of Nigeria, Nsukka)
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements for the degree of Doctor of Philosophy
Department of Chemistry, April 1979 McGill University, Montreal, P.Q. i
ABSTRACT
A physicochemical study of the mechanism of lignin degradation by ozone has been made using spruce periodate and cuoxam lignins and spruce wood protolignin. The ozonization was carried out'in 45% aqueous acetic acid. In all cases, degradation was found to follow first order kinetics indicating a similar reaction mechanism and a single rate controlling process. From
the rate c~4stants obtained, it was concluded that the carbohyd rate moieties in wood do not affect the rate of protolignin degradation and dissolution. Spectroscopic studies of the alkali soluble degradation products of lignin showed that the chemical mechanism is probably electrophilic, involving the attack on phenol ether bonds, methoxyl groups, aromatic and other un saturated structures and the formation of carboxyl groups. De carboxylation occurs in the later stages of the reaction. From a consideration of the possible reactions that may degrade the lignin network, it was concluded that the observed phenol ether bond cleavage is the principal network-degrading reaction during ozonization. A tentative mechanism has, therefore, been proposed for the cleavage of the phenol ether bonds in lignin. Molecular weights and molecular weight distributions of the alkali-soluble degradation products, as studied by gel permeation chromatography and ultracentrifugation, indicate that a random heterolytic degradation mechanism is involved. The significance of the various 0· results in relation to the lignin architecture is discussed. ii
Finally, a study was made of the effect of ozone on cellulose and hemicelluloses during the delignification of wood. The results are discussed in the light of a potential pulping method using ozone. iii
RESUME
Une etude physicoch:imi.que du rrecani.sn:e de degradation des lignines
/ / periodate et cuoxame de 1' epinette et de la protolignine du l:ois d 'epinette a ete' ' effectuee.I L •ozonisation fut acccmplie en solution aqueuse d' acide acetique a 45%. Une cinetique de premier order lors de la degradation fut obtenue dans les trois cas, indiquant un m9can.isrre de reaction similaire et que la degradation e5t controlee par une seuie etape. A partir des constantes de vitesse abtenues, il fut conclu que les hydrates de carbone dans le bois n' influencaient r::as le taux de degradation et de dissolution de la lignine.
Des etudes spectroscopiques sur les produits de de9-radation de la lignine
/ / qui sont solubles en milieu alcalin, ont rrontre que le m=canisrne de la
reaction est fort probablem:mt de type e'lectrophilique 1 CattpJrtant une attaque des liens ether ~lique, des groupes rcethoxyles, arorratiques et autres structures insaturees, ainsi que la forma.tion de groupes carboxyl
iques. La decarroxylation se produit a des stades ulterieurs de la reaction.
/ / / / En considerant les reactions qui peuvent possiblernent degrader le reseau de la lignine, il fut conclu que . la scission du lien ether phenolique est
la reaction principale de degradation du reseau ~t 1 'ozonisation. Un rrecanisrre possible pour la scission du lien ether phenolique de la
\ / / lignine fut des lors propose. Les poids rroleculaires ainsi que les dis-
tributions de poids :rroleculaires des produits de de9radation solubles en milieu basique, furent etudies par chrarnatograrru.e en perm:i'ation de gel et
/ / / / _, par ultracentrifugation. Les resultats on indique un mecanisme heteroly- tique de degradation de la lignine s 'effectuant au hasard. L' i.rrq;x::Jrtance de
/ / ces divers resultats sur 1' architecture de la lignine et consideree. Finale:-
/ rrent, une etude fut faite sur l'effet de l'ozone sur la cellulose et sur iv
/ . ,• ,. 1 'hemicellulose pendant la delignification du bois. I.Bs resultats sont discute's a la lumiere d 1 une rre"t..i']ode fOSSible de reduction en pate qui utiliserant l'ozone. V
FOREWORD
The use of ozone as a delignifying agent has recently attracted attention owing to its potential as an answer to the increasing pollution problems encountered in the pulp and paper industry. A detailed understanding of the lignin-ozone reaction is expected to facilitate efforts to optimize ozone delignification processes. The work described in this thesis is aimed at elucidating the mechanism of lignin degradation by ozone. The work was carried out in the physical chemistry division of the Pulp and Paper Re search Institute of Canada at McGill University, under the super vision of Dr. R.St. John Manley. The thesis comprises five chapters, four appendices and
two sections in which claims to original research and suggestions for further research are outlined. Four of the chapters are written in the form of scientific papers and may be submitted for public ation with minor alterations. The scheme of the thesis is as follows: CHAPTER 1 - a general introduction in which topics relevant to the investigation are discussed. The topics include the chemistry of wood, the structure and architecture of lignin, the theory of gelation, the nature of the reaction between lignin and ozone, the use of ozone in pulping and bleaching and the,degradation of carbohydrates during ozone delignification of wood. CHAPTER 2 - a study of the kinetics of lignin degradation by ozone. CHAPTER 3 - a structural investigation of the alkali-soluble 0 degradation products of lignin obtained during ozonization. CHAPTER 4 - a study of the molecular weights and molecular weight vi
distributions of the alkali-soluble degradation products ob- tained during the ozonization of lignin. CHAPTER 5 - an investigation of the fate of the carbohydrates during the degradation of the protolignin in wood by ozone. APPENDICES - supplementary material such as detailed experimental procedure, data, calculations and theory of the techniques employed in the investigation. The thesis closes with claims to original research and suggestions for further work. A glossary of symbols is also included at the end of the thesis.
R.A.D. Mbachu April, 1979. vii
ACKNOWLEDGEMENTS
The author wishes to express his sincere gratitude to all those who have contributed to the completion of this work. He is particularly indebted to: Dr. R. St. John Manley, for his able guidance, patience and assistance throughout the investigation and in the preparation of the thesis; Dr. H.I. Bolker for valuable critical comments and very useful suggestionsi Drs. D.A.I. Goring, D.G. Gray and A.S. Perlin for helpful discussions: Dr. G.K. Hamer, for proton magnetic resonance spectroscopy; Mr. W.Q. Yean, for the molecular weight determinations; Mr. V. Berzins, for chemical analysis;
Mr. G. Suranyi, for general assistance in the laboratory; Mrs. c. Ewan and s. Shaieb for typing this thesis; Fellow graduate students for their friendship and helpful discussions;
McGill University, for a Demonstratorship, 1975-1976; The Pulp and Paper Research Institute of Canada, for a Studentship, 1976-1979, laboratory accommodation and the use of their facilities and services.
The Federal Nigerian Government for a Scholarship in 1976. viii
Last but not least, I wish to express my indebtedness 0 to Kate for her understanding, moral support and encouragement throughout this work. ix
TABLE OF CONTENTS
CHAPTER 1
GENERAL INTRODUCTION ...... 1
THE CHEMISTRY OF WOOD ••••••••••••••••••••••••••••••• 4
STRUCTURE AND ARCHITECTURE OF LIGNIN...... 7
THEORY OF GELATION . . • . . . . • . . . • • • . . • . . . • . . . • • . . . • • • . . 17 Critical condition for gelation in cross-linked systems ...... 20 Weight fraction of sol in a gelled polymer ...... 23 Degree of polymerization and weight fraction of sol ...... 2 6 Molecular size distribution in infinite networks. 29
THE NATURE OF THE LIGNIN-OZONE REACTION...... 34
Ozone • • • • • • • . . . • • . • • • . • • • • • . . • • . • • • • . • • • • . • • • . . • 34 Ozonolysis of lignin .•...... •..•...... 36
THE USE OF OZONE IN BLEACHING AND PULPING •.•..•.•.•. 41
DEGRADATION OF CARBOHYDRATES BY OZONE ....•....•....• 43
Cellulose ...... 4 3 Hemicelluloses ...... 44
RATES OF SOLUBILIZATION OF WOOD COMPONENTS...... 44
SCOPE AND AIMS OF THE THESIS • ...... • . . . • ...... 4 5 REFERENCES ...... 48
CHAPTER 2
DEGRADATION OF LIGNIN BY OZONE (I) THE KINETICS OF LIGNIN DEGRADATION BY OZONE ...... 56 X
0 ABSTRACT ••••••••••••.•••••••.••••••••••.••.•••••••• 57 IN'rRODUCTION ...... 58 EXPERIMENTAL ...... 61 Ozonization of periodate, cuoxam lignin and spruce protolignin ..•..••...•....•.•....•.••.•. 61
Recovery of the alkali-soluble degradation products ...... 6 4
Estimation of lignin in treated wood samples... 64
RESULTS AND DISCUSSION . . • • • . . . . • ...... • • • ...... 6 6
Effects of stirring on yield of lignin ...... •. 66
Rate of degradation of the isolated lignins .... 69
Consumption of ozone by lignin ....••.•.•.••.••. 72
Rate of degradation of spruce protolignin .•...• 75
Comparison of rate constants of degradation.... 80
CONCLUSION 81
REFERENCES 83
CHAPTER 3
DEGRADATION OF LIGNIN BY OZONE (II) SPECTROSCOPIC STUDIES OF THE ALKALI-SOLUBLE DEGRAD- AT ION PRODUCTS . • . . • . . . . . • ...... • • . • . . • • • • • . . • • . • 8 5
ABSTRACT • • • • • • • • • • • • • • • • • • • • • . • • • • • • • • • • • • • • • • • • • • • 8 6
INTRODUCTION 88 EXPERIMENTAL ...... 91 Infra-red analysis ...... 91
Proton magnetic resonance spectroscopy .•...•... 91
Ultra-violet analysis . . . . • . • . . • . • . . . . . • . • • . . • . . 91
Sodium borohydride reduction...... 92 xi.
0 RESULTS AND DISCUSSION ...... 93 Infra-red analysis of the soluble products ...•... 93 Proton magnetic resonance spectra of soluble products ...... 99 Ultra-violet analysis of the soluble products 106 Phenolic hydroxyl content of soluble products 110 Difference spectra of soluble products .•.•...... 117 Acetic-acid soluble degradation products •...... 121 CONCLUSION 124 REFERENCES ...... 126
CHAPTER 4
DEGRADATION OF LIGNIN BY OZONE (III) MOLECULAR WEIGHT AND MOLECULAR WEIGHT DISTRIBUTION OF THE ALKALI-SOLUBLE DEGRADATION PRODUCTS ...•....•...•.. 130
ABSTRACT • • . . • • . . . • . . . • . . • • . • . . . . • . . • . . • • . . • . • • . • • . • . . 131 INTRODUCTION 132 EXPERIMENTAL 135
Calibration of Sephadex G-100 column 135 Calibration of Sephadex G-200 column ...... 136 Gel permeation chromatography of the soluble prod- ucts ...... 139 Molecular weights of soluble products •.•.•••••..• 141 RESULTS AND DISCUSSION • . . . . . • ...... • . • • • . . • . . . • . . • 142 [1] Molecular weight distributions .....••..•.•....•.. 142 (a) Degradation products of isolated lignins ....• 142 {b) Degradation products of spruce protolignin .•. 144 xii
[2] Interpretation of chromatograms ...•.••.••••••...• 146 [3] Molecular weights of soluble products ...... •...• 148 [4] Elemental analysis 154 [5] Proposed mechanism of lignin degradation by ozone. 156 CONCLUSION 159
REFERENCES ...... •· 160
CHAPTER 5
DEGRADATION OF LIGNIN BY OZONE (IV) THE FATE OF THE CARBOHYDRATE MATRIX DURING THE DEGRAD ATION OF SPRUCE PROTOLIGNIN ••••••••••••••.••••••••••••••• 163
ABSTRACT . . . . • . • . . . • . . . • ...... • . • • . . . . • . • ...... • . . . • 16 4 INTRODUCTION ...... 165 EXPERIMENTAL 168 A) Ozonization of spruce wood meal •••.•••••••.••. 168 B) Determination of lignin • • • . • . • • . . • • • . . • . . • • • • • 16 8 C) Preparation of holocellulose .•••••••.••••••••. 169 D) Carbohydrate determination 170 E) Determination of viscosity 170 RESULTS AND DISCUSSION 171 1) Total yield and lignin content ..•••.••.••.•.•• 171 2) Comparison of the rates of degradation of a-cellulose, hemicellulose and lignin .•.•••••• 176
3) Retention of hemicelluloses and pulp properties 180 4) Degree of polymerization and pulp properties •. 181 CONCLUSION ...... 184 REFERENCES ...... 186 xiii
APPENDIX 1
DETERMINATION OF OZONE CONCENTRATION 189 Determination of ozone consumed by lignin .•...••• 192 REFERENCES 193
APPENDIX 2
CHARACTERISTIC IR BANDS AND CHEMICAL SHIFTS OF PROTON IN LIGNIN ...... • ...... • ...... 19 4
APPENDIX 3
THEORY OF GEL CHROMATOGRAPHY . • • • • • • • • . • • • • • . • • • • • • • • . • • • . 19 8 Resolution in gel permeation chromatography ....•. 204 REFERENCES 208
APPENDIX 4
DETERMINATION OF WEIGHT AVERAGE MOLECULAR WEIGHTS 209
THEORY • • • • • • • • • • • • • • • • • • • • • . • • • • • • • • . . • • • . • • • • • • • • • • • 210 EXPERIMENTAL ...... 212 CALCULATIONS ...... 213 DETERMINATION OF PARTIAL SPECIFIC VOLUME ...•...•••••• 214 DETERMINATION OF THE REFRACTIVE INDEX INCREMENT ••••.• 218
RESULTS • • . • • • ...... • . . • • . . . • ...... • • ...... • . . . 220
REFERENCES . • . • • . • . • ...... • . • • . . • . . . • . . • . . 2 4 5 CLAIMS TO ORIGINAL RESEARCH • • . • • • . . • • . . • • . • • • . . • . • . . . . • • • 246 SUGGESTIONS FOR FURTHER RESEARCH ••.•.••.•..•.••••...••••. 249 GLOSSARY OF PRINCIPAL SYMBOLS .•••••••.•••••••••.•••.•••.• 252 xiv
LIST OF FIGURES
FIG. No.
CHAPTER 1
1.1 Structure of cellulose .•.•••..•.•••.•.....•••• 6
1.2 Structure of the two main softwood hemi- celluloses ...... 6
1.3 The elementary building block of lignin ••.••.• 9 1.4 Resonance stabilised free radicals produced by
the dehydrogenation of 1-p-hydroxypropene .•.•. 10
1.5 Freudenberg's representation of spruce lignin .. 12
1. 6 (a) Basic monomeric unit of softwood lignin •..••.. 13
{b) Basic monomeric unit of hardwood lignin .....•• 13
1. 7 (a) Structural representation of a linear polymer .• 19
(b) Structural representation of a branched polymer. 19 (c) Structural representation of an infinite network-
polymer ...... 19
1.8 A section of a network formed by random poly functional condensation .•..••...... •.•.•..• 21
1.9 Polyfunctional condensation without network
formation ...... 21
1.10 A section of a network of cross-linked primary
chains ...... 2 2
1.11 The weight fraction of sol as a function of
cross-linking density ..•.•••••.•.•..••••••..... 27
1.12 The weight fraction distribution of species of
various complexities in a random trifunctional XV
FIG. No.
condensation ...... 31
1.13 Weight fraction distribution of chains of
various complexities in a system of cross-
linked primary chains .•.••••...•..•.•.••..... 33
1.14 Hybrid structures of the ozone molecule ...... 35 1.15 Mechanism of ozonolysis of lignin model corn-
ponents as proposed by Hatakeyama et al...... 38
1.16 Mechanism of ozonolysis of lignin model corn-
pounds as proposed by Kratzl ~al...... •.... 39
CHAPTER 2
2.1 Diagram of the experimental arrangement used
for the ozonization of lignin...... 63
2.2 Diagram of the Reactor used for ozonization of
lignin ...... 65
2.3 Yield of undissolved periodate lignin as a
function of the duration of ozone treatment 67 2.4 Yield of undissolved cuoxam lignin as a func-
tion of the duration of ozone treatment 68
2.5 Rates of degradation of periodate and cuoxam
lignins during ozonization...... 71
2.6 Amount of ozone consumed by periodate and
cuoxam lignins during ozonization...... 74
2.7 Rate of degradation of spruce protolignin dur-
ing ozonization ...... 79 xvi
FIG. No. CHAPTER 3
3.1 IR spectra of the original sample and the alkali soluble degradation products of spruce periodate
lignin during ozonization .....••...••.•.•.••..... 94 3.2 IR spectra of the original sample and the alkali soluble degradation products of spruce cuoxam
lignin during ozonization •.••••..••..•••••••...•• 96 3.3 IR spectra of the alkali-soluble degradation products of spruce protolignin during ozonization. 98 3.4 N.M.R. spectra of the alkali-soluble degradation products of spruce periodate lignin during ozoniz-
ation ...... 101 3.5 N.M.R. spectra of the alkali-soluble degradation products of spruce cuoxam lignin during ozoniz-
ation ...... 102 3.6 UV calibration curve . . • • ...... • . • . • . • • . • • . • . . • • . . 108 3.7 UV spectra of the alkali-soluble degradation prod ucts of spruce cuoxam lignin during ozonization .• 109 3.8 UV absorption of the alkali-soluble degradation products of lignin in neutral and alkali solutions. 112 3.9 Direct and difference spectra of the alkali-soluble degradation products of lignin during ozonization •• 118 3.10 Proposed mechanism for the cleavage of a phenol
ether bond of lignin by ozone ....•...••..••...... 120 3.11 The IR spectrum of the acetic acid-soluble degrad- ation products during ozonization...... 123 xvii
FIG. No. CHAPTER 4
4.1 Calibration curve for Sephadex G-100 column 138 4.2 Calibration curve for Sephadex G-200 column 140 4.3 Chromatogram of sample SPL-5 on Sephadex G-50
eo 1 umn • • • • .. • • . • . • • . . • . • ...... •. ·• . • ...... • • . • . • 14 3 4.4 Chromatogram of sample SPL-5 on Sephadex G-100 column ...... 143 4.5 Chromatogram of sample SPL-5 on Sephadex G-200
column ...... 143 4.6 Chromatogram of alkali-soluble degradation products of spruce periodate lignin during ozonization ...... 145 4.7 Chromatograms of alkali-soluble degradation products of spruce protolignin during ozoniz-
ation ...... 147
CHAPTER 5
5.1 Yield and lignin content of ozone pulps as a function of ozonization time •...... 173 5.2 Removal of lignin and carbohydrates during the ozonization of spruce wood meal ...... •...•.... 175 5.3 Rates of degradation of alpha-cellulose, hemi
celluloses and lignin during the ozonization of
spruce wood meal ...... 178 xviii
FIG. No. APPENDIX 3
A-3-1 Schematic representation of the elution volume and accessible volume of a gel column •.•••.....•. 200 A-3-2 Schematic representation of band broadening on a
gel column ...... 207
APPENDIX 4
A-4-1 Plot of 1/Mw vs c (lower) and 1/Mw vs Field (upper) for sample SPL-5 ...... 226 A-4-2 Plot of 1/Mw vs c {lower) and 1/Mw vs Field (upper)
for sample SPL-10 228 A-4-3 Plot of 1/Mw vs c (lower) and 1/Mw vs Field (upper)
for sample SPL-15 230
A-4-4 Plot of 1/~ vs c (lower) and 1/Mw vs Field (upper)
for sample SPL-30 ...... 232 A-4-5 Plot of 1/Mw vs c (lower) and 1/Mw vs Field (upper)
for sample SCL-5 ...... 234 A-4-6 Plot of 1/Mw vs c (lower) and 1/Mw vs Field (upper)
for sample SCL-15 . . . • . • . • ...... • ...... 2 36 A-4-7 Plot of 1/Mw vs c (lower) and 1/Mw vs Field {upper)
for sample SCL-30 238 A-4-8 Plot of 1/Mw vs c (lower) and 1/Mw vs Field (upper)
for sample SSL-1 ...... 2 4 o A-4-9 Plot of 1/M vs c (lower) and 1/M vs Field (upper) w w for sample SSL-20 ...... 242 xix
FIG. No.
A-4-10 Plot of 1/Mw vs c (lower) and 1/Mw vs Field (upper) for sample SSL-30 244 XX
LIST OF TABLES
TABLE No. CHAPTER 2
2.1 Yield of undissolved periodate and cuoxam lignins after ozonization and alkali extraction .. 70 2.2 Rate constants for the degradation of periodate and cuoxam lignins by ozone •••••••.••.••.•••..•. 70 2.3 Amount of ozone consumed and the yield of dis solved periodate and cuoxam lignins during ozon-
ization ...... 73 2.4 Yield of alkali-soluble degradation products of
periodate lignin during ozonization .••••••.••.•. 76 2.5 Yield of alkali-soluble degradation products of
cuoxam lignin during ozonization .•.•....•...... 76 2.6 Lignin content of the original and ozone treated wood meal after alkali extraction .•..•.•.•..•... 78
CHAPTER 3
3.1 Signal intensity of the proton magnetic resonance spectra of the alkali-soluble degradation products as a function of the duration of ozone treatment.. 103 3.2 Methoxyl content of the original and alkali-soluble degradation products of lignin as a function of the duration of ozone treatment...... 107 3.3 Phenolic hydroxyl content of the alkali-soluble xxi
TABLE No. page
degradation products of lignin as a function of
the duration of ozone treatment...... 114
CHAPTER 4
4.1 Weight average molecular weights of lignin
fractions used for the calibration of Sephadex
G-100 col'unt.n ...... 137
4.2 Weight and number average molecular weights and polydispersity of lignin fractions used for Sephadex G-200 calibration .••••.••..•••••.••... 137
4.3 Partial specific volume and refractive index
increments of alkali-soluble degradation products
of lignins after ozonization .•.••.••••••••....• 141 4.4 Weight average molecular weights of the alkali
soluble degradation products of the lignins during
ozonization ...... 151 4.5 Elemental analysis of the original samples and the alkali-soluble degradation products of spruce periodate and cuoxam lignins ...•...•.•••...•..• 155 4.6 Weight average degree of polymerization of the
alkali-soluble degradation products of spruce periodate and cuoxam lignins ...... 158
CHAPTER 5
5.1 Yield and lignin content of ozonized spruce
wood meal after alkali extration ... ..•..••..•. 172 xxii
TABLE No.
5.2 a-Cellulose and hemicellulose content of the original spruce wood meal and ozone pulped
samples ...... 177
5.3 Viscosities and degrees of polymerisation of the ozone pulps ...... 182
APPENDIX 2
A-2-1 Characteristic infra-red bands of lignin 195 A-2-2 Chemical shifts of protons found in the n.m.r. spectra of lignin model compounds and
lignin ...... 197
APPENDIX 4
A-4-1 Data for the measurement of the cell constant of Digital Precision Density Meter...... 216 A-4-2 Determination of the density of dimethyl- sulfoxide ...... 217
A-4-3 Results of the molecular weight determination of lignin fraction c2 . • • . • . • • • • . • • . • • • • • • • • . 222 A-4-4 Results of the molecular weight determination
of lignin fraction c5 ..••..••..•...... · 22 3 A-4-5 Results of the molecular weight determination of lignin fraction c6 ••...... •.•••••...••. 224 A-4-6 Results of the molecular weight determination c of sample SPL- 5 ...... 225 xxiii
TABLE No.
A-4-7 Results of the molecular weight determination
of sample SPL-10 ...... 227
A-4-8 Results of the molecular weight determination
of s amp 1 e S PL-15 . . • . • • . . • • . . • . • • • . • ...... 2 2 9
A-4-9 Results of the molecular weight determination
of sample SPL- 30 ...... 2 31
A-4-10 Results of the molecular weight determination
of sample SCL- 5 ...... 2 3 3
A-4-11 Results of the molecular weight determination
of sample SCL-15 ...... 235
A-4-12 Results of the molecular weight determination
of sample SCL-30 ...... 237
A-4-13 Results of the molecular weight determination
of sample SSL-1 ...... 2 39
A-4-14 Results of the molecular weight determination of sample SSL-20 .•.....•..•••.•...••••..•... 241 A-4-15 Results of the molecular weight determination of sample SSL-30 . • . • . • • . . . . • . . • . . • . . • ...... • 243 - 1 -
CHAPTER 1
GENERAL INTRODUCTION - 2
Wood, an interpenetrating system of high polymers 0 (1} is one of the worlds important renewable natural resources, and forms the basis of a huge industry - the Pulp and Paper Industry. Pulp is produced by the chemical or mechanical separation of the wood fibers. Chemical pulps are obtained by the removal of one of the components of wood - lignin, through various chemical processes. One of these, the Kraft process, is especially important, for it furnishes about 75% of the chemical pulp utilized in the industry. The Kraft process employs a solu tionof sodium sulfide and sodium hydroxide as the pulping liquor. Despite the advantages of high strength pulp and rapidity of the process, the yield of pulp is low and the pulps produced are very dark in colour. These dark pulps can be used to make cardboard, boxes, newsprint and other paper products. However, to make white paper products these pulps have to be bleached. Bleaching can be seen as a continuation of the pulping process in which the colouring matter remaining in the chemical pulp is removed with as little degradation of the pulp fibres as possible (2-4). Bleaching of a mechanical pulp involves the chemical modification of the chromophoric groups, e.g., carbonyl or ethylenic groups without removal of the lignin in the pulp {2-4} . The existing bleaching methods for chemical pulps are based mainly on the use of chlorine-containing chemicals like chlorine, chlorine dioxide and sodium hypochlorite. From the point of view of environmental protection these chemicals are - 3 -
0 harmful. The solution to the above problem may be the use of oxygen-containing bleaching agents like oxygen or ozone which may not pose environmental problems. While oxygen-bleaching, has be come a reality, ozone bleaching is still in the pilot plant stage. As for ozone pulping, experiments are still in the laboratory stage. Since ozone is formed from oxygen in an endothenni.c reaction and decomposes into molecular and atomic oxygen, it is a much stronger oxidizing agent than oxygen and bleaches much more rapidly.
Despite the increasing interest in the use of ozone for bleaching, little or no attention has been devoted to the physicochemical study of the mechanism by which lignin is de graded by ozone. A fundamental study of this nature is likely to provide some useful information which could help to optimize the bleaching processes by ozone. More importantly, a study of the degradation of lignin by ozone may provide some insight into the architecture of lignin as it exists in wood. This thesis deals with a physicochemical investigation of the ozone-lignin reaction. Therefore, it seems appropriate that this introductory chapter should begin with a discussion of the chemistry of wood, and the structure and architecture of the lignin macromolecule. The theory of gelation is then discussed since lignin is increasingly regarded as a 3-dimensional network gel. In the latter sections, the topics covered are: the nature of the known reactions between lignin and ozone, the use of ozone in pulping and bleaching, and the removal of - 4 -
0 carbohydrates during lignin degradation in wood. Finally, the introduction closes with an outline of the scope and aims of this thesis.
THE CHEMISTRY OF WOOD
Wood is composed of three chemical systems: poly- saccharides, lignins and extractives. Polysaccharides consist of cellulose and hemicelluloses. Depending on the wood species, they make up 60-80% of the dry wood weight {5).
Cellulose accounts for 43 ± 2% by weight of softwoods and hardwoods (5,6). It is a linear polydisperse polymer of high molecular weight. The average degree of polymerization generally ranges from 1000-17000 (6-8) depending on the wood species and the method of delignification of the wood. The
cellulose chain is built up of e-~-glucopyranose residues
which are linked by (1~4) glycosidic bonds {5) (see Fig. 1.1). In the cell wall, cellulose chains pass through alternate crystalline and amorphous regions (9,10). Cellulose is virtually absent between the fibre cells. The cellulose fibre consists 0 of microfibrils about 100-200 A in diameter (11-13). These microfibrils are made up of much finer fibrils called elementary 0 fibrils or protofibrils that are of about 35 A in diameter (14-18). The ultrastructure of the protofibrils has been a matter of much debate since it is not certain whether they contain straight chains {19-22) or folded chains (23-27). At c present the question is still unresolved. - 5 -
0 Hemicelluloses are lower molecular weight polysac charides, that are structurally more complex than cellulose owing to some degree of branching (28,29). They occur in intimate admixture with lignin in the cell walls. Unlike cellulose, hemicelluloses are soluble in dilute alkali and boiling water. They have a degree of polymerization of 100-200 (30)'. Hemicelluloses consist of a varying combination of galactose, mannose, xylose, glucose, and arabinose units
as well as acid residues (e.g. uronic acids). ~-acetyl galacto-glucomannan and arabino-4-0-methyl glucoronoxylan (30,31) are the two most abundant softwood hemicelluloses (Fig. 1.2). Xylose containing hemicellulose fractions are called xylans or pentosans. Hemicelluloses in which mannose units are linked to each other and to glucose are called glucomannans. The extractives in wood are not part of the cell wall components; they include fats, and resins which are sol uble in neutral organic solvents or inorganic salts. They are volatilized by steam. Softwoods contain about S-8% extractives and hardwoods contain between 2% and 4% extractives (3). Lignin is the second most abundant component of wood next to cellulose. It constitutes 30% by weight of softwood and 20% by weight of hardwoods. It is formed between the fibre cells and acts, besides other functions, as a cement for the fibres thus imparting structural rigidity to wood. Less than 30% of the total lignin in wood is lodged in the middle lamella - 6 -
FIG. 1.1 Structure of cellulose (from Cote (30)).
FIG. 1.2 Structures of the two main softwood hemicelluloses (from Cote (30)). CELLULOSE
H OH H OH -o 0 o-
HEMICELLULOSES
0
-o
0 0-
0-ACETYL-GALACTO-GLUCOMANNAN
OH
0 ~ -o
0 OH 0 4-0- METHYL-GLUCORONOXYLAN - 7 -
(32). The remainder is found in the cell wall intimately as sociated with carbohydrates. Lignin is aromatic in nature and completely insoluble in non-degradative solvents. There are two basic methods of isolating lignin from wood. The first involves the degradation of the lignin with acids followed by extraction with organic solvents such as di oxane (33) or ethanol (34,35). The yield of lignin is usually small (10% for Brauns native lignin) and therefore these lignins may not represent the bulk of the lignin in wood. The second method of isolating lignin involves the degradation of cellulose by acid salts such as sodium paraperiodate (36,37) or the complexing and dissolution of the cellulose by reagents like cuprammonium hydroxide (38). Lignins isolated by this method are insoluble in inert solvents. The yields are usually high (about 60-70% for periodate lignin). These lignins, especially periodate lignin, retain the morphological features of wood (36,37). However, no isolation method has been shown to yield lignin which is identical in structure and architecture to the
lignin in wood. Therefore each lignin is identified by the wood species and the isolation procedure employed, e.g., spruce-cuoxam lignin or birch-dioxane lignin.
STRUCTURE AND ARCHITECTURE OF LIGNIN
Lignin as it occurs in wood is aromatic in nature (39-46) and has been shown to be built up of phenyl propane units (47,48) otherwise called the c -c unit (Fig. 1.3). The 0 6 3 earlier view (49-51) that lignin is a condensation product of - 8 -
0 coniferyl alcohol [1] and hydroxyconiferyl alcohols [II] was confirmed experimentally by Freudenberg (52). It is now generally accepted that enzymatic dehydrogenation of coniferyl alcohol structures leads to the formation of resonance stabilised free radicals (Fig. 1.4) which link in a variety of ways to give the extremely complex lignin macromolecule (Fig. 1.5}. The predominant linkage in lignin is the glycerol aryl ether linkage (53}. The basic unit in softwood lignin is the guaiacyl propane {3-methoxy-4-hydroxyphenylpropane)monomer (Fig. 1.6a). About 70% of these units are etherified; the rest contain free phenolic hydroxyl groups (54,55). In hardwood lignin, syringyl propane (3,5-dimethoxy-4-hydroxyphenyl propane) as well as guaiacyl propane units constitute the basic structural units (56) (Fig. 1.6b) · The general agreement on the precursors of lignin and their mode of polymerization has limited the problem of lignin structure to that of elucidation of the linkages between these monomeric units. Based on the degradation studies with model compounds and the alkaline cupric oxide oxidation of lignin sul fonates carried out by Pearl (57-63), the 5-5 linkages between
two guaiacyl propane units and the a - a linkages of two guaiacyl monomers were established. Subsequent investigations by Adler (55,64-67) and others have established the presence of such link ages as the alkyl-aryl ether linkages, and aryl glycerol s-aryl ether linkages. From the established linkages and groups present in lignin, attempts were made to give a concise structural representation of lignin. Amongst the proposed structures were those of Brauns (68,69), Erdtman (70), Adler, (71, 72), Freudenberg - 9 -
a f3 Y c-c-c
FIG. 1.3 The elementary building block of lignin.
CH20H CH 0H I I 2 CH CH 11 11 CH COH ~ ~
#OCH3 OH - - -I -I I 0 o e
FIG. 1.4 Resonance stabilized free radicals produced by the dehydrogenation of 1-p-hydroxyphenylpropene.
1-' 0 - 11 -
0 (73-76) and Forss and Fremer (77). The Freudenberg formulation of 1965 for spruce lignin is currently the most widely accepted (76). It contains 18 structural units interlinked in a manner analogous to the naturally occurring lignin molecule. Unlike the structure of some other complex macromolecules. the arrange- ment of the monomeric units is completely disordered (Fig. 1.5).
In the Freudenberg formulatio~, certain units (e.g. unit 5) are be- lieved to bind lignin covalently to carbohydrate molecules (78-81) in a lignin carbohydrate complex (LCC). The polymeric structure of lignin was established by indirect evidence through model compound studies and dehydrogen- ation products of coniferyl alcohol (52). However, n.m.r. spectroscopic evidence by Nimz et al. (82) who compared natural lignin with synthetic dehydrogenation products (DHP) reinforced Freudenberg's view that lignin is formed by enzymatic dehydro genation of coniferyl alcohol structures. The insolubility of natural lignin raised the question as to whether the lignin exists as highly branched or cross- linked chains. By comparing experimentally determined values of intrinsic viscosities In], in various solvents with the value for spherical particles of lignin calculated from Einstein's equation (equation 1.1), when the specific volume, V, of bulk lignin is taken to be 0.7 ml g -1 , Goring (83) found a comparatively small increment in the dimensions of the particles in solution. He concluded that lignin exists in solution as compact microgels.
[ n ] = 0 • 0 2 SV • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • [ 1 • 1] - 12 -
0
Ht;OH CH H2COH tH -eH (===--..:.. ~Me -0
FIG. 1.5 Freudenberg's 1965 representation of an 18-unit segment of spruce lignin. 0 - 13 -
c-c-c
(8)
c-c-c
FIG. 1.6 Basic monomeric units of softwood and hardwood lignins. (a) guaiacyl propane (3-methoxy-4- hydroxyphenylpropane) unit. (b) syringy1 propane (3,5 dimethoxy-4- hydroxypheny1propane) unit. - 14 -
Furthermore, the values for the exponent, a, in the Mark-Houwink equation (equation 1.2) determined for soluble lignin in several solvents lie between a= 0 and a= 0.5 (83), indicating a con formation between an Einstein sphere and a non-free draining random coil.
[n] = K nMa ...... • . • ...... • ...... [ 1. 2] (Kn and a are constants and M is the molecular weight) .
The values of the exponent a, obtained are in keeping with the macromolecule being part of a cross-linked network. From diffusion rates, sedimentation rates and vis- cosities of milled-wood lignins, Alekseev et al. (84) calcu lated the values of the exponent a, in the Mark-Houwink equation and obtained results which indicated that the macromolecules exist in solution as rigid spheres. Recently, Pla and Robert (85-86) reported values of less than 0.5 for the exponent, a, in equation 1.2 for dioxane lignin preparations and interpreted the results to indicate that the macromolecules in solution exist as branched or cross-linked chains. Bolker and Brenner {87) fitted the data obtained from the acid catalysed degradation of sulfite and dioxane lignins to theoretical curves calculated from gelation theory. From the results obtained, they concluded that lignin exists as a gel formed by cross-linking primary chains of a weight average degree of polymerization of 18. They calculated the cross-linking density and found a value of 0.277 for intact lignin. The - 15 -
significance of this value is that 5 out of every 18 phenyl propane units bear cross-linking benzyl ether groups. A some what lower value of 7 was found for the average length of the primary chain obtained in the chlorine monoxide degradation of spruce periodate and cuoxam lignins (88). Despite the seemingly general agreement in the concept of lignin in wood existing as a cross-linked network polymer, some workers share the view that lignin as it exists in wood is finite and made up of polyfunctional molecules and that during extraction with solvents, these highly reactive molecules polymerize to result in an infinite network gel {89-93). Brown et al. {94,95) obtained bimodal chromatograms of soluble lignins
liberated by the action of brown~rot fungi and concluded that lignin in wood is finite and consists of two major components that differ in molecular weight. Obiaga et al. (96) interpreted bimodal chromatograms of soluble lignin obtained during Kraft and Soda pulping of milled wood lignin and spruce wood to signify the presence of high and low molecular weight components of lignin. For the milled wood lignin, the low molecular weight peak was established at a weight average molecular weight of about 3000 to 4000. This peak was found to increase at the expense of the peak at the high molecular weight end of the chromatogram. From these results, they postulated that lignin in wood exists as an assembly of modules with a degree of polymerization of 18. It should be mentioned that Brown and 0 Obiaga based their respective conclusions on the apparent bimodal distributions obtained on Sephadex gels. However, as - 16 -
Bolker et al. {88) later showed, their interpretation of the bi modal curves was incorrect since the peak at the high molecular weight end of their chromatograms, represented an abbreviated high molecular weight tail of the low molecular weight maximum. Despite the lack of concordance on the precise architecture of lignin, it has been generally observed that the molecular weight of soluble lignin increases as extraction is continued (88,96-101). This observation has been explained in various ways as follows: (i) that the increase in molecular weight is due to condensation and cross-linking of finite molecules during extraction (89-93), (ii) that the pores in the cell wall exercise a sieving action on the size of the molecules, the pores becoming larger as extraction continues (102) and (iii) that the molecular weight increase is the expected consequence of the degradation of an infinite network gel (87,88). In the present work, an attempt has been made to con tribute to our understanding of the architecture of the lignin macromolecule by a study of the degradation reactions between ozone and spruce periodate and cuoxam lignins and spruce wood protolignin. The use of isolated lignins for this study permitted the evaluation of the effect of the cell wall on the size of the molecules solubilized during degradation of lignin. In order to interpret precisely the results obtained and to understand the physical and mathematical nature of infinite networks, a brief discussion of the theory of gelation is given in the next section of this chapter. - 17 -
0 THEORY OF GELATION Polymers are large molecules composed of monomer units which are joined together by covalent bonds. Architectur ally, polymers can be classified into three categories: linear, branched and network. Linear polymers consist of monomers with a functionality of two (Fig. 1.7a). If the functionality of the monomers in the polymer exceeds two, a more complex structure results since the polymer chain now develops side-chain appendages through which it can also grow. Such a polymer is said to be branched (Fig. 1.7b). A monomer unit possessing a functionality of three or more is called a branch unit. In a branched polymer, a tri functional branch point is considered to terminate a chain and initiate two new chains. A tetrafunctional branch point terminates a chain and initiates 3 new chains. Thus if there are X trifunctional branch points, there are Y = 2 - X + 2X = 2 + X ends in the poly mer. Similarly, tetrafunctional branch points give Y = 2 - X + 3X = 2 + 2X ends. In the absence of intramolecular condensation, the number of ends exceeds the number of branch points. Considering a
polyfunctional system containing trifunctional units, A3 , and hi functional units, B2, the polymer grows by the addition of one A3 unit and two B2 units to a randomly selected starting B2 unit. Any chain terminating in an A3 unit is replaced by two new chains. If both chains also terminate in branched units, 4 more chains are formed. Flory (103) defined a branching coefficient, a, as the probability that a chain ends in a branched unit irrespective - 18 -
of the number of pairs of bifunctional units A2 present in the reaction mixture. When a< 0.5, there·is the possibility that the chain ends at an unreacted functional group. Since the net- work cannot grow indefinitely, termination sets in and imposes a limitation on the size of the molecules, thus a finite
polymer is obtained. However, when a > 0.5, formation of new chains and branching of the successive chains continue until an
infinite network is formed (Fig. 1.7c). Therefore a= 0.5 is the critical condition for incipient formation of infinite net- works in a trifunctional system.
Infinite networks can be formed in two ways. ~he first is the polymerization of polyfunctional monomers (Fig. 1.8).
As mentioned_~arlier, a = 0.5 is the critical condition for the formation of infinite networks in a trifunctionally branched
system. Beyond a = 0.5, however, there are molecules which do not form part of the network. Thus there exists at a = 0.5, a gel phase which is infinite and insoluble in inert solvents and a sol phase consisting of finite molecules. In a system where the monomers have a functionality greater than three, infinite
network formation can also occur. If f is the functionality of a branching unit (greater than two) gelation occurs when a(f-1) exceeds unity. In general, as Flory has shown (103), the critical condition for gelation in polyfunctional systems is given by equation 1.3.
a = 1/(f-1) ...... [1.3] 0 c - 19 -
0
(a) LINEAR
(b) BRANCHED
(c) NETWORK 0
FIG. 1.7 Structural variations of polymers. - 20 -
0 The gel point is marked by a rapid increase in viscosity such that the reaction mixture loses its fluidity. Theoretically, gelation should manifest the formation of an infinite network,
i.e., the observed gel point should occur at a= 0.5, the critical condition for the formation of an infinite network in
for example, a trifunctionally branched system. Howeve~ the . experimentally observed gel point is usually higher than the theoretical extent of reaction. Intramolecular condensation has been implicated as the cause of this discrepancy between theory and experiment. Polymerization of polyfunctional monomers does not always result in network formation. For example in the case of
AB 2 monomers where functional groups A can only condense with B, and B with A, networks cannot be formed since functional groups B cannot condense with each other {Fig. 1.9). Infinite networks also result from chemical bonding be- tween existing or pre-formed primary chains (cross-linking) (Fig. 1.10). The best example of this type of network formation is in the vulcanization of rubber where cross-linkages are introduced through the sulphuJ: present-in the reaction mixture.
Critical condition for gelation in cross-linked systems According to Flory (103-106) the fraction of cross
linked units, p, (cross-linking density) is given as
p = v/N 0 ••••••••••••••••••••••••••••••••••·•••• [1.4]
where N is the total number of units contained in the polymer 0 - 21 .
FIG. 1.8 A section of a network formed by random po1yfunctiona1 condensation.
B
A
FIG. 1.9 Polyfunctiona1 condensation that cannot lead to network formation. - 22 -
FIG. 1.10 A section of a network of cross-linked primary chains.
and v is the number of units for each cross-link (t"i.·TO). If a polymer gel results from cross-linking primary chains of y units which are randomly distributed, and if each primary chain contains at least one cross-linking unit, then the expected number of cross-linked units in a primary chain
(expectancy, E) is given as
e:: = p (y- 1) ...... [1.5]
If E < 1, limited cross-linking results and an infinite net- work cannot be formed. However when E 7 1, infinite networks are formed. The incipient formation of a network gel occurs at an expectancy sc = 1, i.e.
= 1/(y- 1) ~ 1/y (when y is large) ...... [1.6] - 23 -
When the size of the primary chain, y, is large, the number of cross-links required to effect gelation is small. In fact one cross-link per two primary chains is sufficient for gelation to occur. For an arbitrnry distribution of sizes of the primary chains, the expectation value becomes
00 e: r p wy<:r- 1) p(yw- 1) [1.7] = y=l = ··············~··· and the critical cross-linking density is given as
Pc = 1/ Weight fraction of sol in a gelled polymer Flory has shown that beyond the gel point, a fraction of the polymer remains finite in size and soluble (104). There- fore, according to his treatment, if the probability of a randomly selected non cross-linked unit belonging to the sol fraction is ~s' then the probability that a cross-linked unit 2 being part of the sol is $ s· If this cross-linked unit is selected at random, then the probability of its being part of the sol is p~~' where p is the fraction of cross-linked units. The probability that the unit is part of the sol but is not cross-linked is equal to (1- p)~s· The weight - 24 - fraction of the sol, Ws, is the sum of these two probabilities, hence (1-p}q> ••••••••••••••••••••••••••• [1.9] + s Therefore /$ (1- p) + p$ ws s = s If p is small (< 0.3) i.e. y is large, cps and [1.10) - ws ) ...... If each unit is cross-linked once, then the proba- bility that a primary molecule of y units having i cross- linked units is ( y! ) [ 1.11] (y-i) ! i! excluding the i cross-linkages, the probability that none of the members of these cross-linked units belongs to an infinite network is ~~- Hence, the probability, Sy' of a randomly selected primary molecule of y units being part of the sol is s = r p (i)dli. s [1.12] Y i=O Y substituting Eq. 1.11 into Eq. 1.12, for P (i) y - 25 - 5 y = [1 - p { 1 - cp s) ] Y = (W s/ cp s) Y ...... • . [ 1 .13 J (from equation 1.9) Therefore the weight fraction of the sol, W , is the product s of the probability of a y-mer being part of the sol and the weight fraction of the y-mers summed over all values of y w = E w s ...... [1.14] s y=l y y CO = E w (W I q, ) y ••••.••••••••••••••••••••• [1.15] y=l y s s . where Wy is the weight fraction of the primary y-mer molecules. If p is SIP.all then substituting equation 1.10 into equation 1.15 gives equation 1.16 = 2: V<7 [ 1.16] y-1 y Equation 1.16 can be solved bv substituting trial values of Ns in the right hand side and evaluating the summation by a graphical method. If the value of o is known then Ws can be computed. For a distribution of primary chains of varying chain lengths Eouation 1.16 becomes = [1- p(l- Ws)Jy ••..•.••.....•.•....•• [1.17] In a monodisperse system y=y. If the system 0 is polydisperse, equation 1.17 will hold only for one value - 26 - 0 of p since for higher p values y is too low. A plot of ~vs vs p for various values of y is shown in (Fig. 1.11). It can be seen that in a polydisperse system, the higher the cross- linking density, the lower the weight fraction of the sol portion . . Weight average degree of polymerization and weight fraction ·of sol In the preceding section, the probability of a cross linked unit being part of the sol was defined as $~. It follows from Flory's trea~ment, therefore, that $2 P 'W jp (1.18] s = s ...... where p ' is the cress-linking density in t.he sol an:i p is the total cross-linking density p f = Since W /" 1- p(l- W) (from Equation 1.10) s '~'s = s 2 p' = pWS/[1- p (1- W )] •••••••••••••••••••••• [1.19] 6 pWS[l + 2p (1- Ws)] ••••.•.•••••...•.••.... [1.20] On further approximation pI :::: pWS ••••••••••••••••••••••••••••••••••••••• [1.21] The Stockmeyer equation (107) gives the weight average degree of polymerization of the sol X~ as - 27 - 0 1.0 a 0.5 0 0.1 0.2 0.3 p FIG. 1.11 The weight fraction of sol as a function of cross-linking density. (a) y = 18; (b) y = 10; (c) y = 7. (From Rhodes, H.E.W., M.Sc. Thesis ,McGill University, 1975) - 28 - X~ = y ~ ( 1 + p I ) I [ 1 - p I ( y w - 1) J • • • • • • • • • • • • [ 1. 2 2] where y' is the weight average chain length in the sol and w p' is the cross-linking density in the sol. Substituting equation 1.20 for p' into equation 1.22 yields y {1 + pW [1 + 2p(l- Ws))} = w s [1.23] Equation 1.23 can be simplified by considering the primary chains as monodisperse, i.e., Yw = y • • • • • • • • • • • • . • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • [1.24] (as in equation 1.17) Therefore X'w becomes X' = [1.25] w The monodispersity approximation reduces equation 1.25 to two variables p and Ws since y now becomes a constant. The value of p can be obtained in terms of Ws (equation 1.17). Therefore for a given value of W , the corresponding value of s X~ can be calculated. This has been done by Bolker et al. (87,88) who used this equation to determine the average length of the primary chain in the lignin macromolecule. However, chain lengths determined by this method should be taken with caution for the following reasons. First, the values of W determined s - 29 - experimentally for soluble lignins depend to a great extent, on the solvent used. Bolker et al. (88) observed that lignin fragments that were insoluble in tetrahydrofuran dissolved quite well in aqueous O.lN NaOH. The present author further observed that lignin fragments which were soluble in dimethyl- sulfoxide and aqueous 2% NaOH remained insoluble in THF. Secondly, values of X'w are obtained by dividing the weight average molecular weight, Mw, of the fragments by the molecular weight of a lignin basic unit {c unit). This immediately implies 9 that the differences in molecular weight values such as those observed from ultracentrifugation or light scattering methods affect values of xi.w Molecular size distribution in infinite networks One of the questions which the present investigation attempts to answer is whether lignin in wood exists as a three- dimensional cross-linked gel or, is finite but forms an infinite network by the condensation of the reactive bifunctional and trifunctional rnonomers during degradation followed by extraction with solvents. Accordingly, it is now of interest to consider the molecular size distributions expected of these two types of infinite networks. By assuming that intramolecular reactions do not occur in species of finite size and also that the length of an individual chain is independent of the size of the net- work of which it is a part, Flory (104) derived an equation for the weight fraction Wz of a network of Z chains during the polymerization of bi- and trifunctional units as - 30 - 0 2 (1- CL) (Z + l)!B(Z- 1)/2! • • • • • • • • • • • • • • [ 1. 26] [(Z + l)/2)!£(Z + 3)/2)! where Z = 2n + 1, n is the number of branching units, CL is defined as the branching coefficient and B = a(l - CL). In Fig. 1.12, Wz calculated from -equation 1.26 is plotted against Z for several values of the branching eo- efficient, CL. The same curves can apply for the sol fractions in gelled polymers, since the changes in the molecular size distribution in the sol fraction during polymerization follow an exactly opposite course to the size distribution up to the gel point. Therefore the curve for CL = 0.25 in the case of the gel will also represent the distribution for the sol fraction when CL= 0.75. Similarly the curve for CL= 0.15 in the gel portion represents the distribution for the sol fraction when CL = 0.85. It is seen from Fig. 1.12 that there is an abundance of single chains in the distribution and that there is a continuous decrease in weight fraction, Wz, to wards zero as Z increases. Using a statistical method, Flory (lQS) also derived an expression for the weight fraction, Wz, of a network of Z chains in an infinite network formed by cross-linking chains of uniform length. The result is given in equation 1.27, Z-1 wz = z {ye -y) z = z yZ! (8/e) ••••••••••••••••• [1.27] where y is defined as the cross-linking index and is given as - 31 - 0 a a= 0.50 0.15 b a= 0.25 c a= 0.15 9 17 25 33 z FIG. 1.12 The weight fraction distribution of species of various complexities in a random tri functional condensation (104). - 32 - C> y = pv (p = cross-linking density) 'V = y - 1 (y = chain length) 1-y and 13 = ye By applying Stirling's approximation 2 Z! ~ 12TI"Z(Z/e) equation 1.27 reduces to wz = ~ i21T sz;z312 .•.•...••••.•.•.••.•••••.•.•••. [1.28] A plot of WZ versus z is shown in Fig. 1.13 for several values of the cross-linking index y. Like the case of the polyfunctional condensation discussed earlier, Fig. 1.13 also represents the size distribution up to the gel point. Qualitatively, there is no difference in the two distributions (Fig. 1.12 and 1.13) • The abundance of single chains in each case decreases as the molecular complexity, Z, increases. There is, however, a difference in the two cases with respect to the extent of reaction at the gel point. In the random condensation of trifunctional monomers incipient gelation is expected theoretically at a branching probability ac = 0.5, since a ~ P2 (103), (P is the probability that a functional group has reacted) P = 0.70 at a= 0.5. Hence, 70% of the functional groups are expected to react at incipient gelation. For a system of cross-linked primary chains of y units, the critical cross-linked density, pc' is given by equation 1.8 (yw = y). When y is large, pc = 1/y and a ratio of one cross-link for two primary chains is sufficient for - 33 - 0 a Y= 1.0 0.2 b Y=0.5 0.1 0 10 20 z FIG. 1.13 Weight fraction distribution of chains of various complexities in a system of cross linked primary chains (105) . - 34 - gelation to occur. From the foregoing discussion, it is ap propriate to deduce the molecular size distribution which is to be expected of a gel undergoing degradation since this is pertinent to the present investigation. It has been mentioned earlier that the sol which remains after the onset of gelation decreases in molec~lar weight as a result of the preferential incorporation of the larger and more complex species into the gel. If the degradation of a gel is considered the reverse of the post-gel polymerization process, then the molecuiar weight of the sol resulting from the breakdown of the gel is expected to increase with the extent of degradation. Furthermore, a decrease in the average molecular weight of the sol fraction is expected to occur at the later stages of the degradation process, since the sol fraction continues the process of degradation by which it is derived in the first place. The expected molecular weight distribution in the sol fraction is one with a single maximum at the low.molecular weight end and a tail towards the high molecular weight end of the distribution. In the early stages of the reaction, the high molecular weight tail should increase in length as the degradation continues, subsequently this trend is reversed due to secondary degradation reactions. THE NATURE OF THE REACTION BETWEEN LIGNIN AND OZONE Ozone Ozone, o3 , is an unstable, triatomic, allotropic form of oxygen with a characteristic pungent smell.A't high concentration, - 35 - ozone is a blue coloured gas which at -ll2°C condenses to a dark blue liquid. On further cooling, it solidifies to a violet-black substance which melts at -193°C (108). The three atoms of oxygen are believed to form an obtuse angle {116° 45' ± 30') with a bond length of 1.278 ± .002 A.0 The molecule exists as a hybrid of 4 structures (109) (Fig. 1.14) • .. + .. + 0 6 ;f0 \ ....._... ___.,..._- 10\\~ .. .. 1 .. \ .. +...... _ ...... /"\.. - :O 6:- :Q 6: :O 0: :0 0: "' .. -·· -·· + .. FIG. 1.14 Resonance hybrid structures of ozone (109). Ozone is formed from oxygen in a strongly endo- thermic reaction and decomposes easily into molecular and 0 atomic oxygen (108). It is therefore a much stronger oxidizing - 36 - agent than oxygen. In the stratosphere ozone is formed by the photochemical effect of ultraviolet radiation with air (108). In the laboratory, ozone is obtained during the electrolysis of water. More recently ozone has been obtained by subjecting air or oxygen to silent electrical discharges in ozone-gener ating machines called ozonizers or ozonators (108). Industrially the cost of ozone production is minimal, being mainly dependent on the cost of electric power consumed by the generator. Ozone is very soluble in water and is even more soluble in solvents like acetic acid and acetic anhydride (108). Ozonolysis of lignin The oxidative degradation of lignin by ozone was first studied by Doree and Cunningham (110,111) who observed that beechwood is delignified by ozone in the presence of moisture. The products obtained were a mixture of acids. Carbon dioxide was evolved. Konig (112) ozonized spruce wood in an acetic acid suspension and isolated formic and oxalic acids as products. Because the carbohydrates in the wood were degraded, it was difficult to decide which of the degradation products originated from lignin. Much later, investigations with isolated lignins began (112-117), and even then, ozonolysis was carried to the extent that the lignin became completely soluble in the ozoniz ing medium. Accordingly, only low molecular weight compounds like formic, acetic and oxalic acids were isolated, the products depending on the kind of lignin and the reaction medium. - 37 - Although oxalic acid was invariably found among the reaction products, no information was obtained about the mechanism of the reaction. Freudenberg (118) observed that the ozonization of cuoxam lignin proceeded much faster in acetic acid than in water and suggested an electrophilic mechanism as the mode of lignin oxidation by ozone. Because of the complexity of the lignin macromolecule, information about the mechanism of ozonolysis with lignin has mainly been obtained through model compound studies. Using vanillyl alcohol and veratryl alcohol (III) which corres pond to the terminal and internal guaiacyl groups of lignin, respectively, Hatakeyama et al. (119) found that during ozonolysis in an acid medium, aromatic ring cleavage occurred at the c3-c4 bonds in the guaiacyl structure. R = H, vanillyl alcohol R = CH , veratryl alcohol 3 I 11 C) 0 c3 03 ~ I ... COOH muconic acid COOH structure 0 <:;2 l c COOH oxalic 17~ 6ooH acid L.LCOOCH 3 c3 + ~ / c COOH maleic GOOCH 3 0u .... acid COOR - COOH FIG. 1.15 Mechanism of ozonolysis of lignin model compounds (119). w 00 - 39 - The products were mucanic acid derivatives which underwent further degradation to maleic and oxalic acids (Fig. 1.15). De- • methylation was thought to preceding scission although with other substrates containing the veratryl structure, ring opening with- aut demethylatian was observed (120). Kratzl (120) compared the carboxyl content of the products of ozonization of Kraft lignin before and after saponification with aqueous sodium hydroxide. A higher carboxyl content in the saponified products indicated the formation of mucanic ester structures thus supporting the idea of ring cleavage between c3-c4 of carbon atoms carrying oxygen functionalities. In addition, by employing. model compounds of the veratryl type, he obtained on ozonolysis in carbon tetrachloride, crystalline ozanides. These azanides were unstable and decomposed an warming to give smaller fragments (Fig. 1.16). R 03 ------~ R = H, R' = CH 3, veratral, R = CH 3 , R' = CH 3, homoveratral; R H, R' COCH , guaiacyl acetate; R CH CO, R' CH , = = 3 = 3 = 3 acetoveratrane; R = COOCH , R' CH , methyl veratrate; 3 = 3 R = CHO, R' = CH 3, veratraldehyde. FIG. 1.16 Ozonolysis of lignin model compounds ( 120) • - 40 - In aqueous acetic acid ozonolysis of acetoveratrone did not favour scission between the c3-c4 of aromatic unit. It was concluded that ring scission during ozonolysis is de pendent on reaction conditions and the specific structures. Katuscak (121,122) ozonized methanol and hydrochloric acid lignins and obtained an increase in the active functional groups (e.g., hydroperoxide) during ozonization. An increase in carboxyl and carbonyl content of the lignins previously observed by Hatakeyama (119) was confirmed in this investigation. From the foregoing review, it would appear that the breakdown of the lignin macromolecule proceeds mainly through the cleavage of its aromatic rings, preferably between carbon atoms carrying oxygen functionalities. However, a careful examination of the lignin model compounds chosen for previous investigations reveals that they do not contain alkyl-aryl ether bonds. Thus, no information was gained concerning the reaction of the ether bonds in lignin with ozone. Even where isolated lignins were employed in studies of mechanism, the reaction of ozone with the ether bonds was not investigated. Consider ing the increasing speculation of an infinite network archi tecture for the lignin macromolecule, it is surprising that the reaction of the ether bonds of lignin with ozone had been overlooked in previous studies, since these linkages would represent the cross-links in the lignin structure. If lignin is an infinite network gel, then it should degrade principally by the scission of its cross-links. In the present work the effect of ozone on the ether bonds of lignin was studied and - 41 - 0 the results are reported in Chapter 3 of this thesis. THE USE OF OZONE IN PULPING AND BLEACHING OF WOOD Pulping of wood involves the chemical or mechanical separation of individual fibre cells in wood. Once pulped, these fibres become more adaptable for use in a variety of ways, e.g., papermaking. As indicated earlier, separation of the individual fibre cells is achieved by the removal of lignin. Among the most versatile methods of pulping is the Kraft process which supplies three-quarters of the world's chemical pulp. This process utilizes a composite aqueous liquor containing sodium sulfide and sodium hydroxide to delignify the wood. Low yield of pulp (about 50% of wood) coupled with the production of dark coloured pulps limit the utility of Kraft pulps (2,3,4). A less competitive pulping method is the sulfite process whose sensitivity to the type of useable wood limits its versatility (2,3,4). The cooking liquor for the sulphite process is sulphur dioxide absorbed in alkaline base solution. Delignification is achieved through sulphonation and hydrolysis reactions that form soluble lignin sulphonates. The effluent from the sulfite process and the bleach plants of Kraft mills contains chemical compounds which are highly pollutant to natural waters. Besides there are odorous gases, some of them toxic, which escape into· the atmosphere at various stages of the pulping process. A good example is provided by the Kraft process where gases like hydrogen sulfide H 2s, methyl mercaptan (CH SH}, dimethyl disulfide (CH sscH ) are 3 3 3 - 42 - evolved (123) . Moreover, in the chemical bleaching processes where the residual lignin in the pulps is removed to improve bright ness, mainly chlorine containing compounds are employed, e.g., chlorine, chlorine dioxide and sodium hypochlorite. The bleach plant effluents contain highly toxic chemical that present an environmental hazard. An answer to the pollution problems posed by the existing pulping and bleaching methods is likely to be found in the use of the oxygen containing pulping and bleaching agents like oxygen and ozone. In addition to their being harmless to the environment, they degrade extensively, the organic substances being removed from the wood and therefore reduce the pollution load of effluents discharged into the streams. There is the added advantage that the bleaching effluents can be recovered by burning them together with the spent pulping liquors in a recovery furnace. The use of oxygen for bleaching has achieved consider able success in an industrial scale (124). Bleaching with ozone is, however, still in the pilot plant stage (125). In bleaching, ozone has been used alone or together with other bleaching agents {e.g., oxygen, chlorine dioxide, hydrogen peroxide) in multi- stage laboratory bleaching {126,127). Investigations reveal that pulp consistency (110,111, 126-130), time of treatment (130), pH {129), temperature (131), ozone charge {129,131), and the medium (129,132) affect the results of ozone bleaching. - 43 - 0 Exploratory experiments into the use of ozone for pulp ing were started by Schuerch et al. (133), who ozonized Norway spruce wood meal suspended in either water or nitromethane at 0°C. It was reported that after 48 hours of ozonation of Norway spruce wood in water or nitromethane, over 80% of the wood re mained as solid residue. Although the reaction with lignin was faster in nitromethane than in water, no specificity of attack of lignin was observed in the nitromethane. Whether for bleaching or pulping, ozone attacks wood components indiscriminately and leads to a reduction in viscosity of the pulp. As mentioned earlier, this affects the mechanical strength properties of the pulp. Thus, despite the advantages in its use, ozone has not yet been used in the industrial scale. The problem is to find an effective inhibitor of carbohydrate depolymerization during ozone processing. In the continuing search for such an inhibitor several reaction media have been tried. Chapter 5 of this thesis describes the results obtained when wood is pulped with ozone in 45% aqueous acetic acid suspension and at room temperature. DEGRADATION OF CARBOHYDRATES BY OZONE Cellulose Doree et al. {110,111) showed that moist cellulose could be degraded by ozone. Optimum cellulose oxidation was observed at 45-50% water content. The main reactions include the oxidation of terminaL alcoholic groups to aldehydes, carboxylic acids and peroxide groups followed by chain cleavage. Carbon dioxide was also evolved. Samuelson (134) noted that the decrease in the - 44 - 0 degree of polymerization {DP) of cotton and wood pulp was limited in extent, even after the removal of a substantial portion of the cellulose. This led to the conclusion that the DP-limit corresponded to the average length of the cellulose crystallites. A DP-limit of about 400 was observed when Norway spruce and basswood were delignified by ozone (133). Lower values were observed for chemical pulps. T~e rate of oxidation of cellulose by ozone is influenced by organic solvents (135) • A comparison of the rates of ozonization of pure cellulose and wood pulp indicated a somewhat higher rate for the cellulosic material in wood pulp (134). This was explained in terms of the increased accessibility of the cellulose in the wood pulp to ozone resulting from a significant amount of material of inter mediate crystalline order. Hemicelluloses The reaction of hemicelluloses in wood \17ith ozone is similar to that of cellulose, although the rate of degradation depends on the accessibility of these hemicelluloses to ozone. While a portion of the hemicellulose is susceptible to ozonolysis, a small portion is located in regions that are almost inaccess ible to ozone and so resist degradation (136). PATES OF SOLUBILIZATION OF WOOD COMPONENTS The rates of solubilization of carbohydrates and lignin in Norway spruce was studied in two solvent systems - water and 0 nitromethane (133). Lignin solubilization was found to proceed twice as fast as holocellulose when ozonization was carried out - 45 - in water. In nitromethane, lignin removal by ozone was even faster although in this solvent holocellulose removal was slower than in water. In general, ozonolysis in these solvent systems was slow re quiring about three hours to remove 20% of the lignin in nitromethane No specificity of ozone attack on lignin was observed in the solvent media studied. In this thesis, the relative rates of removal of hole cellulose and lignin in spruce wood have been investigated in 45% aqueous acetic acid. The viscosity of the holocellulose at each stage of the delignification was also studied to gain information on lignin specificity. The results are presented in Chapter 5. SCOPE AND AL~S OF THE THESIS This thesis describes a physicochemical study of the mechanism of the degradation of lignin by.ozone. The work is of interest in connection with the potential use of ozone as an alternative bleaching or pulping agent that poses little environ mental hazard. Although the reaction between lignin and ozone has been studied in the past, complete understanding of the reactions has not been achieved so far, and the mechanism of lignin degrad ation by ozone remains obscure. Accordingly, in the present work, the principal aim is to elucidate the mechanism of lignin degrad ation. The work is also aimed at contributing to our knowledge and understanding of (i) the architecture of the lignin macro molecule, {ii) the process of ozone delignification of wood during pulping and bleaching and, indeed, of the processes of delignification in general. In the experimental work it would have been possible to use one of a variety of isolated lignins. However, as - 46 - indicated e~rlier, the choice of spruce periodate and spruce cuoxam lignins was made because both lignins, like the proto lignin in wood, are insoluble and retain the morphological features of the wood from which they are derived. They can, there fore, be assumed to closely resemble the protolignin in wood. In order to test this assumption, the degradation of protolignin in spruce wood by ozone was then studied under identical con ditions for comparison. The ozonization of lignin can be carried out in a variety of reaction media. This has been the case in some of the earlier work on the ozonolysis of lignin. However, none of these other solvent media were found to significantly improve the specificity of attack of ozone on lignin. Therefore, since industrial research has been focused on finding reaction media in which carbohydrate depolymerization is inhibited or minimized during ozone bleaching or pulping, 45% aqueous acetic acid was chosen in the present study. The reasons for choosing acetic acid as the reaction medium will become evident later. The work is presented in four chapters as follows: In Chapter II, th~kinetics of lignin degradation by ozone is described. Besides contributing to the understanding of the overall mechanism of the ozone degradation of lignin, the study was carried out in order to answer the question whether the carbohydrates associated with the protolignin affect the rate of lignin degradation in wood. The rate of ozone consumption 0 per c9 structural unit of lignin was measured and was found to reflect the reactivity of each kind of lignin studied. - 47 - Chapter III deals with a spectroscopic investigation of the structural changes in the alkali-soluble degradation products obtained during ozonization. This aspect of the in vestigation was expected to provide information about the chemical reactions that take place during the degradation of the lignin by ozone. Chapter IV describes a study of the changes in the molecular weight distribution of the alkali-soluble degradation products obtained during the ozonolysis of the lignins. This part of the investigation was of interest in elucidating the mode of lignin degradation by ozone (i.e., random or selective). Gel chromatography was the tool used for this investigation. Chapter IV also contains a study of the weight average molecular weights of the alkali-soluble degradation products as determined by the short column sedimentation equilibrium technique. The trend of the weight average molecular weights of the soluble products during the degradation process proved invaluable in contributing to the understanding of the lignin architecture. Finally, in Chapter 5, the fate of the carbohydrate moiety during the ozone degradation of the spruce protolignin is described. 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Katuscak, A., Hrivik, M., Mahdalik, M., Paperi ja Puu, No. 9, Vol. ~, (1971). - 55 ~ 0 122. Katuscak, A., Rybarik, I., Paulinyova, E., Hahdalik, M., Paperi ja Puu, No. 11, Vol ~' (1971). 123. Ref. 3, Chapter 10. 124. Baczyinska, I., Przeglad Papierniczy 30, No. 11, 409(1974). 125. Maumert, P.A., Fritzvold, B., Soteland, N., in "The Norwegian semi-industrial pilot plant for processing of paper by ozone" presented at the 3rd Congress of the International Ozone Institute, Paris, May 4th, 1977. 126. Secrist, R.B., Singh, R.P., Tappi 54, No. 4, 581(1971). 127. Liebergott, N., Pulp and Paper Mag. of Canada 73, No. 9, T 214 I 71 (1972) • 128. Brabender, G.J., United States Patent 2, 466,633(1949). 129. Procter, A.R., Pulp and Paper Mag. of Canada 75, No. 6, 58(1974). 130. Lindholm, Carl-Anders, Paperi ja Puu, No. 1, 17(1977). 131. Kubayashi, Takeshi, Husakawa, Jun, Kubo, Takamasa, and Kimura Yutaka, Japan Tappi, Vol. lQ, No. 3, 47 (Mar. 1976). 132. Osawa, Z., Schuerch, C., Tappi 46, No. 2, 79(1963). 133. Osawa, z., Erby, W.A., Sarkanen, K.V., Carpenter, E., and Schuerch, c., Tappi, Vol 46, No. 2, 84(1963). 134. Samuelson, Olof; Grangard, Gurnar, Jonsson, Kurt, and Schramon, Kerstin, Svensk Papperstidn. No. 20, l!, 779 (1953). 135. Szymanski, C.D., J. Appl. Polym. Sci.m Vol.~' pp. 1957- 1606 (1964). 136. Moore, W.E., Effland, Marilyn, Sinha, Biswajit, Burdick, M.P., and Schuerch, c., Tappi, Vol. 49, No. 5, 206 (May 1966). - 56 - CHAPTER 2 DEGRADATION OF LIGNIN BY OZONE: (I) THE KINETICS OF LIGNIN DEGRADATION BY OZONE - 57 - ABSTRACT The ozonolysis of spruce periodate and cuoxam lignins and protolignin in spruce wood has been studied in 45% aqueous acetic acid at room temperature. Stirring was found to affect the rate of reaction and a tentative explan- ation of this effect is given. Degradation followed first order kinetics characterized by a rate constant K, with values of 6.95 x 10-4 sec-l for periodate lignin, 5.10 x 10 -4 sec-1 for cuoxam lignin and 5.09 x 10 -4 sec -1 for proto- lignin in spruce wood. The similarity of the rate constants shows (1) that periodate and cuoxarn lignins are good models for wood lignin and (2) that the carbohydrate matrix has an insignificant effect on the rate of delignification of the protolignin by ozone. The observation of first order kinetics implies a single rate controlling process for the degradation of these lignins. The average rate of ozone con- sumption per c9 unit for periodate lignin was determined to be 0.12 moles per minute and 0.08 moles per minute for cuoxam lignin. The implications of the various results are discussed. - 58 - INTRODUCTION In recent years, the reaction of ozone with lignin has attracted considerable interest principally because the industrial bleaching of chemical and mechanical pulps with ozone produces waste effluents which are less hazardous to the environment than conventional bleaching methods. In spite of its potential industrial importance, relatively few detailed studies of the reaction have been carried out (1-4) and even those have been performed with inappropriate lignin samples, as will be discussed later. A physicoche.mical study of the reactivity of lignin with ozone is expected to contribute to the general understanding of the mechanism by which the lignin is removed during ozone bleaching of pulps and in ozone pulp ing of wood. Such work may also contribute to our knowledge and understanding of the architecture of the lignin polymer, and of the processes of delignification in general. In view of the association of the protolignin with the carbohydrate components of wood, the assumption is generally made that the distribution of the protolignin affects both the mechanism and the rate of degradation. One approach to evaluate the effect of this protolignin/carbohydrate as sociation is to compare the reactivity of isolated lignins with that of the protolignin under identical experimental con ditions. There is, however, the problem of choosing isolated lignins that are representative of the protolignin. The bulk of the investigations concerning the - 59 - reactivity of lignins during degradation by ozone (1-4) has been carried out with soluble isolated lignins. These lignins are prepared by solvent extraction from the cellulosic material under drastic conditions - a process which usually results in extensive modification of the protolignin. Moreover, the yield of such lignins is usually small and so their behaviour during degradation is not considered to be representative of the protolignin from which they are derived. To understand the interaction between lignin and ozone, it is essential to employ lignin preparations which retain most of the characteristics of the protolignin, e.g. its insolubility without degradation in all solvents. Accordingly, in the present study spruce period ate and cuoxam lignins were chosen because they are isolated by depolymerising the carbohydrates associated with the lignin in wood, leaving the lignin almost intact. Besides represent ing adequately the total lignin content of wood, these isolated lignins have been shown (5-7) to retain the morphological features of the wood from which they are derived. It is, there fore, justifiable to assume that information obtained from an insoluble lignin of this sort represents the behaviour of protolignin towards ozone. Another advantage of using periodate and cuoxam lignin is that the possible effect of the carbohyd rate matrix on the rate of delignification is eliminated from consideration (8). In an attempt to understand the overall mode of degradation of lignin by ozone, the kinetics of degradation, - 60 - structural studies of the soluble products and the determination of molecular weights and molecular weight distributions of the soluble degradation products were carried out. For these studies, aqueous acetic acid was chosen as the medium for ozonization. This choice was made because earlier findings showed .that . (i) ozonization of the lignin proceeds much more quickly in this medium than in water (9), (il.). ozone molecules are relatively stable in this medium (10), (iii) the lignin samples as well as sawdust are insoluble and unaffected by aqueous acetic acid solution, and (iv) ozonization of cellulose in acidic solutions seems to protect the cellulose chains from cleavage (11) • This has been explained in terms of the decarboxylation of the acidic groups formed during ozonization in an acid medium. The presence of groups like aldehydic and carboxyl groups formed during ozonization of cellulose disturbs the electrical balance of the ring system leading to cleavage of the chain (11). Thus from a practical viewpoint, acetic acid is expected to protect the cellulose chains in wood as well as accelerate the breakdown of the lignin during pulping or bleaching. The present chapter deals with the rates at which the isolated lignins and protolignin are degraded by ozone, the effect of stirring on the rate as well as the consumption of ozone by lignin during treatment. Other aspects of the work will be described in subsequent chapters. - 61 - EXPERIMENTAL Spruce periodate lignin (Klason,93.2%) was supplied by Dr. D.A.I. Goring of the Pulp and Paper Research Institute of Canada. Microanalysis yielded 58.85% C, 5.95% H, 33.60% 0, 12.16% OCH 3. Spruce cuoxam lignin (Klason 87.5%) was provided by Dr. c. Heitner of the same Institute. Microanalysis yielded 60.91% C, 5.99%H, 34.24% O, and 14.12% OCH 3 . Spruce sawdust was obtained from the Pulp and Paper Research Institute of Canada. The Klason lignin content was determined to be 27.6%. Before use, the samples were ground to pass an 80 mesh screen in a Wiley Mill. The sawdust was pre-extracted with benzene-alcohol 2/1 {v/v) for 12 hours after which it was air dried. The lignin samples as well as the sawdust were brought to approximately 50% moisture content with a fine spray of distilled water. The three samples were always stored in the cold when not in use. All reactions were carried out at room temperature {25°C) and in 45% aqueous acetic acid medium. Ozonization of oeriodate, cuoxam lignin and lignin in soruce wood Ozone was produced by passing a stream of oxygen through a Welsbach Laboratory ozonator, Model T-408. The oper 3 ating pressure and voltage were 5.6 x 10 kilograms per square meter gauge and 120 volts respectively. The flow rate was 0.5 1/min and ozone concentration was determined as 49.7 mg ozone per litre oxygen (3.7% by weight) (see Appendix 1). The - 62 - experimental arrangement is illustrated in Fig. 2.1. For the ozone treatmentJthe lignin suspension was supported on a medium-porosity sintered glass filter through which the ozone was passed (Fig. 2.2). The apparatus was equipped with a stirring device which maintained the particles in a well dispersed state in the suspension. Onreacted ozone in the effluent gas was collected in a gas absorber containing 2% potassium iodide solution. Thus the ozone concentration was determined by titrating the 2% KI solution with standard O.lN sodium thiosulphate solution. When the reaction mixture was stirred, the experimental set-up did not permit the determin ation of ozone gas concentration. Lignin and sawdust were ozonized as 4% suspensions in the aqueous acetic acid. After ozonization in suspension, the sample was filtered off, washed with distilled water and then extracted with 20 times its volume of 2% NaOH at 65°C for 1 hr. After filtration, the undissolved lignin was washed with 10 mls of the alkali in the case of lignin and 50 mls of alkali for saw- dust. Washincr of the residue was cont~nued until the ensuing filtrate was colourless. This was taken as an indication that all the degraded material had been extracted. The washings were added to the mother filtrate. The insoluble residue was now washed with distilled water, reacidified with 45% aqueous acetic acid, filtered off, and then washed several times with distilled water. It was then dried overnight in a vacuum oven at room temperature. The residue was further dried over P 2o5 (') e 0 --o o-- A A oxygen source E reactor B flow meter F gas absorber for measurement C silica gel of ozone concentration D ozonator G gas absorber containing K I solution FIG. 2.1 Schematic diagram of the experimental arrangement used for the ozonization of lignin and wood meal. m w - 64 - to constant weight. Recovery of the alkali-soluble products The alkali-soluble degradation products were pre cipitated by titrating the alkali extract with O.lN HCl to a pH of 2-3. The solubilized lignin from spruce wood was dif ficult to precipitate from the 2% NaOH solution. Therefore, the titrated solution was allowed to stand for about 24 hours. Only then did the degraded products gradually precipitate from the titrated solution. The precipitates were centrifuged at 15,000 r.p.m. for 20 minutes. The supernatant solutions were discarded and the precipitates washed several times with dis tilled water and freeze-dried from a dioxane-water solution (9:1). The freeze dried samples were weighed accurately and ·stored in a refrigerator. Estimation of lignin in treated wood samples Klason lignin analyses for the original and ozonized wood samples were carried out according to the TAPPI standard method for wood T222-0S-74 (12). Acid-soluble lignin was estimated spectrophoto metrically by measuring the absorbance at 205 nm of the Klason hydrolysates. An absorptivity of 110 1 g-l cm~(l3) was used to calculate the acid soluble lignin. - 65 - MOTOR t:==== TO 2% Kl SOLUTION PADDLE STIRRER 45% AQUEOUS ACETIC ACID LIGNIN OR SPRUCE WOOD MIXTURE OF OZONE AND OXYGEN FIG. 2.2 Diagram of the Reactor used for the ozoniz ation of lignin and wood meal. (a) without stirring (b) stirring. - 66 - RESULTS AND DISCUSSION Effect of stirring on yield of lignin Ozone attacked spruce periodate and cuoxam lignins as well as the protolignin in spruce wood meal, causing a breakdown of the macromolecular network and rendering the lignin extractable with 2% aqueous sodium hydroxide. The amount of alkali-soluble products increased as a function of time of ozone treatment until the lignin was completely sol uble. Paralleling this increase in the amount of the soluble material was a corresponding decrease in yield of the in soluble residue. A plot of the undissolved lignin versus time of ozone treatment is shown in Figs. 2.3 and 2.4. With out stirring, a slow rate is observed at approximately 15 minutes of ozonization. It was also observed that at this time, the lignin had aggregated into compact clumps, pre sumably due to the formation of charged groups during ozon ization. However, on stirring the reaction mixtures during ozone treatment, the lignin was observed to be well dispersed and the slow rate was replaced by an increased reaction rate. It was concluded that the slow rate was caused by aggregation of the lignin particles in the reaction mixture. This can be explained by the proposition that aggregation reduces the surface area available for attack by ozone. Thus a possible explanation for the effect of stirring is that it retains the lignin particles in a dispersed state and thus facilitates the reaction. - 67 - 100 • NO STIRRING • STIRRING 0~ 80 z CD ....J- 60 0 LU ~ 0 40 Cl) ~ z0 :::> 20 0 20 40 60 TIME FIG. 2.3 Yield of undissolved spruce periodate lignin at various times of ozonization in 45% aqueous acetic acid at room temperature. Ozone concentration, 49.7 mg o 3;1 o 2 (~3.7% by weight). 0 0 100 1\ • NO STIRRING ~ 80 L ~ • STIRRING . z -z C) 60 -__. 0w I ~~ • ~ 40 0 (/) (/) 0 z 20 :::> 0 20 40 60 80 100 TIME, min. FIG. 2.4 Yield of undissolved spruce cuoxam lignin at various times of ozonization in 45% aqueous acetic acid at room temperature. Ozone concentration is 49.7 mg 0'1 o ;1 o (~3.7%by weight). Flow rate 0.5 1/min. CO 3 2 - 69 - In another set of experiments it was observed that aggregation of lignin particles also occurred in water suspen sion. This means that the phenomenon is not peculiar to the aqueous acetic acid medium but is due to the formation of certain charged species during ozonization. Rate of degradation of the isolated lignins The reaction rates were calculated from the yield of undissolved lignin after each treatment and alkali extraction (Table 2.1). Each value represents the average of two determin- ations which agree within ± 0.5%. Assuming a simple first order reaction, the reaction rate R* is given by R* = ddL t i::: - KL ...... {2 .1) where R* is the rate of degradation in grams per gram per minute, L is the amount of undissolved lignin at time t, and K is the rate constant. Rearrangement and intergration of equation 2.1 gives L 0 Kt loglO L = 2.303 ...... (2. 2) where L is initial amount of lignin in grams, and the factor 0 2.303 converts the natural log to common logarithms, Accord- ingly, a plot of log Lo vs. t for periodate and cuoxam L lignins should be linear if the degradation follows first order kinetics. This is in fact the case as shown in Fig. 2.5). Using the method of least squares, the values of the slope of the lines were obtained from which the rate constants - 70 - TABLE 2. Yield of undissolved lignin in grams during ozonization of spruce periodate and cuoxam lignins Time of Treatment Periodate Lignin Cuoxam Lignin (min) 0 1.000 1.062 5 0.733 0.972 10 0.557 15 0.505 0.819 30 0.283 0.402 45 0.146 0.294 60 0.040 90 0.210 TABLE 2.2 Rate constants for the degradation of spruce periodate and cuoxam lignins by ozone 4 -1 Correlation Type of Lignin K x 10 (sec ) Coefficient periodate 6.95 0.999 cuoxam 5.10 0.996 - 71 - 1.0 ,....------r------r------, e PERIODATE LIGNIN o CUOXAM LIGNIN 0.8 ':1' 0.6 ~ 0 ...... __...._J 0 .,... 0.4 Cl 0 _J 0.2 0 20 40 60 TIME, min. FIG. 2.5 Log (L /L) versus ozonization time for spruce 10 0 periodate and cuoxam lignins. - 72- K were calculated (see Table 2.2). A comparison of the rate constants show that the degradation of spruce periodate lignin proceeds about 1.4 times as fast as that of cuoxam lignin. The reason for this differ ence in reactivity is not understood at present. A first order reaction for the dissolution of these lignins is in accordance with the findings of Arrhenius (14) in some of the earliest work on pulping. Binet and Page (15) also showed that the de lignification of Black Spruce by sodium chlorite in an acid medium follows a first order reaction pathway. The implication of a first order reaction is that the rate of degradation of lignin by ozone is controlled by a single process. Consumption of ozone by lignin The amount of ozone consumed by the lignin as a func tion of time is shown in Table 2.3. These data are of interest in attempting to establish whether there is a relation between the rate of ozone consumption and the rate of lignin degrad ation by ozone. This is the question that is now to be addressed. Attention has already been drawn to the course of the reaction between periodate lignin and ozone under quies cent conditions (see Figs. 2.3, 2.4). It might be expected that the rate of ozone consumption would be low at the point where the slow rate begins since the amount of lignin solubilized decreases rapidly with time. This was not observed. The amount of ozone consumed increased with time, despite the decrease in the amount of lignin solubilized (Fig. 2.6). - 73 - TABLE 2. 3 Rate of ozone consumption by periodate and cuoxam lignins for the first 30 minutes of ozone treatment Yield of Time of alkali- Rate of ozone ozone extracted consumption Lignin treatment lignin Ozone consumed moles/C9 . type (min.) (%) moles;c unit unit/min~ 9 Spruce 0 0.0 0.00 0.00 period- ate 5 22.5 0.28 0.06 lignin 10 24.5 1.02 0.10 15 47.6 2.16 0.14 20 46.5 2.79 0.14 25 48.3 3.91 0.16 Spruce 0 0.0 0.00 0.00 cuoxam lignin 5 7.4 0.31 0.06 10 16.0 0.50 0.05 15 26.8 1.18 0.08 30 49.5 4.53 0.15 - 74 - - 6 .------, ·c:...... ::J l:J. cuoxam lignin 0.. ~5 o periodate lignin CIJ .. (]) . 0 ~4 : 0 I i UJ .l ~ ...• :J 3 •.. Cl) .... z .... · 0 ...... ·• 0 2 ·· . ..· .. . UJ ...... z ...... ~ 0 1 0 10 20 30 40 50 60 PERCENT LIGNIN SOLUBILIZED FIG. 2.6 Ozone consumed versus lignin solubilized for spruce periodate and cuoxam lignins at various stages of lignin degradation. Ozonization was carried out under quiescent conditions. - 75 - An explanation for this effect was found when the molecular weights of the soluble products were studied (Chapter 4). It was observed that weight-average molecular weights of the sol uble products increased with time of ozone treatment but de creased after a given time (15 minutes for periodate lignin) indicating that the soluble products are further fragmented in the reaction mixture. Fragmentation of the soluble products requires that ozone be consumed. Therefore the point to be made here is that ozone consumed after 15 minutes under quies cent conditions is utilized in the fragmentation of the soluble products. The breakdown of the soluble products is expected of a gel undergoing degradation. Another argument for further fragmentation of the partly solubilized lignin is provided by the observation that the yield of acid precipitable products in the 2% NaOH extracts de creased with time, despite the increase in yield of the soluble portion (Tables 2.4, 2.5). It would appear that small molecules cannot be precipitated from the alkali solution by acid, and as more of these molecules were produced, the yield of acid pre cipitable products decreased. These simple molecules could in clude some monomeric and dimeric products as well as gaseous products. Rate of degradation of lignin in spruce wood In order to compare the rate of ozonolysis of iso lated lignins with that of lignin in spruce wood, the wood meal was reduced to the same particle size (80 mesh) as the isolated lignin prior to ozone treatment. The total lignin content of - 76 - TABLE 2.4 Yield of soluble products recovered by acid precipitation during the ozonization of spruce periodate lignin 'Nt. of t'lt. of Wt. of soluble % Time of lignin soluble products of soluble treatment used products recovered products . (min) (g) (g) (g) ·recovered 0 1. 00 0 5 1.00 0.267 0.048 18.1 10 1.00 0.443 0.050 11.4 15 1.00 0.495 0.020 4.0 30 1.00 0.717 0.015 2.0 45 1.00 0.854 0.009 1.1 60 1.00 0.960 0 0 TABLE 2.5 Yield of soluble products recovered by acid precipitation during ozonization of spruce cuoxam lignin Wt. of Wt. of Wt. of soluble % Time of lignin soluble products of soluble treatment used products recovered products (min {~:> (g) (g) recovered 0 1.062 0.000 5 1.062 0.090 0.032 35.2 15 1.062 0.244 0.061 25.0 30 1.062 0.536 0.0384 7.2 45 1.062 0.768 0.017 2.2 90 1.062 0.852 0.000 0.0 - 77 - the original and ozonized wood meal was determined as the sum of the Klason lignin and the acid soluble lignin in the Klason hydrolysate. Table 2.6 shows the lignin content of the original and ozone-treated wood meal after each treatment time. Each value of the total lignin is an average of two measurements. The precision was ± 0.2%. The values for two separate ozone treatments for the same time agreed within ± 0.5% From Table 2.6, it can be seen that the ozonization of spruce wood results in a progressive decrease in the lignin content with increasing ozonization time up to 120 minutes of ozone treatment. Longer periods of ozonization produce little or no change in the lignin content of the wood. It is therefore concluded that a small portion of the lignin (~3.6% of the wood) is resistant to degradation. In order to calculate the reaction rate, equation 2.2 was modified to account for the portion of the lignin that is resistant to degradation by ozone. If L is the amount of r resistant lignin in grams, the rate of degradation is given by equation 2.3: L -L o r Kt = • • • • • • • • • • • • • • • • • • • • • • • • • • (2. 3) L-L r 2.303 where L0 Land t have the same meaning as in equation 2.2. The rate of lignin degradation calculated from equation 2.3 is plotted in Fig. 2.7. From the least square slope of the line the value of K was calculated to be 5.09 x 10-4 sec-1 . These results indicate that the degradation of the protolignin - 78 C) TABLE 2.6 Lignin content of the original and treated wood meal during ozonization of spruce wood Acid- Klason soluble Total Time of lignin lignin lignin Total lignin % treatment (% of (% of (% of (% of original Deligni- (rnin. ) pulp) pulp) pulp} sample) fication 0 27.6 0.11 27.7 27.7 0 5 24.6 0.22 24.8 23.0 17.0 10 21.6 0.30 21.9 19.4 30.0 15 20.4 0.31 20.7 17.7 36.0 20 18.6 0.33 18.9 15.3 44.8 30 18.2 0.32 18.5 14.4 48.0 40 13.3 0.29 13.6 9.7 65.0 60 10.3 0.20 10.5 7.1 74.4 80 8.4 0.16 8.6 5.5 80.1 120 6.0 0.14 6.1 3.4 87.7 240 6.3 0.30 * 6.6 3.7 86.6 * Only one determination. - 79 - 0 1.0 0.8 ...... --...,_ ,_ .....J .....J I 0 I .....J .....J ...... __..... 0.6 0,.... O'l 0 .....J 0.4 0.2 0 20 40 60 80 100 TIME ( min) L -L FIG. 2.7 Log { ~-Lr} versus ozonization time for 10 r spruce wood protolignin (Corr. Coeff. 0.99) - 80 by ozone follows first order kinetics. The small portion of the lignin that is resistant to degradation probably follows a different mechanism. Comparison of the rate constants for the degradation of the isolated lignins and protolignin in spruce wood Figs. 2.5 and 2.7 demonstrate that the degradation of spruce periodate and cuoxam lignins and spruce protolignin follow first order kinetics. This indicates that in all cases the degradation process is governed by a similar reaction mechanism. It is interesting to note that the values for the rate constants of the isolated lignins (see Table 2.2) are of the same order of magnitude as that of the spruce protolignin. It is concluded that periodate and cuoxam lignins (especially cuoxam lignin) retain the network structure of the protolignin and are therefore suitable for physicochemical studies of lignin. The somewhat higher reaction rate for spruce periodate lignin is attributed to the slight structural modification that occurred during isolation (16). In this connection, it is interesting to mention that Fleming (17) has recently found that during the cooking of these lignins by the Kraft process cuoxam lignin and protolignin dissolved at the same rate whereas periodate lignin dissolved at a faster rate. Another inference that can be drawn from the present results is that the carbohydrate matrix in wood has an insignifi- cant effect on the rate of delignification of the protolignin by ozone. - 81 - CONCLUSION The results reported in this chapter indicate that the ozone degradation of spruce periodate and cuoxam lignins and the bulk of the protolignin in spruce wood follow first order kinetics. It is concluded that the degradation process in isolated lignins and protolignin is governed by a similar reaction mechanism and that in all cases, there is a single rate controlling reaction. A small portion of the protolignin is resistant to ozonolysis and probably degrades by a dif ferent mechanism. Because the values for the rate constants of iso lated lignins and the protolignin in spruce wood are of comparable magnitude, it is most probable that periodate and cuoxam lignins (in particular cuoxam lignin) retain the net work structure of the protolignin during isolation. The some what higher rate constant for spruce periodate lignin, when compared to cuoxam lignin and protolignin, is believed to result from slight structural modification of the periodate lignin during its isolation. The comparable reaction rates of the insoluble iso lated lignins and the protolignin in spruce wood indicate that the retention of the carbohydrate portion of wood plays no part in controlling the rate of delignification of the prate lignin by ozone. From the yield of the soluble products recovered by precipitation from the alkali extract, it is concluded that - 82 . partly degraded lignin is further being attacked by ozone to produce smaller fragments of lower molecular weights. In the earlier stages of the lignin-ozone reaction, the amount of ozone consumed is utilized primarily for the degradation of the lignin gel. At the later stages, the ozone consumed is used to degrade both the lignin gel and the primary degradation products. The rate of ozone consumption is dependent on the lignin type. Thus, at the early stages of the reaction, periodate lignin consumes ozone at an average rate of 0.12 moles;c9/minute, whereas for cuoxam lignin the average rate of ozone consumption was found to be 0.08 moles;c9/minute. The rates of ozone consumption reflect the respective rates of degradation of these lignins. For instance, the ratio of the rate constants, K (for periodate lignin to cuoxarn lignin) is 1.4. The ratio of their rates of ozone consumption is 1.5. It can be assumed that protolignin in spruce wood absorbs ozone at the approximate rate of 0.08 moles;c9;rninute since its rate of degradation is similar to that of cuoxam lignin. - 83 REFERENCES 1. Katuscak, S., Hrivik, A., Mahdalik, H., Paperi ja Puu Vol. 53, No. 9, 519-524{1971). 2. Katuscak, s., Rybarik, I., Paulinyova, E., Mahdalik, M., Ibid. No. 11, 665-670{1971). 3. Kratzl, K., Claus, P., Reichel, G., Tappi, Vol. 59, No. 11 (NOV• 1976). 4. Hatakeyama, H., Toonoka, T., Nakano, J., and Migita, N., Kogyo, Kagaku Zasshi, Vol. 71, 1214(1967). 5. Wald, W.J., Ritchie, P.F., Purves, C.B., J. Am. Chem. Soc., _§1, 1371(1947). 6. Ritchie, P.F., Purves, C.B., Paper Mag. Can., 48, No. 12, 74 (Nov. 1947). 7. Freudenberg, K., Zocher, H., and Durr, w., Ber., g, 1.814 ( 1929) . 8. Bolker, H.I., Rhodes, H.E.W., Lee, K.S., J. Ag. Food Chem., 25 (4) 708 (1977). 9. Freudenberg, K., Sohns, F., Janson, A., Ann., 518, 62(1935). 10. Walter, H. Reginald, Sherman, M. Ruth, J. Food Science, Vol. 41(5), 993-995(1976). 11. Doree, Charles, Healey, A.C., J. Textile Inst. T27-42, (March 19 38) • 12. Tappi Standard, T222-0S-74. 13. Tappi Standard (Useful methods) 250. 14. Arrhenius, s., Svensk Papperstidn., £1, 189(1924). 15. Binet, R., Page, D.H., Transactions by the Tech. Sec. Vol. 3 (3) 73-75 (Sept. 1977). - 84 - 16. Ad1er, E., Hernestam, S., Acta. Chem. Scand., ~, 319 (1955). 17. Fleming, B., private communication. 0 - 85 CHAPTER 3 DEGRADATION OF LIGNIN BY OZONE: (II) SPECTROSCOPIC STUDIES OF THE ALKALI-SOLUBLE DEGRADATION PRODUCTS - 86 ABSTRACT The alkali-soluble degradation products obtained during the ozonization of spruce periodate and cuoxam lignins and lignin in spruce wood, have been studied by spectroscopic methods. The results show that carboxyl groups are formed dur ing ozonization in 45% aqueous acetic atid. In tHe latter stages of the reaction, decarboxylation occurs as shown by the lower carboxyl content of the alkali-soluble degradation prod ucts. Evidence was obtained indicating the cleavage of aromatic rings and the attack of methoxyl groups during lignin degrad ation by ozone. Absorptivity measurements (~E) of the alkali-soluble degradation products of the lignins reveal an increase in the phenolic hydroxyl content as the time of ozone treatment in creased. Since ozone attacks phenolic structures, it is deduced that phenolic hydroxyl groups are regenerated through the cleavage of phenol ether bonds of lignin by ozone. From UV difference spectra, it was deduced that both unconjugated phenols and phenols conjugated with carbonyl groups are formed during the lignin-ozone reaction. Based on the known reactions of ozone with other structures in lignin, it is suggested that the principal network-degrading reaction involves the cleavage of ether bonds and that the aromatic ring cleavage is second ary. A tentative mechanism is proposed for the cleavage of the phenol ether bonds in lignin durinq ozonization. - 87 - NMR spectra of the alkali-soluble degradation prod ucts show signals due to highly shielded protons. In the case of the degradation products from periodate lignin, the intensity of these signals increases with time of ozone treat ment. The origin of these signals is not understood at present. - 88 INTRODUCTION In the preceding chapter, the kinetics of ozone degradation of spruce periodate and cuoxam lignins and proto lignin in spruce wood were studied. The results indicated that, in all cases, degradation followed first order kinetics suggesting a similar chemical degradation mechanism and a single rate controlling process. The closeness of the values of the rate constants of the isolated lignins to that of the protolignin led to the conclusion that the carbohydrate portion of wood plays no part in controlling the rate of degradation of the protolignin, nor does it restrict the rate of solution of the protolignin after degradation. Furthermore, it was concluded that periodate and cuoxam lignins (especially. cuoxam lignin) retain the network structure of the proto lignin from which they are derived. The amount of ozone con sumed by each lignin type was found to be a measure of its reactivity towards ozone. In order to gain insight into the chemical reactions that lead to the oxidative degradation of the lignins by ozone, it was considered necessary to study the structural . changes that occur in the alkali-soluble products as a function of the duration of ozone treatment. Spectroscopy was chosen as the tool for this aspect of the study because it has, in the past, provided answers to difficult problems in lignin structure elucidation and identification of reaction products. Infra-red spectroscopy (IR) has long been employed - 89 in the study of lignins and lignin reaction products (1-13). Recently Katuscak {14) and coworkers followed the changes in the structure of methanol lignin during ozonization by infra- red spectroscopy. In the present study, changes in the content of aromatic rings, carbonyl, carboxyl, and hydroxyl groups, during ozonization have been followed by infra-red spectres- copy. The first application of nuclear magnetic resonance spectroscopy (n.m.r.) to the study of liqnin was carried out by Ludwig (15,16) who determined the chemical shifts of n.m.r. resonance signals from protons in a number of lignin related model compounds. Signals with certain ranges of chemical shift from acetylated softwood lignins were then related to character- istic proton signals from the model compounds. N.m.r. has since been employed in the estimation of lianin structural features {17) and the studv of liqnin reaction products {18-21). Ozone- , - ' treated methanol lignin samples have been studied by n.m.r. as a function of time of treatment (14). In that study, an un- identified signal at 1.24o was attributed to highly shielded aliphatic protons. The proton signals in the successive samples became progressively sharper and in accordance with Hrutfiord and ~1cCarthy (22), the authors interpreted this to indicate a progressive decrease in molecular weight of the methanol lignin. In the present study, the trend in signal intensity of protons attached to particular functional groups has been utilized to confirm the observations made from the IR analysis. More im- portantly, the changes in signal intensity of the a and s - 90 - protons of the side chain in the guaiacyl skeleton have neen used to follow the reaction of ozone with the ether groups attached to the a and 8 carbons of the side chain. Since Herzog and Hillmer's (23) discovery of the characteristic ultraviolet {UV) absorption of lignin solutions, the literature is replete with information concerning the UV spectra of lignins and lignin reaction products (24-27). The (UV) difference spectroscopy of Aulin-Erdtman (28) and Gold schmid (29) has been successfully applied in monitoring changes resulting from reactions of lignins with various re agents. Recently it has been applied in the estimation of the phenolic hydroxyl groups in the different morphological regions of wood (30). In the present work, UV spectroscopy has been used to follow the changes in the structure and in particular the phenolic hydroxyl content of the alkali-soluble degradation products of the lignins under investigation. The main purpose of investigating the phenolic hydroxyl content was to cast some light on the different reactions that lead to lignin degradation, since, in a previous study (31), it was concluded that ozone degrades lignin mainly by way of its aromatic rings. An increase in the phenolic hydroxyl content of the successive alkali-soluble products would result from the cleavage of ether bonds during lignin degradation, and therefore would provide another pathway for the breakdown of the lignin macromolecule. - 91 - EXPERIMENTAL ~·he alkali-soluble degradation products used in all investigations were prepared by ozonization of periodate and cuoxam lignins and spruce wood meal. The experimental method has been described earlier {32). Infra-red analysis IR spectra were recorded in a Unicam SP 200G grating spectrophotometer using the KBr disc technique. Each disc con- tained 1.5 mg lignin per lOO mg KBr. Proton magnetic resonance spectrosCOEY Since the original samples were insoluble in d6- dimethylsulfoxide, proton magnetic resonance spectra could only be obtained from the alkali-soluble products of lignin degradation by ozone. Spectra were ' obtained with a Bruker WH90 Proton Magnetic Resonance Spectrometer, frequency 90.2 MHz. Samples were measured as 2.5% solutions in d6-dimethylsulfoxide solvent, at a constant temperature of 35°C. Ultra-violet analysis uv spectra of the soluble products were obtained with a Unicam SPlOO grating spectrophotometer. All spectra were ob- tained at a constant temperature of 25°C. The insolubility and in some cases the partial solubility of the products in con- ventional organic solvents like chloroform, tetrahydrofuran, and benzene left buffer solutions as the only choice for the UV study. Standard buffer solutions supplied by Anachemia were - 92 used tor the UV study. The buffer solution, pH 6, consisted of O.lM monobasic potassium phosphate and sodium hydroxide, whereas the buffer solution, pH 12, was made by dissolving 220 grams of sodium phosphate (Na HP0 .7H 0) and 120 mls of lN NaOH in 5 2 4 2 litres of distilled water. Although, during the preparative stage these soluble products were freeze-dried from dioxane water (9:1) solution; once dry, they were only partially soluble in this solvent. All UV measurements were made at a concentration of 1 mg lignin per 20 ml buffer. This concentration was arrived at by a series of trial runs. The validity of the Beer-Lambert law in the buffer solution at pH 6 was tested by calibration over the range of concentration likely to be used. Reduction with sodium borohydride Ozone-treated lignin samples were reduced by suspend ing 0.1 g of the sample in 20 mls of an aqueous solution of O.lN sodium borohydride for 5 hrs at room temoerature. At the end of the borohydride treatment the sample was filtered off and dried under reduced pressure before use for the IR analysis. - 93 . RESULTS AND DISCUSSION Infra-red analysis Because lignin monomeric units are complex and are linked in a random manner, it is impossible to apply group theory to interpret a lignin spectrum. However the general con- cordance in assignment of bands by various workers has made it possible to relate certain frequencies to characteristic groups in lignin. Band assignments have been given by Browning (33) (see also Appendix 2) . The original periodate and cuoxam lignins shmv the characteristic lignin IR bands (Appendix 2). However, the cuoxam lignin showed no absorption in the 1705-1720 cm-l region, in- dicating the absence of unconjugated carbonyl groups. The pres ence of ~ small shoulder at 1715 cm-l in periodate lignin is presumed to be due to the formation of these unconjugated carbonyls during isolation since they are not present in proto- lignin. The alkali-soluble degradation products exhibited marked structural changes during ozonization. In the first few minutes of treatment, infrared analysis of the alakli-soluble degradation products showed an increase in intensity of the 1715- 1720 cm-l band compared to the original samples {Fig. 3.1, 3.2, 3.3). Reduction with aqueous sodium borohydride caused the 1715- 1 1720 crn:- band to shift to 1590-1600 cm-1. This is an indication of the formation of aliphatic carboxylic acids during ozonization (34}. Accordingly, an increase in the intensity of the 1715-1720 - 94 FIG. 3.1 Infra-red spectra (KBr wafers) of the original sample (SPL-0) and the alkali-soluble degradation prod.ucts (SPL-5 to SPL-45) obtained during the ozonization of spruce periodate lignin in 45% aqueous acetic acid. A- SPL-0 original sample B- SPL-5 5 rnin C - SPL-15 15 min D- SPL-30 30 min E - SPL-45 45 min 0 c 1750 1500 1250 1000 750 A 8 UJ 0 z c ~ 1- ~ Cl)z <( D a: 1- E COOH.( 1750 1500 1250 1000 750 WAVENUMBER , cm-1 - 95 - cm-1 band would be expected as the ozonization time increased. This trend was reported by Katuscak (14) during the ozonization of methanol lignin in the solid state. On the contrary, in the present study, at longer times of treatment, successive alkali- soluble degradation products showed a decrease in the intensity of this band. The same observation was made both for periodate and cuoxam lignin products and also for the soluble lignin from spruce wood. The explanation of this behaviour was obtained when copious quantities of carbon dioxide were detected in the effluent gas using barium hydroxide; this led to conclusion that de- carboxylation takes place during ozonization of lignin in an acid medium. Further evidence in support of decarboxylation has been recently supplied by Balashov et al. (35) who observed this effect during the oxidation of lignin model compounds with ozone. During the ozonization of methanol lignin (14) an enhanced absorption was observed in the 1680-1660 cm-l region and was attributed to the formation of quinones. In the present study, such enhanced absorption was not observed. In all three lignin types, the alkali-soluble degrad- ation products showed a progressive decrease in intensity of the 1600 cm-l and 1510-1515 cm-l bands. As these bands are typical of aromatic structures, it was deduced that ozonization of lignin is accompanied by 'Cleavage of aromatic rings •. This is in agreement with earlier findings (14,11,34,36,37) on ozonization of lignins and wood. -1 . The broad band centred at 1600 cm deserves special attention. This band was usually thought to be due to aromatic - 96 - FIG. 3.2 Infra-red spectra (KBr wafers) of the original sample (SCL-0) and the alkali-soluble degradation products (SCL-5 to SO.r-90) obtained during the ozonization of spruce cuoxam lignin in 45% aqueous acetic acid. A- SCL-0 original sample B - scr...-5 5 rnin c - SO.r-30 30 min D- SO.r-90 45 rnin 1500 1250 1000 750 .._,. A B c D Ar. r!ng \"'" \ J-oH COnjug. \p guaiacyl ring 1750 1500 1250 1000 750 WAVE NUMBER, cm-1 - 97 - 0 rings conjugated with carbonyl groups. Kolboe and Ellefsen (38) have demonstrated that a large proportion of this band is due to diphenyl groups. In the present investigation it was ob- served that the original lignins showed a band extending from 1585-1600 cm-1 • This is also true for the alkali-soluble lignin products obtained at the early stages of ozone treatment of wood (see Fig. 3.3, SSL-1). At longer treatment times, the alkali-soluble products lost the 1600 cm-l contribution and showed bands centered at 1585 cm-1 . The 1585-1590 cm-l con- tribution in lignin has been shown to be due to a benzene nucleus conjugated with a carbonyl group (38). The loss of the 1600 cm-l contribution is attributed to ozone attack on diphenyl structures of the lignin. The loss of this absorption early in the ozonization process indicates that diphenyl type aromatic rings are very susceptible to ozone attack. Increasing time of ozone treatment resulted in the gradual loss of the 1585 cm-l band, indicating the attack by ozone on the aromatic rings conjugated with carbonyl groups. The conclusion that is drawn from the foregoing observations is that in an acidic medium there is a preferential attack, by ozone, of aromatic rings in diphenyl structures as compared to ozone attack on aromatic rings conjugated with carbonyl groups. The reactivity of these chemical species to- wards ozone provides indirect evidence for the electrophilic nature of the ozone-lignin reaction as previously suggested by Freudenberg (39). The phenyl group is electron releasing and so tends to stabilize the aromatic ring towards an electrophilic - 98 - FIG. 3.3 Infra-red (KBr wafers) spectra of the alkali soluble lignin degradation products (SSL-1 to SSL-30) obtained during ozonization of spruce wood in 45% aqueous acetic acid. A- SSL-1 1 min B - SSL-20 20 rnin C - SSL-30 30 min 1750 1500 1250 1000 750 A 8 c guaiacyl ~ ring COOH 1750 1500 1250 1000 750 WAVENUMBER , cm -1 - 99 - attack. On the other hand, a destabilization effect results from the electron-withdrawing tendency of the carbonyl group. It should be mentioned here that only recently, studies on the oxidative degradation of softwood lignin model compounds with ozone indicated that biphenyl structures of lignin were more reactive than phenyl coumarans and aryl ethers (40). The order of reactivity was explained in terms of the electron- donating tendency of the phenyl group. The generation of hydroxyl groups during ozone treat ment was particularly evident in the alkali-soluble degradation products from the periodate lignins. This was manifested by the development of a small band at 1380 cm -1 (virtually absent in the original samples, see Fig. 3.1,3.2). Deuteration studies (38) have revealed that the 1380 cm-l band is due to·free phenolic hydroxyl groups. The increase in intensity of this band in the alkali-soluble products during ozonization has, therefore, been attributed to the cleavage of phenol ether bonds in lignin. The phenolic hydroxyl content of the alkali- soluble products was therefore investigated in more detail by UV spectroscopy. The results are described in this chapter. Semi-quantitative interpretation of the nuclear magnetic reson ance spectra of the alkali-soluble products The chemical shifts for typical protons found in lignin model compounds and adjusted for related signals from lignin have been reported by Ludwig et al. (15,16) (see also Appendix 2}. Spectra of the original periodate and cuoxam lignins could not be obtained for comparison because these lignins - 100 - are insoluble in all solvents including the d6-DMSO used for this study. The n.m.r. spectra of the alkali-soluble degrad ation products after ozonization of the isolated lignins are shown in Figs. 3.4 and 3.5. Because of difficulty in integrat ing the peaks, a proton distribution could not be calculated for these samples. The apparent lack of success in integration of the peaks is not peculiar to these samples. Similar dif ficulties were encountered by Lenz (21) who failed to obtain an integration of the peaks in his spectra of milled wood lignin in d6-DMSO solvent. This may be related to the use of d 6-DMSO as the solvent. All the spectra of the alkali-soluble degradation products in d 6-DMSO showed a drift in baseline, particularly in the region where highly shielded protons give n.m.r. signals. This made it difficult for the machine to inte grate the peaks. Although proton distribution could not be calculated, a semi-quantitative picture of the trend in signal intensity with increasing ozonization time for the alkali soluble products was determined from Figs. 3.4 and 3.5 and is given in Table 3.1. The decrease in intensity of the signals in the range 8.08-8.36o due to carboxyl protons reinforces the IR observation that decarboxylation takes place in the later stages of the reaction. Furthermore, the decreasing intensity of the broad signals at 6.37-7.83o agrees with IR observations and in dicates that degradation of lignin by ozone is accompanied by cleavage of aromatic rings. It was also observed that there is a marked decrease - 101 - 0 FIG. 3.4 90 MHz proton magnetic resonance spectra (in d - 6 dimethylsulfoxide) of the alkali-soluble degradation products (SPL-5 to SPL-45) obtained during the ozonization of spruce periodate lignin in 45% aqueous acetic acid. A- SPL-5 5 min B- SPL-15 15 min C - SPL-30 30 min D- SPL-45 45 min N G J: 0 (.) c;' < ,._ ! 0 M J: (.) ~ _) 0 m l ~ !.. I Cl l ~ I ~ N ~ ,:s 1j ~; ~ -E l:j ~ a. -a. 1.() ro I \ ( i ! ~ ~ ~ ' I i ' \ t . l f ~ l 'I ) I O'l c: ~ / \ \ $ \ I \) 1 ~ ! } 0 } ; 1 o-~ CO (.) ( <) \ ' ~ ~ 1 J t f I fl l I I l <' ( l\ I j 1 ; ' I I •1 ' i Ql ol o:n FIG. 3. 5 90 MHz proton magnetic resonance spectra (in d - 6 dirrethylsulfoxide) of the alkali-soluble degradation products (SCL-5 to SCI.r-90) obtained during the ozonization of spruce cuoxam lignin in 45% aqueous acetic acid. A- SCL-5 5 min B - SCL-30 30 min c- SCL-90 45 min (/) ~ 1- ~ 0 ('11 :t: () i I- 0 0 (") ---too- (/) :t: ~ () Q C\1 ~ -E 0. -0. 0> .i::c: ....:- <( :t: 0- 0 () Q) CO <( I - 103 - TABLE 3.1 Trend in signal intensity for successive alkali-soluble degradation products obtained during the ozonization of spruce periodate and cuoxarn lignins Chemical Trend in signal shift Proton intensity in o (ppm) types successive products Comments 8.08-8.36 carboxyl decreasing protons 6. 3-7. 83 aromatic decreasing more rapid decrease protons for cuoxam lignin products 3.95-4.91 a,a,y decreasing not significant for protons y protons; more of the pronounced for a side chain and Cl protons 2.75-3.91 methoxyl decreasing protons (aliphatic and aromatic) 2.5 d6-DMSO peak 0.50-1.58 highly shield rapid increase in no discernible tren• ed aliphatic periodate lignin for cuoxam lignin protons degradation degradation product: products - 104 - in the content of side chain protons (a,S) during ozone re action with the lignins. The side chain protons showed signals in the range 3.95-4.918. The decrease in the intensity of the signals in this range is a clear manifestation that functional groups attached to the a and S carbon atoms of the side chain are attacked by ozone. Thus aryl glycerol-B-aryl ether type structures (I) and other ether structures (II, III) as well as a and S hydroxyl groups are attacked by ozone. These results make it reasonable to suggest that ether-bond cleavage plays an important role in the degradation of lignin. Ludwig (15,16} assigned the signals occurring between 5.18-5.788 to protons on an a-carbon in the phenyl coumaran ring system (III) (see Appendix 2). The absence of any signals in this range suggests that protons attached to the a-carbon of phenyl coum aran type rings are attacked during ozonization. Changes in the methoxyl content of the soluble products are indicated by the decrease in intensity of the signals between 2.75-3.918. Proton signals from water in the ~ 6 -DMSO may interfere in this region. However, the attack of methoxyl groups is evidenced from methoxyl analysis of the pro ucts (Table 3.2). Perhaps the most interesting observation in the n.m.r. spectra of these alkali-soluble degradation products was the occurrence of signals between 0.5-1.588. Specifically two signals centered at 0.5d (broad) and 1.28o (sharp) were observed. Similar proton signals have been reported from the n.m.~ spectra of acetolysis products of lignin (19), the spectra - 105 - M 0 I () 0 I 0 I I-o-o- o I I I I I I M () I I 0 0 I I I-()-()~() I I I I I I M () I · I 0 0 0 I I I I -0-()-() 0- 1-1 I I I I I I ).... CQ. ~ - 106 - of acetolysis products of lignin (19), the spectra of the nondistillable lignin fractions from the hydrogenation of maple wood (18), the spectra of the bark lignin fraction in western red cedar (20), and even from the solid state ozoniz ation products of methanol lignin (14). Protons which are known to give signals in this range (15,16) are bonded to methyl or methylene carbons which are not attached directly to oxygen functions, carbonyl functions, aromatic systems and other deshielding groups. The present work provides additional evidence that such groups exist in lignin. What the nature of these protons is and how they fit into the structure of lignin is still a puzzling question. It is even more so since, in the case of periodate lignin degradat~on products, the in tensity of this signal increased markedly with the time of ozonization (Fig. 3.4). Ultra violet spectra of the alkali-soluble degradation products It was mentioned earlier in this chapter that the alkali-soluble degradation products obtained during ozonization of the lignins are insoluble in most organic solvents. Conse quently UV spectra were obtained in acid-neutral buffer solution, pH 6. The validity of the Beer-Lambert law in this solvent over the concentration range employed was confirmed by the linearity of the calibration curve (Fig. 3.6). Unlike a typical lignin spectrum which shows maxima near 205 nm and 280 nm and a shoulder at 230 nm, all the samples 0 had similar absorption bands, showing maxima at 227-230 nm and 280 nm and a minimum at 265 nm (Fig. 3.7). The 205 nm absorption - 107 TABLE 3. 2 Methoxyl content of the original and alkali-soluble degradation products of spruce periodate lignin {SPL) and spruce cuoxam lignin {SCL) as a function of the duration of ozone treatment Time of ozone treatment Methoxvl content Sample No. {min.) (%) SPL-0 0 '12.17 SPL-5 5 8.58 SPL-15 15 8.11 SPL-30 30 9.09 SCL-0 0 14.12 SCL-5 5 9.70 SCL-15 15 7.51 SCL-30 30 5.41 was absent in all spectra. The shape of the spectra is similar to that obtained for the products of the degradation of methanol lignin by ozone in the solid state (14), a-lthough the later spectra were taken in dioxane~ Since all the spectra were obtained under identical conditions of concentration and temperature, the decrease in absorption throughout the entire 225-350 nm region in successive alkali-soluble degradation products indicates the progressive destruction of the aromatic conjugated systems of periodate, and cuoxam lignins. The 280 nm band in lignins arises from guaiacyl, syringyl and related structures with a preponderance of aromatic methoxyl over aromatic hydroxyl and so the decrease in absorbance of this - 108 0 1.2 .------, 1.0 0 A 250nm UJ 0.8 0 A 280nm 0z tii a: 0.6 0 (/) CJ <( 0.4 0.2 0 0.05 0.10 0.15 CONCENTRATION ( g /I) FIG. 3.6 W Calibration (in buffer solution, pH 6) at 250 and 280 rm. - 109 . FIG. 3. 7 tJV spectra (in buffer solution pH 6) of the alkali-soluble degradation products (SCL-5 to SCL-45) obtained during the ozonization of spruce cuoxa.m lignin in 45% aqueous acetic acid. SCL-5 5 min SCL-15 ------15 min SCL-30 -· ·-· ·- 30 min SCL-45 - -- - 45 min 1.0 ~--~--~----~~.~~N j C') t. I 0 /J I 0 ,/) I C') ,. , I 1.0 E ( ( \ ('... c N ... } "j I I t- i • J (!) z .. / ;· J 0 UJ 1.0 _J ... ··:/·· I N w ·············· . / .. ·················· /. I s:~ .,...... / .. 1.0 C\J ( : I C\1 ··... ··. l '·,. .. ( ~-. N CO 0 ,...: d 38N\18tJOS8V - 110 - band indicates that methoxyl groups are attacked by ozone. Phenolic hydroxyl content of alkali-soluble degradation products As indicated earlier it was of interest to estimate the amount of free phenolic hydroxyl groups in the soluble products in order to gain some insight into the nature of the degradation mechanism. The ~€ method developed by Aulin-Erdtman (27,28) and later applied by Goldschmid (29) was employed in the present study. This method is based on the fact that the UV absorption of phenols and phenoxide ions are different. The absorption maximum given by a phenoxide ion is generally higher than that of non-ionized phenol; moreover it is located at a longer wavelength. In alkali solutions {pH 12) the phenolic hydroxyl groups are ionized whereas under neutral conditions they are not ionized. Thus the increase in ultraviolet ab- sorption produced by the addition of alkali to the system is related directly to the concentration of ionizable phenolic groups in the lignin. UV spectrograms of the alkali-soluble degradation products were measured in the buffer solutions. For each sample, spectra were obtained at pH 6 and pH 12 (Fig. 3.8). The absorb- ance of these solutions was obtained at the same concentration. Repeat scans in the same cells were reproducible within 0.005 absorbance unit. Measurements taken within 48 hours gave the same values showing that the samples did not interact with the buffer solutions in this time interval. The difference in absorbance, ~A, at pH 6 and pH 12 gives a measure of the phenolic - 111 - hydroxyl content for each sample. Absorptivity values were calculated from the solvent zero absorbance tracings. The calculation of the phenolic hydroxyl content of the samples was carried out according to the procedure outlined by Yang and Goring (30). It is based on the assumption that all phenolic hydroxyl groups are non-ionized at pH 6 and ionized at pH 12. According to the Beer-Lambert law: (A)6 €: Ph-OH) ( c Ph-OH) 1 + ( €: Ph-O-R) ( c Ph-O-R) 1..... [ 1 ] = ( 6 6 6 6 €: c €: c {A)l2 = ( Ph-0) 12 ( Ph-0) 121 + ( Ph-0-12) ( Ph-O-R) 12 1. ... [2] Where A = absorbance at 250 nm c = concentration of lignin in g/litre €: = absorptivity 1 = thickness of cell in cm ) 6 = measured at pH 6 )12 = measured at pH 12 Ph-OH = ionizable free phenolic hydroxyl groups Ph-0 = ionized free phenolic groups Ph-o-R = etherified phenolic groups If it is further assumed that absorptivity and concentration of the etherified phenolic groups do not change in going from an acid-neutral solution to alkali solution, ( e; Ph-O-R) ( e:oH-0-R) 6 = .. 12 ...... [3] c = { Ph-0-R} •.••..•.•••.•...... •..• [4] 12 - 112 - FIG. 3.8 W absorbance (in buffer solutions, pH 6 and pH 12) of the alkali-soluble degradation products of spruce cuoxam lignin during ozonization in 45% aqueous acetic acid. :treasured at pH 6 ------:treasured at pH 12 A- SCI.r-5 5 min B - SCirl5 15 min c - ~45 45 rnin 0 .... A 1.2 ···· ...... : .. 0.8 .; .. i 0.4 ...... 0 B 1.2 ..····· .. .. : ··. .... --~·...... ·\.. Q) 0.8 \ (.) ···... c ...... CO ..c 0.4 ······ ··············· ... !.... ······ ...... 0 ...... (J) ··········-· ..c <( 0 c 1.2 ...., 0.8 i ...... :.: '·· .. . .~ ...... 0.4 ...... ············ ·····- 0 225 250 275 300 325 Wavelength nm - 113 - and the concentration of the phenolic groups is equal to that of the ionized free phenolic groups, c c Ph-OH = Ph-0 •••.•••.•.•••.••.•.••••.••.•..•••••.• [5] then the concentration of the free phenolic groups can be calculated by substracting [1] from [2], thus (A) - (A) = 8A • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • [ 6] 12 6 All measurements were made with a solution concentration of 0.05 g/litre and with matched 1-cm cells thus enabling ~e values to be calculated. Goldschmid (29) determined the percent phenolic hydroxyl content by multiplying the difference in absorptivity of the alkali and acid-neutral solutions at 300 nm by a factor of 0.414 which was determined from model compound studies. This value is, however, mainly applied to aromatic compounds that are not conjugated at the a-position of the side chain. Wexler (41) has shown that for both conjugated and non-con- jugated elements, the short wavelength maximum in the difference spectrum is preferred. The conversion factor to percent phenolic hydroxyl is 0.192. Wexler's method has been adopted in the present work. A phenolic hydroxyl content of 2% in a purified lignosulphonate corresponds to about 0.25 phenolic groups per unit (41). Using this information, the phenolic c9 hydroxyl content was calculated for the alkali-soluble degrad- ation products of periodate and cuoxam lignins. The results are shown in Table 3.3. - 114 - TABLE 3.3 Phenolic hydroxyl content of the alkali-soluble degrad ation products obtained during the ozonization of spruce cuoxam lignin (SCL) and spruce periodate lignin (SPL) Time of ozone A 250 nm Sample treatment No. (min.) (A) 12 (A) 6 ~E % Ph-OH Ph-OH/C9 SCL-15 15 0.515 0.385 0.13 2.6 0.50 0.06 SCL-30 30 0.475 0.245 0.23 4.6 0.88 0.11 SCL-45 45 0.385 0.085 0.30 6.0 1.15 0.14 SPL-5 5 0.515 0.385 0.13 2.6 0.50 0.06 SPL-10 10 0.585 0.395 0.19 3.8 0.73 0.09 SPL-15 15 0.525 0.295 0.23 4.6 0.88 0.11 SPL-25 25 0.635 0.365 0.27 5.4 1.04 0.13 - 115 - The data indicates that there is an increase in the phenolic hydroxyl content of the soluble products as the time of ozonization and hence the weight fraction, Ws, of the soluble products increases. The values of the phenolic hydroxyl per c9 unit may be somewhat in error since carboxyl groups, which have been shown to be produced during ozonization, are apt to interfere with the measurements of absorbance at pH 12. However, the fact that the ph-OH values continue to increase despite the decreasing trend in the carboxyl content of the successive soluble degradation products (see Figs. 3.1-3.5) shows that the carboxyl groups have no significant effect on the absorbance values. It is well known that ozone attacks phenolic struc tures according to Criegee's mechanism (42) resulting in aromatic ring opening. It is therefore logical to expect the initial amount (if any) of the phenolic hydroxyl groups to decrease as the time of ozonization advances. The observed in crease in the content of phenolic hydroxyl groups in the soluble products suggests that these groups must be regenerated during the ozonization of the lignin. This is possible only by the cleavage of a simple phenol ether linkage or by the opening of a five or six-membered ring. The former would involve the cleavage of aryl glycerol-a-aryl ether structures and benzyl aryl ethers linkages (I,II). In the latter case, phenyl coumaran structures (III) would be involved. Here, a carbon to carbon linkage must also be cleaved at carbon atom 5 of the benzene ring and the carbon chain replaced by a hydrogen atom. This is - 116 - quite possible in an oxidation reaction where a carboxyl group is first formed, followed by the splitting off of carbon dioxide. N.m.r. spectra discussed earlier suggest that ether structures of the type mentioned here are cleaved by ozone. These results lead to the conclusion that in addition to the aromatic ring degradation reported earlier by other workers (21,37,43-45) and substantiated by the present work, ozone degrades lignin by way of phenol ether bond cleavage. In fact the main reaction leading to lignin degradation by ozone is believed to be that involving ether-bond cleavage. The reason for this conclusion should become clear from the following considerations. In previous studies on the course of aromatic ring scission by ozone (31,37,45) it was found that ozone attacks carbon atoms carrying oxygen functionalities preferentially. In the guaiacyl unit of lignin, scission was found to occur mainly between the c3 and c4 positions of the aromatic ring, resulting in the formation of muconic acid structures (31,37, 45). If Freudenberg's formulation (46) (see Fig. 1.5) is assumed to give a close representation of spruce lignin, it can be seen that ring opening by ozone cannot affect the integrity of the lignin macromolecule. On the other hand, scission of an ether linkage will result in the fragmentation of the lignin. In view of the predominance of the ether linkages in lignin (47), it is proposed that the observed ether cleavage by ozone is the principal reaction leading to the degradation of the lignin network. It should be mentioned, however, that the - 117 - ultimate fate of the muconic acid structures resulting from aromatic ring opening, is complete fragmentation but this re action is secondary and occurs after extensive degradation of the products of reaction (31,45). Difference spectra of alkali-soluble products The difference spectrogram obtained by using an acid neutral solution (pH 6) of sample SCL-15 in the reference cell of the double beam spectrophotometer and the alkali solution (pH 12) in the sample cell is shown in (Fig. 3.9). The spectro gram shows three maxima at 250 nm, 300 nm and 350-360 nm. From model compound studies and studies on lignosulphonates, non-conjugated guaiacyl units without branching at the carbon in the side chain para to a hydroxyl group, exhibit a strong peak in the difference spectra at about 250 nm and a weak band at 300 nm. In such units the ~ nm value, which gives the spacing in nm between the ~ 250 nm peak and the ~ 300 nm peak, ranges from 45 to 51 nm. The ~ nm value for sample SCL-15 is 50 nm. The difference spectrogram therefore indicates that simple, non-conjugated guaiacyl units of this type are formed during the degradation of these lignins under the reaction conditions. In contrast, conjugated guaiacyl derivatives such as vanillin and isoeugenol with an a-ethylenic group absorb above 300 nm. The effect of conjugation of the aromatic hydroxyl with a carbonyl group in the para position,is a displacement of the long wavelength maximum at 300 nm by 50 to 60 nm thus giving 0 a band at about 350-360 nm. The difference spectra of the - 118 - FIG. 3.9 Direct and differential spectrograms of sample SCL-15. pH 6 pH 12 Difference spectrum ;I il :I :l: :::> ! , a::: iJ 1-u l LLI •I/ a.. •: I CJ) ••.. I LLI . u .•l I z .: I LLI 0 a::: ..l I 1.{) CV) (D ~ tt J I :I: :I: LL. ..: I Q. Q. 25 .... I _.: I .... / E .... / c .... / .:· I I .l· I .-l I t5 .: I z ..·/ 0 UJ •' .·· / 0 ....J ..·· ""' UJ .. ·· ..._, "" et) ~ ..;;; ....., ···~"" ~ .,~ ~ I' •\ !\ ! 1 . I •.. .. ·· I .... ; ••• ····· .,1 "' 0 ····· . ~ 1.{) --····· •••••••-- .,. .., .... (fill' C\J C\J 0 38N'v'8tjQS8V - 119 - soluble fractions as exemplified by sample SCL-15 also exhibit a peak at 360 nm indicating the formation of carbonyl con jugated guaiacyl derivatives during ozonization. The possibility of an ethylenic group conjugated with the guaiacyl ring such as in isoeugenolis dismissed by the absence of a negative absorp tion at 250 nm in the difference spectrum. Mechanism of phenol ether bond cleavage in lignin by ozone The molecular weight distributionsof the alkali soluble degradation products are reported in Chapter 4 of this thesis. The results indicate a random stepwise degradation mechanism and thus the possibility of a radical mechanism as the mode of lignin network degradation by ozone can be dismissed. The IR observation that biphenyl aromatic rings are attacked faster than aromatic rings conjugated with carbonyl groups suggests an electrophilic mechanism. Price and Tumolo (48) proposed a polar mechanism for the attack on ethers by ozone. Based on their work, a mechanism is proposed to explain the cleavage of phenol ether bonds and the formation of free phenolic groups#and aromatic rings conjugated with carbonyl groups (Fig. 3.10). The most plausible mechanism is believed to involve a concerted 1,3 dipolar insertion into the C-H bond of the ether function (48-52). This would be accomplished through a hydride transfer from the ether function to the ozone. An unstable hydrotrioxide intermediate would be formed which breaks down to give the products (Fig. 3.10). The proposed mechanism is tentative and probably - 120 - I -9--c- ~ I . ·0 0 -c------:•...- -c-• c...,o H,o 0 e=> e OCH 0-0-0 ~~~ 3 -c-I -c-• o o C·~) H ~ 0 £c;'\QOCH3 FIG. 3.10 Proposed macha:nism for the cleavage of a phenol ether bond in lignin by ozone. - 121 - represents the limit of the present understanding of the re action between the ether bonds of lignin and ozone. It is possible that other factors, especially the reaction medium, may influence the course of the reaction. Earlier investigations (39,44) have shown that ozonization of lignin proceeds much faster in acidic media than in water. It is also well known (53,54) that ozone i~ more stable at low pH values. I.t is, therefore, reasonable to believe that the enhanced reaction rate in acidic solutions is a physical effect {as opposed to one involving a chemical reaction) since the concentration of ozone in the reaction mixture appears to be an important factor affecting the re- action rate. Obviously, further work is required before a com plete understanding of the reactions of lignin with ozone is possible. Acetic acid-soluble degradation products It should be mentioned here that in the absence of ozone treatment, periodate, cuoxam and wood lignins are in soluble in 45% aqueous acetic acid. However when ozone treat ment was done over a considerable period, (say 20 minutes} some degraded material dissolved in the acetic acid as evidenced by the faint yellow calor of the solution. However, the amount of acetic acid-soluble products was comparatively small. For in stance, from a 90-minute ozonization of spruce cuoxam lignin only 0.3% of the soluble portion was recovered from the aqueous acetic acid medium. The acetic acid-soluble material was recovered by - 122 - vacuum evaporation of the filtrate from the lignin suspension after ozonization. The concentrate was diluted with about 10 times its volume of water, and then centrifuged at 15000 rpm for 20·minutes. The precipitate was washed with distilled water and freeze-dried from a dioxane-water solution. The material is greyish white in color and is insoluble in water. It dis- solves in dioxane-water, DMSO, and dilute alkali. The IR spectrum of this product (Fig. 3.11) is not typical of lignin. The absence of any bands in the 1510-1515 cm -1 and 1590-1600 cm -1 regions show that these products have lost their aromaticity. A carbonyl band (possibly acetyl carbonyl) is evident at 1735 cm-l A broad band centered at 1075 cm-l is possibly due to a mixture of primary, secondary and tertiary aliphatic alcohols. Because only a negligible amount of the total degraded material was soluble in the acetic acid medium, this portion of the soluble products \-Tas not studied in detail. It is, however, mentioned here for the sake of completeness. () 0 100~------~------.------.------.------~ ~ w {) 50 z .,._~ ~ Cl) z <( .,._a.: 0 1750 1500 1250 1000 WAVENUMBER , cm-1 FIG. 3.11 Infra-red spectrum of the acetic acid-soluble degradation products of spruce cuoxam lignin. 1-' 1\.} w - 124 - 0 CONCLUSION The results presented in this chapter show that o:zonization of lignin results in the formation of unconjugated carboxylic acids. This is possibly the result of aromatic ring opening and t~e oxidation of terminal primary alcoholic groups or the attack of unsaturated structures of the side chain in the lignin c9 unit. During ozonization in an acidic medium, carboxyl groups are initially formed; subsequently decarboxylation occurs and this results in alkali-soluble degradation products having progressively lower carboxyl content as the reaction proceeds. N.m.r. spectra of the alkali-soluble products suggest that ozone attacks methoxyl groups and ether structures of the lignin macromolecule. a-Protons of phenyl coumaran type ether linkages were absent in the alkali-soluble products pointing to the rapid cleavage of such ether structures. N.m.r., IR, and UV show that aromatic rings of the lignin macromolecule are attacked by ozone. Aromatic rings con jugated with ethylenic groups (as in diphenyl structures} appear to be more rapidly attacked than those conjugated with carbonyl groups. This is indirect evidence supporting the electrophilic nature of the lignin-ozone reaction. N.m.r. spectra show signals due to highly shielded protons. The intensity of these signals increases with time in the case of the alkali-soluble products of spruce periodate - 125 - lignin. The origin of these signals is unknown. UV spectra show that free phenolic groups are re generated during lignin degradation by ozone. This is ascribed to the cleavage by ozone of simple phenol ether linkages or the opening of a furan or pyran ring in lignin. This led to the conclusion that the main reaction leading to the degradation of the lignin network by ozone is the cleavage of the ether linkages and that aromatic ring cleavage is secondary. Finally, the UV spectra show that both unconjugated phenols as well as phenols with a carbonyl group in the a position of the guaiacyl ring are formed during lignin degrad atio.n by ozone. 0 - 126 - REFERENCES 1. Jones, E.J., J. Am. Chern. Soc., IQ, 1984(1948). 2. Jones, E.J., Tappi, ~, 167(1949). 3. Freudenberg, K., Siebert, w., Heirnberger, w. and Kraft, R., Chern. Ber., ~' 533(1950). 4. Freudenberg, K., Dietrich, H., Siebert, W., Ibid.,~, 961 (1951) • 5. Kratzl, K. and 'I'sharnlerh, Monatsh. Chern., ~~ 786 (1952). 6. Kratzl, K., Ibid., 84, 406(1953). 7. Schubert, W.J. and Nord, F.F., J. Am. Chern. Soc., ~, 3835 (1950). 8. Kudzin, S.F., Debaun, R.M., Nord, F.F., Ibid., ll' 4615 (1951) . 9. Kudzin, S.F., Nord, F.F., Ibid., ll, 690, 4619(1951). 10. Destevens, G., Nord, F.F., Ibid., ll, 4622(1951). 11. Hess, C.L., Tappi, ~, 312(1952). 12. Sarkanen, K.V., Chang, H.M., Ericsson, B., Tappi, 50, 572 (1967). 13. Sarkanen, K.V., Chang, H.M., Allan, G.G., Ibid., 50, 587 {1967). 14. Katuscak, s., Rybarik, I., Paulinyova, E., Mahdalik, M., Paperi ja Puu, Vol. 53, No. 11, 665(1971). 15. Ludwig, G.H., Nist, B.J. and McCarthy, J.L., J. Am. Chem. Soc.,~, 1186(1964). 16. Ludwig, G.H., Nist, B.J. and McCarthy, J.L. 1 J. Am. Chem. Soc.,~, 1196(1964}. - 127 - 17. Bland, D.E. and Sternhell, S., Australian J. Chem., 18, 401 (1965). 18. Parker, P.E., Ph.D. Thesis, State University College of Forestry, Syracuse University, Syracuse, N.Y. (June 1965). 19. Fukuzumi, T., Sakuma, S., Takahashi, H., Tomita, K., Fugihara, K., Isome, Y., and Shibamoto, T., Bolzforschung, 20 (4}, 51(1966). 20. Swan, E.P., Pulp Paper Mfg. Can. 67: T456(1966}. 21. Lenz, L. Bernard, Tappi, Vol. 51, 511 (Nov. 11, 1968). 22. Hrutfiord, B.F. and McCarthy, J.L., Advances in Chemistry Series, No. 59, Washington, D.C., American Chemical Society, 1966, Chapter 15, pp. 226-237. 23. Herzog, R.O., and Hillmer, A., Chem. Ber., 60, 365(1927); Z. Physical Chem., 168, 117(1927). 24. Patterson, R.F. and Hibbert, H., J. Am. Chem. Soc.,~, 1872 (1943) . 25. Glading, R.E., Paper Trade J., 3, Nov. 23,32(1940). 26. Patterson, R.F. and Hibbert, H., J. Am. Chem. Soc., 65, 1869 (1943). 27. Aulin-Erdtman, G., Svensk Papperstidn., 47, 91(1944). 28. Aulin-Erdtman, G., Tappi, 32, 160(1949). 29. Goldschmid, o., Anal. Chem., ~, 1421-23(1954). 30. Yang, J.M. and Goring, D.A.I., Pulp and Paper Can. Trans actions, Vol. 4{1) TR2, March 1978. 31. Hatakeyama, H., Tonooka, T., Nakano, J., and Migita, N., Kogyo Kagaku Zasshi, Vol. 71, 1214(1967). 0 32. Chapter 2 of this thesis. - 128 - 33. Browning, B.L., "Methods of Wood Chemistry", Vol. 2, Interscience Publishers, N.Y., London, Sydney, p. 750 (1967). 34. Sarkanen, K.V. and Ludwig, C.H. {eds.) "Lignins: Occur- rence, Formation, Structure and Reactions", Wiley Inter- science, New York (1971), Chapter 7. 35. Balashov, C.V., Mandrykin, Yu.l: Tregubov, B.A., Kovalenko, E.I., Mater- Vses, Mezhvus, Konf. Ozonu, 2nd 156-157 (1977), Maltsev, Yu., A, Moskovskii Gos (Eds.). 36. Soteland, N., Norsk Skogind, 25, No. 3, 61 and No. 5, (1971). 37. Kratzl, K., Claus, P. Reichel, G. Tappi, Vol. ~, No. 11, 86 (Nov. 1976) . 38. Kolboe, S. and Ellefsen, Q., Tapoi,. -45, 163(1962). 39. Freudenberg, K., Johns, F., Janson, A., Ann. Chem., 518, 62 (1935). 40. Kojima, Y., l1iura, I 41. lvexler, S. Arthur, Analytical Chem. Vol. ~, No. 1, 213 {Jan. 1964) . 42. Criegee, R., Record of Chemical Progress, 18, 111(1957). 43. Katuscak, A., Hrivik, M., Mahdalik, M., Paperi ja Puu Vol. 53, No. 9, 519(1971). 44. Katuscak, A., Rybarik, H., Paulinyova, E., Mahalik, M., Paperi ja Puu, Vol. 53, No. 11, Vol. 665(1971). 45. Hatakeyama, H., Tonooka, T., Nakano, I., Migita, N., Ibid., Vol. 2Q, 2348(1967). - 129 46. Freudenberg, K., Science, 148, 595(1965). 47. Adler, E., Wood Sci. Technol., 11, 169-218(1977}. 48. Price, C.C., Tumolo, L.A., J. Am. Chem. Soc.,~, 4691 (1964) . 49. White, H.M., Bailey, P.S., J. Org. Chem., 30, 3037(1965). 50. Batterbee, J.E., Bailey, P.S., J. Org. Chem., 32, 3899 (1967}. 51. Erickson, R.E., Hansen, R.T., Harkins, J., J. Am. Chem., Soc., 2Q, 6777(1968). 52. Deslongchamps, P., Atlani, P., Frehel, D., Malaval, A., and Moreau, c., Can. J. Chem., ~' 3651(1974) 53. Adler, M.G., Hill, G.R., J. Am. Chem. Soc. 72, 1884-6 (1950). 54. Stumm, w., Helv. Chim. Acta 37, 773(1954). 0 - 130 - 0 CHAPTER 4 DEGRADATION OF LIGNIN BY OZONE: (III) MOLECULAR WEIGHT AND MOLECULAR WEIGHT DISTRIBUTIONS OF THE ALKALI-SOLUBLE DEGRADATION PRODUCTS - 131 - ABSTRACT The molecular weights and molecular weight distributions of the alkali-soluble degradation products from the ozonolysis of spruce periodate and cuoxam lignins and spruce protolignin have been studied by gel permeation chromatography and ultracentrifugation. The bimodal distribution previously reported for soluble lignins was found to be an artifact; the cor rect distribution has one broad low molecular weight maximum, with a long tail extending towards the high molecular weight end of the distribution. Weight average molecular weights of the alkali soluble degradation products, as obtained by sedimentation equilibrium measurements, increased with time of ozonization up to about 15 minutes. Beyond this time, fragmentation of the partly degraded products results in a decrease in molecular weight. Lignin degradation was found to follow the pattern expected of a three-dimensional, infinite network polymer gel undergoing breakdown. Based on the molecular weights and the molecular weight distributions, a random stepwise mechanism is suggested as the mode of lignin degradation by ozone. - 132 - 0 INTRODUCTION In previous chapters of this thesis, the kinetics of lignin degradation by ozone at room temperature and the struc- tural changes in the alkali-soluble degradation products were studied. It was found that lignin, whether isolated or in wood, degraded by a first order reaction pathway, a result which suggested a single rate controlling degradation process. Structurally an increase in the carboxyl and phenolic hydroxyl groups and a decrease in the aromatic and methoxyl content of the soluble products accompany lignin degradation by ozone. The increase in the phenolic hydroxyl content of the sol- uble products was demonstrated to result from the cleavage of phenol ether bonds and the conclusion was reached that the break- down of the lignin macromolecule results mainly from the cleavage of the phenol ether bonds. Ozonization was found to result in the formation of aromatic rings conjugated with carbonyl groups. The more rapid attack of biphenyl type aromatic rings, compared to aromatic rings conjugated with carbonyl groups provided evidence in support of the electrophilic nature of the ozone-lignin re- action. The present chapter deals with studies of the molecular weights and molecular weight distributions of the alkali-soluble degradation products obtained from the ozonolysis of spruce periodate and cuoxam lignins and spruce protolignin. The molecular weight distribution was studied primarily to distinguish between the possible random and selective modes of lignin degradation by - 133 ozone. The trend in the molecular weights of the soluble products as a function of ozonization time was expected to cast light on the architecture of lignin as it exists in wood. The molecular weight distribution of soluble lignins has been widely studied by gel permeation chromatography (1-10). Basically two types of distributions have been reported namely bimodal (1-10) and unimodal (2,10). Of the various studies, the following are of particular interest since they are represent ative of the current views on the subject. McNaughton et al. (2} obtained bimodal chromatograms for spruce lignin fractions on Sephadex G-25 and G-100. However, after summing the distributions for all the fractions, they concluded that the dual peak was an artifact, and that the correct distrib ution should have one maximum in the chromatogram. Wayman and Obiaga (6), reported bimodal distributions in Sephadex G-100 chromatograms of isolated milled wood lignins and lignins dissolved during Kraft and soda pulping of spruce wood. Based on these chromatograms, they proposed a chain reaction de polymerisation mechanism for the degradation of lignin during alkaline pulping. They suggested that lignin in wood exists as modules and degradation entails the'unzippering'of these modules to yield material of the same molecular weight as the primary repeating unit of Forss and Fermer (1) or the average chain length of Bolker and Brenner {7). Rhodes et al. (10), using a Sephadex G-50-DMSO system, obtained bimodal chromatograms for the soluble products from - 134 . chlorine monoxide degradation of spruce periodate and cuoxam lignins. The same samples gave unimodal chromatograms on a Styragel-tetrahydrofuran system. From Flory's theory (11), they obtained by calculation, the expected size distribution of the sol of a three-dimensional infinite network gel. Based on this distribution, they concluded that the 'high peak' in their Sephadex chromatogfams is an artifact anq merely represents the accumulation of all macromolecules of hydrodynamic volume greater than that separable by the Sephadex gel. It was argued that with a gel of sufficiently wide separation range, the correct distrib ution of the soluble lignin will be one with a single low molecular weight maximum as was observed on the Styragel-tetra hydrofuran system. Although the present study was undertaken primarily to determine the mode of degradation of lignin by ozone, it was also expected to resolve the disagreement about the correct molecular weight distribution of soluble lignins. Unlike previous studies, the same samples \vere studied on different gels (Sephadex G-50, G-100, G-200) having a wide range of sizes and therefore separation capabilities. The position of the exclusion limit of each Sephadex gel, which has been so much neglected in most prev ious studies, was determined and clearly defined, thus enabling a more precise interpretation of the chromatograms. - 135 . EXPERIMENTAL Calibration of Sephadex G-100 column The G-100 column was calibrated with narrow molecular weight lignin fractions obtained by degrading spruce periodate lignin with alkali. The lignin fractions were prepared as follows: The sample of periodate lignin (93.2% Klason) was cooked for 12 hours with O.SN NaOH in a steel bomb immersed in an oil bath at 160-170°C. The product was filtered through a glass fiber filter to remove a small amount of insoluble residue. 2 ml of the filtrate was made up to 10 ml with 0.2N NaOH. The solution was layered on a preparative Sephadex G-100 column (Pharmacia Canada Ltd. Column K25/45) and eluted at a flow rate of 1.1 ml/min. The column effluent was collected in 10 ml aliquots with the aid of a fraction collector. Each fraction was titrated with 0.05N HCl to pH 3. The precipitated lignin was centrifuged at 15,000 rpm for 20 min, washed several times with distilled water and freeze-dried from a dioxane-water solution. The fractions were run on the analytical Sephadex G-100 column (Pharmacia, K25/45). The Sephadex G-100 was pre swollen in dimethylsulfoxide. The lignin fractions were dissolved in dimethylsulfoxide at a concentration of 5 mg/ml. 100 ~1 of solution was used for each analysis. From the shape of each chromatogram, fractions of relatively narrow polydispersity (c1 ,c2,c5 ,c6) were selected to be run on the ultracentrifuge for molecular weight determination. These fractions were used to - 136 calibrate the Sephadex G-100 colum. The short column sediment- ation equilibrium technique of Van Holde and Baldwin (12) was used to determine the molecular weights of c1 ,c2 ,c5 and c6 • Details of this technique are given in Appendix 4. The partial specific volume of the fractions was deter mined as 0.676 ml g-l with the aid of a digital precision density meter (see Appendix 4); The refractive index increment, dn/dc, was determined to be 0.133 ml g-l (also see Appendix 4) using a Zeiss microdiffusion interferometer. Table 4.1 shows the values for the weight average molecular weight Mw of these fractions. The void volume of the column was determined with a fraction, • The position of the c1 6 void volume was confirmed by using Blue Dextran 2000, Mw = 2 x 10 • The total permeation volume of the column was determined by using vanillin (Mw = 152). The logarithm of the molecular weights were plotted against the peak elution volumes to obtain the calibration curve (Fig. 4.1). Calibration of Sephadex G-200 column The Sephadex G-200 column was calibrated with narrow molecular weight dioxane-lignin fractions kindly supplied by Dr. F. Pla of the Ecole Francaise de Papeterie in Grenoble, France. The molecular weights and polydispersity of these fractions are given in Table 4. 2. The void volume and total 0 permeation volume of the Sephadex G-200 column were determined - 137 - 0 TABLE 4.1 Weight average molecular weights of lignin fractions employed in calibration of the Sephadex G-100 column M Fraction No. w cl 504,000 c2 173,000 CS 11,500 c6 4,900 TABLE 4.2 Weight average (Mw)' number average (Mn), and polydispersity (Mw/Mn} of fractions used to calibrate Sephadex G-200 M M Fraction w n MwfMn F6 9,800 7,900 1.24 F7 7,700 6,200 1.24 F9 3,640 3,060 1.19 Fll 1,600 1,450 1.10 - 138 5 0 50 100 150 ELUTION VOLUME, ml FIG. 4.1 Calibration curve for the Sephadex G-100 column. - 139 - as in the case of Sephadex G-100 using fraction c1 , Blue Dextran and vanillin. These fractions were supplemented by fraction c2 , ~ = 173,000. The calibration curve for the G-200 column is shown in Fig. 4.2. The justification for the use of dioxane lignin fractions for the calibration of the G-200 column is provided by the observa tion that periodate lignin fraction c5 , eluted through the G-200 column gave a maximum at an elution volume which, from the G-200 calibration curve, corresponded to its molecular weight. Gel chromatography of the alkali-soluble degradation products of lignin degradation by ozone The alkali-soluble products from the degradation of periodate, cuoxam and spruce wood lignins were isolated by acid precipitation at pH 3. Details are described in Chapter 2 of this thesis. Chromatograms of the soluble products were obtained on the analytical Sephadex G-100 column attached to a Waters Associate Liquid Chromatograph Model 301. All samples were injected as 0.5% solutions (5 mg/ml) in dimethylsulfoxide~ 10 ~1 of solution was used for each analysis. Injection was done by means of a microliter syringe to a continuously flowing stream of dimethylsulfoxide. A constant flow rate of 0.4 ml/min was maintained for the Sephadex G-50 and G-100 columns by means of a peristaltic pump. The elution rate for the G-200 column was 0.2 ml/min. In all cases, the column effluent was continuously monitored at 280 nm using a differential 0 UV detector. The outlet was connected to a 5 ml siphon attached to an electrical device which triggered an event marker each time - 140 - c 6 ~------~ 5 s I~ 4 ,.-0 0) 0 .....J 3 \ \ \ \l I 2 0 100 150 200 ELUTION VOLUME , ml FIG. 4.2 Calibration curve for the Sephadex G-200 column. - 141 - the siphon was filled. Spikes were therefore recorded on the chromatograms at 5 ml intervals. Molecular weights of the alkali-soluble lignins Weight average molecular weights of the soluble products were determined by the short column sedimentation equilibrium technique (12). The partial specific volume V,and the refractive index increments dn/dc,of the alkali-soluble products from the ozonization of the different lignins are given in Table 4.3 (see also Appendix 4}. TABLE 4. 3 Partial specific volume and refractive index increments of the alkali-soluble products from the ozonization of periodate, cuoxam lignins and protolignins from spruce wood Degradation products V -1 dn/dc from the ozonization of (ml g } (ml g-1) Periodate Lignin 0.638 0.065 Cuoxam Lignin 0.591 0.062 Spruce Protolignin 0.612 0.062 Elemental Analysis Elemental analysis of the alkali-soluble products from lignin degradation by ozone was done by Schwarzkopf Micro analytical Laboratory, New York. - 142 . RESULTS AND DISCUSSION [1] Molecular Weight Distributions (a) Degradation products of the isolated lignins Chromatographed on Sephadex G-50, the ozone treated periodate and cuoxam lignin degradation products showed a large peak at the exclusion limit of the column with a tail extending to higher elution volumes (see for instance, sample SPL-5, Fig. 4.3). The area under this peak indicated the exclusion of a substantial fraction of the total material; accordingly no further study was carried out with this gel. An apparent bimodal distribution was observed in the • chromatograms of the same samples on Sephadex G-100 (see, for instance, sample SPL-5, Fig. 4.4). By comparison with the G-50 column, the area under the exclusion peak of the Sephadex G-100 was considerably reduced in favour of a low molecular weight peak, the maximum of which was situated between elution volumes of 110 ml and 113 ml. From the calibration curve for the G-100 column, the position of the low molecular weight peak indicated a pre- ponderance of material with a weight average molecular weight Mw , of 6000-6600. However, the presence of a substantial fraction of excluded material for the alkali-soluble products on the G-100 gel prompted the use of Sephadex G-200. It was expected that the G-200 gel would provide a better separation of the products and consequently give a closer approximation to the true molecular weight distribution of the products. On Sephadex G-200, the periodate lignin products also - 143 - FIG. 4.3 Sephadex G-50 chromatogram of the alkali soluble degradation products obtained after 5 min ozonization of spruce periodate lignin (SPL-5} FIG. 4.4 Sephadex G-100 chromatogram of the alkali soluble degradation products obtained after 5 min ozonization of spruce periodate lignin (SPL-5) FIG. 4.5 Sephadex G-200 chromatogram of the alkali soluble degradation products obtained after 5 min ozonization of spruce periodate lignin (SPL-5) l Q E c: !VOID VOLUME 0 CO C\1 SEPHADEX-G -50 w uz ~ FIG. 4. 3 a: 0 (J) c::J <( 0 50 100 150 200 ELUTION VOLUME , ml E c: 0 CO SEPHADEX - G -100 C\1 UJ !VOID VOLUME uz ~ FIG. 4. 4 a: !TOTAL 0 VOLUME (J) c::J <( 0 50 100 150 ELUTION VOLUME , E c: 0 CO SEPHADEX- G -200 C\1 uUJ z j10fAL ~ jVOID VOLUME VOLUME a: FIG. 4.5 0 (J) c::J <( 0 50 100 150 ELUTION VOLUME , - 144 gave an apparent bimodal distribution, but with the exclusion peak now reduced to a mere shoulder in the chromatogram (Fig. 4.5, 4.6). The low molecular weight peak ranged between elution volumes of 163-166 ml, corresponding to Mw's between 5600-7500. At the void volume of the column the peak height was increased in going from sample SPL-5 to SPL-15. This increase in height corresponds to the formation of a longer high molecular weight tail in the chromatogram. Sample SPL-30 showed a very broad peak in the low molecular weight region and it was difficult to establish the centre of this peak. It should be noted that the positions of the low molecular weight maximum on the G-100 and G-200 gels correspond to the same molecular weight on the different calibration curves (for instance, sample SPL-5). Similar results were obtained for the ozone-treated cuoxam lignin products. {b) Degradation products of spruce protolignin The shape of the chromatograms of the alkali-soluble degradation products of spruce protolignin after ozonization were essentially similar to those of periodate and cuoxam lignin degradation products (Fig. 4.7). However, the low molecular weight peaks in the spruce protolignin products occurred at lower elution volumes. For instance, for sample SSL-1 and SSL-20, the low molecular weight maximum ranged between elution volumes of 154-158 ml (Mw = 12,500-17,700). Longer periods of ozonization {for example 30 min) yield products which showed a shift of the low molecular weight maximum to a higher elution volume of - 145 - FIG. 4.6 Sephadex G-200 chromatograms of the alkali soluble degradation products of spruce periodate lignin (SPL) obtained at various times of ozone treatment of the periodate lignin. SPL-5 5 min SPL-15 15 min SPL-30 30 min 0 0 N I ~ I 0 X w 0 0 C\1 0 ------~0 WU08G'3~NV8~0S8V - 146 . 166 ml (Mw = 5600). Unfortunately it was not possible to isolate the degradation products solubilized beyond 30 min of ozone treatment. It is believed that extensive degradation of the products occur beyond 30 min of ozonization. [2] Interpretation of chromatograms A characteristic feature of these chromatograms (worthy of special mention) is the relative proportion of the excluded material compared to the total material in going from Sephadex G-50 to G-200 (Fig. 4.3, 4.4, 4.5). On G-200, the alkali-soluble products were nearly completely separated. It can be expected that with a gel of higher exclusion limit, complete separation would be achieved resulting in a distribution with a single low molecular weight maximum. In an effort to obtain complete separation of these products, unsuccessful attempts were made with controlled porosity glass packings (CPG) of 120/200 mesh size and different pore diameters. A combination of pore diameters was chosen to cover 6 a separation range between molecular weights (Mw = 500 - 1 x 10 ). The alkali-soluble products were strongly adsorbed on to the glass. Attempts to circumvent the adsorption problem by treating the glass with Carbowax 20M proved abortive. The present results obtained on the Sephadex gels demon strate that the apparent bimodal distribution previously reported for soluble lignins is an artifact, and that the correct distribu- tion of soluble lignins is one with a single maximum at the low ~ molecular weight end of the molecular weight spectrum. The results strongly reinforce those of Bolker et al. (10) and McNaughton et al. - 147 - FIG. 4.7 Sephadex G-200 chromatograms of the alkali soluble degradation products of spruce wood protolignin (SSL) obtained at various times of ozone treatment of spruce wood. SSL-1 1 min SSL-20 ...... 20 min SSL-30 30 min 0 L.U 0 ...J ~ N :J I ~ _J C) 0 I f2 > X 0 w ... 0 0 <( C\1 J: 0..w V) 0 E LO ccc or- ·- ·-·- u.J EEE ~ 00 ::> Cl -r-'C\JCV) _J z . LU I . t ~ 00 ta_J -r-'C\JCV) 0 z 0 0 ~~~ L.U or- - (/)(/)(/) ~ 1- (/)(/)(/) 0 :J ::> - ...J _J ~~ UJ ------~0 WU08G'38NV8~0S8V - 148 - (2), which suggested that soluble lignins have a single low molecular weight maximum in the distribution curve. The pattern exhibited in the distribution on G-200 demands further discussion. By assuming that degradation follows the reverse path of poly merization, Flory's mathematical treatment of gelation (11) can be applied to the degradation of lignin. Rhodes (10) has pointed out that according to t~is treatment, the solqble products obtained early in the degradation process should have a broad distribution with a single low molecular weight maximum, the abundance of fractions decreasing as the complexity (molecular weight) increases. As the degradation proceeds the high molecular weight tail should increase at the expense of the low molecular weight maximum. From Flory's treatment, the above pattern of molecular weight distribution is expected of the degradation of any infinite network gel, whether it is formed from the random condensation of bi-and trifunctional monomers or from the polymerization of al ready existing primary chains. The correspondence of the dis tributions of the lignin degradation products observed on Sephadex G-200 (Fig. 4.6, 4.7) with theory suggests that lignin as it exists in wood is a three~dimensional infinite network polymer gel. l3J Molecular weights of alkali-soluble degradation products Direct calculation of molecular weights from the chromate grams on Sephadex G-200 was not possible owing to the presence of some excluded material in the distributions. Weight average molecular weights were,therefore,determined by the short column sedimentation equilibrium technique (12). In Table 4.4, the weight - 149 - average molecular weights, M , of the alkali-soluble products w obtained during the ozonolysis of spruce periodate and cuoxam lignins and protolignin in spruce wood are shown together with the weight fraction of the sol, ws. From Table 4.4,it is evident that in the early stages of the reaction (up to about 15 min) the molecular weight of the soluble products increases with the time of ozonization and the weight fraction of the soluble fraction. The same trend hac been reported for other lignin degradation reactions (2,5,6,10,13-15), although substantially different interpretations had been given to explain its origin. One of these, conveniently called the "pore size theory" (17) proposes a model in which lignin in the middle lamella is finite and of higher molecular weight than lignin in the secondary wall. According to this model, the molecular size of soluble lignin diffusing out of the wood is controlled by the sizes of the pores in the cell wall. Since these pores were shown to increase in size during delignification, the sizes of the ensuing lignin molecules increases as the extraction proceeds. In the present work, the trend of increasing molecular weight with increasing solubility was observed both with the isolated lignins {periodate and cuoxam lignins} and lignin in spruce wood. This parallels the results obtained by Rhodes (10) on the chlorine monoxide degradation of spruce periodate and cuoxam lignins. Ob- viously, the argument relating the controlling influence of the pores in the cell wall on the molecular weight of the soluble products does not apply in the case of the isolated lignins. Furthermore, it has been shown in this thesis (18) that the rate - 150 - of solubilization of the periodate and cuoxam lignins are about the same as the rate of delignification of spruce proto lignin by ozone, indicating that the carbohydrate components of the wood do not act as physical barriers restricting the dif- fusion of the degraded lignin macromolecules out of the wood. In connection with the observed increase in molecular weight of the soluble ·products, there remains to be considered the explanation adduced by the proponents (19-23) of the 'condensation theory'. According to this theory, lignin in wood consists of finite macromolecules with reactive sites that con- dense to form larger macromolecules when the lignin is extracted. Condensation is believed to occur under acidic and alkaline conditions. It is implicit in this theory that so long as there are reactive sites, the molecular weight of the soluble lignin would continue to increase. When the reactive sites are exhausted, no further increase in molecular weight is observed, even after long periods of extraction. However, as evidence against the condensation in an acidic medium, a decrease in the molecular weight of milled wood lignin upon mild hydrolysis (24), and a decrease in the molecular weight of soluble sulfite lignin upon a second cooking (25), have been reported. Furthermore, Rhodes --et al. observed a decrease in molecular weight of soluble chlorine monoxide treated periodate lignins on extensive degradation. Returning to Table 4.4, it is seen that beyond a certain time of ozonization, the molecular weight of the soluble products begins to decrease with increasing time of ozonization. This observation is contrary to the postulation of the condensation - 151 - TABLE 4. 4 Weight average molecular weights of the alkali-soluble products obtained during the degradation of spruce perio date and cuoxam lignins and spruce protolignin by ozone Time of ozone Weight fraction Sample No. treatment (min) M of sol, Ws w SPL-5 5 0.27 78,900 SPL-10 10 0.44 133,300 SPL-15 15 0.50 153,800 SPL-30 30 0.72 52,300 SCL-5 5 0.08 108,330 SCL-15 15 0.23 126,700 SCL-30 30 0.50 21,200 SSL-1 1 11,700 SSL-20 20 0.44 16,900 SSL-30 30 0.48 13,300 SPL - Soluble periodate lignin SCL - Soluble cuoxam lignin SSL - Soluble spruce protolignin - 152 - theory. The overall trend of the molecular weights of the soluble products (i.e. the initial increase and the subsequent decrease) can be explained if lignin is a three-dimensional in finite network polymer gel undergoing degradation by a random process (7,10,26). In such a network, the liberation of a frag ment requires that all bonds connecting it to the rest of the network be cleaved. At any stage of the degradation process, cleavage of a bond is expected to result in any one of three effects - a) liberation of a portion of the network b) decrease of the number of closed loops within the network by one c) division of an already liberated portion into two. The first effect (a) permits the dissolution of a part of the net work, whereas (b) results in a gradual increase in the molecular weight of the portions that are liberated. In the absence of (c), the molecular weight of the soluble portion continues to increase until complete dissolution of the gel is achieved. This is probably the case in the continuous flow delignification process where the solubilized lignin is not given time to undergo further degradation (2). However, in a random process, the effect of (c) is to decrease the average molecular weight of the soluble portion. In the specific case of the ozone-lignin reaction, the decrease in the average molecular weight of the soluble products is observed in the later stages of the reaction. It is presumed that in the early stages of the reaction the amount of the liberated portion of the lignin is small and the effect of - 153 - secondary degradation is negligible. An alternative explanation and that favoured by the author,is that the various bonds in the lignin macromolecule have different susceptibilities to cleavage by ozone. A closer look at the values of the weight average molecular weights of the soluble products (Table 4.4) reveals that the degradation products of periodate and cuoxam lignins are of comparatively much higher molecular weights than those of the protolignin. Bearing in mind that isolated lignins are never recovered in 100% yield from wood (64% yield for periodate lignin used in this study), it is clear that a significant portion of the lignin gel is lost during isolation. Rhodes et al. (10) have estimated that the loss of only the sol portion of lignin during isolation would result in molecular weight values. of the soluble products, which are less than 5% higher than those ex pected by degrading the intact protolignin. However, from Table 4.4,it can be seen that the weight average molecular weights observed for the isolated lignins are about 700% higher than those obtained from the protolignin at corresponding values of {Compare, for instance, samples SPL-10 and SSL-20.) This ws . indicates that in addition to the loss of the original sol, the sol from the degradation of the lignin network is lost during the isolation procedure. Goring {26) has suggested the existence of two types of lignin gels in wood that differ only in their rate of degradation during chemical pulping. Bolker {27) has modified this two gel concept to apply to the existence of two gels - 154 - differing only in their crosslinking density. This two gel concept appears to be more appropriate in explaining the observed molecular weights of the soluble products, since the loss of a portion of the gel (the portion yielding the low molecular weight lignin fragments) should result in an isolated lignin whose degradation products would be of very high molecular weights. It is reasonable to assume that the gel that would yield the low molecular weight lignin fragments on degradation should be that with a lower crosslinking density. Recent evidence by Yang and Goring, (28) that the number of phenolic hydroxyl groups in the secondary wall is about twice as that in the middle lamella suggests that the secondary wall lignin may have a lower cross linking density than the middle lamella lignin. If this picture is true, it is probable that the periodate and cuoxam lignins are rich in middle lamella lignin. [4] Elemental analysis Results of the elemental analysis of the soluble products are shown in Table 4.5. It is obvious that the loss of methoxyl groups and the general trend toward an increase in oxygen content are consequent upon·ozonization of the lignins. No trend in the hydrogen content is discernible in the two series of products. The empirical formulae in Table 4.4 are of limited significance since ozone is known to attack aromatic structures suggesting the possibility of cleavage of the phenyl propane (c9 unit) back bone. Also certain volatile products which are formed may have been lost and not accounted for in the overall picture of the ~ e TABLE 4.5 Elemental analysis of the alkali-soluble degradation products during the ozonization of spruce periodate and cuoxam lignins Time of ozone Molecular Sample treatment Analysis of soluble products Empirical formula weight (min) %C %H %0 %0CH based on No. 3 c9 c9 unit SPL-0 0 58.85 5.95 33.60 12.16 C9Hl0.3°3.86(0CH3)0.72 203 SPL-5 5 46.70 5.45 39.70* 9.58 C9Hl3.77°5.67(0CH3)0.65 233 SPL-10 10 43.74 4.84* 43.31* 8.11 C9Hll.94°6.68(0CH3)0.65 247 SPL-15 15 45.24 4.96 40.5 7.96 C9Hll.B4°6.04(0CH3)0.62 236 SPL- 30 30 47.44 5.35 38.12* 9.09 C9Hl2.19°5.43(0CH3)0.66 228 SCL-0 0 60.91 5.99 34.24 14.12 C9Hl0.61°3.79(0CH3)0.82 205 SCL-5 5 51.00 5.76 33.54* 9.70 C9Hl2.20°4.44(0CH3)0.66 212 SCL-15 15 42.68 4.61 40.2 7.51 C9Hll.66°6.35(0CH3)0.61 240 SCL-30 30 32.39 3.69 58.51* 5.41 C9Hl2.30°12.20(0CH3)0.57 333 * Determined by difference SPL - Soluble periodate lignin SCL - Soluble cuoxam lignin 1-' Samples SPL-0 and SCL-0 are the original periodate and Ul cuoxam liqnins,respectively Ul - 156 - phenyl propane backbone. However, the reported values serve to provide a basis for the calculation of the molecular weights of the lignin units as degradation proceeds. By dividing the weight average molecular weights Mw's (Table 4.4) by the molecular weights of the basic units (Table 4.5), the weight average degrees of polymerization of the successive sol fractions are obtained. Results which are shown in Table 4.6 indicate increasing degrees of polymerization (DP) in the soluble products up to a DP of about 650 (for periodate lignin) prior to subsequent re duction in the average molecular weights due to secondary degrad ation reactions. Thus a rapid release of huge lignin macromolecules, even in the initial stages of lignin degradation, is consequent upon ozone treatment of the isolated lignins. Soluble products with the highest degrees of polymerization are found at about 15 min of ozone treatment. [5] Proposed mechanism of lignin degradation by ozone There are two principal mechanisms by which degradation of a polymer can occur. The first is the step reaction mechanism in which cleavage of the bonds occurs at random throughout the macromolecule. The result of this random cleavage is a mixture of products covering a wide range of molecular weights (29,30) and the character of the distribution is expected to change as the degradation proceeds. In contrast, a polymer can be degraded by a chain reaction depolymerization mechanism. This mechanism can be considered as an "unzippering' of the polymer backbone (29,30). The products consist largely of the original monomers, - 157 - and show a narrow molecular weight distribution, the character of which does not change as the degradation proceeds. The results presented in this chapter indicate that the soluble products of lignin degradation by ozone have broad molecular weight distributions and therefore consist of a poly disperse mixture of products. Each sample showed one true maximum in the distribution curve. The peak at.the void volume of the Sephadex G-200 (Fig. 4.6, 4.7) was shown to be an artifact representing a high molecular weight tail of the low molecular weight maximum. Changes in the height of this peak with time of ozone treatment in successive samples reflect changes in the length of the high molecular weight tail and therefore changes in the molecular weights of the samples. The character of the molecular weight distribution of the alkali-soluble products, taken together with the trend in molecular weights, permit the suggestion that ozone degrades lignin by a random step reaction mechanism. Chemically, degradation of the lignin macromolecule was found to occur through the random heterolytic cleavage of the phenol ether bonds {31). Ozone also attacks aromatic rings and other unsaturated linkages in lignin. The initial products of degradation undergo further fragmentation until the lignin is completely degraded into low molecular weight compounds. - 158 - TABLE 4.6 Weight average degree of polymerization of the soluble products from the degradation of spruce periodate and cuoxam lignins by ozone Weight Weight Time of average average Weight ozone Molecular degree of fraction Sample treatment mol. wts. weights of polymer l-1 of sol, W No. (min) w basic unit ization 5 SPL-5 5 78,900 233 339 0.27 SPL-10 10 133,300 247 540 0.44 SPL-15 15 153,800 236 652 0.50 SPL-30 30 52,300 228 229 0.72 SCL-5 5 108,330 212 511 0.08 SCL-15 15 126,700 240 528 0.23 SCL-30 30 21,200 333 64 0.50 - 159 - CONCLUSION The results presented in this chapter show that the correct molecular weight distribution of soluble lignins on Sephadex gels is essentially unimodal; the 'high' molecular weight peak in the distribution is an artifact resulting from the accumulation of molecules of a hydrodynamic volume greater than the maximum acceptable to the gel. The wide poly dispersity exhibited by the alkali-soluble degradation products is characteristic of a non-linear polymer (32). Furthermore, the increase in molecular weight with increasing solubility and the subsequent decrease in molecular weight resulting from further fragmentation are consistent with the random scission of a three-dimensional infinite network polymer gel. The broad distribution obtained for the soluble degradation products, taken together with the observed trend in weight average molecular weights of these products suggest a random stepwise mechanism for the degradation of lignin by ozone. - 160 - REFERENCES 1. Forss, K. and Fremer, K.E., Paperi ja Puu, !2, 443(1965). 2. McNaughton, J.G., Yean, W.Q., and Goring, D.A.I., Tappi, Vol. 50, No. 11, 548 (1967) . 3. Brown, W., Falkehag, S.I., Cowling, E.B., Nature 214, 410 (1967) . 4. James, A.N., Pickard, E., Shotton, P.G., J. Chromatog. 32' (1971). 5. Katuscakova, G., Oltus, E., Zbornik, 95-104(1971). 6. Obiaga, I., Wayman, Morris, Svensk Papperstidning No. 18, 699 (1973). 7. Bolker, H.I. and Brenner, H.S., Science, 173, 170(1970). 8. Wegener, Gerd, Fenge1 Dietrich, Wood Sci. Technol. 11, 13 3-14 5 (19 7 7) . 9. Albrecht, J.S., Nicholls, G.A., Paperi ja Puu, Vol. 56, No. 11, 927(1974). 10. Bolker, H.I., Rhodes, H.E.W., Lee, Kuen Sing, J. Agric. Food Chem., Vol. 25, No. 4, 708(1977). 11. Flory, P.J., "Principles of Polymer Chemistry" (Cornell University Press, Ithaca) Chap. 9 (1953). 12. Van Holde, K.E., and Baldwin, R.L., J. Phys. Chem. 62, 734(1958). 13. Yean, W.Q., and Goring, D.A.I., Pulp and Paper Mag. Can. 65, T-217 (1964). 14. Rezanowich, A., Yean, W.Q. and Goring, D.A.I., Svensk Papperstidn. ~' 141(1963). - 161 - 15. Kosikova, B, andSkamla, J., Drevarsky Vyskum 1968, 59 (Abstr. Bull. Inst. Paper Chem., 41, 297). 16. Bogomolov, B.D., Babikova, N.D., Pivovanover, V.A., and Stepovaya, L.P., Khim Ispol'Zovanie Lignina 1974, 102 (Abstr. Bull. Inst. Paper Chem., 45, 104/3). 17. Ahlgren, P.A., Yean, W.Q., and Goring, D.A.I., Tappi 54, 737 (1971). 18. This thesis, Chapter 2. 19. Alekseev, A.D., Reznikov, V.M., Senko, I.V., Khim Drev (Riga) 1969, }, 91 (Abstr. Bull. Inst. Paper Chem. 43, Abstr. 283). 20. Lacan, M., Matasovic, D., kern. ind. (Zogreb) 15, 475(1966) (Abstr. Bull. Inst. Paper Chem. 40: Abstr. 227). 21. Alekseev, A.D., Reznikov, V.M., Sovrem Metody Iss led. Khim lignina Arkhangel'sk 1970, 34 (Abstr. Bull. Inst. Paper Chem. 42: Abstr. 7868). 22. Alekseev, A.D., Rezniko, V.M., Slvamental, L.G., Khim Drev (Riga) 1971, ~, 1977 (Abstr. Bull. Inst. Paper Chem. 43: Abstr. 10620). 23. Chupka, E.I., Obolenskaya, A.V., Nikitin, V.M., Khim Drev (Riga) ~, 103(1970) (Abstr. Bull. Inst. Paper Chem. 43: Abstr. 7195). 24. Adler, R., Miksche, G.E., and Johanson, B., Holzforschung ~, 71 (1968). 25. Felicetta, V.F. and McCarthy, J.L., J. Am. Chem. Soc. 79, 4499 (1957). 26. Szabo, A., Goring, D.A.I., Tappi, Vol. 51, No. 10 (Oct. 1968). - 162 27. Bolker, H.I. and Berry, R.M., Unpublished work. 28. Yang, J.M. and Goring, D.A.I., Pulp an9 Paper Can. Trans actions, Vol. 4(1), TR2 (March 1978). 29. Jellinek, H.H.G., in 'Polymer Degradation Mechanisms' National Bureau of Standards Circular No. 525, Nov. 16, 1953, p. 1. 30. Williams, D.J., 'Polymer Science and Engineering', Prentice Hall, N.J., (1971), p. 16. 31. This thesis, Chapter 3. 32. Adler, E., Pepper, J.M. and Eriksoo, E., Ind. Eng. Chem., 49, 1391(1957). - 163 . CHAPTER 5 DEGRADATION OF LIGNIN BY OZONE: (IV) THE FATE OF THE CARBOHYDRATE MATRIX DURING THE DEGRADATION OF SPRUCE PROTOLIGNIN BY OZONE - 164 ABSTRACT ?re-extracted spruce wood meal was ozonized in 45% aqueous acetic acid at room temperature. The ozone-treated wood meal was then extracted with dilute alkali at 65°C, for one hour. Lignin, a-cellulose, and hemicellulose content as well as the viscosities of the pulped wood meal samples were measured as a function of the time of ozonization. Results indicate that although the attack on the wood components by ozone is not selective in this medium, cellulose and hemicelluloses are very slowly degraded compared to lignin. Lignin degradation was found to occur approximately four times as fast as that of the carbohydrates. At the fiber liberation point, the resulting pulp retained 78% of the original hemicelluloses and about 90% of the a-cellulose compared to 25% of the lignin. During ozonization of the wood meal the resulting pulp samples showed a slow decrease in the average degree of polymerization (DP), attaining a limit near 350. The DP-limit was attributed to the inaccessibility of the ordered regions in native cellulose to ozone. The feasibility of commercializing this method of pulping is briefly discussed. 0 - 165 - 0 INTRODUCTION The previous chapters in this thesis dealt with physicochemical studies aimed at elucidating the mechanism of lignin degradation by ozone. The degradation of lignin was found to follow first order kinetics, implying a single rate controlling process. Structural investigation of the alkali soluble degradation products revealed that the degradation of lignin by ozone is principally due to the cleavage of ether bonds and aromatic rings. The molecular weight dis tribution of the alkali-soluble degradation products indicated the formation of polydisperse fragments with a single low molecular weight maximum. In the early stages of the degradation process, this maximum shifted to higher elution volumes with in crease in the weight fraction of the soluble products. Mo lecular weights of the alkali-soluble products increased up to a certain limit as the weight fraction of the soluble portion increased; beyond this limit extensive degradation resulted in lower molecular weights. It was concluded that these ob servations are consistent with the stepwise random degradation of a three-dimensional crosslinked infinite network polymer gel. It is noteworthy, however, that in the previous chapters, the practical application of the lignin-ozone reaction was not discussed. Ozone has attracted a great deal of attention for the bleaching of pulps (1-20) but not as a pulping agent. In the present work, it was considered of interest to evaluate - 166 ~ the possibility of pulping wood by treatment of the wood sus pension in stirred 45% aqueous acetic acid with ozone, followed by alkali extraction of the treated wood meal. Pulping of the wood involves the separation of the fibers through delignifi cation by chemical or mechanical means. In spruce wood the point of fiber liberation occurs at about 60% yield of the pulp or about 10% Klason lignin content of pulp. Earlier studies concerning the use of ozone for pulp ing were carried out in water and nitromethane (5). Results were not promising as delignification was slow and cellulose was extensively degraded. Reactions involving ozone are solvent dependent and since the rate of oxidation of lignin is enhanced in an acetic acid medium (21-23) it was hoped that in this medium, considerable delignification would occur before the ozone had time to cause extensive carbohydrate depolymerization. In the present study the possibility of obtaining a good yield during ozone pulping was also explored. The yield of a pulping process is determined by the selectivity of the pulping chemicals. Accordingly, the total yield and carbohydrate content of the pulps resulting from ozone treatment have been determined during delignification. In most pulping processes, the retention of hemi celluloses leads to pulps with good paper making properties. Schuerch et al. (24) reported the resistance of a significant portion of the hemicelluloses in a number of wood species when treated with ozone. In the present work, the amount of the total hemicelluloses retained in the pulp particularly at the - 167 - 0 point of fiber liberation was studied .. During pulping reactions it is possible that the un dissolved carbohydrates including cellulose may have been de graded to some extent. This in turn results in deterioration of the strength properties of the pulp. The extent to which the undissolved carbohydrate was degraded by .ozone, when th~ lignin-ozone reaction is carried out in 45% acetic acid, was followed by the determination of the viscosity of the result ing pulps as a function of time of ozonization. Finally, from the yield of pulp obtained by the present method and the extent of cellulose depolymerization at the fiber liberation point, the utility of this pulping process was eval uated. - 168 - 0 EXPERIMENTAL A) Ozonization of spruce wood meal Spruce wood meal, ground to pass an 80 mesh screen in a Wiley mill, was pre-extracted with benzene-alcohol 2/l(v/v) for 12 hours, after which it was air dried. The wood meal was brought to approximately 50% moisture content with a fine spray of distilled water. The sample was kept in a low- temperature room for 48 hours before use for the ozonization. Prior to ozone treatment, the exact consistency of the wood meal was determined. 5 g (oven dry) wood meal was suspended in 45% aqueous acetic acid and ozonized for varying time intervals. Details of the procedure are given elsewhere in this thesis (25). After treatment with ozone, each sample was filtered, washed with distilled water, and extracted with 100 ml of 2% NaOH solution at Gsac for one hour. In order to remove all the de- graded material, the sample was again washed with 50 ml of cold 2% NaOH. Finally it was washed several times with distilled water until the effluent became colorless. The residue was dried overnight under vacuum at room temperature and then over P2o5 to a constant weight. B) Determination of lignin content of the ozone pulped wood meal samples Klason lignin analyses were carried out on the pulp samples according to the Tappi standard method (26). Acid-soluble lignin was estimated spectrophotometrically by measuring the absorbance of the Klason hydrolysates at 205 nm. - 169 - 1 An a b sorpt ~v~. 't y va 1 ue o f 110 1 g- cm-l was used to calculate lignin concentration (27) . C) Preparation of holocellulose from the ozone-treated wood meal samples Each pulp sample was transformed into a holocellulose sample in order to determinate its a-cellulose and hemicellulose content, as well as its viscosity. The sodium chlorite method developed by Wise et al. {28) was adopted for the preparation of the holocellulose samples. This method specified the use of 5 g air-dry wood meal, 160 ml of distilled water, 0.5 ml of glacial acetic acid and 1.5±0.1 g of sodium chlorite in an Erlenmeyer flask. In the present study, the amount of wood meal used was generally less than 5 g depending on the yield of pulp after ozonization and alkali extraction. However, the amount of reagents and pulp were in the same proportion as used in Wise's method. The temperature was maintained at 75°C during chlorite treatment. At hourly intervals and without cooling, a fresh charge of chemicals was added. The contents of the flask were mixed by occasional swirling. Where the residual lignin in the starting wood meal was above 20% of the sample, four hours of chlorite treatment were considered adequate to remove almost all the lignin. Below 20% lignin content of the starting material, chlorite treatment was stopped at the end of the third hour. For samples of 10% or less lignin content, only two hours of 0 chlorite treatment were carried out. At the end of chlorite treatment, each flask was cooled - 170 - 0 in an ice bath below l0°C. The holocellulose was filtered in a medium porosity fritted funnel and washed with ice water until the color and odor of chlorine had been removed. It was then washed with acetone, air dried and weighed. D) Carbohydrate determination The a-cellulose content of the holocellulose samples from (C) was determined by the Tappi-standard method (29). In general, the holocellulose plus the lignin yields 100% of the weight of the extractive free wood. The hemicellulose content of the resulting pulps was therefore calculated by substract ing the sum of the total lignin content and the a-cellulose from the total yield of the ozone treated wood meal sample. E) Determination of viscosity The viscosities of the different holocellulose prepar ations from (C) were measured in 0.5% cupriethylenediamine so lution according to the Tappi-standard method (30). The intrinsic viscosity, [n]C.E.D.' in cupriethylenediamine solution was calculated from the 0.5% C.E.D. viscosity according to equation 5.1 (31) [nlc.E.D. = 954 log A- 325 ..•..•....••..•...•. [5.1] where A is the 0.5% C.E.D. viscosity. Viscosity average degrees of polymerization were then calculated from the intrinsic viscosity values according to equation 5.2 (31). + • • . • • . • • • • [ 0. 9 0 5 log DP = log 0. 7 5 log [ n] C. E. 0 5. 2] - 171 - RESULTS AND DISCUSSION (1) Total yield and lignin content On a pre-extracted, untreated wood meal, alkali ex- traction for one hour at 65°C dissolved 9.5±0.2% of the.wood. Thus a starting point is taken at 90.5% yield for the control sample. It was observed that no lignin was removed by the alkali extraction. Total yield was determined from equation 5.3. = oven dry wt. of wood meal after ozonization and % yield oven dry wt. of original wood meal alkali extraction x lOO ••..••••..••.•••..•••• [5.3] after alkali extraction The total lignin content of the pulp samples was determined as the sum of Klason lignin and acid-soluble lignin. The total lignin values for each treatment time represents the average of two determinations. The precision was ±0.2%. The non-lignin yield is the difference between the total yield and the total lignin content. Since the wood had been pre-extracted to remove resins and fatty acids, the total holocellulose content was taken to equal the non-lignin yield (32). Furthermore, by correcting for the 9.5% loss in yield of the wood meal following alkali extraction, a non-lignin yield of 62.8% was assumed to constitute the entirety of carbohydrates. Table 5.1 shows the pulp yields and their lignin content as a function of ozonization time. Fig. 5.1 shows a plot of the total yield and non- lignin yield versus percent delignification. Evidently the - 172 - 0 TABLE 5.1 Yield and lignin content of ozone-treated and extracted spruce wood meal as a function of time of ozonization Acid- Total Time of Klason soluble Total lignin ozone Total lignin lignin lignin (% of % treatment yield (% of (% of (% of original Delignifi- (min) (%) pulp) pulp) pulp) sample) cation 0 90.5 27.6 0.11 27.7 27.7 0 5 83.8 24.6 0.22 24.8 23.0 17.0 10 80.0 21.6 0.30 21.9 19.4 30.0 15 77.6 20.4 0.31 20.7 17.7 36.0 20 73.1 18.6 0.33 18.9 15.3 44.8 30 70.4 18.2 0.32 18.5 14.4 48.0 40 64.4 13.3 0.29 13.6 9.7 65.0 60 61.1 10.3 0.20 10.5 7.1 74.4 80 58.3 8.4 0.16 8.6 5.5 80.1 120 50.6 6.0 0.14 6.1 3.4 87.7 240 50.5 6.3 0.30 * 6.6 3.7 86.6 * only one determination 0 - 17 3 - 0 100 .------. o TOTAL YIELD Cl NON- LIGNIN YIELD 80 Cl _J 60 Cl w Cl >-- 1-z w 40 (.) 0::w CL 20 0 20 40 60 80 100 PERCENT DELIGNIFICATION FIG. 5.1 Total yield and non-lignin (carbohydrate) yield versus percent delignification during the ozonization of spruce wood meal in 45% aqueous acetic acid. - 174 - results indicate that in 45% acetic acid, lignin is not select ively attacked by ozone since the carbohydrate moiety is attacked simultaneously. However, they indicate a very slow removal of the carbohydrate component compared to the lignin. In Fig. 5.2, the percent lignin solubilized is plotted against the percent carbohydrate solubilized during the ozoniz ation of wood in 45% acetic acid. For comparison, the data ob tained for other delignification processes (33-35) are plotted in the same figure. From the slope of the linear plots, it can be seen that the ratio of the lignin to carbohydrate solubilized is approximately 4:1 for the present method of delignification {ozone/acetic acid)~ For the other methods of delignification, this ratio is generally lower, ranging from 3:1 for the oxygen/alkali process {34) to 2:1 for the ozone/water and ozone/nitromethane methods (33). In the Kraft process (35) {NaOH + Na2s), lignin removal proceeds at approximately the same rate as the carbohydrate until 30% of the carbohydrates are removed, thereafter the Kraft liquor tends to be more selective on the lignin. The significance of a higher ratio of lignin to carbohydrate solubilization, is that at comparable extents of delignification, the present method of pulping results in pulps with a higher carbohydrate content than the other delignification processes discussed. Consequently, a higher yield of pulp is obtained. Perhaps, equally important as the yield is the rate at which lignin is removed from the wood in the different methods of delignification. Because the conditions of reaction are - 175 - FIG. 5.2 Plot of lignin versus carbohydrate solubilized during the ozonization of spruce wood meal in 45% aqueous acetic acid (03/CH 3COOH) . Values obtained for other delignification processes are plotted in the same figure for comparison. ozonization of spruce wood meal {80 mesh) in 45% aqueous acetic acid at room temperature {present work). Ozonization of spruce wood meal {80 mesh) in nitromethane at 0°C ( 33} . Ozonization of spruce wood meal (80 mesh) in water at room temperature (33). Oxygen delignification of spruce wood in presence of base. NaOH charge on wood is 12%. Temp., 159°C. Pressure, 500-800 psi ( 34) • NaOH+Na2s Kraft pulping of spruce wood at 140°C (35). 0 100 ,.....------~ 80 0 UJ N ::J 60 cc :::::> _J @ z z (!) 40 ::J 1- z 0 03jCH COOH UJ 3 () a: 0 3/CH3N02 u.J • c.. Cl 20 0 3/H20 6 0 2/NaOH lOt NaOH+ Na2S 0 20 40 60 c PERCENT CARBOHYDRATES SOLUBILIZED - 176 - somewhat similar, direct comparison can be made between the present method of pulping and the other methods involving the use of ozone. In nitromethane, for instance, the solubilization of 20% lignin by ozone is accomplished in 180 minutes (33), compared to 5 minutes required by the present method. In water, the removal of 16% lignin is achieved in 420 minutes (33). Rapid.solubilization of lignin implies low ozone charge and consequently low cost of pulp production. (2) Comparison of the rates of degradation of a-cellulose, hemi cellulose and lignin in spruce wood The hemicellulose content of each sample was calculated from the experimentally determined values of a-cellulose and lignin in the pulp sample, according to the equation 5.4 % Hemicellulose = total yield (%) - (% a-cellulose + % total lignin) ...... [5.4] Table 5.2 shows the a-cellulose and hemicellulose content of the original and ozone treated samples. The rates of degrad- ation for the first 80 minutes of reaction, measured from the yield of undissolved component (Table 5.2) after the ozonization and alkali extraction, is plotted in Fig. 5.3. Results indicate that lignin is rapidly degraded in the initial stages of the reaction, the rates decreasing with increasing time. On the other hand, cellulose and hemicelluloses are less susceptible to degradation by ozone in 45% acetic acid. After 60 minutes of ozonization only 10% cellulose is lost compared to 22% loss c in hemicelluloses and 74% decrease in total lignin content. The - 177 - TABLE 5.2 a-Cellulose and hemicellulose content of the original spruce wood meal and ozone pulped samples Time of ozone- a-Cellulose Hemicelluloses treatment a-Cellulose {% of original {% of original (min) (% of pulp} sample) sample} 0 60.9 42.3 20.5 5 63.6 41.5 19.3 10 63.7 40.9 19.7 15 66.2 41.6 18.3 20 67.5 40.4 17.4 40 67.8 38.0 16.7 60 67.1 37.9 16.1 240 75.3 36.5 10.3 0 - 178 - ~- 1=100 z w z 0a.. 80 ~ 0 (.) a- CELLULOSE 60 • Cl 0 HEMICELLULOSE 0 0 • LIGNIN ~ .40 Cl LU ~ 0 20 (j) (j)- z0 :J 0 20 40 60 80 100 . TIME , m1n FIG. 5.3 % Yield of undissolved lignin, cellulose and hemicellulose as a function of the duration of ozone treatment of spruce wood in 45% aqueous acid at room temperature. - 179 - relative rates of degradation of the different wood components is therefore in the order lignin > hemicellulose > ~-cellulose. The relative rates of degradation of the various wood components can be explained in terms of the chemical mechanism of degradation and the physical structure of the respective wood polymers. The rapid rate of degradation of the lignin by ozone compared to the cellulose and hemicelluloses is probably a consequence of both the chemical mechanism of degradation and the physical structure. Lignin is an amorphous polymer built up of aromatic and unsaturated structures, which are rapidly attacked by ozone according to the Criegee mechanism (36). Besides, ozone attacks and cleaves phenol ether linkages in lignin (37). All these reactions lead to the extensive breakdown of the lignin network. In cellulose and hemicelluloses, the units are linked essentially by (1~4) glycosidic bonds. The chemical reactions of these carbohydrates with ozone involve (a) ozone-initiated oxidation of aliphatic functional groups leading to the formation of aldehydic, acidic and ketonic groups (38} , (b) cleavage of glycosidic bonds presumably through peroxide formation (39-41), and (c) cleavage of acetal linkages through a hydrotrioxide intermediate (42-44). In view of the very similar reactions of cellulose and hemicelluloses with ozone, the faster rate of degradation of the hemicelluloses by ozone is more probably a consequence of the physical structure rather than the chemical mechanism. In contrast 0 - 180 - to cellulose, the hemicelluloses in situ are amorphous. Cellulose chains are linked laterally by hydrogen bonds, and this association of chains results in crystallinity. The cellulose chains pass alternatively through crystalline and amorphous regions. Wood cellulose is considered to have a region of intermediate order in addition to the crystalline and amorphous regions (45,46). The physical structure of the cellulose wood is expected to limit its accessibility to the ozone gas molecules. In fact earlier investigations (5,33) suggest that ozone cannot penetrate the crystalline lattice of cellulose and that attack in this region is superficial. (3) Retention of hemicelluloses and pulp properties Fig. 5.3 indicates that during ozonization of wood in 45% acetic acid followed by alkali extraction, the removal of cellulose and hemicellulose is very slow. The result is that the pulps produced are rich in hemicelluloses. At the point of fiber liberation (60% yield of pulp or 10% Klason lignin of pulp} the pulp resulting from this process still retains 78% of the original hemicelluloses (23% of pulp) and about 90% of the a-dellulose content (Fig. 5.3). The yield of pulp is 61% (Table 5.1) The effect of hemicelluloses on the properties of pulps has been discussed in detail (47). Apparentl~hemicelluloses, by virtue of their location in the outer part of the fiber walls and their easy accessibility to wate4 play an important 0 role in improving interfiber bonding and hence the strength - 181 - properties of the paper. However, a very high content of hemi celluloses decreases the tearing strength of paper. This has been attributed to the decreased amount of fibers in the pulp. To make paper types where high tear strength, sheet softness and opacity are desired, it is necessary to remove part of the hemi celluloses from the pulp. Retention of hemicelluloses is undesir able in "dissolving pulps" where the fiber component is converted to make films, plastics, etc. Here the emphasis is on purity of the cellulose. (4) Degree of polymerization and pulp properties Investigation of paper strength (48) reveal that tear ing and folding strength of paper decrease with a decrease in the degree of polymerization of the cellulose in the pulp. There fore it is expected that the properties of the paper produced from the pulp depend not only on the quantity of the cellulose retained but also on its quality. In Table 5.3 the viscosities and corresponding vis cosity average degrees of polymerization of the resulting pulps are shown for different values of yield and ozonization time. It should be mentioned that the values of viscosity reported here are not precise since the holocellulose samples from the pulps did not dissolve to give clear solutions in cupriethylene diamine. Besides,it is well known (49,50) that the method of holocellulose preparation employed in the present study (sodium chlorite delignification) produces holocellulose samples that 0 are to some extent degraded by the chlorite treatment. All these - 182 - TABLE 5.3 Viscosities and viscosity average degrees of polymerization of the original sample and pulps obtained during the ozonization of spruce wood meal Time of Yield of Degree of ozone- pulp (% of 0.5% C.E.D. polymerization treatment original viscosity (viscosity (min) sample) (centipoise) average) 0 90.5 15.3 1183 5 83.8 12.9 1068 10 80.0 12.8 1063 15 77.6 11.6 998 20 73.1 11.2 975 40 64.4 9.6 873 60 61.1 7.6 722 80 58.3 4.6 551 120 52.0 4.2 353 150 4.2 353 0 - 183 - notwithstanding, the viscosity values reported here have an orienting significance. By assuming a uniform error in these values, some useful deductions can be made. It is clear from Table 5.3 that there is a gradual decrease in the average length of the cellulose chains in the pulp with increasing time of ozonization. However, at the point of fiber liberation (60% yield of pulp or 10% Klason lignin of pulp) there is only a 38% drop in the average DP of the wood meal. It should be borne in mind that the DP values of the carbohydrate fraction is an average for the degradation of the cellulose and hemicelluloses fractions. Since hemicellulose is more rapidly attacked by ozone, it is expected to contribute more than the cellulose to the decrease in DP of the carbohydrate fraction. The cellulose chains are the strength bearing elements in the pulp and a decrease in the DP of the cellulose is thus more significant in terms of the strength properties of the pulp. Another observation is that the degree of polymerization decreases with increasing ozonization time and reaches a limit at about 350 (Table 5.3). A similar observation was made by Schuerch and coworkers {33) who observed a DP-limit near 400 during the ozonization of Norway spruce and bass wood in nitre methane. The DP-limit was assumed to correspond to the length of the ordered regions in native wood cellulose. As indicated already, these ordered regions are inaccessible to ozone and c attack is mainly superficial. - 184 - CONCLUSION The results presented in this chapter indicate that the degradation of protolignin by ozone is not selective as cellulose and hemicelluloses are also depolymerized. However, by the simple expedient of ozonizing wood in 45% aqueous so lution of acetic acid, followed by extraction with 2% aqueous sodium hvdroxide, it is possible to produce pulps that are not severely degraded at the fiber liberation point. The pulp at this point has a fairly good yield and retains about 78% of the original hemicelluloses. These pulps may be useful in making some paper products where paper strength is not very crucial, e.g. newsprint. Because these pulps are bright little bleaching is required. In the course of the present investigation an average DP-limit of 350 was found for spruce wood cellulose, close to the value of 400 originally found for Norway spruce and bass wood (33). The DP-limit is attributed to the average length of the ordered regions in native wood cellulose. Although the present pulping method produces pulps with properties suitable for making certain grades of paper, it is not as yet being suggested as a feasible industrial process for the following reasons. Spruce wood meal employed for the present work was of 80 mesh particle size, thus only very short fibers were produced. It shollld be borne in mind that the overriding aim of this thesis was to study the mechanism 0 of lignin degradation by ozone. To realize this aim, it was - 185 - considered necessary to study the degradation of relatively unmodified isolated lignins and to compare the results obtained with those for the degradation of protolignin. In order to maintain uniformity in reaction conditions the isolated lignins and spruce wood were all reduced to the same particle size. In commercial processes, wood pulps are produced from wood chips to obtain fibers of about 1-3 mm in length. This is because paper strength is related to fiber length. It is believed that the use of wood chips in the present system may affect the reaction rates but not the properties of the pulp. Besides the use of the wood chips, an important factor which has to be considered alongside with the commercialization of this process is the economics. This aspect has not been given attention in this thesis. 0 - 186 - REFERENCES 0 1. Doree, C.H., Cunningham, M., J. Chem. Soc., 101, 497(1912). 2. Doree, C.H., Cunningham, M., J. Chem. Soc., 103, 677(1913). 3. Brabender, G.J., Bard, J.W., Daily, J.M., U.S. Pat. 2,466,633 (1949). 4. Whi tner, T. C. , U. S. Pat. 2, 4 3 8, 10 0 ( 19 4 8) . 5. Osawa, z., Schuerch, C., Tappi, ~, No. 2, 79(1963). 6. Moore, W.E., Effland, M., Sinha, B., Burdick, M.P. 7. Schuerch, C., Tappi, Vol.~, No. 5, 206(1966). 8. Goring, D.A.I., Pulp and Paper Mag. of Canada,~' No. 8, T372 (1967). 9. Soteland, N., Kringstad, J., Norsk. Skogindustri, ~' No. 2, 46 (1968). 10. Ancelle, B., Plancon, M., U.S. Pat. 3451,881 (1969). 11. Secrist, R.B., Singh, R.P., Tappi, ~, No. 4, 581(1971). 12. Soteland, N., Norsk Skogindustri, ~' No. 3, 61(1971). 13. Liebergott, N., Pulp and Paper Mag. of Canada, llr No. 9, 70 (1972). 14. Soteland, N., Norsk Skogindustri, ~' No. 10, 274(1973). 15. Soteland, N., Paper presented in Vancouver (1973). 16. Soteland, N., Loras, V., Norsk Skogindustri, ~' No. 6, 165(1974). 17. Procter, A.R., Pulp and Paper Mag. of Canada, ~' No. 6, 58(1974). 18. Kamishima, H., Fujii, T., Akamatsu, I., Japan Tappi, lQ, ~ No. 7, 381-39l(July 1976). [Abstr. Bull. Inst. Paper Chem. Vol. 47 Abstr. 4977] - 187 - 19. Soteland, N., Paper presented at the Canadian Wood Chem. Symposium, Mont Gabriel. Que., Montreal. Sept. 1976. 20. Lindholm, Carl-Anders, Paperi ja Puu, No. 1, 17(1977}. 21. Freudenberg, K., Sohns, F., and Janson, A., Ann., 518, 62 (1935}. 22. Katuscak, S. Hrivik, A. Mahdalik, J., Paperi ja Puu, Vol. 53, No. 9, 519-524(1971}. 23. Katuscak, S., Rybarik, I., Paulinyova, E., Mahdalik, M., Ibid., 665-670(1971). 24. Moore, W.E., Effland, M., Sinha, B., Burdick, M., Schuerch, C., Tappi, VoL 49, No. 5, 206(May 1966}. 25. Chapter 2 of this thesis. 26. Tappi Standard T222-0S-74. 27. Tappi, UM (Useful methods) 250. 28. Wise, L.E., Murphy, M., D'Addieco, A.A., Paper Trade J., 122, No. 2,35{1946). 29. Tappi Standard T203-0S-74. 30. Tappi Standard T230-0S-76. 31. Sihtola, H., Kyrklund, B., Laamanen, L., Palerius 1, Paperi ja Puu, Vol. 45, 4a 225(1963). 32. Ahlgren, P.A., and Goring, D.A.I., Can. J. Chem., 49, 1272 (1971) . 33. Osawa, z., Erby, W.A., Sarkanen, K.V., Carpenter, E., Schuerch, C., Tappi, Vol.~, No. 2, 84{1957). 34. Renard, J. J., Mackie, D.M., Bolker, H. I., Clayton, D. W., Transactions of the Technical Section 1{1): 1-11 (Mar. 1975). c 35. Enkvist, T. ' Svensk Papperstidning, 60, No. 17, 616 (1957). - 188 - 36. Criegee, R., Record of Chemical Progress, 18, 111(1957). 37. This thesis, chapter 3. 38. Mester, L., and Major, A., Chem. Ind., 469(1957). 39. Doree, Charles, Healey, A.C., J. Textile Inst., T27 (March 1938). 40. Kargin, V.A., Usmanov, Kh.U., Alkhodzhaev, Vysokomole kulycornye Soedinerinya, 1, 149(1959). 41. Katai, A.A., Schuerch, Conrad, J. Poly. Sci., Part I, Vol. 4, 2683(1966). 42. Deslongchamps, P., Moreau, C., Can. J. Chem., ~, 2465 (1971) • 43. Deslongchamps, P., Moreau, C., Frehel, D., Atlami, P., Can. J. Chem., 50, 3402(1972). 44. Deslongchamps, P., Atlani, P. Frehel, D., Malavel, A., Moreau, C., Can. J. Chem., 2, 3651(1974). 45. Jorgensen, L., Acta. Chem. Scand., !' 185(1950). 46. Samuelson, Olof, Grangard, Gunnar, Jonsson, Kurt, Schrarnon. Kerstein, Svensk Paperstidning Nr., 20,3l(Oct. 1953). 47. Rydohlm, S.A., "Pulping Processes", Interscience, New York (1965), Chapter 21. 48. Rydohlm, S.A. Ibid., pp. 1152-1160. 49. Time11, T.E., Jahn, E.C., Svensk Paperstidning, 1!, 831(1951). 50. Timell, T.E., Pulp and Paper Mag. Can., 60, T26 {1959). c - 189 - APPENDIX 1 DETERMINATION OF THE CONCENTRATION OF OZONE IN AN OZONE/OXYGEN MIXTURE 0 - 190 - 0 There are several methods for the determination of the concentration of ozone in oxygen or air. In the present stud~ the iodometric method has been adopted because it has been found to give reliable results (1,2). The concentration of ozone was determined as follows: oxygen (Medigas 99.97%) was passed through a Welsbach laboratory ozonator (Model T-408) operated . 3 2 at 120 volts and 5.6 x 10 kg/m gauge pressure. The flow of ozonized oxygen was 0.5 lit/min. The mixture of ozone and oxygen was trapped in 250 ml of 2% aqueous potassium iodide {KI) con- tained in a gas absorber. After a given time, the flow of the ozonized oxygen was stopped and the absorber flushed with nitre- gen. The contents of the absorber were transferred quantitively into a one litre beaker. The absorber was rinsed several times with distilled water and the washings were added to the contents of the beaker. The solution in the beaker was acidified with 10 ml of lM H2so4 and then titrated against standard O.lN sodium thio sulphate (Na2s2o3 .5H20) solution, using starch as an indicator until the starch-iodine colour just disappeared. The reactions involved are as follows: The concentration of ozone,c,was calculated from equation A-1-1 cmg/l = Normality of thiosulphate x vol of thiosulphate x 24 0 volume of ozonized oxygen in litres A-1-1 - 191 - 0 In the above equation, 24 is the equivalent weight of ozone. The volume of ozonized oxygen was determined from the flow rate of the ozone/oxygen mixture and the time for which the gas was allowed to flow into the gas absorber. Model determination of the ozone concentration in oxygen In all the experiments reported in this thesis, the flow rate of the ozone/oxygen mixture was maintained at 0.5 1/min. Several determinations of ozone concentration were carried out and the results averaged. Temperature = 74°F 3 Ozonator pressure = 5.6 x 10 kg/m2 Ozonator voltage = 120 volts Flow rate of ozone/oxygen mixture·= 0.5 1/min Readings Initial Final Ozone Number burette burette Vol. of thio concentra of titra reading reading sulfate used tion tions (ml) (ml) (ml) mg/1 1 10.8 21.15 10.35 49.6? 2 21.15 31.5 10.35 49.68 3 0.0 10.4 10.40 49.92 Average 49.76 0 - 192 0 Determination of the amount of ozone consumed by lignin The amount of ozone consumed by lignin is calculated from the difference between the initial ozone concentration (from a blank determinationt and the concentration of the ef fluent gas a~ter the ozonization of lignin for a known treatment time. Model calculation: Ozonization of cuoxam lignin for 5 minutes: Weight of lignin ozonized = 0.151 g Initial ozone concentration= 49.76 mg ozone/1 oxygen Concentration of ozone in effluent gas = 45.41 mg/1. The decrease in ozone concentration= 49.76- 45.41 = 4.35 mg/1 After 5 minutes, 2.5 litres of ozonized oxygen has been delivered. Therefore the decrease in ozone concentration in 2.5 litres = 4.35 x 2.5 = 10.875 mg This is equal to the amount of ozone consumed by 0.151 g of lignin in 5 minutes. 0.010875 Therefore ozone consumed per gram of lignin = = 0.072 g. 0.1510 Ozone consumed in moles per gram of lignin = 0.0015 = 1.5 m moles/g of lignin. Ozone consumed in moles;c of lignin (mol. wt. of c unit 205) 9 9 is = 0.0015 X 205 = 0.3075 moles. Ozone consumed per c unit per minute 0.062 moles;c unit/min. 9 = 9 0 In calculating the amount of ozone consumed by lignin, it was assumed that any decrease in ozone concentration in the effluent - 193 - 0 gas after each reaction time was due to the uptake of ozone by the lignin. REFERENCES 1. Boelter, E.D., Putnam, G.L., and Lash, E.I., Analytical Chemistry, Vol. 22, No. 12, 1533(1950). 2. Birdsall, C.M., Jenkins, A.C., and Spadinder, E., Analytical Chemistry, Vol. 24, No. 4, 662(1952). 0 - 194 - 0 APPENDIX 2 CHARACTERISTIC INFRA-RED BANDS OF FUNCTIONAL GROUPS AND CHEMICAL SHIFTS OF PROTONS IN LIGNIN 0 - 195 - 0 TABLE A-2-1 Characteristic Infra-red Bands of Lignin -1 Wave number cm Structural Assignment 3400-3300 Stretching vibrations of OH (hydrogen bonded} 3200-2900 -C-H stretching vibrations 3190 -OH 2890 Tertiary C-H groups 2860-2850 Methoxyl (OCH 3) groups 1725 Acid or ester carbonyl groups 1725-1650 Carbonyl (C=O) stretching frequencies 1712-1705 Nonconjugated ketone carbonyl groups 1668 Aldehyde or ketone carbonyl 1660 Ketone carbonyl a- to an aromatic ring (p-position etherified) 1603-1595 Aromatic stretching bands (typical unconjugated guaiacyl nucleus) 1600-1510 General C=C skeletal vibrations in an aromatic ring 1585-1580 Vibrations of rings conjugated with an a-carbonyl 1515-1510 Aromatic stretching bands (typical unconjugated guaiacyl nucleus) 1480-1350 C-H deformation 0 1462-1452 Bending of C-H bonds - 196 - 1430 Aliphatic groups 0 1425-1420 Partyl - C-H bonds of methyl groups 1370 Bending vibrations of OH bonds 1270-1268 e-o stretching aromatic methoxvl 1230-1215 e-o stretching aromatic (phenyl) 1220 OH group vibration (C-O stretching mode in phenolic hydroxyl groups) 1150 1,3,4-substituted aromatic ring 1140 Dialkyl ether linkages 1087 OH group vibrations 1092-1076 Aliphatic ether linkages and secondary OH groups 1043 OH group vibrations 1040 Dialkyl ether linkages 1035-1030 e-o deformation (methoxyl group) 970 C-H out of plane deformation in ethylenic double bonds 585 1,3,4-substituted aromatic ring (monohydrogen out of 'plane deformation) 835 Trisubstituted aromatic ring 817 1,3,4-substituted aromatic ring (two adjacent ring hydrogen out of plane deformation) 0 - 197 - 0 TABLE A-2-2 Ranges of ppm values for chemical shifts of signals from protons found in model compounds adjusted for lignin preparations Chemical shifts Chemical shift rang• Protona from adjusted for lignin Range types models (a) preparations (a} 1 carboxylic and 11.50 to 9.48 lL. 50 to 8. 00 aldehydes 2 aromatic 7.70 to 6.67 8.00 to 6.28 ortho to a-9arbonyl 7.70 to 7.32 all others 7.20 to 6.67 a-vinyl 6.72 to 6.47 3 e-vinyl 6.32 to 5.86 6.28 to 5.74 a -acetylated 1benzylic 8.18 to 5.94 4 a3 5.58 to 5.65 5.74 to 5.18 5a methoxyl 3.90 to 3.70 5.18 to 2.50 5b a2,s,y, other than 4.90 to 2.72 those in ranges 3,4,8 6 Aromatic acetoxyl 2.29 to 2.27 2.50 to 2.19 except those ortho to biphenyl link 7 Aromatic acetoxyl, 2.13 2.19 to 1.68 ortho to biphenyl link Aliphatic acetoxyl 2.09 to 1.98 8 Highly shielded 1.91 to 0.78 1. 58 to 0. 38 aliphatic a a1-Protons on alpha carbons in arylgylcerol - aryl ether systerr a 2-All protons on alpha carbon except those in 1 and 3. 0 a 3-Protons on alpha carbon in acetylated coumaran system. - 198 - 0 APPENDIX 3 THEORY OF GEL CHROMATOGRAPHY 0 - 199 - 0 Gel chromatography is a method that separates molecules by size. Separation is carried out on columns that are packed with a gel or some other porous material and com pletely filled with solvent. This technique is termed 'gel filtration' when the solvent is water and 'gel permeation' with organic solvents. The same solvent is employed to dissolve the sample and also for elution through the column. While large molecules are excluded from the pores of the gel, small molecules completely diffuse into them. Molecules of intermediate size penetrate the larger pores of the gel. The molecules are constantly diffusing back and forth between the pores and the interstices. As the solvent flows through the interstices, the molecules in the pores stay behind until they diffuse back out. The larger molecules are eluted first and the smaller molecules last. A species is eluted at a volume exactly equal to the volume available to it in the column. The elution volume, Ve' of excluded molecules is equal to the interstitial or void volume, V , (Fig. A-3-1). The elution volume of small molecules 0 which completely penetrate the pores of the gel is equal to the total liquid volume, Vt' of the column. For molecules of intermediate size, the elution volume is given by the expression in equation A-3-1 = V + Kd Vi ••••••.••••••• • •••••••••••• Eq. A-3-1 0 0 where Kd is the partition coefficient and is equal to the ratio 0 0 I I I - I I I I I I . I . . I . I I I I I I I I I I . I I I . I . I I I I I I I I I I Total I I Void 1 I I Volume to o: 0 I Volume : I I I I Vt lo ol I I Vo : I I I I I I I I I I I I 1- I I I I I • I I I l Ve = Vo Ve = Vt = Vo + Vi Ve = Vo' + Vi, ace FIG. A-3-1 Schematic representation of the elution volume and accessible column volume of a gel column (From Billmeyer, F.W. and Altgelt, K.H., in "Gel permeation chromato graphy". Altgelt, K.H., Segal, L., (eds). Marcel Dekker Inc., New York 1971). N 0 0 - 201 - 0 of the accessible pore volume, v.1.,acc to the total pore volume v.1. v. = 1.,acc ...... Eq. A-3-2 v.1. Equation A-3-1 is of general application since for complete exclusion Kd = 0 and for total permeation Kd = 1. The elution volume ve,is measured experimentally. The value of Kd can be calculated if V and Vi are known. The value of 0 Kd is seldom reliable owing to the uncertainties in the determination of vi. In order to overcome the uncertainty in the deter- mination of the constant Kd, Laurent and Killander (1) have defined another constant Kav (the volume fraction of the stationary phase available to molecules of a given size) which is generally applicable in gel chromatography (Equation A-3-3). = Eq. A-3-3 In the above expression, (Vt-V ) is the total 0 volume of the gel bed and is easily measured. Kav is related to the radius of spherical particles of a solute according to equation A-3-4 2 Kav = exp [•c (r+ R') _}· ••••••••••••••••••• Eq. A-3-4 0 where r is the radius of the spherical solute particle. R'and Care the radius and concentration of the particles in - 202 - the gel matrix. At low Kav (< 0.15) the value of r is high, 0 and the material does not readily diffuse into the gel. Conversely at high Kav (> 0.75), r, is low and ready diffusion of the material into the gel occurs. Between these lower and upper limits fractionation through partition diffusion into the gel occurs readily and to varying extents. Several theories have been proposed to interpret the separation of molecules of different sizes on a gel bed. The Exclusion principle is based on the assumption that a swollen gel contains regions which the solute molecules can not penetrate unless they are below a certain size. The volume accessible for the spherical solute particles is governed by their radius r; the accessible volume in turn is determined by the radius R and concentration C, of the rods in the gel matrix. Ackers (2) proposed a theory of restricted diffusion according to which the elution volume of macromolecules is determined by their rate of diffusion in the gel phase. The experimental observation that the elution volume is not depend- ent on flow rate stands in the way of this interpretation. The partition theory considers the gel with its solvent as a stationary phase. According to Bronsted (3) the I term A, the potential energy difference between the two phases in the Boltzman equation (Eq. A-3-5), is proportional to the molecular weight. = = e .••.•••...•.••••.... Eq. A-3-5 0 In equation A-3-5, c1 and c2 are the concentration or number of - 203 - molecules per unit volume in the two phases and k is the 0 Boltzman's constant. In gel chromatography the significance of the proportionality of A1 to the molecular weight, is that the gel phase has a greater affinity for small molecules than for larger ones. Larger molecules are therefore eluted before smaller molecules. Equation A-3-5 would require that the elution volume would depend on temperature.. However experimental. evidence shows that elution volume is independent of temperature, and so the partition theory does not account for all the events that take place in gel chromatography. Because the elution volume has been found to be independent of temperature, concentration and flow rate, the exclusion mechanism is favoured as providing the closest inter- pretation of the events that take place during separation of molecules according to size on a gel matrix. It should be mentioned nevertheless, that the exclusion principle does not interpret all phenomena in gel chromatography since interaction between the gel and solute has been observed in several cases making separation partially dependent on molecular size. To give a precise interpretation of a chromatogram the column needs to be calibrated. Peak elution volume has long been shown to depend not only on molecular weight of the solute but also on the molecular structure. For a calibration to be meaningful, therefore, structural features of the sample under investigation and that used for calibration have to be taken into account. A practical approach is to calibrate the column c with reference solutes that are structurally similar to the sample - 204 - being studied. This gives the familiar plot of the logarithm of the molecular weight, log M, vs elution volume Ve. A second approach is to employ the universal calibration of Benoit (4,5} which plots the logarithm of the hydrodynamic volume, log n (M] against the elution volume. For lignins, the Benoit calibration may not be adequate since Pla and Robert (6,7) have observed that the exponent, a, in the Kuhn-Mark Houwink equation, [n] KMa, decreases as the molecular weight = V increases. Resolution in gel permeation chromatography GPC like any other chromatographic technique is limited in its resolution. It is expected that when a monodisperse polymer solution is injected as a discrete pulse into a column, the chromatogram appears as a rectangle with a width of unit increment of molecular weight. This is however not observed experimentally. Instead,spreading occurs as a result of molecular diffusion and convection along the axis of the tube and the chromatogram appears as a bell shaped curve. The area under the curve is still proportional to the concentration of the entire sample, but the height of the curve does not reflect directly the relative abundance of the components at the corresponding elution volume since it depends also on the amount of the neighboring components. There are two main types of band broadening (8,9): (a) Symmetrical band broadening is the consequence of axial dispersion. Its effect is to lower the calculated M and raise 0 n - 205 - c the calculated Mw (Fig. A-3-2). (b) Unsymmetrical band broaden ing otherwise known as skewing is attriliuted to an interaction between a non-uniform velocity profile and the simultaneous radial and axial dispersion. The effect of skewing is to lower both M and M • Any precise interpretation of a chromatogram n w should account for band broadening. Tung (10) corrected for band broadening by assuming a Gaussian chromatogram for a single species and represented the Gaussian-shaped chromatogram F (v) by an equation A-3-6. where v is the elution volume of the sample, v is the peak 0 elution volume, A is a constant related to the area and weight of polymer injected, and h is the resolution factor given as 2 h =! cr ,where cr is the variance. In his view the chromatogram of a multicomponent polymer system of m species is the summation of m Gausian distributions given as 2 F(v) = I A.r;;:j'lT exp ·[-h.(v-v i) l. . ••••••.•..••. Eq. A-3-7 i=l ~J --j_' . ~ 0 J For a very large number of species, a continuous distribution function W (y) is employed to account for the abundance of components in the mixture. The chromatogram F (v) is then represented by the Tung's integral dispersion equation A-3-8 as given vb r 2 F (v) =J w(y)J h/lT exp [-h (v-y) ] dy ..•..•.•.••••.•. Eq. A-3-8 V ;:! - 206 - 0 va - initial eluent volume vb - final eluent volume Most of the methods for correcting band broadening are based on different ways of solving this equation. Methods have been developed by Smith (11}, Hess and Kratz (12}, Pickett, Cantow and Johnson (13) and Balke and Hamielec (14). c - 207 - 0 SYMETRICAL BAND BROADENING Starting Dispersed Pulse Pulse Symetrical ARI Chromatogram ELUENT VOLUME UNSYMETRICAL BAND BROADENING Starting Dispersed Pulse Pulse Unsymetrical ARI Chromatogram ELUENT VOLUME FIG. A-3-2 Schematic diagram of band broadening in a gel column. (From Duerksen, J.H.; in "Gel permeation c Chromatography", Altgelt, K.H., Segal, L. (eds). Marcel Dekker Inc., New York 1971). - 208 - 4:) REFERENCES 1. Laurent, T.C., and Killander, J., J. Chromatog., 14, 317 (1964). 2. Ackers, G.K., Biochemistry, l, 723(1964). 3. Bronsted, J.N.Z .• , physikd. chem. Bodenstein Festband, 257-266 (1931). 4. Benoit, H., Grubisic, Z., Remp, P., Decker, D., and Zilliox, J.G., J. Chem. Phys., &l, 1507(1966). 5. Grubisic, Z., Remp, P., and Benoit, H., J. Polym. Sci., Part B , 5 , 7 5 3 {19 6 7 ) • 6. Pla, F., Robert, A., Cellulose Chem., Technol. ~, 11-19 {1974). 7. P1a, F., Robert, A., Ibid.~' 3-10(1974). 8. Tay1or, G.I., Proc. Roy. Soc., A225,473(1954). 9. Bi1lmeyer, F.W., and Kelley, R.N., J. Chromatog., 34, 322 (1968). 10. Tung, L.H., J. Appl. Polym. Sci., 10, 375(1966). 11. Smith, W.N., J. Appl. Polym. Sci., 11, 639(1967). 12. Hess, M., and Kratz, R.F., J. Polym. Sci., Part A-2, !' 731 (1966). 13. Picket, H.E., Cantow, J.R., and Johnson, J.F., J. Polym. Sci., Part C, 21, 67(1968). 14. Balke, S.T., and Hamielec, A.E., J. App1. Polym. Sci., c 13, 1381(1969}. - 209 - 0 APPENDIX 4 DETERMINATION OF WEIGHT AVERAGE MOLECULAR WEIGHTS c - 210 - 0 The short coluron sedimentation equilibrium technique of van Holde and Baldwin (1), which was later applied for lignin molecular weight studies by Goring (2,3), was used to determine the molecular weights of all the samples studied. This technique has the advantage over the usual sedimentation equilibrium technique of reducing considerably the time required for attain- ment of equilibrium (1). THEORY The short column sedimentation equilibrium technique is based on Goldberg's (4) differential equation for the sedi- mentation equilibrium for a multicomponent system containing q non-ionizing solutes which is given as de. - 2 RT dei q ( alny.1 _l M. (1-V.p)w X RT .E_ ,.. • ) ...... A-4-1 1. 1. = c. dx + dx 1. J-1 -J T,p,ck k=l=o where x is the distance from the centre of rotation, c is the concentration, w is the angular velocity, M is the molecular weight, p is the density of the solution, R is the gas constant, T is the absolute temperature, V is the partial specific volume and y is the activity coefficient of the solute in terms of the concentration. In the above equation, concentration but not pressure dependent factors are taken into account. For a two component system (q=l), equation A-4-1 reduces to 2 c M(l-Vp)w x = ~T ~~( 1 + c a;~y) ..•.•.•...•...... •.. A-4-2 - 211 . 0 In the limit of zero concentration, the activity coefficient becomes ind~pendent of concentration, and the term c ~EYac becomes negligible. Equation A-4-2 further reduces to ~ 2 RT de M(l-vp)w x = c dx •.••••..•...... •...... A-4-3 Rearranging equation A-4-3 gives - l, ___,de ...... A- 4-4 XC dX Molecular weights can be determined from equation A-4-4 if a positio~ x,is found in the column where the concentra- tion is equal to the original concentration c , by deter 0 mining the concentration gradient at that point. The equation A-4-4 at such a position is then written as l RT M= , (de) A-4-5 Ox x=x 2 - ...... xco w (1-'Vp) For a 1 mm column, Van Holde and Baldwin (1), defined the - position x by the following equation, -2 2 2 x = (b + a ) /2 ...... A-4-6 and the concentration at that position as H2 c (x) = c H/sinh H 1 + + ) ••••••• A-4-7 0 = c 0/ ( 6 and 2 - 2 2 H M { 1- p ) ( b - = c 0 v a ) 14 RT ... : ...•...•..•...... • A-4-8 - 212 where a and b are distances from the centre of rotation to 0 the meniscus and base of the solution~ For a 1 mm column, x is within 0.2% of the column midpoint and the concentration at this midpoint for the short column is within 1% of the original concentration if H < 0.25. EXPERIMENTAL The ultracentrifuge experiments were carried out by Mr. W.Q. Yean of the Pulp and Paper Research Institute of Canada. Runs were made at25°C· Schlieren observations were made at a phase plate angle of 65°. Dimethyl sulfoxide was used as the solvent for the lignin samples. Before use, the solvent was freshly distilled and further dried with molecular sieves. The samples were dried over silica gel under vacuum for 24 hr before use for the ultracentrifuge experiments. For all runs, a double sector centrepiece was used. The centrepiece compartments were filled with liquid using a precision microsyringe. The solution compartment of the centre piece was first loaded with 0.28 ml of FC-43 (fluorochemical compound used to raise the bottom of the fluid in the column) followed by 0.03 ml of the solution under investigation. For the solvent compartment, 1 ml of the solvent (DMSO) was loaded over 0.26 ml of the FC-43 solution. Each sample was run at three d1fferent speeds and three or four different concentrations. At each speed a solution of the highest concentration was first run. Subsequently, lower c concentrations were obtained by dilution. Exposures were taken - 213 . 0 on Royal pan films from Kodak. The photographic negatives were magnified eight times. The heigh~ ym,of the displacement of the light on the Schlieren pattern was read midway between the meniscus and the base of the cell. The actual height, ye,was obtained by dividing the measured height,ym,by 8. This height was converted into concentration terms after calibrating the Schlieren system with the calibration cell for the Beckman Model E Ultracentrifuge. CALCULATIONS Molecular weights were calculated from equation A-4-4. In terms of the measured parameters, this equation is simplified as follows. For any phase plate angle,e and rotor speed, the linear displacement of the light passing through the column of solution is given as Tl I dC dn ...... A-4-9 Ye = .ua dx • de cotem1 where L is the optical lever arm, m is the cylindrical 1 magnification, dn/dc is the refractive index increment and a' is the cell thickness. From equation A-4-9, de = A-4-10 dx L'. m . a'. dn/dc ...... 1 Equation A-4-5 can therefore be written as 1 RT M A-4-11 = 2 - L' .m .a.· ' d n /de· · · · • • · • • • · · · · · • · c XC w (l-vp) 1 0 - 214 - 1 1 = 2 -· • ...... A-4-12 xco w (1-vp) RTtane where K'= L' m1a•. dx and X = X ...... ~ ...... A-4-13 - m2 where is the camera magnification, x is the distance m2 from the reference line to the centre of rotation, x is the distance of the centre ordinate from the centre of the rotor and dx is the distance on the film from the reference line to the centre ordinate. R, T, w, v, p have the same meaning as in equation A-4-1. To calculate weight average molecular weights, x, y, L'and a are given in cm, p and c 0 in g/ml, v and dn/dc in ml/g. The values of v and dn/dc were determined by separate experiments which are described later. DETERMINATION OF PARTIAL SPECIFIC VOLUME The partial specific volume of the various samples were determined with the aid of the Digital Precision density Meter, DMA02C. The density meter measures the variation of the natural frequency of a hollow glass oscillator (Duran 50) with changing solution density. The oscillator is sealed in a metal block and its temperature is controlled by a constant temperature bath. The temperature of the bath was measured by means of an immersion thermometer (± 0.02°C). The cell block temperature was held at 2S.OO±O.Ol°C. Slightly more than 0.5 ml of solution was required for each measurement. - 215 - Before use, the instrument was first calibrated with dry air (density= 0.001169 at 24.95°C and 749.7 mm pressure). The density of pure water at the same temperature and pressure -1 is 0.997044 g ml • Since P, the period of oscillation of the hollow glass oscillator is the basic experimental parameter, a cell constant A, was obtained from the equation p H 0 - p . 2 a~r ...... A-4-14 A = 2 2 PH .o- Pair 2 where PH and P . are the respective periods of oscillation 0 a~r 2 in water and air and pH 0 , pair their respective densities (see 2 Table A-4-1). The density of a liquid (in this case DMSO) was determined by the following relation PDMSO = p~20 + A [ p~MSO - p~2~ ••••••• • •••• • •••• .!l.-4-15 (see Table A-4-2). The density of the lignin sample in DMSO was also determined by an equivalent relationship, P 1 =PH 0 +A ~p2 1 t' - PH2 o] ...... A-4-16 samp e 2 Lso u 1on 2 Before carrying out measurements on the density meter, the calibration materials were allowed to reach thermal equilibrium. Readings were taken several times and the results averaged. This method of density determination gives a pre 5 cision of ± 2 x 10- g ml-l in dimethylsulfoxide. Data for 0 the calibration and measurements of the densities of DMSO - 216 - 0 and some of the samples are given in Tables A-4-1 and A-4-2. TABLE A-4-l Determination of cell constant (24.95°C) Measured periods (P) Measurement No. of oscillation p . PH 0 a~r 2 1 1635492 2257079 2 90 79 3 94 77 4 89 79 5 91 77 6 93 79 7 91 77 8 89 77 9 92 78 Average P 1635491 2257078 Barometric pressure = 749.7 mm Hg p -1 dry air at 24.95°C = 0.001169 g ml P.H 0 -1 2 at 24.95°C = 0.997056 g m1 11 A = 4115971 X 10- 0 - 217 - 0 TABLE A-4-2 Determination of the density of dimethylsulfoxide (24.95°C) . Measured Eeriods of oscillation Measurement No. PDMSO Pc 2 1 2309366 23133 2 70 30 3 68 27 4 66 33 5 69 28 6 69 32 7 72 29 8 60 33 9 68 Average P 2309367 23100314 Barometric pressure= 749.7 mm Hg, Concentration of c 2 in OMSO = 5 mg/ml The density of DMSO is calculated as follows 2 2 PPMSO = PH o +. 0000411597 [ (2309367) - (22570776) ) 2 ~DMSO = 0.997056 + 0.09828053649 = 1.09533653649 = 1.0953 c - 218 - Density of fraction C2 0 2 2 = 0.997056 + 0.000041159 [(23100314) - (22570776) ] = 0.997056 + 0.099543 "' 1.09659 "' 1.0966 Model calculation Having measured the densities of the samples, the partial specific volume v of the various samples were then calculated from the densities according to the relationship c- (·o - P > solution ·nMSO .•..•••..•.•.•••...•• A-4-li V = P.DMSOC where C = concentration of lignin in DMSO (g/ml) 0 solution = density of sample in DMSO = density of DMSO For the samples used for column calibration (say c2 > the v was determined as follows 0.005 - (1.0966 - 1.0953) V= 1.0953 X 0.005 = 0.6756 "' 0.676 ml/g. DETERMINATION OF THE REFRACTIVE INDEX INCREMENT The refractive index increments for the various samples were measured in a Zeiss microdiffusion interferometer. After filling the solvent and solution compartment with di- 0 methyl sulfoxide and the lignin solution, respectively, the - 219 - cells were allowed to equilibrate to a constant temperature of 23°C. Before diffusion commenced, the boundary between the solution and solvent was formed and the initial interference pattern was photographed. After the commencement of diffusion, the progress of diffusion was followed using white light until the fringes in the solvent and solution were matched. Photo- graphs of the matched fringes were taken. Both the fringe shift and the unit fringe distance were measured on enlarged pro- jections of the photographic negatives.~he refractive index increment, dn/dc, was then calculated from equation A-4-18 dn/dc = A-4-18 wher-e h = number of fringes displaced L = depth of the cell = 0.5 cm A = wavelength = 546 nrn C = concentration in g/ml The determination of h was done with green mono- chromatic light. For each series of samples (e.g., C, SCL, SPL, SSL), trial runs indicated that within the series, the dn/dc values of individual samples were quite close, therefore the average of three determinations carried out with one sample was taken as the dn/dc value of the series. MODEL CALCULATION 0 Calibration fraction C~ - 220 - shift/distance of N fringes 7.s;1io2 6.70 (1) h = I N = = where N = number of fringes L = 0.5 cm 7 ). = 546 X 10- cm -3 c = 5.45 X 10 g/m1 -7 dn 546 X 10 X 6.7 = 0.134 de = 3 5.45 X 10- X 5 X 10-l (2) h = 7.5/(557} = 6.58 7 546 X 10- X 6.58 dn = 0.132 de = 3 5.45 X 10- X 5 X 10-l 0.134 + 0.132 0.133 ml g-l Average value of dn/dc = 2 = RESULTS The result of the ultracentrifuge sedimentation measurements of the various samples are presented in Tables A-4-3 to A-4-15. Since measurements were made at several solute con- centrations and rotor speeds, the reciprocal of the calculated values of the weight average molecular weights were plotted against concentration and the plots extrapolated to zero con- centration. The extrapolated values were then plotted against 2 the square of the angular velocity, w • Extrapolation to zero c centrifugal field and zero speed gave reciprocal values of - 221 - weight average molecular weights independent of concentration and speed. (Figs. A-4-1 to A- 4-10) • c - 222 - 0 TABLE A-4-3 1. Sample: c2 7 Speed: 24K T = 298°K K' ·= 21.3 X 10 -1 -1 dn/ = 0.133 ml.g V = 0.676 ml.g a = 65° de Measured Actual Cone. disp1. displ. 4 mg/ml y y dx dx X X 1/M X 10 m e m 3.93 2.6 0.325 5.65 0.706 7.3 6.971 0.811 2.94 2.0 0.25 5.5 0.688 7.3 6.979 0.793 1.96 l. 25 0.156 17.1 2.137 7.3 6.303 o. 769 2. Speed: 28K 3.93 2.1 0.263 5.65 0.706 7.3 6.971 1.367 2.94 1.6 0.200 5.5 o. 688 7.3 6.979 1.349 1.96 1.0 0.125 17.1 2.137 7.3 6.303 1.300 3. Speed: 32K 3. 93 2.15 • 269 5.65 0.706 7.3 6. 971 1.744 2.94 1.65 0.206 5.5 0.688 7.3 6.979 l. 708 1. 96 1.0 0.125 17.25 2.156 7.3 6.294 1.695 4. Speed: 36K 3. 93 2.1 0.263 5.6 0.7 7.3 6.974 2.262 c 2.94 1.65 0.206 5.55 0.694 7.3 6.976 2.161 1. 96 1.0 0.125 17.25 2.156 7.3 6.294 2.145 - 223 - 0 TABLE A-4-4 1. Sample: CS 7 Speed: 28K T = 298°K K' = 21.3 X 10 -1 a 65° dn/ -1 V = 0. 676 ml. g = de = 0.133ml.g Measured Actual Cone. disp1. displ. - 1 4 mg/m1 y y dx dx X X X 10 m e m /M 6.134 3.35 0.419 5.7 0.713 7.3 6.968 1.268 4.601 2.475 0.309 15.0 1.875 7.3 6.425 1. 254 3.067 1.85 0.231 5.6 0.7 7.3 6.974 1.215 2. Speed: 32K 6.134 3.925 0.491 5.7 0. 713 7.3 6.968 1.492 4.601 2.9 0.363 15.0 1.875 7.3 6.425 1.140 3.067 2.2 0.275 5.6 0.7 7.3 6.974 1.335 3. Speed: 36K 6.134 4.700 0.588 5.75 0.719 7.3 6.965 1.577 4.601 3.500 0.438 14.90 1.863 7.3 6.431 1.468 3.067 2.650 0.331 5.50 0.688 7.3 6. 979 1.404 c - 224 - 0 TABLE A-4-5 1. Sample: c6 7 Speed: 28K T = 298°K !(' = 21.3 X 10 -1 -1 dn/ 0.133 ml.g V = 0. 676 ml.g e = 65° de = .Measured Actual displ. COne. displ. - 1 4 .mg/ml y y 4~ dx X X /M X 10 m e m 6.67 2.50 0.313 5.250 0.656 7.3 6.994 1.957 5.003 l. 725 0.216 15.95 1.994 7.3 6.370 1.940 3.335 1.15 0.144 5.55 0.694 7.3 6.976 2.038 2.501 0.875 0.109 16.4 2.05 7.3 6.344 1.963 2. Speed: 32K 6.67 3.25 0.406 5.35 0.669 7.3 6.988 2.026 5.003 2.15 0.269 16.3 2.038 7.3 6.349 2.025 3.335 1. 575 0.197 5.55 0.694 7.3 6.976 2.028 2.501 1.05 0.131 16.3 2.038 7.3 6.349 1.982 3. Speed: 36K 6.67 4.10 0.513 5.35 0.669 7.3 6.988 1.971 5.003 2.6 0.325 16.3 2.038 7.3 6.349 1.952 3.335 2.0 0.25 5.55 0.694 7.3 6.976 2.028 2.501 1. 35 0.169 16.2 2.025 7.3 6.355 2.046 0 - 225 - 0 TABLE A-4-6 Sample: SPL-5 7 21.3 X 10 1. Speed: 14K T = 298°K K'= -1 -1 dn/ 0. 065 ml.g -V ::: 0.638 ml.g a ::: 65° de = Measured Actual Cone. displ. disp1. y y dx X X mg/ml m e (cm) (cm) (cm) (cm) (cm) 8.24 2.5 0.3125 6.05 o. 756 7.3 6.947 3.404 6.18 2.0 0.25 6.05 o. 756 7.3 6.947 3.191 3.09 0.95 0.12 17.30 0.160 7.3 6.291 3.044 2. Speed: 17K 8.24 2.88 0.36 6.05 0.76 7.3 6.947 4.364 6.18 2.25 0.28 6.10 0. 76 7.3 6.944 4.180 3.09 1.15 0.14 17.2 2.15 7.3 6.297 3. 711 3. Speed: 20K 8.24 3.25 0.41 6.05 0.76 7.3 6.947 5.342 6.18 2.55 0.32 6.10 0.76 7.3 6.944 5.11 3.09 1. 25 0.16 17.2 2.15 7.3 6.297 4.72 0 - 226 SPL-5 0 4 3 ,..---....._ l() 0.,- X 2 Mw = 78,800 .,-~ ,____...- 1 w 2 x10-6 1 2 3 4 20K 5 ,..---....._ 4 l() 14 K 0.,- X 3 .,-~ "---"""' 2 0 2 4 6 8 10 CONCENTRATION (mgjml) c FIG. A-4-1 Plot of 1/Mw vs c (lowe~ and 1/Mw vs Field (upper) for sample SPL-5. - 227 - TABLE A-4-7 Sample: SPL-10 7 l. Speed : l4K T = 298°K K'= 21.3 X 10 -1 -1 V 638 ml.g 65° dn/ = o. 065 ml.g • o. e = de Measured Actual Cone. displ. displ. mg/m1 y y dx dx X X 1/M X 105 m e m (cm) 7.79 3.13 0.39 16.95 2.11 7.3 6.318 2.341 2.60 1.25 0.16 16.5 2.10 7.3 6.338 l. 957 5.20 2.5 0.31 6.1 0.76 7.3 6.944 2.144 10.38 4.35 0.54 5.5 0.69 7.3 6.979 2.469 2. Speed: 17K 7.79 3.90 0.49 16.80 2.10 7.3 6.321 2.766 2.60 1.50 0.19 16.50 2.06 7.3 6.338 2.405 5.20 3.05 0.38 6.10 0.76 7.3 6.944 2.592 10.38 5.4 0.68 5.5 0.69 7.3 6.979 2.932 3. Speed: 20K 7.79 4.25 0.53 16.80 2.10 7.3 6. 321 3.515 2.60 1.6 0.20 16.5 2.06 7.3 6.338 3.120 5.20 3.38 0.42 6.1 0.76 7.3 6.944 3.241 10.38 5.85 0.73 5.5 0.69 7.3 6.979 3.746 0 - 228 - 4~------~ 0 SPL-10 3 ,---..._ 1.0 0.,... 2 X Mw = 133,300 E ""'-,.- 1 ...... ____.., w2x10-6 0 1 2 3 4 5 4 20K ,.---...... 3 17 K 1.0 14K 0,.- X E 2 ...._...,~ 1 0 2 4 6 8 10 12 CONCENTRATION ( mgj ml) 0 FIG. A-4-2 Plot of 1/Mw vs c (lower) and 1/Mw vs Field (upper) for sample SPL-10. - 229 - TABLE A-4-8 Sample: SPL-15 1 7 1. Speed: llK K = 21.3 X 10 - -1 -1 V = 0.638 ml.g = 0.065 ml.g Measured ·Actual Cone. displ. displ. mg/ml y y dx X X 1/M X 105 m e dxm (cm) (cm) (cm) (cm) (cm) (cm) 11.65 3.6 0.45 5.5 0.69 7.3 6.979 2.067 8.74 2.85 0.36 6.05 0.76 7.3 6.947 1.956 5.83 1.8 0.23 17.65 2.21 7.3 6.271 1.864 2. Speed: 14K 11.65 4.25 0.53 5.9 0.74 7.3 6.956 2.827 8.74 3.5 0.44 6.05 0.76 7.3 6.947 2.580 5.83 2.16 0.27 16.7 2.09 7.3 6.326 2.580 3. Speed: 17K 11.65 5.2 0.65 5.9 0.74 7.3 6.959 3.406 8.74 4.0 o.so 6.05 0.76 7.3 6.947 3.329 5.83 2.5 0.31 16.7 2.09 7.3 6.326 3.23 c - 230 - c 5~------, SPL-15 4 ,...... __ 3 lO 0,.... X 2 E Mw=153,800 ,...."'-...... __.... 1 w2 x 10-6 0 1 2 3 4 17K 3 ,...... __ 14K lO 0,.... 11 K X 2 E ,...."'-...... __.... 1 0 2 4 6 8 10 14 CONCENTRATION (mgjml) 0 FIG. A-4-3 Plot of 1/Mw vs c (lower) and 1/Mw vs Field (upper) for sample SPL-15. - 231 - TABLE A-4-9 Sample: SPL-30 1 7 1. Speed : 17K T = 298°K K = 21.3 X 10 -1 -1 65° dn/ 0.065 ml.g V= 0. 638 ml.g e ... de "" Measured Actual Cone. displ. displ. mg/ml y dx dx X ·-X 1/M X 105 m Ye m (cm) (cm) (cm) 9.94 3.85 0.48 6.0 0.75 7.3 6.950 3.921 7.45 2.7 0.34 16.7 2.08 7.3 6.326 3.830 4.97 2.05 0.26 5.9 0.74 7.3 6.956 3.698 2. Speed: 20K 9.94 4.45 0.56 6.0 0.75 7.3 6.950 4.695 7.45 3.56 0.45 16.75 2.09 7.3 6.323 4.615 4.97 2.35 0.29 5.9 0.74 7.3 6.956 4.465 .2.48 1.05 0.13 19.4 2.43 7.3 6.169 4.446 3. Speed: 24K 9.94 5.1 0.64 6.0 0.75 7.3 6.950 5.899 7.45 3.58 0.45 16.75 2.09 7.3 6.323 5.762 4.97 2.75 0.34 5.9 0.74 7.3 6.956 5.495 c 2.48 1. 25 0.16 19.4 2.43 7.3 6.169 5.378 - 232 - SPL-30 Mw=52,300 2 -6 W X 10 0 1 2 3 4 5 6 5 ~ 20K !,() 0 T"'" 4 17 K X T"'"~ ~ 3 0 2 4 6 8 10 12 14 CONCENTRATION ( mg jml ) c FIG. A-4-4 Plot of 1/Mw vs c (lower) and 1/Mw vs Field (upper) for sample SPL-30. - 233 - 0 TABLE A-4-10 Sample: SCL-5 7 1. Speed: 14K T = 298°K K'= 21.3 X 10 -1 -1 dn/ 0.062 ml.g V= 0. 592 ml.g 6 = 65° de = Measured Actual Cone. displ. displ. mg/ml y y dx dx X X 1/M X 105 m e m 7.90 2.3 0.286 5.90 0.738 7.3 6.956 3.954 5.93 1. 63 0.203 16.95 2.119 7.3 6.312 3.810 3.95 1.30 0.163 6.3 0.788 7.3 6.933 3.487 2. Speed: 17K 7.90 2.55 0.319 5.90 0.738 7.3 6.956 5.260 5.93 1. 85 0.231 16.95 2.119 7.3 6.312 4.934 3.95 1.40 0.175 6.3 0.788 7.3 6.933 4.774 3. Speed: 20K 7.90 2.80 0.35 5.90 0.738 7.3 6.956 6.628 5.93 1.98 0.25 16.95 2.119 7.3 6.312 6.395 3.95 1. 55 0.19 6.30 0.788 7.3 6.933 5.967 0 - 234 - 8 SCL-5 6 ...... --...... 1.0 0 ~ X 4 Mw = 108,300 ~ ~ ...... __..... 2 w2x 1CJ6 0 1 2 3 4 5 8 ...... --...... 1.0 20K 0 ~ 6 X 17K E ~ ...... __..... 4 14K 2 4 6 8 10 12 CONCENTRATION ( mg/ml) c FIG. A-4-5 Plot of 1/Mw vs c (lower) and 1/Mw vs Field (upper) for sample SCL-5. - 235 - 0 TABLE A-4-11 Sample: ScL-15 7 1. Speed: 14K T = 298°K K'= 21.3 X 10 -1 -1 6 = dn/ = 0. 062 ml.g V= 0.592 ml.g 65° de Measured Actual Cone. displ. displ. y y dx dx X X 1/M X 105 mg/ml m e m 9.27 3.0 o. 375 6.2 o. 775 7.3 6.938 3.536 6.95 2.18 0.272 17.1 2.138 7.3 6.303 3.333 4.63 1. 73 0.216 6.85 0.856 7.3 6.901 3.068 2. Speed: 17K 9.27 ~.55 0.444 6.2 0.775 7.3 6.938 4.407 6.95 2.55 0.319 17.1 2.138 7.3 6.303 4.193 4.63 2.0 0.25 6.85 0.856 7.3 6.901 3.902 3. Speed: 20K 9.27 4.0 0.5 6.2 0. 775 7.3 6.938 5.413 6.95 2.8 0.35 17.1 2.138 7.3 6.303 5.284 4.63 2.2 0.275 6.85 0.856 7.3 6.901 4.908 c - 236 - 0 6 SCL-15 ,.---.... 4 1.0 0 T- X Mw =126,700 2 .....___....~,... w2x 10-6 1 2 3 4 5 0 6 ,...... --.... 20K 1.0 0 17 K T- 4 X 14 K .____...T-~ 2 0 2 4 6 8 10 CONCENTRATION ( mg / ml ) ~ FIG. A-4-6 Plot of 1/Mw vs c {lower) and 1/Mw vs Field (upper) for sample SCL-15. - 237 - 0 TABLE A-4-12 Sample: ScL-30 1 7 1. Speed: 17K T = 298°K K = 21.3 X 10 -1 -1 V= 0. 592 ml.g 65° dn/ 0. 062 ml.g e = de = Measured· Actual Cone. disp1. displ. mg/m1 y y dx dx X X ·1/M X 104 m e m 9.94 1.6 0.20 6.5 0.813 7.3 6.92 1.046 7.47 1.2 0.15 17.4 2.175 7.3 6.286 0.953 4.97 1.0 0.125 6.05 0.756 7.3 6.947 0.843 2. Speed: 20K 9.94 2.05 0.256 6.5 0.813 7.3 6.921 1.130 7.47 1. 55 0.194 17.4 2.175 7.3 6.286 1.021 4.97 1. 30 0.163 6.05 o. 756 7.3 6.947 0.897 3. Speed: 24K 9.94 2.7 o. 338 6.5 0.813 7.3 6.921 1. 236 7.47 2.0 0.25 17.4 2.175 7.3 6.286 1.140 4.97 1. 65 0.206 6.1 0.763 7.3 6.944 1. 018 c - 238 - 0 SCL- 30 1 Mw = 21,200 2 8 10 0 2 4 6 8 10 12 CONCENTRATION ( mg / ml) ~ FIG. A-4-7 Plot of 1/Mw vs c (lower) and 1/Mw vs Field (upper) for sample SCL-30. - 239 . TABLE A-4-13 .Sample: SSL-1 7 1. Speed: 32K K'= 21.3 X 10 -1 -1 V = 0. 612 rnl. g = 0.062 ml.g Measured Actual Cone. displ. displ. rng/rn1 y y dx dx X X rn e rn 9.58 2.6 0.325 16.75 2.09 7.3 6.323 1.88 7.18 2.3 0.278 6.25 0.78 7.3 6.936 1.81 4.79 1.5 0.188 5.85 0.73 7.3 6.959 1.80 2.. Speed: 3 6K 9.58 3.0 0.375 16.75 2.09 7.3 6.323 2.06 7.18 2.5 0.313 6.25 0.78 7.3 6.936 2. 04 4.79 1. 73 o. 216 5.85 o. 731 7.3 6.959 1.98 3. Speed: 40K 9.58 3.2 0.4 16.75 2.09 7.3 6.323 2.39 7.18 2.7 0.338 6.25 0.78 7.3 6.936 2.34 4.79 1. 85 0.231 5.85 0.73 7.3 6.959 2.28 c - 240 - 3~------, SSL-1 2 ~ 'l:t 0,... >< E Mw =11,700 ~ "'---"' 1 0 14 18 12 16 20 22 ,....--...... _ 40K 'l:t 0,... 36K 2 X 32K E ..._.....~ 10 2 4 6 8 10 12 14 CONCENTRATION ( mg / ml ) FIG. A-4-8 Plot of 1/Mw vs c (lower) and 1/Mw vs Field (upper) for sample SSL-1. - 241 - 0 TABLE A-4-14 Sample; SSL-20 7 1. Speed: 20K T = 298°K K'= 21.3 X 10 -1 -1 V= 0. 612 ml.g 65° dn/ :: 0. 062 ml.g e = de Measured Actual Cone. displ. displ. 4 mg/ml y y dx dx X X 1/M X 10 m e ·m 8.15 1. 95 0.24 6.0 0.75 7.3 6.950 0.92 6.11 1.35 0.17 16.55 2.07 7.3 6. 335 0.91 4.08 1. 03 0.13 6.15 0.77 7.3 6.941 0.87 2. Speed: 24K 8.15 2.5 0.31 6.0 0.75 7.3 6.950 1.03 6.11 1. 78 0.22 16.55 2.07 7.3 6. 335 0.99 4.08 1. 325 0.17 6.15 0.77 7.3 6.941 0.97 3. Speed: 28K 8.15 3.0 0.38 6.0 0.75 7.3 6.950 1.17 6.11 2.1 0.26 16.55 2.069 7.3 6.335 1.14 4.08 1. 58 0.2 6.15 0.77 7.3 6.941 1.11 0 - 242 - 3~------~ SSL- 20 2 Mw=16,900 ....---...... ~ 0,... X E 1 ...... __...... ~ 0 2 4 8 10 ,...--...,_ ~ 28K 0.,... 1 24K X 20K E ...... _.....~ 0 2 4 6 8 10 12 CONCENTRATION ( mg j ml ) 0 Plot FIG. A-4-9 of 1/Mw vs c (lower) and 1/Mw vs Field (upper) for sample SSL-20. - 243 - TABLE A-4-15 Sample: SSL-30 7 1. Speed : 24K T = 298°K K'= 21.3 X 10 -1 -1 dn/ 0. 062 ml.g V= 0. 612 ml.g e = 65° de = Measured Actual Cone. displ. displ. dx dx X X 1/MX 104 mg/ml y y m m e 10.14 1.8 0.23 6.1 o. 76 7.3 6.944 1. 78 7.60 1. 25 0.16 17.2 2.15 7.3 6.297 1. 75 5.07 0.95 0.12 6.05 0.76 7.3 6.947 1.69 2. Speed: 28K 10.14 2.0 0.25 6.1 o. 76 7.3 6.944 2.18 7.60 1.4 0.18 17.15 2.14 7.3 6.300 2.12 5.07 1.1 0.13 6.05 0.76 7.3 6.947 2.08 ·3. Speed: 32K 10.14 2.3 0.29 6.1 0.76 7.3 6.944 2.47 7.60 1.6 0.20 17.15 2.14 7.3 6.300 2.12 5.07 1.2 0.15 6.05 o. 76 7.3 6.947 2.39 0 - 244 - 0 3~------~ SSL-30 2 X E 1 Mw =13,300 ...... __..~ 0 2 4 8 10 12 32K ....---... 28K 'o;t 0,..... 2 >< 24K E ....____'""-,..... 1 0 2 4 6 8 10 12 14 CONCENTRATION ( mgjml) FIG. A-4-10 Plot of 1/M vs c (lower) and 1/M vs Field (upper) 0. w w for sample SSL-30. - 245 - ~ REFERENCES 1. Van Holde, K.E., and Baldwin, R.L., J. Phys. Chem., ~, 734 (1958). 2. Yean, W.Q. and Goring, D.A.I., Pulp and Paper Mag. Can. ~, T-127(1964). 3. Rezanowich, A., Yean, W.Q. and Goring, D.A.I., Svensk Paperstidn. ~, 141(1963}. 4. Goldberg, R.J., J. Phys. Chem., 57, 194(1953}. 0 - 246 - CLAIMS TO ORIGINAL RESEARCH CHAPTER 2 (1) The ozonization of isolated lignins and protolignin in 45% aqueous acetic acid at room temperature. In this system spruce periodate and cuoxam lignins were studied as models for the spruce wood protolignin. (2) The observation that lignin degradation by ozone follows first order kinetics and that the carbohydrate moieties in wood do not affect the rate of delignification. (3) The observation that during ozonization, the rate of degrad ation of spruce protolignin is the same as that of spruce cuoxam lignin but slightly slower than that of spruce periodate lignin. This reinforces earlier suggestions that the periodate and cuoxam lignins retain the networK structure of protolignin. CHAPTER 3 (4) The IR analysis of the alkali-soluble degradation products of lignin after ozonization. (5) The UV analysis of the alkali-soluble degradation products of lignin after ozonization. (6) The N.M.R. analysis of the alkali-soluble degradation products of lignin after ozonization. {7) The demonstration that the phenolic hydroxyl content of 0 lignin increases during ozone treatment. (8) The suggestion that the principal lignin network-degrading - 247 - reaction during ozonization involves the cleavage of the phenol ether bonds. (9) The proposal of a mechanism for the cleavage of the phenol ether bonds of lignin. (10) The observation that during the ozonization of lignin in 45% aqueous acetic acid, decarboxylation occurs in the later stages of the reaction. CHAPTER 4 (11) The determination of the molecular weight distribution of soluble degradation products of lignin obtained during ozonization. The distribution is broad with a low molecular weight maximum and a high molecular weight tail. (12) The proposal of a heterolytic step-reaction (random) degrad ation for the breakdown of the lignin macromolecule by ozone, {13) The determination of the weight average molecular weights of soluble degradation products of lignin obtained during ozonization. CHAPTER 5 (14} The discovery that during the ozonization of wood in 45% aqueous acid, lignin is solubilized 4 times faster than carbohydrates. (15) The observation that in the above reaction, a significant fraction of the original a-cellulose and hemicelluloses are retained in the pulp even at the fiber liberation point. - 248 - 0 (16) The comparison of the rate of lignin and carbohydrate removal in various delignification processes. This comparison shows that carbohydrates are degraded to a lesser extent in the present ozonization process than in the other delignification processes involving carbohydrate depolymerization. This observation finds its use in obtain ing high yield pulps from ozone treatment. 0 - 249 - 0 SUGGESTIONS FOR FURTHER RESEARCH While the main features of lignin degradation during ozonization appear to have been essentially elucidated by the present work, certain gaps in our knowledge of the chemistry still need to be filled. Investigations based on the following suggestions should further improve our understanding of the subject. [1] It has been shown in the present work that phenol ether bonds of lignin are cleaved during ozonization. The scission of the phenol ether bonds has been identified as the main network-degrading reaction of lignin during ozoniz ation. It should be interesting to confirm both the observed ether cleavage and the proposed reaction mechanism with appropriate lignin model compounds like the a-aryl ether or s-aryl ether models. The reactions can be followed by spectroscopy and/or chromatography. [2] Starting with a lignin model compound having both the a-aryl and s-aryl ether linkages, a comparison can be made of the relative susceptibilities of these ether bonds to cleavage by ozone. This information should cast light on the observed first order degradation kinetics. [3] Because the work described in this thesis was primarily concerned with a study of the degradation of lignin as a macromolecular network, no attempt was made to isolate 0 and identify the individual degradation products. While this could be a difficult task, there is no doubt that the - 250 - identification of some of the degradation products of the lignins or models should contribute to a more detailed understanding of the mechanism of lignin degradation by ozone. [4] In Chapter 5 of this thesis, it was observed that the use of 45% aqueous acetic acid as a reaction medium for ·wood is effective in inhibiting extensive carbohydrate degradation while at the same time promoting the rapid removal of lignin. This observation has a significant bearing on the practical aspects of delignification because of the possibility of obtaining ozone pulps in which carbohydrate degradation is minimized. According to the method reported in this thesis, the wood meal is suspended in a 45% aqueous acetic acid solution prior to ozone treatment. Lignin removal can be enhanced by impregnating wood with aqueous acetic acid be fore ozone treatment. Rapid delignification should be achieved with a minimum loss in viscosity. Besides pulping of wood, the present method can be applied to the bleaching of pulps either by suspending the pulp in the aqueous acetic acid or better still, by spraying the pulp with 45% aqueous acetic acid solution before ozone treatment. Exploratory experiments of the type suggested have been carried out with Kraft pulp at the Pulp and Paper Research Institute of Canada. The results proved the superiority of the present method to the existing methods using ozone, in terms of 0 lignin removal and carbohydrate protection. [5] The choice of 45% aqueous solution of acetic acid was made - 251 - to achieve sufficiently low pH while at the same time hav ing water in the system. The presence of water is necessary for the reaction to occur. It is probable that a lower concentration of aqueous acetic acid solution could yield better results. It is desirable to find the optimum con centration of acetic acid for best results. Besides, other organic acids should be tried as reaction media for lignin or wood. 0 - 252 - c GLOSSARY OF PRINCIPAL SYMBOLS A Absorbance (A)6 Absorbance at pH 6 (A)l2 Absorbance at pH 12 A250 Absorbance at 250 nm ~A Difference in absorbance at pH 6 and pH 12 a Constant characteristic of a polymer molecule in sol a' Cell thickness in the ultracentrifuge experiments (cm) c Concentration of rods in the gel matrix c Concentration g/liter DP Degree of polymerisation dn/dc Refractive index increment (ml g-l) F(v) Distribution function of single species in a chromatogram f Functionality of a monomer h Resolution factor of a chromatogram K Rate constant (sec-1 ) K' Ultracentrifuge constant Kav Volume fraction of stationary phase available to molecules of a given size in gel chromatography Kd Partition coefficient K An empirical constant that is dependent on the n size and shape of polymer molecule k Boltzmann's constant L Amount of undissolved lignin at time t during the ozonization of lignin (gm) 0 L' Optical lever arm of the ultracentrifuge (cm) Lo Initial amount of lignin (gm) - 253 - Amount of lignin resistant to solubilization L c r by ozone treatment (gm) L Thickness of UV cell (cm) Molecular weight Number average molecular weight Weight average molecular weight Ultracentrifuge cylindrical magnification Ultracentrifuge camera magnification Total number of units in a crosslinked polymer p Probability that a functional group has reacted Ph-0 Ionized free phenolic hydroxyl groups Ph-OH Ionizable free phenolic hydroxyl groups Ph-OR Etherified free phenolic hydroxyl groups p (.) y ~ Probability of a primary chain of y units with i crosslinked members Rate of degradation of lignin (g/g min) R Gas constant R' Radius of particles of a gel matrix r Radius of a spherical solute particle SCL Soluble products obtained during ozonization of periodate lignin SPL Soluble lignin obtained during the ozonization of spruce wood SSL Soluble lignin obtained uring the ozonization of spruce wood Probability of a randomly selected chain of y units being part of the sol fraction T Absolute temperature (°K) Partial specific volume (ml g-l) Elution volume (ml) Total pore volume of the gel matrix - 254 Accessible pore volume of gel particles Void volume of interstitial volume (ml) Total volume of gel bed (ml) Weight fraction of sol Weight fraction of primary chains of y units Continuous distribution function Weight fraction of a network of Z chains in an infinite network Weight average degree of polymerisation of the sol fraction of a gel X Number of branch points in a polymer X Distance between the midpoint of column and centre of rotation in the ultracentrifuge Position in ultracentrifuge column where concentration is equal to original concentration C 0 y Number of ends in a polymer y Number of units in a primary chain y Average number of units in a primary chain Actual height of the ordinate midway between the meniscus and base of the ultracentrifuge cell (cm) V Measured height of the ordinate midway between - nl the meniscus and base of the ultracentrifue cell (cm) Yw Weight average chain length of primary chains a Branching coefficient ac Critical branching coefficient for gelation in polyfunctional system 1-y s = a{l -a) or Ye y pv crosslinking index 0 E Absorptivity ~E Difference in absorptivity of a solution at pH 6 and pH 12 - 255 - E: I Expected number of crosslinked units in a ·~ primary chain E: I Expected number of crosslinked units at incipient c formation of a network gel [nl Intrinsic viscosity (dl g-l) e Schlieren angle used in ultracentrifuge experiments A Wavelength A I Potential energy difference between two phases \) Number of units per crosslink (two) p Density of solution, crosslinking density p I Crosslinking density in the sol fraction p I Critical crosslinking density c <~>s Probability of a non-crosslinking unit being part of the sol w Angular velocity of the ultracentrifuge rotor c