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Kinetic and mechanistic studies of polyoxometalate (POM) reaction with lignin and model compounds Kim, Yong Sik 2007

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KINETIC AND MECHANISTIC STUDIES OF POLYOXOMETALATE (POM) REACTION WITH LIGNIN AND MODEL COMPOUNDS by Yong Sik K i m A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF D O C T O R O F P H I L O S O P H Y i n The Faculty of Graduate Studies (Forestry) THE UNIVERSITY OF BRITISH COLUMBIA March, 2007 ©Yong Sik Kim, 2007 Abstract Polyoxometalates (POMs) are a rapidly growing class of metal-oxygen-cluster anions. The properties of P O M s can be modified by altering the P O M s chemical composition and structure. Due to low cost, commercial availability, and synthetic tractability P O M s have found application in various fields of chemistry and technology. P O M s are reusable and thermally stable to oxidative conditions, making them an attractive alternative to chlorine for the delignification of wood pulp. The research addressed in this dissertation deals with detailed kinetic and mechanistic studies of K 5 [ S i V W u 0 4 o ] 1 2 H 2 0 , a P O M used in the delignification of wood pulp, oxidation of phenolic lignin model compounds and milled wood lignin ( M W L ) . Results from lignin model studies suggest an overall second-order reaction rate; first order with respect to both P O M and phenolic substrate. It was observed that electron-transfer from neutral phenols was slower than that from the corresponding phenoxide anions. Hammett studies revealed the reaction involved the formation of an electron-deficient radical intermediate where the rate-determining step is electron-transfer from a neutral substrate. The structure of the substituted phenol, in terms of its electron donating/withdrawing character, along with the position of the substituent on ii the aromatic ring heavily influences the reaction rates. Increasing the number of ortho methoxyl groups dramatically increased the reaction rate, e.g. phenol < guaiacyl < syringyl model structures. The ortho methoxyl group(s) resonance stabilizes and delocalizes the forming phenoxyl radical intermediate. Similarly, the reaction rate of para-substituted guaiacyl and syringyl model compounds showed a dependence on the nature of the para-substituent; inductive or resonance conjugated electron withdrawing effects and inductive donating effects. The effect of POM oxidation on the chemical structure of a Lodgepole pine MWL is investigated. 1 3 C nuclear magnetic resonance (NMR) spectroscopic data revealed an approximate 28 % decrease in J3-0-4 inter-unit linkages after POM treatment, the decrease in P-O-4 inter-unit linkages being accompanied by an increase in carbonyl content. These results suggest that POM oxidation involves side-chain (such as a-OH/P-O-4) oxidation. 1 3 C NMR spectroscopy along with gel permeation chromatography revealed an increase in the degree of condensation which supports the idea that radical coupling is a major reaction pathway in this process. in Table of Contents Abstract ii Table of Contents iv List of Figures vii List of Schemes xiii List of Tables xiv List of Abbreviations xvi Acknowledgements xviii Dedication xix Co-Authorship Statement xx 1. Introduction 1 1.1 B a c k g r o u n d 1 1.2 L i g n i n S t r u c t u r e 3 1.3 L i g n i n I s o l a t i o n b y the B j o r k m a n M e t h o d 9 1.4 N u c l e a r M a g n e t i c R e s o n a n c e ( N M R ) S p e c t r o s c o p y o f L i g n i n 11 1.4.1 ' H N M R 12 1.4.2 1 3 C N M R 13 1.4.3 Two-Dimensional Heteronuclear Correlation Experiments 16 1.4.3.1 HSQC (Heteronuclear Single Quantum Coherence) 16 1.4.3.2 H M B C (Heteronuclear Multiple Bond Coherence) 17 1.5 P o l y o x o m e t a l a t e s 18 1.5.1 Polyoxometalate Delignification of Wood Pulp 20 1.5.1.1 Two-stage technology 20 1.5.1.2 One-stage (catalytic) technology 23 1.6 P O M R e a c t i o n M e c h a n i s m w i t h L i g n i n M o d e l s C o m p o u n d s 26 1.6.1 Phenolic Lignin Model Compounds 26 1.6.2 Non-Phenolic Lignin Model Compounds 31 1.7 R e s e a r c h O b j e c t i v e s 34 1.8 R e f e r e n c e s 37 2. Polyoxometalate (POM) Oxidation of Phenols: Effect of Aromatic Substituent Groups on Reaction Mechanism 43 2.1 I n t r o d u c t i o n 44 2.2 E x p e r i m e n t a l 4 6 2.2.1 Materials 46 2.2.2 Kinetic Measurements 47 2.2.3 Product Analysis 50 iv 2.2.4 Analytical Methods 51 2.2.5 Identification of Reaction Products 52 2.3 R e s u l t s a n d D i s c u s s i o n 55 2.3.1 Kinetics and Mechanism 55 2.3.2 Activation Parameters 66 2.3.3 P O M Oxidation of Substituted Phenols 70 2.3.4 Product Analysis 74 2.4 C o n c l u s i o n s 82 2.5 R e f e r e n c e s 84 3. Polyoxometalate (POM) Oxidation of Lignin Model Compounds 87 3.1 I n t r o d u c t i o n 88 3.2 E x p e r i m e n t a l 89 3.2.1 Materials 89 3.2.2 Syntheses of Lignin Model Compounds 92 3.2.3 Kinetic Measurements 101 3.2.4 Reaction Conditions for Product Analysis 103 3.2.5 Analytical Methods 104 3.2.6 Identification of Reaction Products 105 3.3 R e s u l t s a n d D i s c u s s i o n 108 3.3.1 Kinetics 108 3.3.2 Reactions of p-Hydroxylphenyl, Guaiacyl and Syringyl Lignin Model Compounds 113 3.3.3 Reactions of Guaiacyl Lignin Model Compounds with Different para-Position Substituent Groups 119 3.3.4 Reactions of Syringyl Lignin Model Compounds with Different para Position Substituent Groups 121 3.3.5 Reactions of Alkylated Guaiacyl and 5-5 Dimer Lignin Model Compounds 123 3.3.6 Reactions of 13-0-4 Lignin Model Compounds 128 3.4 C o n c l u s i o n s 130 3.5 R e f e r e n c e s 133 4. Polyoxometalate (POM) Oxidation of Milled Wood Lignin (MWL).. 136 4.1 I n t r o d u c t i o n 137 4.2 E x p e r i m e n t a l 138 4.2.1 Materials 138 4.2.2 Planetary Ball Milling Method 138 4.2.3 Milled Wood Lignin (MWL) Isolation 139 4.2.4 POM Treatment of MWL 141 4.2.5 Acetylation of MWL 141 4.2.6 Determination of Lignin content in MWL 142 • 4.2.7 Gel Permeation Chromatography (GPC) and Fourier Transform Infrared (FT-IR) analysis 142 4.2.8 Nuclear Magnetic Resonance (NMR) analyses 143 4.2.8.1 Quantitative 'H NMR spectroscopy 143 4.2.8.2 'H- 1 3 C two-dimensional Heteronuclear Single Quantum Coherence (HSQC) NMR spectroscopy ' 144 4.2.8.3 Quantitative 1 3 C NMR spectroscopy 144 4.3 R e s u l t s a n d D i s c u s s i o n 145 4.3.1 Structural Analyses of MWL and POM-MWL using NMR Spectroscopy 145 4.3.2 Functional Groups (Methoxyl, Hydroxyl and Carbonyl Groups) 154 4.3.3 (3-0-4 Inter-unit Linkage 156 4.3.4 Aromatic Carbons and Degree of Condensation 159 4.3.5 Other Lignin Structures 160 4.3.6 FT-IR Analyses of MWL and POM-MWL 161 4.3.7 Molecular Mass Distributions 166 4.4 C o n c l u s i o n s 168 4.5 R e f e r e n c e s 170 5. Conclusions and Future Work , 172 5.1 C o n c l u s i o n s 172 5.2 F u t u r e W o r k 174 6. Appendix 176 6.1 R e a c t i o n O r d e r D e t e r m i n a t i o n b y I n i t i a l R a t e L a w i n C h a p t e r 2 176 6.2 R e a c t i o n O r d e r D e t e r m i n a t i o n b y I n i t i a l R a t e L a w i n C h a p t e r 3 177 6.3 D e v i a t i o n o f E q u a t i o n 3.2 179 6.4 M e t h o x y l C o n t e n t A n a l y s i s 181 6.5 K l a s o n L i g n i n C o n t e n t A n a l y s i s 183 6.6 N M R S p e c t r u m o f N o n - A c e t y l a t e d M W L 185 6.7 A n a l y s i s o f the S e c o n d D e r i v i a t i v e S p e c t r a o f H y d r o x y l a n d C a r b o n y l S t r e t c h i n g r e g i o n s o f M W L a n d P O M - M W L 187 6.8 V i s i b l e S p e c t r a A b s o r b a n c e - T i m e D a t a ( in a t t a c h e d C D ) 188 6.9 R e f e r e n c e s 189 List of Figures Figure 1.1 The primary phenylpropane precursors of lignin 4 Figure 1.2 Prominent inter-unit linkages found in softwood (SW) and hardwood (HW) lignin [18] 5 Figure 1.3 A structural scheme for a typical softwood lignin [19] 7 Figure 1.4 H S Q C N M R spectra of acetylated M W L s [43]; a) pine acetylated M W L ( p i n e - A c - M W L ) and b) Poplar acetylated M W L (polpar A c - M W L ) 16 Figure 1.5 H M B C N M R spectra of (a) Poplar acetylated M W L and (b) Aspen acetylated C W [48] 17 Figure 1.6 The cc-Keggin P O M anion [ S 1 V W 1 1 O 4 0 ] 5 " in polyhedral notation. The Si04 is located in the center of the black tetrahedral, M O x units are depicted; W 0 6 octahedra are shown in gray, while the V O ^ octahedron is shown in white [56]. 19 Figure 1.7 Oxidation of A benzyl alcohol, and B cyclohexanol by [PV2M010O40] 5 " in toluene; alkyl-substituted phenols C and D by [ P V n Moi2- nC>4o] ( n + 3 )" in acetic acid under aerobic conditions; and E 3,3',5,5'-tetra-tert-butyldiphenyl-4,4'-diol by a-K 5 [ S i V W i o 0 4 0 5 " ] in 0.1 M L i O A c / H O A c buffered water/t-BuOH solution (2:3), p H 4.8 under anaerobic conditions [50, 66-68] 27 Figure 1.8 Proposed reaction pathway for the oxidation of a syringyl p-O-4 dimer by oc-[SiVWi 004o] 5~ at room temperature under anaerobic conditions [60] 28 Figure 1.9 Reaction scheme for lignin oxidation in the presence of heteropolyanions, [PM07V5O40] 8 " under aerobic oxidation ( V v and represent V 0 2 + and V 0 2 + in the oxidized and reduced forms of the P O M , respectively [9] 29 Figure 1.10 Products formed from the reaction of vanil lyl alcohol with HPA-5 or H P A - 5 - M n 1 1 under aerobic conditions (30 min, p H 3, 90 °C, 0 2 pressure 0.5 MPa) [64] 30 Figure 1.11 Oxidation products (% yield) detected from the reaction of Na 5 ( + 1 . 9 ) [SiV 1 ( .o . i ) MoW 1 o( + o. i )0 4 o] (POM) with l-(3,4,5-trimethoxylphenyl)ethanol at 160°C; 1 (3,4,5-trimethoxylacetophenone), 2 (3,4,5-triethoxylbenzaldehyde) and 3 (3,4,5-trimethoxylbenxoic acid) [63] 32 vn Figure 1.12 Products of the reaction of veratryl alcohol with H P A - 5 or H P A - 5 - M n 1 1 under aerobic conditions (30 min, pH 3, 90 °C, O2 pressure 0.5 M P a ) [64] 33 Figure 2.1 Phenols studied 46 Figure 2.2 Relationship between concentration of P O M and absorbance at a) 350 nm ( S i V W u O 4 0 5 ~ ) and b) 520 nm ( S i V W n 0 4 o 6 ' ) 48 Figure 2.3 Schematic representation of the U V - V i s stopped f low apparatus (1 cm U V path-length) used for kinetic analyses : 49 Figure 2.4 Reaction products formed during the P O M ( S i V W n O V ) oxidation of 1 (p-methoxylphenol), 4 (m-methylphenol) and 5 (phenol). *dimers formed during the P O M oxidation of 4 54 Figure 2.5 Change in P O M ( S i V W u 0 4 o 5 " ) absorbance during reaction with a) 2 and b) 5 in sodium acetate buffer (I = 0.2 M , pH 5.0) at 25 °C and 45 °C, respectively. ([2] = [5] = 12.25 mmol L"', [POM] = 0.25 mmol L"1). The arrows indicate the decrease (i) in absorbance of the oxidized P O M at 350 nm and the increase (T) in absorbance of the reduced P O M at 520 nm, respectively. 2 (p-methylphenol) and 5 (phenol) 56 Figure 2.6 Plot of initial rate versus a) [POM] and b) [5] at 25 °C in sodium acetate buffer (I = 0.2 M , p H 5.0). 5 (phenol) and P O M ( S i V W ^ o 5 " ) 59 Figure 2.7 Absorbance-time plots for the reaction of P O M ( S i V W n 0 4 o 5 " ) with 1 at different temperatures (°C) in sodium acetate buffer (I = 0.2 M , p H 5.0). [1] = 12.25 mmol L" 1, and [ S i V W n 0 4 o 5 " ] = 0.25 mmol L ' 1 . 1 (p-methoxylphenol) 60 Figure 2.8 Kinetic analysis of S i V W 1 1 O 4 0 5 " oxidation of 1 at different temperatures (°C) in sodium acetate buffer (I = 0.2 M , p H 5.0): a) init ial rate plot and b) pseudo-first order plot. [1] = 12.25 mmol L"\ and [ S i V W n O 4 0 5 1 = 0.25 mmol L" 1. 1 (p-methoxylphenol) 61 Figure 2.9 Effect of acidity (1/[H +]) on the second order rate constant (k) for the P O M ( S i V W n O 4 0 5 " ) oxidation of a) 1, and b) 2, 5, 8 at 20 °C, 35 °C, 45 °C, and 80 °C, respectively in sodium acetate buffer (I = 0.2 M , p H 3.9 - 6.0). [phenols] = 12.25 mmol L"1, [ S i V W n O 4 0 5 " ] = 0.25 mmol L" 1. 1 (p-methoxylphenol), 2 (p-methylphenol), 5 (phenol), and 8 (m-chlorophenol) 64 V l l l Figure 2.10 Isokinetic relationship (Arrhenius plots) of various substituted phenols. 1 (p-methoxylphenol), 2 (p-methylphenol), 4 (m-methylphenol), 5 (phenol), 7 (p-bromophenol), 8 (m-chlorophenol) and 9 (m-nitrophenol) 67 Figure 2.11 Activation enthalpy versus entropy for the oxidation of phenols with SiVWnO-40 5" in sodium acetate buffer (I = 0.2 M , p H 5.0) at 25 °C. 1 (p-methoxylphenol), 2 (p-methylphenol), 4 (m-methylphenol), 5 (phenol), 7 (p-bromophenol), 8 (m-chlorophenol) and 9 (m-nitrophenol) 69 Figure 2.12 Hammett plots for the oxidation of substituted phenols with S i V W n 0 4 o 5 " in sodium acetate buffer (I = 0.2 M , p H 5.0) at room temperature. The rate constants, and substituent constants (c and o+) are those listed in Table 2.5. 1 (p-methoxylphenol), 2 (p-methylphenol), 4 (m-methylphenol), 5 (phenol), 7 (p-bromophenol), 8 (m-chlorophenol) and 9 (m-nitrophenol) 73 Figure 2.13 FT-IR spectra of precipitates formed during the P O M (S iVWn0 4 o 5 ~) reaction (red line) and the initial phenolic compounds (blue line): a) 1 (p-methoxylphenol); b) 4 (m-methylphenol); and c) 5 (phenol). A l l of these reactions were run using sodium acetate buffer (I = 0.2 M , p H 5.0) 75 Figure 2.14 Gel permeation chromatography (GPC) elution curves of the precipitated materials formed during P O M ( S i V W n O 4 0 5 " ) reaction with 1 (25 °C for 1 h), 4, and 5 (60 °C for 2 h and 5 h, respectively) in sodium acetate buffer (I = 0.2 M , p H 5.0). 1 (p-methoxylphenol), 4 (m-methylphenol) and 5 (phenol) 76 Figure 2.15 M A L D I - T O F - M S spectra of polymeric materials formed during the P O M (SiVWnCvtt)5") reaction with 1 (25 °C for 1 h), 4 , and 5 (60 °C for 2 h and 5 h, respectively) in sodium acetate buffer (I = 0.2 M , p H 5.0). 3,5-dimethoxyl-4-hydroxylcinnamic acid was the matrix for 1 and 2,5-dihydroxylbenzoic acid was the matrix for 4 and 5. 1 (p-methoxylphenol), 4 (m-methylphenol) and 5 (phenol). 78 Figure 2.16 ' H N M R spectra of initial phenolic compounds (upper) and oligomers (lower) formed during the reaction of P O M ( S i V W n 0 4 o 5 " ) in sodium acetate buffer (I = 0.2 M , p H 5.0) with a) 1 at 25 °C for 1 h, b) 4 at 60 °C for 2 h, and c) 5 at 60 °C for 5 h. *Unknown signals or impurities. 1 (p-methoxylphenol), 4 (m-methylphenol) and 5 (phenol) 80 Figure 3.1 Lignin model compounds studied 91 ix Figure 3.2 Schematic representation of the stainless steel UV-Vis reaction cell. The cell was equipped with two 1 cm thick quartz windows, a void volume of 10 cm 3 and a 4 cm U V path-length 102 Figure 3.3 Reaction products identified from the P O M oxidation of 1-3, 5-8, 10, and 13 108 Figure 3.4 Plots of a) log[initial rate] versus log[POM] or b) log[model compounds] at 45 °C (1), 25 °C (2 and 3), and 40 °C (11), in sodium acetate buffer (I = 0.2 M , pH 5.0) under argon. 1 (1-(4-hydroxylphenyl)-ethanol), 2 (l-(4-hydroxyl-3-methoxylphenyl)-ethanol), 3 (l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol) and 11 (4-[2-hydroxyl-2-(4-hydroxyl-3-methoxylphenyl)ethoxyl]-3-methoxyl-benzoic acid) 110 Figure 3.5 Absorbance-time plot for the reaction of P O M (SiVWnO 4 0 5 ~) with 3 (1-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol) at 40 °C in sodium acetate buffer (I = 0.2 M , pH 5.0) under argon. The open circles represent the experimental data points and the red curve represents the calculated data using equation 3.2. The sum of the squares of the standardized residuals was 0.2 x 10"4 I l l Figure 3.6 Absorbance-time plots for the reaction of P O M (SiVWn0 4o 5~) with compounds 1, 2, and 3. Reaction conditions: [model compounds 1-3] =0.1 mmol L" 1 and [SiVW,|O 4 0 5 "] = 0.25 mmol L" 1 in sodium acetate buffer (I = 0.2 M , pH 5.0) under argon at 50 °C (1) and 40 °C (2 and 3). 1 (l-(4-hydroxylphenyl)-ethanol), 2 (l-(4-hydroxyl-3-methoxylphenyl)-ethanol) and 3 (l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol) 113 Figure 3.7 Absorbance-time plots for the reaction of P O M (SiVWnO 4 0 5 ") with compounds 4 and 5. Reaction conditions: [model compounds 4 and 5] = 0.1 mmol L"' and [S iVW n O 4 0 5 " ] = 0.25 mmol L" 1 in sodium acetate buffer (I = 0.2 M , pH 5.0) under argon at 40 °C. 4 (l-(4-hydroxyl-3-methoxylphenyl)-ethyl methyl ether) and 5 (l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethyl methyl ether) 115 Figure 3.8 Absorbance-time plots for the reaction of P O M (SiVWn0 4o 5~) with compounds 2, 4, 6 and 9. Reaction conditions: [model compounds 2, 4, 6 and 9] = 0.1 mmol L" 1 and [SiVWnO^ 5"] = 0.25 mmol L" 1 in sodium acetate buffer (I = 0.2 M , pH 5.0) under argon at 50 °C (6) and 40 °C (2, 4 and 9). 2 (l-(4-hydroxyl-3-methoxylphenyl)-ethanol), 4 (l-(4-hydroxyl-3-methoxylphenyl)-ethyl methyl ether), 6 (4-acetyl-2-methoxylphenol) and 9 (4-ethyl-2-methoxylphenol) 119 Figure 3.9 Absorbance-time plots for the reaction of POM (SiVWnO4 0 5") with compounds 3 , 5 and 7. Reaction conditions: [model compounds 3 , 5 and 7] = 0.1 mmol L"1 and [SiVWnO^5"] = 0.25 mmol L"1 in sodium acetate buffer (I = 0.2 M , pH 5.0) under argon at 40 °C. 3 (l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol), 5 (l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethyl methyl ether) and 7 (4-acetyl-2,6-dimethoxylphenol) 121 Figure 3.10 Absorbance-time plots for the reaction of POM (SiVWnO405~) with compounds 8, 9 and 10. Reaction conditions: [model compounds 8 - 10] = 0.1 mmol L"1 and [SiVW uO 4 0 5"] = 0.25 mmol L"1 in sodium acetate buffer (I = 0.2 M , pH 5.0) under argon at 20 °C. 8 (4-methyl-2-methoxyl-phenol), 9 (4-ethyl-2-methoxyl-phenol) and 10 (3,3'-dimethoxyl-5,5'-dimethyl-biphenyl-2,2'-diol).124 Figure 3.11 Absorbance-time plots for the reaction of POM (SiVWnO4 0 5") with compounds 11, 12 and 13. Reaction conditions: [model compounds 11 - 1 3 ] = 0.1 mmol L"1 and [SiVWHO 4 0 5"] = 0.25 mmol L"1 in sodium acetate buffer (I = 0.2 M , pH 5.0) under argon at 30 °C. 11 (4-[2-hydroxyl-2-(4-hydroxyl-3-methoxylphenyl)ethoxyl]-3-methoxyl-benzoic acid), 12 (4-[2-hydroxyl-2-(4-hydroxyl-3,5-dimethoxylphenyl)ethoxyl]-3-methoxyl-benzoic acid) and 13 (4-[2-hydroxyl-2-(4-hydroxyl-3,5-dimethoxylphenyl)ethoxyl]-3,5-dimethoxyl-benzoic acid) .- 129 Figure 4.1 Preparation of milled wood and milled wood lignin (MWL) 140 Figure 4.2 Quantitative *H NMR spectra for a) acetylated MWL and b) acetylated POM-MWL. *Unknown signals or impurities 147 Figure 4.3 Quantitative 1 3 C NMR spectra for acetylated MWL and acetylated POM-MWL 149 Figure 4.4 Lignin substructures (from Capanema et al. 2004) [16] 153 Figure 4.5 'H- 1 3 C HSQC spectra of a) acetylated MWL and b) acetylated POM-MWL. Peak labels correspond to Figure 4.4. The yellow color indicates methoxyl groups [19] 158 Figure 4.6 FT-IR spectra of MWL and POM-MWL 161 Figure 4.7 Deconvoluted FT-IR spectra of the hydroxyl stretching region ( VOH ) of a) MWL and b) POM-MWL. * value in parenthesis indicates IR band area 163 xi Figure 4.8 Deconvoluted FT-IR spectra of the carbonyl stretching region (vc=o) of a) MWL and b) POM-MWL. *value in parenthesis indicates IR band area 166 Figure 4.9 Molecular weight distributions of acetylated MWL and acetylated POM-MWL 167 Figure 6.1 Plot of ln[initial rate] versus a) ln[POM] and b) ln[5] at 25°C in sodium acetate buffer (I = 0.2 M , pH 5.0). 5 (phenol) 176 Figure 6.2 Plot of initial rate versus [POM] at 45 °C (1), 25 °C (2 and 3 ) , and 40 °C (11), in sodium acetate buffer (I = 0.2 M , pH 5.0) under argon. 1 (l-(4-hydroxylphenyl)-ethanol), 2 (l-(4-hydroxyl-3-methoxylphenyl)-ethanol), 3 (l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol) and 11 (4-[2-hydroxyl-2-(4-hydroxyl-3-methoxylphenyl)ethoxyl]-3-methoxyl-benzoic acid) 177 Figure 6.3 Plot of initial rate versus [model compound] at 45 °C (1), 25 °C (2 and 3) , and 40 °C (11), in sodium acetate buffer (I = 0.2 M , pH-5.0) under argon. 1 (l-(4-hydroxylphenyl)-ethanol), 2 (l-(4-hydroxyl-3-methoxylphenyl)-ethanol), 3 (l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol) and 11 (4-[2-hydroxyl-2-(4-hydroxyl-3-methoxylphenyl)ethoxyl]-3-methoxyl-benzoic acid) 178 Figure 6.4 Quantitative l 3 C NMR spectrum for non- acetylated MWL 185 Figure 6.5 FT-IR spectra (upper) and second-derivative spectra (lower) of the hydroxyl stretching region of a) MWL and b) POM-MWL. *the number of peaks was determined from the second-derivative spectra; local minima with values that tend to be above or near zero indicate hidden peaks 187 Figure 6.6 FT-IR spectra (upper) and second-derivative spectra (lower) of the carbonyl stretching region in a) MWL and b) POM-MWL. *the number of peaks was determined from the second-derivative spectra; local minima with values that tend to be above or near zero indicate hidden peaks 188 xii List of Schemes Scheme 1.1 Proposed two-step process for convention of lignin in wood to CO2 and H 2 0 21 Scheme 2.1 Possible reaction mechanism for POM (SiVW|iO4 0 5~) oxidation of phenols 57 Scheme 2.2 Possible structures of polymeric materials formed from the reaction of POM (S1VW11O40 5") with 1 (25 °C for 1 h), 4 , and 5 (60 °C for 2 h and 5 h, respectively) in sodium acetate buffer (I = 0.2 M , pH 5.0). 1 (p-methoxylphenol), 4 (m-methylphenol) and 5 (phenol) 79 Scheme 2.3 Possible mechanism for the POM (SiVWiiO 4 0 5") oxidation of 1 under anaerobic conditions 81 Scheme 3.1 Synthetic pathway for the preparation of lignin model compound 11. 101 Scheme 3.2 Possible conjugative delocalized resonance structures of the phenoxy radical intermediate 115 Scheme 3.3 Possible reaction mechanisms for POM (SiVW u0 4o 5") oxidation of 1 (1-(4-hydroxylphenyl)-ethanol) and 2 (l-(4-hydroxyl-3-methoxylphenyl)-ethanol). 116 Scheme 3.4 Possible reaction mechanisms for the POM (SiVWnO^5") oxidation of 3 (l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol) 118 Scheme 3.5 Possible reaction mechanism for POM (SiVWnO405~) oxidation of 5 (1-(4-hydroxyl-3,5-dimethoxylphenyl)-ethyl methyl ether) 123 Scheme 3.6 Possible reaction mechanism for POM (SiVWnO405~) oxidation of 8 (2-methoxyl-4-methyl-phenol) and 10 (3,3'-dimethoxyl-5,5'-dimethyl-biphenyl-2,2'-diol) 127 Scheme 3.7 Products detected from the POM (SiVWnO405") oxidation of 13 (4-[2-hydroxyl-2-(4-hydroxyl-3,5-dimethoxylphenyl)ethoxyl]-3,5-dimethoxyl-benzoic acid) at 25 °C '. '. 130 xiii List of Tables Table 1.1 Frequency of inter-unit linkages in spruce and birch lignin [18] 6 Table 1.2 Functional groups in softwood and hardwood lignin [18, 22-26] 8 Table 1.3 ' H N M R chemical shifts of common lignin inter-unit linkages [26, 40, 44, 45] 13 Table 1.4 l 3 C N M R chemical shifts of the major inter-unit linkage in acetylated softwood lignin [41, 43, 47] 15 Table 2.1 Dimers (14*) formed during the P O M ( S i V W u O 4 0 5 " ) oxidation of 4 (m-methylphenol) 54 Table 2.2 Second-order rate constants (k) for the oxidation of 1 by S i V W n 0 4 o 5 ~ in sodium acetate buffer (I = 0.2 M , p H 5.0). [1] = 12.25 mmol L " 1 , [ S i V W n O 4 0 5 " ] = 0.25 mmol L" 1 62 Table 2.3 Calculated rate constants ki and k 2 for the oxidation of 1, 2, 5, and 8 by SiVWn04o 5 "in sodium acetate buffer (I = 0.2 M , p H 3.9 - 6.0). [phenols] = 12.25 mmol L " 1 , [ S i V W n O 4 0 5 " ] = 0.25 mmol L ' 1 65 Table 2.4 Observed kinetic constants for the oxidation of substituted phenols by S i V W , i O 4 0 5 " in sodium acetate buffer (I = 0.2 M , p H 5.0) 68 Table 2.5 Second-order rate constants for the reaction between S i V W | | 0 4 o 5 " and substituted phenols in sodium acetate buffer (I = 0.2 M , p H 5.0) at 25 °C. Included are logarithm values of each rate constant for the substituted phenol (k x ) versus phenol (kH), as well as Hammett o~ and o+constants for the series of substituted phenols 71 Table 2.6 Oligomerization of 1 (25 °C for 1 h), 4, and 5 (60 ° C for 2 h and 5 h, respectively) using P O M ( S i V W n O 4 0 5 " ) in sodium acetate buffer (I = 0.2 M , p H 5.0). 1 (p-methoxylphenol), 4 (m-methylphenol) and 5 (phenol) 77 Table 3.1 Second-order rate constants (k) calculated by either the initial rate law or the nonlinear regression equation. [Substrate] = 0.1 mmol L " 1 , [S iVWn0 4 o 5 ~] = 0.25 mmol L" 1 in sodium acetate buffer (I = 0.2 M , p H 5.0) under argon 112 xiv Table 4.1 Conditions for NMR experiments 143 Table 4.2 Yield and Klason lignin content of the Lodgepole pine and purified MWL. 145 Table 4.3 Signal assignment in the ! H NMR spectra of acetylated MWL and POM-MWL samples [16-19] 148 Table 4.4 Signal assignment in the 1 3 C NMR spectrum of acetylated MWL and POM-MWL samples [14, 15, 21] 151 Table 4.5 Quantification of inter-unit linkages and functional groups in MWL and POM-MWL via quantitative l 3 C NMR [14] 152 Table 4.6 Signal assignment in the FT-IR spectra of MWL and POM-MWL [24]. 162 Table 6.1 Signal assignment in the 1 3 C NMR spectrum of non-acetylated MWL sample [3-5] 186 xv List of Abbreviations e P o ( + ) °C A Acetone-de ATR BF3-MeOH CDCI3 Da DMSO-d6 EDG EWG eV FT-IR g AG GC GC-MS GPC AH HOMO h HPA HMBC HSQC IKR K k k, k2 k<>bs K„ extinction coefficient reaction constant substituent constant degrees Celsius frequency factor deuteroacetone attenuated total reflectance boron trifluoride-methanol deuterochloroform Dalton deuterodimethyl sulfoxide activation energy electron donating group electron withdrawing group electron volt fourier transformed infrared gram change in Gibbs free energy gas chromatography gas chromatography - mass spectrometry gel permeation chromatography change in enthalpy highest occupied molecular orbital hour(s) heptamolybdopentavanadophosphate anion heteronuclear multiple bond coherence heteronuclear single quantum coherence isokinetic relationship kelvin temperature (absolute temperature) rate constant rate constant rate constant observed rate constant acid dissociation constant MALDI-TOF matrix-assisted laser desorption ionization time-of-flight MHz megahertz MW molecular weight MWL milled wood lignin m/z mass per charge NaBH 4 sodium borohydride NaHS0 4 sodium bisulfate NaHS0 4»Si0 2 silica gel-supported sodium bisulfate NaOMe sodium methoxide nm nanometer NMR nuclear magnetic resonance POM polyoxometalate pK a -log(Ka) rpm revolutions per minute r2 correlation coefficient RI refractive index AS change in entropy SOMO single occupied molecular orbital T temperature TLC thin layer chromatography v/v volume per volume wt. weight w/w weight per weight xvii Acknowledgements The author would like to express his sincere appreciation to Dr. John F. Kadla and Dr. Hou-min Chang for their guidance and support throughout his studies. Their constant encouragement, invaluable advice, and constructive criticism were always available when suggestions or stimulating discussions were needed. Their sincere friendship and guidance have made this study enjoyable and memorable. Sincere gratitude is also extended to my committee member Dr. Brian R. James for his continuing advice, guidance, and support. In addition, the author would like to thank Drs. Kevin M . Holtman, Tomoya Yokoyama, Satoshi Kubo, Jennifer L. Braun, Qizhou Dai, Fadi Asfour, Batia Bar-Nir, and Mr. Kyle Hope-Ross for their suggestions, discussions, and friendship. The author expresses his deepest gratitude to his wife, Su Kyung Choi, for her love, patience, sacrifice and assistance which have made this study an enjoyable experience. Special appreciation is also given to the author's family for their constant support and encouragement throughout this study. Finally, the author wishes to thank all those faculty members, colleagues, and friends who in one way or another have contributed to making this study a successful experience. X V 1 L 1 Dedication / woutd tike to dedicate this dissertation to my parents and" my betoved, Su Kyung Choi, alt of whom had a major influence on my Cife, goats and ambitions. X I X Co-Authorship Statement All experiments were designed and performed by the PhD candidate, Yong Sik Kim. The thesis committee members (Dr. Hou-min Chang and supervisor, Dr. John F. Kadla) assisted with data analysis and manuscript preparation. Because the vast majority of the work was done by the PhD candidate, he should be listed as first author; Drs. Chang and Kadla should be listed as second and third authors, respectively. X X 1. Introduction 1.1 Background Due to environmental concerns, current research and industrial trends in pulp bleaching deal essentially with the progressive substitution of environmentally hazardous chlorine-based bleaching chemicals with oxygen-based reagents (O2, O 3 , H2O2) [1-3]. Unfortunately, a primary limitation of these oxygen-based reagents is their lower reactivity and poor selectivity. Oxygen delignification in aqueous alkaline media is now widely utilized throughout the industry. However, the so-called 50 % delignification barrier hinders the progressive application of this technology. Beyond 50 % residual lignin removal the delignification selectivity decreases dramatically and significant polysaccharide degradation occurs leading to a decrease in pulp quality [3-6]. In oxygen delignification, as well as other 02-based reagents, numerous secondary oxidative species form as a result of reaction with lignin. Of particular concern is hydroxyl radical (HO»), which is capable of reacting with carbohydrates in addition to lignin [3]. As delignification is extended and the amount of lignin available in the pulp to react with HO* decreases, carbohydrate degradation occurs. Therefore, if the generation of these secondary oxidative species, particularly H0», could be minimized or eliminated substantial improvement in the O2 delignification process could be made. One particular area of focus is the utilization of regenerative oxidation catalysts. These compounds 1 avoid the free radical chain oxidation of lignin and represent a promising approach to overcome the drawback of oxygen delignification. In addition to improving the 0 2 delignification process through the addition of catalysts, new oxidative reagents are being developed which are both environmentally benign and highly selective towards lignin. One such class of reagents is polyoxometalates (POMs), which have been reported to efficiently remove lignin from wood pulps without severe polysaccharide degradation [7-9]. POMs are a rapidly growing class of metal-oxygen-cluster anions. POMs are synthetic inorganic compounds that contain highly symmetrical core assemblies of MO(x) units (M = vanadium, molybdenum, tungsten) and react as outer-sphere electron-transfer oxidants and catalysts [10, 11]. POMs offer a safe and environmentally benign alternative to traditional bleaching reagents such as elemental chlorine. Efficient and selective removal of lignin, an aromatic polyol, from wood pulps without severe degradation of carbohydrates, such as cellulose can be accomplished using POMs under both anaerobic and aerobic conditions [7-9, 12]. The catalytic mechanism involves a series of reduction-oxidation cycles wherein the reduction of the POM(O X ) by the lignin substrate is accompanied by subsequent reoxidation of the POM( r ed) by 0 2 . The sum of electron transfer is from lignin to 0 2 . In addition, the selective oxidation of lignin in pulps with oxygen catalyzed by POMs is 2 possible due to the suppression of free radical chain oxidation reactions, and to the lower oxidation potential of lignin as compared to polysaccharides [9]. In the following section, a review of the literature of P O M oxidation of wood pulps and lignin model compounds in aqueous solutions is given. In addition, a general background of lignin and some spectroscopic techniques used for lignin macromolecular characterization is given. Emphasis is on the P O M oxidation of lignin model compounds to provide a better understanding of the oxidation chemistry of lignin-based materials. 1.2 Lignin Structure Wood structural components consist primarily of cellulose, hemicellulose and lignin [13, 14]. Lignin is a main component in wood, and constitutes roughly 15-35 % by weight. Lignin is a crosslinked polymer arising from enzymatic dehydrogenative polymerization of hydroxylated and methoxylated phenylpropane units [15]. Figure 1.1 depicts the three primary phenylpropane precursors of lignin, p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. 3 O H O H .OH O C H 3 H 3 C Q - O C H 3 O H O H O H p-coumaryl alcohol (Hydroxyphenyl) coniferyl alcohol (Guaiacyl) sinapyl alcohol (Syringyl) Figure 1.1 The primary phenylpropane precursors of lignin. The lignin macromolecule is formed by coupling reactions of p-coumaryl, coniferyl, and sinapyl alcohol-based phenoxy radicals (precursors for H, G and S, respectively) [16]. The complex nature of the inter-monomeric linkages found in lignin is based on the 5 resonance-stabilized mesomeric forms of the phenoxy radical intermediates produced by peroxidase-H^Oi oxidation [17]. Coupling of two monomers results in the formation of a dimer, which can further undergo dehydrogenative polymerization and coupling with another monomer to form a trimer or another dimer to form a tetramer. This process of dehydrogenation and radical coupling continues to form the three-dimensional lignin macromolecule. Figure 1.2 shows the most prominent inter-unit linkages in lignin. 4 Biphenyl ( 4 - 0 - 5 ' ) (5-5') Figure 1.2 Prominent inter-unit linkages found in softwood (SW) and hardwood (HW) lignin [18]. Table 1.1 summarizes the relative differences in distribution of inter-unit linkages between spruce (softwood) lignin and birch (hardwood) lignin. It can be seen that there are higher levels of uncondensed, etherified structures such as ]3-0-4' linkages and lower levels of condensed structures, such as 5-5' and {3-5' in hardwood lignin. This is directly attributable to the additional methoxyl group at the C-5 position of the aromatic ring in 5 syringyl units ( F i g u r e 1.1) [18]. In hardwoods, approximately 10 % of the inter-unit linkages involve the C-5 carbon in carbon-carbon bonds, while in softwoods it is approximately 20 - 25 %. F i g u r e 1.3 illustrates a structural scheme for a typical softwood lignin. Recently, dibenzodioxocin structures (a new 8-membered ring structure) were discovered in softwood lignins using 2-D NMR techniques [19]. Dibenzodioxocins serve as the main branching point in softwood lignin ( F i g u r e 1.3), and constitute approximately 5 per 100 aromatic rings in native lignin [20, 21]. T a b l e 1.1 Frequency of inter-unit linkages in spruce and birch lignin [18]. Inter-monomeric linkages per 100 C9-units Lignin p-O-4' a-O-4' p-5' p- p' 5-5' p-1' 4-0-5' Spruce 48 6-8 9- 12 2 9.5-11 7 3.5-4 Birch 60 6-8 6 3 4.5 7 6.5 The lignin ' macromolecule contains a variety of functional groups; primarily methoxyl, hydroxyl and carbonyl groups. A small proportion of the phenolic hydroxyl groups are free, with the majority participating in ether linkages to neighboring phenylpropane units. During enzymatic dehydrogenase polymerization carbonyl and aliphatic hydroxyl groups are incorporated into the lignin structure, the latter making up a significant portion of side-chain functionality ( F i g u r e 1.3). T a b l e 1.2 illustrates the 6 frequency of some of the common functional groups found in lignin. Table 1.2 Functional groups in softwood and hardwood lignin [18, 22-26]. Abundance per 100 Co-units Functional group Softwood lignin Hardwood lignin Carbonyl 6-23 17-31 Aliphatic alcohol Primary 68-78 70-86 Secondary 16-25 20 - 55 Phenolic hydroxyl 20-32 10-15 Methoxyl 92-96 136-160 Lignin is found between cells and within cell walls, providing both resistance to biological attack and structural rigidity [27]. Although lignin plays an important role in the growth of the cell wall as well as the growth of a tree, it causes severe problems for the pulp and paper industry. For instance, in high yield pulps residual lignin is responsible for the low level of brightness as well as the reversion of brightness and corresponding deterioration in pulp quality [3]. Thus, for the production of high quality pulps lignin removal is required. This is typically accomplished using chemical pulping processes such as kraft pulping (alkali degradation of wood in the presence of sodium sulphide) and soda or alkali pulping, followed by extensive bleaching processes to decrease lignin content and increase the brightness of the pulp. Pulp is typically made by mechanical processes, chemical processes, or some 8 combination thereof [14]. Mechanical pulping is the separation of wood fibres through mechanical action. As a result, some fibre damage occurs due to fibre cutting. Mechanical pulps retain a majority of lignin and other constituent components, and as such are high yield pulps. Mechanical pulps are bleached using oxidative or reductive agents that remove chromophoric groups but do not delignify the fibres [3]. By contrast, chemical pulps utilize acidic or alkaline conditions to selectively remove lignin. During kraft pulping, roughly 80 - 90 % of the original lignin is removed though nucleophilic cleavage of primarily fi-O-4 linkages. Residual kraft lignin is structurally different from native lignin. While it possesses structures already present in native lignin, it is enriched in condensed structures that either survive the kraft pulping process or arise from reactions of the lignin structures during pulping [3, 28, 29]. The free phenolic content of residual kraft lignin is about 40 % [30], whereas that of the native lignin is roughly 10 - 20 % [31]. In addition, some conjugated structures form, which confer a brown color to the kraft pulp. Therefore, before kraft pulp can be used in the manufacture of high quality papers such as printing, writing, or tissue papers, it must first be bleached. 1.3 L i g n i n I s o l a t i o n b y t h e B j o r k m a n M e t h o d It is desirable to work with isolated lignins when studying the properties, chemistry, and reaction of lignins. Ideally, isolated lignins should be relatively pure, i.e. do not 9 contain non-lignin substances, and the isolation procedure sufficiently mild that the chemical nature of the lignin remains relatively unchanged [32, 33]. Unfortunately, no method has yet to be developed which isolates lignin in high yield and structurally unchanged [33]. Of the various isolated lignin preparations, milled wood lignin (MWL) is considered the most representative of native lignin. Although MWL represents only part of the native lignin in the wood cell wall, and undergoes some structural changes during ball milling and isolation, it is widely used to study the chemical structure and reactivity of native lignin [34-38]. Bjorkman first developed the isolation of lignin from finely divided wood meal by extraction with neutral solvent in 1954 [32, 39]. This procedure extracts a significant portion of the lignin, and the MWL yield is dependent upon the extent of milling. Generally, the original method of Bjorkman is utilized with some minor modifications. Typically, air-dried wood is first Wiley milled (< 60 mesh) and Soxhlet extracted with acetone or ethanol/toluene (1:2, v/v). The dried, extracted Wiley wood is subsequently ball milled in a vibratory or planetary mill. The time of ball milling can vary from 1 h to 15 h depending on the efficiency of the ball mill [32]. It is significant to note that the milling method can affect both the yield and structure of the MWL [-34-38]. The next step is to suspend the milled wood in 96 % dioxane and stir at room temperature for 24 - 48 h. The solution is centrifuged and the supernatant carefully 1 0 collected. The remaining solid is again dispersed in 96 % dioxane and the above procedure repeated. The combined supernatants from centrifugation are concentrated and freeze-dried to give crude MWL in about 20 - 30% yield; this lignin may contain up to about 10 % residual carbohydrates. In most cases, it is desirable to purify the crude MWL. Purification is accomplished by dissolving the crude MWL in 90 % acetic acid at a concentration of 50 mg mL"1 and precipitated into deionized water (1 g / 220 mL H2O). The precipitated lignin is centrifuged, the supernatant discarded and the lignin washed with deionized water and dried (freeze-dried or air-dried). The dried lignin is redissolved in a 2:1 (v/v) 1,2-dichloroethane/ethanol solution (0.5 - 1 g / 20 mL), centrifuged to remove any undissolved material, and precipitated into anhydrous diethyl ether (0.5 - 1 g / 230 mL). After centrifugation, the insoluble MWL is washed three times with diethyl ether and dried (air-dried or dried in a vacuum desiccator over phosphorus pentoxide (P2O5)). The yield of the purified MWL may be half that of the crude MWL but its residual carbohydrate content is only about 4 % [33]. 1.4 Nuclear Magnetic Resonance (NMR) Spectroscopy of Lignin Nuclear magnetic resonance (NMR) spectroscopy is an indispensable tool for the structural characterization of organic and biological molecules. Unlike degradative 11 methods, the main advantage of NMR spectroscopy is that it is able to examine the entire polymer rather than make assumptions based upon monomelic products. One-dimensional solution-state NMR experiments such as 'H and 1 3 C can be implemented to estimate the hydroxyl content and the inter-unit linkages in lignin [40, 41]. Two-dimensional techniques such as heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond coherence (HMBC) provide further inter-unit linkage information, and can be utilized in the search for new structural features in the lignin polymer [20, 42, 43]. 1.4.1 * H N M R 'l-I NMR spectra are commonly collected using acetylated lignins, as information concerning the phenolic and aliphatic hydroxyl groups can be obtained [44, 45]. Table 1.3 lists the chemical shifts identified in an acetylated spruce lignin. Acetylated lignins are typically dissolved in CDCI3 at a concentration of -20 mg / mL. A relatively high relaxation delay (Di) of 5 - 7 s is used to ensure complete relaxation of all protons, and a minimum of 128 scans are acquired. As a result, the total experimental time for 'H NMR analysis of isolated lignins is very short. Quantification of the various functional groups can be determined by integration of a specific peak and ratioed to either the methoxyl peak at 8H 3.81 ppm [26] or an internal standard [46]. 12 Table 1.3 'H NMR chemical shifts of common lignin inter-unit linkages [26, 40, 44, 45]. Chemical Shift, ppm Inter-unit linkage H a H p H y P-O-4' 6.0-6.1 4.9-5.0 4.1-4.3/4.2-4.4 P-5' 5.5-5.6 3.5-3.8 4.4 P-P' 4.6 - 4.7 -3.1 3.8-3.9/4.2-4.3 Dibenzodioxocin 4.9 4.1 -4.2 4.0-4.1/4.5 a-C=0 in P-O-4' - 5.4-5.5 4.4-4.5 Ar-CH=CH-CH2OAc -6.60 -6.2 - 6.3 .4.7 - 4.8 Ar-CH=CH-CHO 7.6 6.6 9.7 ArCHO 9.9 - -p-r 6.0 - 6.2 3.5 4.4 - 4.4 Ar-CH 2-CH 2-CH 2-OAc 2.5 - 2.7 1.8-2.0 -4.0 Spirodienone -5.2 3.1-3.3 4.0-4.2 Ar-CO-CH 2-CH 2OAc - 3.2-3.3 4.3-4.4 Ar-CH(OAc)-CH2-CH2OAc 5.8 2.1-2.2 4.1 Aliphatic OAc -2.0 - -Aromatic OAc 2.2 - 2.3 - -1.4.2 1 3 C NMR 1 3 C NMR is a powerful tool in the study of lignin structure as it allows for direct analysis of the carbon skeleton structure and provides a comprehensive view of the entire lignin macromolecule [40, 41]. The quantitative abundance of each inter-unit linkage in 13 lignin can be determined from l 3 C NMR if the pulse delay between acquisitions is sufficiently long to ensure that all carbon nuclei have relaxed to their initial Boltzmann distribution. The pulse delay required to achieve these conditions is generally considered to be 5 times the longest rate of longitudinal relaxation (5T0 [40]. As the Tl for the various carbons found in lignin can be quite long, the addition of a relaxation agent, e.g. c hromium (III) acetylacetonate (0.01 M), can dramatically reduce experiment time. Despite several attempts to quantify the inter-unit linkages in lignin using l 3 C NMR, there still exists a large overlap in the 1 3 C NMR spectrum, making estimation of structural information difficult. Recently, there have been reports of the use of a combination of acetylated and non-acetylated lignin spectra to estimate inter-unit linkages [41]. Overlapping signals in non-acetylated spectrum may separate using an acetylated sample due to the alteration of the chemical environment. Capanema et al. [41] utilized both l 3 C and 'H NMR spectroscopy to make quantitative estimations. Table 1.4 lists the chemical shifts of important carbons in the acetylated 1 3 C NMR spectrum. 14 Table 1.4 1 3 C NMR chemical shifts of the major inter-unit linkage in acetylated softwood lignin [41,43, 47]. Inter-unit linkage Chemical Shift, ppm C a Cp Cy Other P-O-4' 73.5 -75.4 79.3 - 80.6 62.8 - 63.5 P-5' 88.1 - 88.6 50-51.5 65.0-66.1 P-P' 85.1 - 86.1 55.0 71.8-72.5 Dibenzodioxocin 84.2 - 84.9 82.5 - 83.4 64.5 a-C=0 in P-O-4' 194- 195 79.7-81.6 63.3 - 64.3 Ar-CH=CH-CH2OAc 133.5 -134.8 123-2-123.7 64.8 - 65.6 Ar-CH=CH-CHO 151.8 128.8 - 132 193.4 ArCHO 191 - -p-r 74.8 - 76.0 50-51.2 64.0 - 64.9 Ar-CH 2-CH 2-CH 2-OAc 32.2 30.2 63.8 Spirodienone 83 - 84 57-58 62-63 180.7 (C=0) Ar-CO-CH 2-CH 2OAc 198 37-38 60-61 Ar-CH(OAc)-CH2-CH2OAc 72.5 -73.0 35.3 60.6-61.0 Methoxyl - - - 56.3 Primary OAc (y-hydroxyl) - - - 171.5 Secondary OAc (a-hydroxyl) - - - 170.8 Phenolic OAc - - - 169.6 15 1.4.3 Two-Dimensional Heteronuclear Correlation Experiments 1.4.3.1 HSQC (Heteronuclear Single Quantum Coherence) HSQC is one of the most useful NMR techniques available for the analysis of lignin structure [20, 42, 43]. HSQC is able to detect the correlations between attached pairs of protons and carbons. The HSQC spectrum is a 2-D spectrum consisting of *H and 1 3 C axes. The structural elucidation of lignin using HSQC analysis is complementary to quantitative 1 3 C NMR, as it can identify overlapping signals in either of the J H or 1 3 C spectra. However, as it is a proton-carbon correlation experiment, it cannot detect quaternary carbons, and thus, no structural information about lignin condensation can be obtained. Figure 1.4 shows examples of HSQC spectra of acetylated MWLs. 6.0 5.5 5.0 45 4.0 15 10 Ii PP"1 6.0 5.5 5.0 4.5 AO 3.5 3.0 2.5 PP m A B C D X 0-ary) ether phenyicoumaran resinol dibenzodioxocin (E)-cinnamyl alcohol (P-O-4') (0-5) (0-0') (5-5'/0-O-4'/ a-O-4) end-group Figure 1.4 HSQC NMR spectra of acetylated MWLs [43]; a) pine acetylated MWL (pine-Ac-MWL) and b) Poplar acetylated MWL (polpar Ac-MWL). 16 1.4.3.2 HMBC (Heteronuclear Multiple Bond Coherence) HMBC is similar to HSQC in that it is a 2-dimensional 'H-13C correlation experiment, but is used for the determination of long range, typically two or three bond I 13 H- C correlations. HMBC is especially useful to confirm the presence of specific structural moieties [48]. Figure 1.5 shows examples of HMBC spectra of acetylated MWL and CW (cell wall). (a) Poplar AoMWL (ta) Aspen Ac-CW I II t»c t~i—i—i—i—i—i—I I—i—i—i—i—i—i—r fi„ 6.2 6.0 S.8 6.2 6.0 5.8 ppm Figure 1.5 HMBC NMR spectra of (a) Poplar acetylated MWL and (b) Aspen acetylated CW [48]. 17 1.5 P o l y o x o m e t a l a t e s Polyoxometalates (POMs) are a rapidly growing class of metal-oxygen-cluster anions [10, 49-51]. POMs are synthetic inorganic compounds that contain highly symmetrical core assemblies of MO(x) units (M = vanadium (V), molybdenum (Mo), tungsten (W)) which react as outer-sphere electron-transfer oxidants and catalysts [10, 11, 52]. The properties of POMs can be controlled by altering the POM composition and structure [11, 53]. As a result, POMs have found applications in analytical and clinical chemistry, catalysis (including photocatalysis), biochemistry (electron transport inhibition), medicine (anti-tumor, anti-viral, and even anti-HTV activity), and solid-state devices [54]. Over the last decade there has been an increasing interest in the application of POMs in the catalytic delignification of wood for the production of paper [8, 12]. POMs offer a safe and environmentally benign alternative to traditional bleaching reagents such as elemental chlorine. Efficient and selective removal of lignin, an aromatic polyol, from wood pulps without severe degradation to carbohydrates such as cellulose can be accomplished using POMs under anaerobic and aerobic conditions [7-9, 12]. The catalytic mechanism involves a series of reduction-oxidation cycles wherein the reduction of the POM(OX) by the substrate (lignin) is accompanied by subsequent reoxidation of the POM ( r e d ) by O2. The sum of electron transfer is from lignin to O2. 18 The heteropolyanions, such as the Keggin structures, are widely used in the POM bleaching of wood pulp. They are a larger class of POM than the isopolyanions; they are more versatile, and more easily modified. The Keggin-type POM anion [SiVWnO40]5~ is presented in Figure 1.6 [10, 52, 55]. Figure 1.6 The a-Keggin POM anion [SiVWn04o]5" in polyhedral notation. The Si0 4 is located in the center of the black tetrahedral, MO x units are depicted; W O 6 octahedra are shown in gray, while the V06 octahedron is shown in white [56]. POMs are easily prepared from acid condensation of common inorganic precursors such as W0 4 2", M0O4 2 " , V O 3 2 ' , PO43", Si0 3 2" [49, 51]. Varying the structure and composition of the POM affects the physical (size, shape, charge, solubility, and thermal stability) and chemical (acidity and redox potential) properties [10, 49]. POMs are inherently resistant to oxidative degradation and many of the reduced POM anions are readily reoxidized by molecular oxygen. As a result, these attributes make POMs attractive for the delignification of wood pulp [7-9, 56-60]. 19 The first generation POMs, a-[PV2Moio040]5~ and a-[SiVWii0 4o] 5\ were shown to be very effective in the delignification of wood pulp [12, 56]. The a-[PV2Moio04o]5" anion was only stable at low pH (around 2) while the a-[SiVWn04o]5" anion was stable at neutral pH. The modification of these POMs produced the second generation of delignification reactants, that include a-[SiV 2Wi 0O 4 0] 6", a-[AlVWn04o]6" and a-[SiV„Moio04o]5" [61, 62]. These second generation POMs were stable under neutral and even basic conditions and were efficient in both the delignification and wet oxidation stages. 1.5.1 P o l y o x o m e t a l a t e D e l i g n i f i c a t i o n o f W o o d P u l p In 1994, Hill and Weinstock first proposed the use of polyoxometalates (POMs) as reusable oxidants to selectively delignify chemical pulps. Following this, Evtugin and Neto proposed a catalytic POM delignification process. Based on these and other works, the chemical selectivity and economic feasibility of pulp delignification by POMs appears competitive with other chlorine-based and totally chlorine free (TCF) deliginfication technologies [7, 12, 56-60]. 1.5.1.1 T w o - s t a g e t e c h n o l o g y The Forest Products Laboratory of the U.S. Department of Agriculture has 20 developed a reusable POM delignification technology. The overall wood pulp bleaching process is accomplished in 2 steps [12, 56]. First, unbleached pulp is heated in an aqueous solution of a fully oxidized POM (POMox) under anaerobic conditions to yield soluble oxidized lignin fragments (Ligninox) and reduced POM (POM r e d) (Scheme 1.1, Step 1). The bleached cellulose is separated and the reduced bleaching liquor is treated with 0 2 at elevated temperature and pressure (Scheme 1.1, Step 2). During this POM reoxidation step, the POM catalyzes the O2 oxidation of the dissolved lignin fragments to CO2 and H2O (wet oxidation). [SiVWuO^]6 Scheme 1.1 Proposed two-step process for convention of lignin in wood to C 0 2 and H 2 0. 2\ The net reaction for POM-mediated delignification is the selective transfer of electrons from lignin to O2. Wood(Cellulose + Lignin) + O 2 P 0 M ° * > Cellulose + C O 2 + H 2 O (1.1) The P O M must have a specific electrochemical potential to 1) effectively oxidize lignin in Step 1 and 2) spontaneously (i.e., thermodynamically favorable) undergo reoxidation by O2 in Step 2. Several P O M s have been examined as selective delignification reagents. O f these, a-[PV 2 MO]o0 4 o] 5 " and a - [S iVWi 0 04o] 5 " were found to be very effective in wood pulp delignification [56, 60]. However, these P O M s were not practical for industrial use. The a-[PV 2 Moio04o] 5 " anion is only stable at low pH; conditions where cellulose hydrolysis occurs, and the oc-[SiVWio04o]5" anion, although stable at neutral p H , could not be easily reoxidized with O2 even at elevated temperature and pressure [12, 56]. In addition, a high concentration of polyoxometalate (0.05 - 0.1 mol L" 1 ) was used as the reaction between the P O M and lignin is stoichiometric. The most promising results were obtained with 0t-[SiVW, i O 4 0 ] 5 " and a-[PV 2 Mo, 0 04o] 5 " [7, 12, 56]. A unique feature of these systems was the effective self-buffering. During 20 delignification-reoxidation cycles, the p H of the a - [ P V 2 M o i o 0 4 0 ] 5 " solution was p H 4.0 ± 0.2 p H units. The buffering capacity was found to be a result of the shifting of the 22 hydrolysis / condensation equilibria between POM species in solution [61]. More recently, an equilibrating metal-oxide cluster ensemble, Na5 (+i 9)[SiVi(_o.i)MoWio(+o.i)04o] has demonstrated high activity in the oxidation of lignin model compounds [63]. Under optimum pH 5 - 6, the active species are reported to be Na5[SiVMoWi0O40] and Na5[SiVWn04o]. 1.5.1.2 One-stage (catalytic) technology In contrast to the anaerobic reaction conditions required by the system of Weinstock and co-workers, Evtuguin et al. have actively developed POM catalysts for delignification under aerobic conditions. The overall catalytic process is very similar to the two-stage strategy represented in Scheme 1.1, except that both the delignification and wet reoxidation and mineralization steps are carried out simultaneously under aerobic conditions. The obvious advantage of this technology is the economic efficiency. First of all, the whole process would be much simpler to design and operate than the two-stage process. Secondly, the amount of the POM used would be significantly reduced [8, 9, 59]. Most one-stage delignification studies have focused on heptamolybdopenta-vanadophosphate anions (HPA-n), e.g. [PMoi2- n V n 0 4 o] ( 3 + n ) \ where n is the number of vanadium atoms and is typically 4 - 6 . The POM ([PV5M07O40]8") is a very effective delignification catalyst. A high degree of delignification was achieved using only 1-3 mM 23 solutions of this POM [9, 57, 58]. In these systems, substrate oxidation occurs via electron transfer involving the vanadium (V) ions of the POM. However, in acidic media partial dissociation of the POM leads to release of V 0 2 + ions, which are very reactive delignification catalysts and play a dominant role in lignin oxidation. Unfortunately, they can also lead to decreased selectivity, thus factors such as solvent, pH and partial reduction of the catalyst prior to use are critical parameters. H* PV v 5 Mo 7 O40 8 - - PV V5- x Mo 70 4 o ( 8 " x ) " + x V v 0 2 + (1.2) The generated V v 0 2 + ions have a higher redox potential than the vanadium atoms in the POM, which dramatically affect both the degree, as well as the rate of delignification. The free vanadium (V v 02 + ) ions initiate the radical-chain oxidation of lignin generating organic radicals that are further oxidized with O2 to yield carbon dioxide and water. Lignin + x V v 0 2 + + y 0 2 + xH + m C 0 2 + nH 2 0 + x V I V 0 2 + (1.3) The reduced vanadium Y w0 2+ species formed upon lignin oxidation can then be incorporated back into the POM framework, which upon reoxidation with 0 2 afford the initial catalyst [9]. 24 PV v 5 . x Mo 7 O 4 0 ( 8 - x ) - + xV^O 2 * P V v 5 . x V I v x M o 7 O 4 0 ( 8 + x ) - (1.4) PV V 5 -xV I Y xMo 7 O 4 0 ( 8 + x ) " + ^ 0 2 + xH + • PV v 5 Mo 7 O 4 0 8 " + ^ H 2 0 ..(1.5) The pH of the HPA-n systems, which affects free vanadium (Vv02+) ion concentration is critical to the delignification rate and efficiency. However, at the optimal acidic pH conditions cellulose degradation and viscosity losses occur. To avoid such detrimental cellulose degradation reactions new polyoxometalates which are stable at higher pH are being developed. It has been reported that POMs such as [SiWnMnm(H20)039]n" and HPA-5-Mn11 with manganese as addenda atoms are stable at close to neutral conditions [64, 65]. The [SiWiiMnni(H20)039]"~ system is reactive under slightly acidic conditions, pH 4 (catalyst concentration 5.9 mmol L" 1, 2 h, 100 °C, 0 2 pressure 5 bar), but compared to oxygen-alkaline delignification there is only minor improvements in delignification and pulp viscosity (kappa number 7.9 versus 8.1, viscosity 1230 cm3g"' versus 1170). Despite some success in developing an economically viable technology for wood pulp delignification, the catalytic process has one key drawback. In order to improve the efficiency of the process, mixed ethanol/water or acetone/water solutions must be used as reaction media [59]. Therefore, in order to make this technology economically and environmentally acceptable, aqueous POM systems that operate at neutral to alkaline 25 conditions need to be developed. 1.6 POM Reaction Mechanism with Lignin Models Compounds 1.6.1 Phenolic Lignin Model Compounds Neumann et al. [66] reported the oxidation of benzyl alcohol and 1-phenylethanol to benzaldehyde and acetophenone, respectively, using [PV2M010O40] 5 " and oxygen. After reaction for 22 h at 100 °C in organic solvent near quantitative conversions were obtained. By contrast, only 4% of cyclohexanol was oxidized to cyclohexanone under the same reaction conditions. In 1992 Lissel et al. and Kholdeeva et al. [50, 67] independently reported the [PV„ Moi2.n04o](n+3)~ catalyzed aerobic oxidation of alkyl-substituted phenols to monomeric 1,4-benzoquinones and dimeric biphenols and diphenoquinones. Similarly, Grigoriev et al. [68] reported the anaerobic oxidation of a biphenol to the corresponding diphenoquinone by a-K5[SiVWio04o] at 60 °C. Figure 1.7 shows some of the oxidation products obtained from POM oxidation of various phenolic compounds. It can be seen that the POM oxidation of phenolic compounds involves an electron-transfer mechanism to generate phenoxy radical intermediates which can further reacted with a second POM or couple with second radical intermediate to yield dimeric products. 26 97% Figure 1.7 Oxidation of A benzyl alcohol, and B cyclohexanol by [PV2M010O40] 5 " in toluene; alkyl-substituted phenols C and D by [PVn Moi2-n04o](n+3)"in acetic acid under aerobic conditions; and E 3,3',5,5'-tetra-tert-butyldiphenyl-4,4'-diol by cc-K5[SiVW10 4o5"] in 0.1 M LiOAc/HOAc buffered water/t-BuOH solution (2:3), pH 4.8 under anaerobic conditions [50, 66-68]. Detailed mechanistic studies by Weinstock et al. [60] reported phenoxy radicals as likely intermediates in the oxidation of lignin model compounds by Ot-[SiVWio04o]5" under anaerobic conditions. In the oxidation of a guaiacyl P-O-4 dimer lignin model 27 compound an organic-soluble precipitate was observed and determined to be a dimeric coupling product. However, reaction of the syringyl {3-0-4 dimer ( F i g u r e 1.8), did not form any coupling products, rather undergoing rapid oxidation and ring fragmentation. At room temperature, the syringyl {3-0-4 dimer was rapidly oxidized by a-[SiVWi 0O 4 0] 5" to yield'an approximately 1:1 mixture of 2,6-dimethoxyl-p-benzoquinone (1) and ring fragment (2) as shown in F i g u r e 1.8 [60]. o OH 1 F i g u r e 1.8 Proposed reaction pathway for the oxidation of a syringyl (3-0-4 dimer by Ot-[SiVWi004o]5" at room temperature under anaerobic conditions [60]. Evtuguin et al. [8, 9, 58, 59] have extensively studied the reactions of lignins with heteropolyanions. F i g u r e 1.9 illustrates their proposed lignin oxidation scheme in the 28 presence of [ P M O T V S O ^ ] 8 " under aerobic conditions. The initial steps involve one-electron oxidation of the lignin aromatic group with V v02 + of the catalyst to generate cyclohexadienyl cation type structures, which undergo subsequent oxidation and hydrolytic cleavage reactions. The reduced vanadium species V ^ O 2 " 1 " formed upon lignin oxidation is then incorporated back into the P O M framework followed by reoxidation with O2 to afford the initial catalyst (as per equations 1.4 and 1.5). Ortho- and para-quinone type structures Figure 1.9 Reaction scheme for lignin oxidation in the presence of heteropolyanions, [PM07V5O40] 8 " under aerobic oxidation (V v and V™ represent V0 2 + and V0 2 + in the oxidized and reduced forms of the P O M , respectively [9]. 29 Recently, detailed mechanistic studies on heptamolybdopentavanadophosphate anion, HPA-5 or HPA-5- M n n oxidation of vanillyl alcohol under aerobic conditions have been reported [64]. The rate-limiting step is the first one-electron oxidation and the formation of a cation radical intermediate. The next step is proton elimination and either i) a second one-electron oxidation by the catalysts to produce various oxidation products or ii) reaction with other radical intermediates to form coupling products (Figure 1.10). In either case, further oxidation by the catalysts occurs to produce a large variety of oxidized aromatic compounds. OH OH Figure 1.10 Products formed from the reaction of vanillyl alcohol with HPA-5 or HPA-5-Mn" under aerobic conditions (30 min, pH 3, 90 °C, 0 2 pressure 0.5 MPa) [64]. 30 1.6.2 N o n - P h e n o l i c L i g n i n M o d e l C o m p o u n d s Although there are a number of studies on the reactions of POMs with wood pulp and phenolic lignin compounds, reactions with non-phenolic lignin structures have not been widely reported. Compared to phenolic structures, non-phenolic lignin moieties are much more resistant to POM oxidation. Nevertheless, the role of non-phenolic structures in the oxidative hydrolysis of lignin is very important as they represent 40 - 55 % of the structural moieties in the native lignin structure. Eberson reported the mechanism of oxidation of p-methoxyltoluene (PMT) by the inorganic catalyst, Co n iWi2O4 0 5" in 55:45 (w/w) acetic acid and water at 50 °C under aerobic conditions [69]. The initial rapid and reversible electron-transfer reaction is followed by the rate-limiting reaction of the radical cation intermediate with base to give a benzylic radical. The benzylic radical is rapidly oxidized by a second equivalent of ConIWi204o5" to the corresponding cation, which further reacts with acetic acid. PMT+ + ConW1204o6- (1.6) PMT + AcOH (1.7) PMT+ + ConWi2O406- (1.8) PMTOAc + H+ (1.9) 31 PMT + ComW12O405-PMT+ + AcO' PMT + ComWi2O405-PMT+ + AcOH Recently, Yokoyama et al. [63] studied the kinetics and reaction mechanism of non-phenolic model compounds [l-(3,4,5-trimethoxylphenyl)ethanol] and [l-(3,4-dimethoylphenyl) ethanol], with the anaerobic non-reversible equilibrated POM mixture, Na5(+i.9)[SiVi(.0.i)MoWio(+o.i)04o]. The rate-determining step was determined to be proton abstraction and formation of a benzylic radical intermediate. Figure 1.11 shows the reaction products obtained from Na5(+i.9)[SiVi(_o.i)MoWio(+o.i)04o] oxidation of [1-(3,4,5-trimethoxylphenyl)ethanol]. Figure 1.11 Oxidation products (% yield) detected from the reaction of Na5 ( + 1.9)[SiVi (. o.i)MoW10(+o.i)04o] (POM) with l-(3,4,5-trimethoxylphenyl)ethanol at 160°C; 1 (3,4,5-trimethoxylacetophenone), 2 (3,4,5-triethoxylbenzaldehyde) and 3 (3,4,5-trimethoxylbenxoic acid) [63]. In the reaction with l-(4-ethoxyl-3,5-dimethoxylphenyl)ethanol, 4-ethoxyl as compared to 4-methoxyl, a substantial increase in the calculated activation entropy was 32 observed. As a result, it was proposed that a pre-association of the more hydrophobic substrate with the metal-oxygen cluster (POM) likely occurs prior to proton transfer. It was further suggested that pre-association might play an important role in the POM oxidation of larger lignin fragments and residual lignin in wood pulp. However, as this was an equilibrated POM oxidation system, detailed kinetics were not reported as several reactive species were involved during the reaction, complicating the kinetic analyses. C H 2 O H C H 2 O H O C H 3 OCH3 C H 2 O H COOH C H 2 O H CHOH CHOH C=0 OH OCH3 OCH3 I 1 I Further oxidation Figure 1.12 Products of the reaction of veratryl alcohol with HPA-5 or HPA-5- Mn 1 1 under aerobic conditions (30 min, pH 3, 90 °C, 0 2 pressure 0.5 MPa) [64]. Recently, Gaspar et al. [64] reported the detailed oxidation mechanism of vertaryP alcohol under aerobic conditions with HPA-5 or HPA-5- Mn 1 1. Figure 1.12 shows 33 products of the reaction with veratryl alcohol by HPA-5 or HPA-5- Mn" under aerobic conditions (30 min, pH 3, 90 °C, 0 2 pressure 0.5 MPa). They proposed the rate-limiting oxidation step to be the first one-electron oxidation, and formation of a cation-radical intermediate, similar to that proposed in the reaction mechanism of phenolic model compounds (Figure 1.10). Based on the various investigations involving lignin model compounds, POMs such as a-[SiVW,o04o]5", [PM07V5O40] 8 ", Na5(+1.9)[SiV1(.0.1)MoW io(+o.i)04o] and HPA-n react as single-electron oxidants, capable of removing residual lignin by reacting primarily with aromatic moieties under either anaerobic or aerobic conditions. 1.7 Research Objectives Lignin is a complex aromatic biopolymer. Lignin has a complex chemical structure consisting of a variety of inter-unit linkages and functional groups. Although widely studied, the structures of native lignins are still an area of active research. The oxidation of lignins by POMs has been studied. Reactions with lignin model compounds have been performed, and pulp bleaching experiments have been conducted on a laboratory scale. However, a comprehensive understanding of the effect of lignin molecular structure on the POM reaction mechanisms and kinetics is far from complete. As more and more interest grows around environmentally benign alternatives to current pulp bleaching 34 chemicals, detailed studies into POM oxidation of lignin and lignin model compounds are needed. Recent work has shown equilibrated POM mixtures such as Na5(+i.9)[SiVi(. o.i)MoWio(+o.i)04o] (Na5[SiVMoW|0O4 0] + Na 5 [SiVW n 0 4 o]) to be very effective in the delignification of wood pulp. Therefore, to better understand the mechanism and kinetics of the POM reactions with lignin, we have chosen to study one of the primary POM species, a-[SiVWn04o]~5 and its reaction with lignin model systems. Two approaches are taken in this study; one involving lignin model compounds under anaerobic POM bleaching conditions, and the other involving milled wood lignin (MWL). The first part of this thesis deals with the effect of phenol and lignin model compound substituent groups on the reaction with POM (a-[SiVWn04o]~5) under anaerobic conditions. The emphasis is on understanding the basic relationship between the electron density of substituted phenols and the POM oxidation rate, and the detailed kinetics and reaction mechanisms of POM oxidation of various lignin structures. The second part of this thesis focuses on understanding the behaviour of lignin, i.e. MWL, during POM oxidation. Using spectroscopic techniques changes in the chemical structure of MWL arising from POM oxidation are investigated. In particular, the structure of MWL before and after POM oxidation is analyzed and compared to results obtained from lignin model compounds. 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Polyoxometalate (POM) Oxidation of Phenols: Effect of Aromatic Substituent Groups on Reaction Mechanism 1 1 A version of this chapter has been submitted for publication. Kim, Y.S., Chang, H.-M. and Kadla, JK. (2007) Polyoxometalate (POM) Oxidation of Phenol: Effect of Aromatic Substituent Groups on Reaction Mechanism, Canadian Journal of Chemistry. 43 2.1 I n t r o d u c t i o n Polyoxometalates (POMs) are a rapidly growing class of metal-oxygen-cluster anions [1-4]. They are synthetic inorganic compounds that contain highly symmetrical core assemblies of MO(x) units (M = vanadium, molybdenum, tungsten) and react as outer-sphere electron-transfer oxidants and catalysts [4-7]. The properties of POMs can be controlled by altering the POM composition and structure [5, 8]. As a result, POMs have found applications in analytical and clinical chemistry, catalysis (including photocatalysis), biochemistry (electron transport inhibition), medicine (anti-tumor, anti-viral, and even anti-HIV activity), and solid-state devices [9]. Unlike many metallo-organic catalysts, POM anions are oxidatively stable and are reversible oxidants. The heteropolyoxometalates, e.g. Keggin-type (general formula X n + Mi 2 0 4 o ( 8 " n ) " (M/X =12)), are the most well known and studied compounds for catalytic applications [1, 9]. Of these, the vanadium-containing complexes are the strongest oxidants. The replacement of one or more molybdenum atoms in the primary structure of Keggin heteropolyoxometalates-Mo with vanadium leads to an enhancement of the oxidation potential due to vanadium's enhanced reducibility [1], As a result, the electrochemical potential for the one-electron reduction of the POM can be tuned according to the substrate being oxidized. Over the last decade there has been an increasing interest in the application of POMs 44 in the catalytic delignification of wood for paper production [10]. POMs offer a safe and environmentally benign alternative to traditional bleaching reagents such as elemental chlorine. Efficient and selective removal of lignin, an aromatic polyol, from wood pulps without severe degradation to carbohydrates (e.g. cellulose) can be accomplished using POMs under anaerobic and aerobic conditions [5, 11-13]. The catalytic mechanism involves a series of redox cycles wherein the reduction of the POM(OX> by the substrate (lignin) is accompanied by subsequent reoxidation of the POM ( reci) by 0 2 - the total electron transfer is from substrate to 0 2 . In the oxidation of aromatic compounds such as lignin, the reduction potential of the POM should be between the oxidation potential of cellulose and lignin. Depending on the structure of the aromatic moieties the oxidation potential, and subsequent oxidation rates can span a wide range of values. In this paper, we report the results of a study on the kinetics and mechanisms of the oxidation of phenols in aqueous solutions by POM (Ks[SiVWn04o]12H20). Of particular interest is the relationship between phenol substitutent groups and reaction kinetics. Using the Hammett equation, the relationship between electron density of substituted phenols and POM oxidation rate is reported. 45 2.2 E x p e r i m e n t a l 2.2.1 M a t e r i a l s K5[SiVWii04o]12H20 (POM) was provided from the USD A Forest Products Laboratory (Madison WI, U.S.) [11]. The aromatic compounds (Figure 2.1): p-methoxylphenol (1), p-methylphenol (2), m-methoxylphenol (3), m-methylphenol (4), phenol (5), p-chlorophenol (6), p-bromophenol (7), m-chlorophenol (8), p-nitrophenol (9) and 1,4-benzoquinone (10) were purchased from Sigma-Aldrich. CDCI3, DMSO-d6, and acetone-d6 were purchased from Cambridge Isotope Laboratories. N,0-bis(trimethylsilyl)acetamide, tetrahydrofuran and pyridine were purchased from Sigma-Aldrich. Acetic acid, acetone, chloroform and sodium acetate were purchased from Fisher Scientific. All chemicals and solvents were used as received. 2: = C H 3 , 3: Rt - H , 4: R 1 = H , 5: R , = H , R 2 = O C H 3 R 2 = C H 3 R 2 = H R 2 = H R 2 = H 6: RT = 7: R , = 8: R , -9: R 1 = R 2 = H R 2 = H R 2 = C I R 2 = N0 2 O H Figure 2.1 Phenols studied. 46 2.2.2 Kinetic Measurements Kinetic reactions were carried out using a dual-syringe stop-flow apparatus equipped with a mixing chamber and attached to a 2 cm3 cuvette (quartz micro flow cell). The temperature was maintained over a range of 4 - 90 ± 1 °C by a Haake Dl /G recirculating refrigerated bath. Absorbance measurements were performed on a PerkinElmer Lambda 45 UV/VIS spectrophotometer with a cell pathlength of 1 cm. Reactions involving compounds 1 -9 were run in a sodium acetate-acetic acid buffer system (I = 0.2 M , pH 3.9 - 6.0). pH values were measured using a Fisher pH meter, model Accumet AR 15. The extinction coefficient (s) of the oxidized POM at 350 nm (e = 2630 M'cm"1) and reduced POM at 520 nm (e = 610 M^cm"1) were determined by linear regression as shown in F i g u r e 2.2. The reaction kinetics were spectrophotometrically recorded by monitoring the absorbance of the oxidized POM at 350 nm or the reduced POM at 520 nm. Data were recorded every 0.1 - 600 s for 0.5 to 50 h, depending on the experiment. The phenolic compounds and POM concentrations were 12.25 mmol L"1 and 0.25 mmol L"1, respectively (49 molar equivalents of phenolic substrate). Al l reactions were run in triplicate and rate constants reported as average values. 47 POM (mol/L) Figure 2.2 Relationship between concentration of POM and absorbance at a) 350 nm (SiVW n0 4o 5") and b) 520 nm (SiVWu04o6"). In a typical kinetic experiment, equal volumes (3 mL) of the phenolic substrate (49 pmol) buffered solution and POM (1 umol) buffered solution were mixed using a dual-syringe stop-flow apparatus (Figure 2.3). Prior to mixing all phenolic and POM buffer solutions were thoroughly purged with argon and equilibrated for at least 2 h at the 48 reaction temperature in a M a x Q ™ 4000 Incubated and Refrigerated Shaker (Barnstead/Lab-Line co.)- For slow reacting model compounds, e.g. 8 and 9, 1.9 m L of degassed phenolic compound (24.5 umol) was loaded into a 5 m L quartz cuvette and sealed with a rubber septum. The cuvette was put into the U V and equilibrated at the reaction temperature for at least 2 h. Reactions were initiated by injecting 0.1 m L of a P O M (0.5 umol) stock solution. The final concentration of phenols and P O M were 12.25 mmol L" 1 and 0.25 mmol L " 1 , respectively. V Jr Light source Observation Cell Figure 2.3 Schematic representation of the U V - V i s stopped flow apparatus (1 cm U V path-length) used for kinetic analyses. 49 2.2.3 Product Analysis In a typical product analysis experiment the substituted phenol (~ 0.4 mmol) was dissolved in ethanol (1 mL) and added via syringe to a sealed reaction flask containing P O M (~ 1.6 mmol) dissolved in 0.2 M sodium acetate buffer (80 mL, pH 5.0) under argon. In the case of 1 the reaction mixture was agitated for 1 h at 25 °C, while 4 was reacted for 2 h at 60 °C and 5 for 5 h at 60 °C. At the end of the reaction, the precipitated polymeric products were collected by filtration using a Nylon membrane filter (0.45 um, 47 mm) and freeze-dried using a VirTis E X freeze-dryer. The filtrate was acidified to pH 2 with concentrated HCI and diluted with a 2:1 (v/v) mixture of chloroform and acetone (70 mL). The organic phase was separated, and the aqueous layer was extracted again with a 2:1 (v/v) mixture of chloroform and acetone (2 x 70 mL). The organic phases were combined, dried over anhydrous MgS04, filtered and concentrated under reduced pressure to approximately 3 mL as the crude product mixture. A portion of the crude product mixture was then analyzed by GC-MS; one sample (-1 mL) was directly analyzed, while the second sample (- 1 mL) was first silylated by reacting with N,0-bis(trimethylsilyl)acetamide (200 uL) in pyridine (0.5 mL) at room temperature for 24 h. The remaining reaction product mixture was separated, if possible, by thin layer chromatography (eluent: 5 % acetone in CHCI3) and analyzed by NMR. 50 2.2.4 Analytical Methods Gas chromatography-mass spectroscopy (GC-MS) analyses were conducted using a ThermoFinnigan TraceGC and PolarisQ ion-trap mass spectrometer. GC analyses were performed with a J&W Scientific Inc. DB-5 column (30 m x 0.32 mm x 0.25 um). The injection temperature was set to 200 °C, the transfer line temperature was set to 200 °C, and the ion source was set to 300 °C. Helium flow was 1 mL min"1. After a 5 min solvent delay at 70 °C, the oven temperature was increased at 5 °C min"' to 280 °C and held at temperature for 5 min prior to being cooled down to 70 °C. Mass spectra were recorded from m/z = 50 to 650 at 0.58 s scan"1 with an electron ionization of 70 eV. When available, commercial samples of identified products were used to verify chromatographic retention time and spectral data. 'H and 1 3 C nuclear magnetic resonance (NMR) analyses of isolated products and polymeric materials were conducted on a Bruker AVANCE 300 MHz spectrometer at 300 K using CDCI3, acetone-d6 or DMSO-d6 as the solvent. Chemical shifts were referenced to tetramethylsilane (TMS; 0.0 ppm). Fourier transform infrared (FT-IR) analysis was performed using a Perkin Elmer Spectrum One spectrometer. The polymeric solids and initial phenolic compounds were dissolved in THF and CHCI3, respectively, at a concentration of 1 mg mL"'. Approximately 100 uL of this solution was spread between ZnSe plates separated by a 51 0.015 mm Teflon spacer; the solvent was evaporated from this apparatus by means of a heat gun. A total of 16 scans per sample were acquired at a spectral resolution of 4.0 cm"'. The average molecular mass and distribution of the polymeric samples were determined by gel permeation chromatography (GPC; Agilent 1100, U V and RI detectors). Chromatographic separation was performed using styragel columns (Styragel H R 4 and H R 2) at 35 ° C , T H F ( H P L C Grade) as the eluting solvent (0.5 m L min"') and U V detection at 280 nm. Sample concentration was 1 mg mL" 1 and the injection volume was 75 uL. The G P C system was calibrated using standard polystyrene samples (Showa Denko) with molecular weights ranging between 580 and 1,800,000 Daltons. M A L D I - T O F (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry analyses were obtained on an Applied Biosystems Voyager System 4311 with linear geometry (positive polarity). Matrices were prepared using 2 mg samples dissolved in 10 m L of T H F . A 1 p L sample solution was mixed with 3,5-dimthoxy-4-hydroxylcinnamic acid (sinapic acid) and/or 2,5-dihydroxylbenzoic acid (gentistic acid) matrix, in a matrix-to-analyte ratio of 1:1. One u L of the mixture was spotted on a stainless steel plate and inserted into the instrument. 2.2.5 I d e n t i f i c a t i o n o f R e a c t i o n P r o d u c t s 1,4-Benzoqunione (10): E I - M S m/z (low resolution) 108(M + , 75), 80(76), 52(100). A 52 commercial sample (Aldrich) was used to compare the chromatographic retention time and spectral data. 2-(2-Hydroxyl-5-methoxyl-phenyl)-[l,4]benzoquinone (11): E I - M S m/z (low resolution) 230 (M + , 100), 215(10), 202(30), 187(45), 174(9), 159(14), 148(10), 131(8), 107(6), 95(5), 79(11), 63(5), 51(9); ' H - N M R : (DMSO-d 6 ) 5 3.78(3H, s, O C H 3 ) , 5.48(1H, d, OH) , 6.78(1H, dd, J=3.0, 10.2, A rH) , 6.91(1H, d, J=10.2, A r H ) , 7.03(4H, m, A rH) ); 1 3 C - N M R : (DMSO-de) 5 , 55.67, 110.79 115.32, 121.81, 134.53, 137.07, 145.65, 157.86, 159.09, 181.76, 187.61. 5,5'-Dimethoxyl-biphenyI-2,2'-diol (12): E I - M S m/z (low resolution) 246 (M + , 100), 108(40), 78(7); ' H - N M R : (CDC1 3 ) 5 3.81(6H, s, O C H 3 ) , 5.24(2H, d, OH) , 6.84(6H, m, ArH) ; 1 3 C - N M R : (CDC1 3 ) 5 55.97, 115.57, 115.91, 117.80, 124.62, 146.56, 154.19. The assignments are in agreement with literature data [14]. Biphenyl-2,2'-diol (13): E I - M S m/z (low resolution) trimethyl si lyl ether: 330 (M + , 71), 315(42), 242(7), 227(19), 147(7), 73(100); ' H - N M R : (acetone-d6) 5 6.95(4H, m, ArH) , 7.22(4H, m, A r H ) ); l 3 C - N M R : (acetone-d6) 5 116.86, 120.39, 126.23, 128.69, 131.65, 153.96. A commercial sample (Aldrich) was used to compare the chromatographic retention time and spectral data. 53 Table 2.1 Dimers (14*) formed during the POM (SiVWn04o5") oxidation of 4 (m-methylphenol). Mass spectral data 214(M+, 86), 199(100), 181(50), 171(13), 153(14), 128(8), 115(6), 107(2), 77(3) 214(M+, 100), 199(19), 181(12), 171(6), 153(5), 122(50), 94(16), 77(6), 66(8) 214(M+, 100), 199(61), 181(26), 171(25), 153(11), 128(10), 115(8), 91(3), 77(3) 214(M+, 57), 199(100), 184(8), 171(53), 143(14), 128(25), 115(11), 91(3), 77(3) 214(M+, 100), 199(92), 181(36), 171(16), 152(12), 128(8), 115(9), 77(4) 214(M+, 100), 199(64), 181(35), 153(12), 141(8), 128(4), 115(8), 91(2), 77(3) Figure 2.4 Reaction products formed during the POM (SiVWn04o5~) oxidation of 1 (p-methoxylphenol), 4 (m-methylphenol) and 5 (phenol). *dimers formed during the POM oxidation of 4. 54 2.3 R e s u l t s a n d D i s c u s s i o n 2.3.1 K i n e t i c s a n d M e c h a n i s m Figure 2.5 shows the change in absorbance for aqueous solutions (pH 5.0) of POM (0.25 mmol L"1) and 2 and 5 (12.25 mmol L"1) at 25 °C and 45 °C, respectively. On mixing with excess phenol, there is a decrease in absorbance at 350 nm (peak of oxidized POM) with a concurrent increase in absorbance at 520 nm (reduced POM). For 2 an isosbestic point is observed at 446 nm, while for 5 it is not clearly seen. In 5 the point of intersection was observed at 446 nm early in the reaction and 457 nm late in the reaction (Figure 2.5b). A similar phenomena was observed in the reaction of phenol with a platinum (III) dinuclear complex [15]. This shift in isosbestic point was attributed to a reaction system that proceeds via at least two steps. POM oxidation with phenols is known to proceed via an electron-transfer mechanism [5, 16, 17]. In aqueous conditions neutral phenols and phenoxide anions exist in a rapid-equilibrium, where the concentration of each is pH dependent [18]. Both neutral phenols and phenoxide anions can react with the POM to give a phenoxy radical intermediate. Possible reaction pathways are shown in Scheme 2.1. 55 a) 300 400 500 600 700 800 Wavelength, nm b) Wavelength (nm) Figure 2.5 Change in P O M (SiVWn04o5") absorbance during reaction with a) 2 and b) 5 in sodium acetate buffer (I = 0.2 M , pH 5.0) at 25 °C and 45 °C, respectively. ([2] = [5] = 12.25 mmol L"', [POM] = 0.25 mmol L"'). The arrows indicate the decrease (i) in absorbance of the oxidized P O M at 350 nm and the increase (T) in absorbance of the reduced P O M at 520 nm, respectively. 2 (p-methylphenol) and 5 (phenol). 56 OH P O M o ^ S i V W ^ O ^ ) 5 -P O M r e d ( S i V W „ 0 40) POM„ POM, + other organic products fast Radical coupling + polyphenols R = H, O C H 3 . C H 3 , Cl, Br, N02 Scheme 2.1 Possible reaction mechanism for POM (SiVWu04 u 5~) oxidation of phenols. According to Scheme 2.1 two equivalents of POM are required to fully oxidize phenols. The overall reaction can be represented by equation 2.1 [5, 16]. x - C 6 H 4 - OH + 2(SiVW, ,O405~) —products + 2(SiVW, ,O 4 0 6") + H + (2.1) Accordingly, equation 2.1 can be written as the rate law equation 2.2. d[Sivw„o4 0 ] = 2 k [ S i V W n 0 4 o 5 - r [ x _ c H _ 0 H ] b dt (2.2) 57 The first step in evaluating the reaction kinetics of POM oxidation of the various phenols was to determine the respective reaction orders. This was accomplished by determining the initial rates of reaction between POM and the phenols using UV-VIS spectroscopy [19]. The reaction order of each reactant was calculated from the initial rate equation 2.3 v = k '[SiVW u O 4 0 5 ~]\ k ' = 2 k [ x - C 6 H 4 - O H ] 0 b or v = k"[x - C 6 H 4 - OH] b , k" = 2k[SiVW, ,O 4 0 5 ' ] 0 a (2.3) In a typical experiment, the POM concentration was held constant (0.25 mmol L"1) while the concentration of the phenolic compound was varied, e.g. [5] was varied from 9.0 to 45 mmol L" 1. Then, the phenolic concentration was held constant (15 mmol L~') and the concentration of the POM varied (1.25 to 6.25 mmol L"1). Using equation 2.3, the initial rates were plotted against the concentration of phenol or POM, and a straight line through the origin was obtained (Figure 2.6 and Appendix). This indicates first-order kinetics with respect to both phenol and POM, and second order kinetics overall. 58 0.00 0.0 1.0 2.0 3.0 4.0 POM (mM) 5.0 6.0 7.0 Figure 2.6 Plot of initial rate versus a) [POM] and b) [5] at 25 °C in sodium acetate buffer (I = 0.2 M , pH 5.0). 5 (phenol) and POM (SiVWnGV"). Accordingly, the second-order rate expression can be expressed as equation 2.4. d[SiVW nO 4 0 6-] dt - = 2k[SiVW nO 4 0 5-] [ x - C 6 H 4 - O H ] (2.4) Under pseudo-first order conditions, i.e. >10-fold excess of phenolic substrate equation 59 2.4 can be written as e q u a t i o n 2.5. d[SiVW„O4 0 6-] s T - k o b s L S l V W n 0 4 U J dt Further, based on the relationship between e q u a t i o n s 2.4 and 2 .5 , the observed pseudo-first order rate constant (kobs) can be expressed according to e q u a t i o n 2.6. k o b s = 2 k [ x - C 6 H 4 - O H ] (2.6) F i g u r e 2.7 shows the absorbance-time plots for the reaction of POM with 1 at different temperatures under pseudo-first order conditions. 0 i . , , , 1 0 10 20 30 40 50 Reaction time (sec) F i g u r e 2.7 Absorbance-time plots for the reaction of POM (SiVWnO^5") with 1 at different temperatures (°C) in sodium acetate buffer (I = 0.2 M , pH 5.0). [1] = 12.25 mmol L"1, and [SiVWu04o5"] = 0.25 mmol L ' 1 . 1 (p-methoxylphenol). 60 The pseudo-first order rate constants (k0bS) are determined by fitting a straight line through the initial 25-50 % of ln[(POM0)/(POM)] versus time plots. F i g u r e 2.8 shows the kinetic data using both initial rates ( F i g u r e 2.8a) and pseudo-first order ( F i g u r e 2.8b) kinetic analyses. F i g u r e 2.8 Kinetic analysis of SiVW u 0 4 o 5 " oxidation of 1 at different temperatures (°C) in sodium acetate buffer (I = 0.2 M , pH 5.0): a) initial rate plot and b) pseudo-first order plot. [1] = 12.25 mmol L" 1, and [SiVWnCW"] = 0.25 mmol L " 1 . 1 (p-methoxylphenol). 61 T a b l e 2.2 lists the pseudo-first order rate constants ( k 0 b s ) for the reaction between POM and 1 in sodium acetate buffer (I = 0.2 M , pH 5.0) at various temperatures. From the pseudo-first order rate constant (kob S ) , the second order rate constant ( k ) can be calculated using e q u a t i o n 2.6 or determined according to e q u a t i o n 2.4 using the initial rate method, as shown in F i g u r e 2 .8a . The calculated ( e q u a t i o n 2.7 - pseudo-first order) and measured ( F i g u r e 2 .8a - initial rate law) second order rate constants ( k ) are included in T a b l e 2.2. It can be seen that values obtained by both methods are in good agreement with each other, and indicate an overall second-order behaviour under these conditions. T a b l e 2.2 Second-order rate constants ( k ) for the oxidation of 1 by SiVWuO^ 5" in sodium acetate buffer (I = 0.2 M , pH 5.0). [1] = 12.25 mmol L"', [SiVWnO^5"] = 0.25 mmol L"1. Temp. Initial rate law Pseudo-first order rate law °C k(M"'s'') a k o b s (s"')b x 10'2 k (M"'s"')c 4 2.48 ± 0.42 5.84 ± 0.51 2.38 ±0.21 10 3.10 ± 0.35 7.46 ±0.14 3.04 ± 0.06 15 3.65 ± 0.09 9.15 ± 0.11 3.74 ± 0.04 20 4.76 ±0.19 11.76 ± 0.10 4.80 ± 0.04 25 6.68 ± 0.47 15.64 ±0.11 6.38 ± 0.04 d second-order rate constants calculated from F i g u r e 2 .8a . b pseudo-first order rate constants calculated from F i g u r e 2 .8b . c second-order rate constants calculated by e q u a t i o n 2.6 using k o b s b . 62 According to Scheme 2.1 the oxidation of phenols can occur through reaction with either the neutral phenol or phenolate ion. Using a steady-state approximation for the initial oxidized intermediates, i.e. -d[x-C6H4-OH+]/dt = -d[x-C6H4-0]/dt = 0, the following expression can be obtained [20, 21]: d[SiVWnO 40 J _ ' dt k1 + * & ' [H+] [S iVW u O 4 0 5 - ] [x -C 6 H 4 -OH] (2.7) where K a is the acid dissociation constant of the phenol and kj and k2 are the rate constants for the oxidation of X-C6H4 -OH and X-C6H4-O", respectively (Scheme 2.1). Accordingly, the rate of POM reduction (phenol oxidation) is dependent on the pH of the reaction system, and the observed second-order rate constant can be expressed as equation 2.8. k: k . l O k, + " 2 " ' v [ H + ] , (2.8) To further understand the mechanism of POM oxidation of phenols, the impact of acidity on k| and k 2 (equations 2.6 and 2.7) for the reaction of POM with 1, 2, 5, and 8 was studied over the pH range 3.8 - 6.0 at 20, 35, 45 and 80 °C. Figure 2.9 shows that the rates of oxidation of 1, 2, 5, and 8 increase linearly with decreasing [H+]. 63 Figure 2.9 Effect of acidity (1/[H+]) on the second order rate constant (k) for the POM (SiVW,i04o5") oxidation of a) 1, and b) 2, 5, 8 at 20 °C, 35 °C, 45 °C, and 80 °C, respectively in sodium acetate buffer (I = 0.2 M , pH 3.9 - 6.0). [phenols] = 12.25 mmol L"', [SiVWii04o5"] = 0.25 mmol L"1. 1 (p-methoxylphenol), 2 (p-methylphenol), 5 (phenol), and 8 (m-chlorophenol). According to equation 2.7, ki is the y-intercept and k 2 K a is the slope of the line generated from plotting k versus 1/[H+]. The rate constants, k 2 were calculated using pK a 64 values at 25 °C: 10.21, 10.14, 9.98, and 9.02 for 1, 2 , 5, and 8, respectively [22]. T a b l e 2.3 lists the calculated rate constants ki and k2 for the POM oxidation of 1, 2 , 5 , and 8. It can be seen that k 2 » ki, indicating the reaction mechanism involves a rate-determining electron transfer from neutral phenol to POM ( S c h e m e 2.1). Likewise, k2/ki is = 106, consistent with the k Phenoiate/k Phenoi ratio reported for the bromination of phenols and phenolate ions [21]. T a b l e 2.3 Calculated rate constants ki and k2 for the oxidation of 1, 2, 5 , and 8 by SiVWn04o5"in sodium acetate buffer (I = 0.2 M , pH 3.9 - 6.0). [phenols] = 12.25 mmol L"1, [SiVW„O405"] = 0.25 mmol L" 1 . Compound* k, k 2 a K0bsb Temp. s"1 M-V 1 M-V1 °C 1 1.59 1.21 x 106 0.22 20 2 8.61 x 10"4 5.88 x 104 1.07 x 10"3 35 5 7.85 x 10"4 5.30 x 103 1.55 x 10"4 45 8 0.79 x 10"4 1.42 x 101 3.53 x 10"5 80 T l (p-methoxylphenol), 2 (p-methylphenol), 5 (phenol), 8 (m-chlorophenol) a estimated using [Ka] at 25°C b pseudo-first order rate constants calculated using e q u a t i o n s 2.6 and 2 .8. It should be noted that the pK a values used in the determination of k 2 are for room temperature conditions, therefore some error may be associated with these values. However, as k 2 is ~ 107 times larger than k], any error associated with the calculation of 65 k2 would not substantially affect the value, and would not be of the same order of magnitude or less than k,. Finally, although these data indicate the reaction mechanism involves a rate-determining electron transfer from neutral phenol to POM, these results do not differentiate between hydrogen atom transfer and proton-coupled electron transfer mechanisms. 2.3.2 A c t i v a t i o n P a r a m e t e r s The temperature dependence of reactions can expressed according to the Arrhenius equation k r = A e ~ E ' / R T (2.9) where k, the rate constant, A is the pre-exponential factor or frequency factor, E a is the activation energy, R is the gas constant and T is absolute temperature. The temperature dependence of reaction rates also permits the evaluation of the enthalpy (AH*) and entropy (AS*) components of the free energy of activation (AG*) according to the Erying equation K ^ ^ - A H V R T ^ A S V R ) ( 2 1 0 ) h 66 where K is the transmission coefficient, which is usually taken to be 1, k is the Boltzmann constant, and h the Planck constant. The effect of temperature on k o b s was studied from 4 - 90 °C at pH 5.0 (I = 0.2 M) for a series of substituted phenols. Activation energies (Ea) were calculated from the slope of ln(kobs) versus 1/T plots (Arrhenius plot, Figure 2.10), and activation parameters (AH 1 and AS*) were determined from ln(kobs/T) versus 1/T plots (Erying plots) [19, 23]. Table 2.4 lists the observed kinetic constants determined for the various substituted phenols. Figure 2.10 Isokinetic relationship (Arrhenius plots) of various substituted phenols. 1 (p-methoxylphenol), 2 (p-methylphenol), 4 (m-methylphenol), 5 (phenol), 7 (p-bromophenol), 8 (m-chlorophenol) and 9 (m-nitrophenol). 67 Table 2.4 Observed kinetic constants for the oxidation of substituted phenols by S i V W n O 4 0 5 ~ in sodium acetate buffer (I = 0.2 M , p H 5.0). Temp. Rate constants AH* AS* Ea Compound °C kobs(s"') kJ-mor1 J'mor'K-' kJ»mol"V 4 (5.84 ± 0.51) x 10" 2 10 (7.46 ± 0.14) x 10" 2 1 15 20 25 (9.15 ± 0.11) x 10" 2 (11.76 ± 0.10) x 10" 2 (15.64 ± 0 . 1 1 ) x 10" 2 29 ± 2 -162 ± 7 32 ± 2 15 (4.00 +0.26) x 10" 4 2 25 35 45 (7.65 +0.33) x 10" 4 (1.54 + 0.04) x 10" 3 (2.61 +0.06) x 10" 3 44 ± 1 -158 + 3 4 6 + 1 25 (1.90 + 0.26) x 10" 4 4 35 45 55 (4.08 ± 0.88) x 10" 4 (7.38 +0.33) x 10" 4 (1.18 ± 0.02) x 10" 3 47 + 3 -158 + 9 49 + 3 25 (4.17 ± 0.06) x 10" 5 5 35 45 55 (9.00 ± 0.1 l ) x 10 - 5 (1.63 ± 0.03) x 10" 4 (3.13 + 0.04) x 10" 4 52 + 1 -156 ± 4 54 ± 1 25 (1.21 +0.06) x 10" 5 7 35 45 55 (3.33 + 0.11) x 10" 5 (6.67 +0.03) x 10" 5 (1.20 + 0.04) x 10 - 4 59 ± 1 -140 ± 4 62 ± 1 25* 2.38 x l O " 7 60 (5.78 ± 0.08) x 10 - 6 8 70 80 90 (1.01 ± 0 . 0 3 ) x 10" 5 (2.29 ± 0.04) x 10" 5 (5.00 ± 0.02) x 10" 5 70 ± 1 -136 ± 3 73 ± 1 25* 4.13 x 1 0 s 60 (1.56 ± 0.02) x 10" 6 9 70 80 90 (2.56 ± 0.04) x 10" 6 (9.00 ± 0.04) x 10" 6 (1.60 ± 0.02) x 10" 6 80 ± 1 -120 ± 3 83 ± 1 extrapolated from a plot of ln(k o b s ) versus 1/T. 68 From F i g u r e 2.10 an isokinetic relationship (IKR) can be seen for this series of reactions. This common point or small area of intersection in the Arrhenius lines (ln(k0bS) versus 1/T) indicates that the same reaction mechanism is present in this series of compounds [24]. This was also confirmed by the linear relationship or compensation effect is also observed between the activation enthalpy (AH*) and entropy (AS*) ( F i g u r e 2.11) [25]; changing the phenol substituent increases A H * while decreasing the degree of order in the transition state (AS*). However, in both cases 1 does not seem to agree with the rest of the data, despite good experimental reproducibility. This may be due to errors associated with data collection, as this compound was extremely reactive, completely consuming the POM within seconds of addition to the system. 120 0 I , , , . 1 -1.8 -1.6 -1.4 -1.2 -1 AS*, kJmor1K"1 F i g u r e 2.11 Activation enthalpy versus entropy for the oxidation of phenols with S i V W n 0 4 o 5 " i n sodium acetate buffer (I = 0.2 M, p H 5.0) at 25 °C. 1 (p-methoxylphenol), 2 (p-methylphenol), 4 (m-methylphenol), 5 (phenol), 7 (p-bromophenol), 8 (m-chlorophenol) and 9 (m-nitrophenol). 69 2.3.3 POM Oxidation of Substituted Phenols One of the most widely employed relationships between substituents and reaction mechanisms/rates is the Hammett equation [26, 27] : log(k x/kH) = ap (2.11) where kx and ku are the rate constants for substituted and unsubstituted substrate, respectively, p is the reaction constant and a is the substituent constant. The reaction constant (p) is the slope of a plot of log (kx/kn) versus O", and reflects the sensitivity of a particular reaction to substituent effects. The substituent constant (a) value reflects the interaction of the substituent with the reacting site by a combination of resonance and field interactions. In reaction systems where there is direct resonance interaction between the substituent and an electron-deficient (o"+) or electron-rich (o~) reaction centre, modified substituent constants are employed . Table 2.5 lists the second-order rate constants observed for the various substituted phenols. For compounds 8 and 9, wherein high reaction temperatures were required to obtain satisfactory reactions, room temperature rate constants were determined from extrapolation of results obtained from a plot of ln(kobS) versus 1/T (Figure 2.10). The reaction rates were very sensitive to the nature of the substituent group; electron-donating 70 groups accelerated the reaction, whereas electron-withdrawing groups decreased the reaction rate. The observed order in rate constants was 1>2>3>4>5>6>7>8>9. Table 2.5 Second-order rate constants for the reaction between SiVWnO^ 5" and substituted phenols in sodium acetate buffer (I = 0.2 M , pH 5.0) at 25 °C. Included are logarithm values of each rate constant for the substituted phenol (kx) versus phenol (kn), as well as Hammett o~ and o + constants for the series of substituted phenols. Compound* Rate constants log[kx/kH] Substituent constants [26] k (MTV) G a + 1 6.38 ± 0.04 3.57 -0.27 -0.76 2 (3.12 ± 0.14) x 10~2 . 1.26 -0.17 -0.31 3 (2.61 ±0.01)x 10"3 0.19 0.12 0.05 4 (7.76 ± 0.93) x 10"3 0.66 -0.07 -0.07 5 (1.70 ± 0.02) x 10"3 0.00 0 0 6 (5.83 + 0.29) x 10"4 -0.46 0.23 0.11 7 (4.94 ± 0.45) x 10"4 -0.54 0.23 0.15 8 9.71 x 10"6a -2.24 0.37 0.37 9 1.69 x 10"6a -3.00 0.71 0.67 a extrapolated from a plot of ln(kobs) versus 1/T (Figure 2.10). 1 1 (p-methoxylphenol), 2 (p-methylphenol), 3 (m-methoxylphenol), 4 (m-methylphenol), 5 (phenol), 6 (p-chlorophenol), 7 (p-bromophenol), 8 (m-chlorophenol) and 9 (m-nitrophenol). 71 To quantify the substituent effects, the corresponding reaction constants (p) were determined from a plot of log (kx/kH) against the corresponding Hammett O" and a + constants (Figure 2.12); p = -6.0 (r2 = 0.88) using a values, and -4.7 (r2 = 0.98) for a + values. The negative reaction constants imply that the reaction rate is favored as a result of an increase in electron density at the reaction site [27, 28], i.e. electron donating substituents enhance the reaction rate. This is consistent with an electron-transfer reaction mechanism [18]. The better correlation between rate data and a + (r2 = 0.98) values as compared to c (r2 = 0.88) support an electronic deficient intermediate in the transition state and a reaction mechanism leading to the formation of an electron-deficient phenoxy radical (scheme 2.1). The strong correlation with o+ is further an indication that the reaction mechanism of all phenols oxidized by POM is essentially the same. 72 -6 1 ' 1 1 -0.4 0 0.4 0.8 a . 6 I , , , -1.0 -0.5 0.0 0.5 1.0 <7+ Figure 2.12 Hammett plots for the oxidation of substituted phenols with SiVWuC^n5" in sodium acetate buffer (I = 0.2 M , pH 5.0) at room temperature. The rate constants, and substituent constants (o and a+) are those listed in Table 2.5. 1 (p-methoxylphenol), 2 (p-methylphenol), 4 (m-methylphenol), 5 (phenol), 7 (p-bromophenol), 8 (m-chlorophenol) and 9 (m-nitrophenol). 73 2.3 .4 P r o d u c t A n a l y s i s Depending on the reaction concentrations, different products were detected. Under the dilute POM conditions used for UV kinetic analysis ([phenols] = 12.25 mmol L"' and [SiVWn04o5"] = 0.25 mmol L"'), no precipitated solids, e.g. polymeric products were detected. However, in the reaction of 1, 4 , and 5 at higher POM concentration, ([phenols] = 6.6 mmol L"1 and [S1VW11O40 5"] = 26.4 mmol L"1) precipitates formed. This is likely the result of subsequent electron transfer of the initial oxidized and oxidatively coupled dimeric products. To collect sufficient amounts of precipitated solids for detailed analysis 1 was reacted with POM for 1 h at 25 °C, while 4 was reacted for 2 h at 60 °C and 5 for 5 h at 60 °C. The yields of solid materials for 1, 4 , and 5 were approximately 40, 50 and 70 wt. %, respectively. F i g u r e 2 .13 shows the FT-IR spectra (red line) of the precipitated solids formed during the POM reactions with 1 ( F i g u r e 2.13 a) , 4 ( F i g u r e 2 .13 b ) , and 5 ( F i g u r e 2.13 c), respectively. The FT-IR spectra (blue line) of the initial phenolic compounds, 1, 4 , and 5 are included for comparison. The spectra of the precipitated solids clearly differ from those of the initial phenolic compounds; in particular the aromatic C-H out-of-plane bending (the ring substitution pattern) region at 800 - 600 cm"1, the aromatic C=C stretching region at 1600 - 1475 cm"1 and the O-H stretching region at - 3300 cm"1. 74 i 1 I I 1 I I 3600 3100 2600 2100 1600 1100 600 Wavenumberfcrrf 1) Figure 2.13 FT-IR spectra of precipitates formed during the POM (SiVWuO^5") reaction (red line) and the initial phenolic compounds (blue line): a) 1 (p-methoxylphenol); b) 4 (m-methylphenol); and c) 5 (phenol). All of these reactions were run using sodium acetate buffer (I = 0.2 M , pH 5.0). 75 The decrease in 0-H stretching indicates the formation of phenyloxide-type structures in the resulting polyphenol [29-31]. This is further supported by the increase in the C-O-C stretching region (1100-1200 cm"1). Likewise, the increase in wavenumber of the C-H out-of-plane bending, along with the broadening of the C=C stretching region also suggests the formation of phenolic polymers [29-31]. GPC analysis (Figure 2.14) of the precipitated solids confirmed they were oligomeric materials; relative average molecular masses (Mw) were 428, 741, and 549 Daltons for 1, 4, and 5, respectively (Table 2.6). Figure 2.14 Gel permeation chromatography (GPC) elution curves of the precipitated materials formed during POM (SiVW u0 4o 5") reaction with 1 (25 °C for 1 h), 4, and 5 (60 °C for 2 h and 5 h, respectively) in sodium acetate buffer (I = 0.2 M , pH 5.0). 1 (p-methoxylphenol), 4 (m-methylphenol) and 5 (phenol). 76 Table 2.6 Oligomerization of 1 (25 °C for 1 h), 4, and 5 (60 °C for 2 h and 5 h, respectively) using POM (SiVWn04o5") in sodium acetate buffer (I = 0.2 M , pH 5.0). 1 (p-methoxylphenol), 4 (m-methylphenol) and 5 (phenol). Compounds M w a # of units6 1 428 4 4 741 7 5 549 6 a Determined by GPC using THF as eluent with polystyrene standards. b number of units calculated from M w divided by molecule weight of each monomer. MALDI-TOF spectra (Figure 2.15) further confirmed the precipitated products to be phenolic oligomers. Positive-ion linear mode MALDI-TOF spectra of the solid precipitate from 1, 4, and 5 contained masses corresponding to oligomeric series of each phenolic unit. The mass difference between peaks in Figure 2.15 (red numbers 122, 106 and 92) represent the molecular weight of the respective phenolic units. Scheme 2.2 shows the possible structures of the oligomeric materials formed during POM reactions of 1, 4, and 5. 77 -1 8 6 5 K M - 8 5 0 1 2 3 1 9 6 9 1 2 2 J i Al 1 0 9 2 »n fi"AAi^w.»„,.>.<<fc/s^iit< 800 900 1000 1100 1200 1300 1400 1500 1600 Mass/Charge 4 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 Mass/Charge 5 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 Mass/Change Figure 2.15 MALDI-TOF-MS spectra of polymeric materials formed during the POM (SiVWu04o5") reaction with 1 (25 °C for 1 h), 4, and 5 (60 °C for 2 h and 5 h, respectively) in sodium acetate buffer (I = 0.2 M , pH 5.0). 3,5-dimethoxyl-4-hydroxylcinnamic acid was the matrix for 1 and 2,5-dihydroxylbenzoic acid was the matrix for 4 and 5.1 (p-methoxylphenol), 4 (m-methylphenol) and 5 (phenol). 78 1: R i = H, R 2 = O C H 3 4: R! = C H 3 , R 2 = H 5'. R-| = H, R 2 = H Scheme 2.2 Possible structures of polymeric materials formed from the reaction of POM (SiVWnOV") with 1 (25 °C for 1 h), 4, and 5 (60 °C for 2 h and 5 h, respectively) in sodium acetate buffer (I = 0.2 M , pH 5.0). 1 (p-methoxylphenol), 4 (m-methylphenol) and 5 (phenol). 'H-NMR spectra of the initial phenolic compounds and the resulting oligmeric products are presented in Figure 2.16. The NMR spectra of 1, 4, and 5 have sharp individual peaks, while those of the products are broad, particularly in the aromatic region (7.5 - 6.6 ppm), the methoxyl region (3.8 - 3.2 ppm) and the methyl region (2.3 -1.8 ppm). In addition, the intensities of the phenolic proton peaks were significantly decreased in 'H-NMR spectra of the oligmeric materials. This is in agreement with the observed decrease in the O-H stretching region (~ 3300 cm"1) of the corresponding FT-IR spectra (Figure 2.13). The broad and multiple peaks observed in the 'H-NMR spectra of the oligomeric materials are similar to results reported for the 'H-NMR analysis polyphenols [31]. These results suggest the formation of oligomers consisting of a mixture of phenylene and oxyphenylene units, as shown in scheme 2.2. 79 a) b) Phenolic O H Aromatic protons .1 Methoxyl group Oligomeric phenols r Phenolic O H Aromatic protons H 2 0 D M S O L Methyl group Oligomeric phenols r Phenolic O H Aromatic protons c) 1 1 Oligomeric phenols u 10 ppm Figure 2.16 'H NMR spectra of initial phenolic compounds (upper) and oligomers (lower) formed during the reaction of POM (SiVWnO^5") in sodium acetate buffer (I = 0.2 M , pH 5.0) with a) 1 at 25 °C for 1 h, b) 4 at 60 °C for 2 h, and c) 5 at 60 °C for 5 h. *Unknown signals or impurities. 1 (p-methoxylphenol), 4 (m-methylphenol) and 5 (phenol). 8 0 In addition to oligomer formation, analysis of the reaction systems revealed oxidation and oxidative coupled products. In the reaction of POM with 1 the detected products were p-benzoquinone, 10 ( - 3 %), 5,5'-2-(2-hydroxyl-5-methoxyl-phenyl)-[l,4]benzoquinone, 11 (~ 27 %), and dimethoxyl-biphenyl-2,2'-diol, 12 (~ 0.7 %). Scheme 2.3 illustrates a possible reaction pathway leading to the observed reaction products. , Scheme 2.3 Possible mechanism for the POM (SiVWuO^5") oxidation of 1 under anaerobic conditions. 81 The first step in the reaction of 1 with POM is oxidation of the phenolic substrate [2, 4, 5, 16, 32]. This likely involves either hydrogen atom transfer or proton-coupled electron transfer mechanisms. The resonance stabilized phenoxy radical intermediate then undergoes (i) a second oxidation step to the corresponding cation and benzoquinone formation (10), or (ii) radical coupling with a second oxidized phenol and dimer formation, (12). The resulting dimeric compound is then rapidly oxidized, and undergoes the same oxidative reaction steps as the initial phenol, which depending on the reaction conditions (concentrations etc.), lead to oligomeric and polymeric products and/or oxidized biphenols (11). In the case of 4 and 5 the primary reaction products were dimers (biphenyl compounds). 2 .4 C o n c l u s i o n s The reaction kinetics and mechanism of POM oxidation of a series of substituted phenols were examined under anaerobic conditions. Using an initial rates method and pseudo-first order kinetics the overall reaction order was determined to be second-order in this series of phenols, first-order in substrate and oxidant. Both the Hammett equation and activation kinetic data indicate the same reaction mechanism exists in this series of phenols, the rate of which is highly dependent on the nature of the substituent group: electron donating groups (EDG) accelerated reaction rates whereas electron withdrawing 82 groups (EWG) retarded reaction rates. The rate-determining step appears to involve an electron-transfer from a neutral substrate by POM. At high POM concentrations, oxidative polymerization of the phenol was observed. This is likely the result of subsequent electron transfer of the initial oxidized and oxidatively coupled dimeric products. Based on these results, POM oxidation of phenols proceeds via either hydrogen atom transfer or proton coupled electron transfer mechanisms. 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De Jesus, Oxidative delignification 84 in the presence of molybdovanadophosphate heteropolyanions: mechanism and kinetic studies. Applied Catalysis A-General, 1998. 167(1): p. 123-139. 14. S. Rayne, R. Sasaki, and P. Wan, Photochemical rearrangement of dibenzo-1,4-dioxins proceeds through reactive spirocyclohexadienone and biphenylquinone intermediates. Photochemical & Photobiological Sciences, 2005. 4(11): p. 876-886. 15. M . Ochiai, K. Fukui, S. Iwatsuki, K. Ishihara, and K. Matsumoto, Synthesis of aryl-platinum dinuclear complexes via ortho C-H bond activation of phenol and transmetalation of arylboronic acid. Organometallics, 2005. 24(23): p. 5528-5536. 16. I.A. Weinstock, K.E. Hammel, M.A. Moen, L.L. Landucci, S. Ralph, C E . Sullivan, and R.S. Reiner, Selective transition-metal catalysis of oxygen delignification using water-soluble salts of polyoxometalate (POM) anions. Part II. reactions of a-[SiVWn04o]5" with phenolic lignin-model compounds. Holzforschung, 1998. 52(3): p. 311-318. 17. D.V. Evtuguin, CP. Neto, H. Carapuca, and J. Soares, Lignin degradation in oxygen delignification catalyzed by [PM07V5O40]8" polyanion. Part II. study on lignin monomeric model compounds. Holzforschung, 2000. 54(5): p. 511-518. 18. D.T.Y. Yiu, M.F.W. Lee, W.W.Y. Lam, and T.C Lau, Kinetics and mechanisms of the oxidation of phenols by a trans-dioxoruthenium(VI) complex. Inorganic Chemistry, 2003. 42(4): p. 1225-1232. 19. P.W. Atkins, The rates of chemical reactions. In Physical Chemistry, Oxford university press, New York, 1998: p. 761-775. 20. J.E. Wajon, D.H. Rosenblatt, and E.P. Burrows, Oxidation of phenol and hydroquinone by chlorine dioxide. Environmental Science & Technology, 1982. 16(7): p. 396-402. 21. O.S. Tee, M . Paventi, and J.M. Bennett, Kinetics and Mechanism of the bromination of phenols and phenoxide ions in aqueous-solution - diffusion-controlled rates. Journal of the American Chemical Society, 1989. 111(6): p. 2233-2240. 22. K.C. Gross and P.G. Seybold, Substituent effects on the physical properties and pK a of phenol. International Journal of Quantum Chemistry, 2001. 85(4-5): p. 569-579. 23. F.A Carey and R J . Sundberg, Advanced organic chemistry part A - structure and mechanism. Kluwer Academic and Plenum Publishers, New York, Boston, Dordrecht, London and Moscow, 2000: p. 204-215. 24. W. Linert, Mechanistic and structural investigations based on the isokinetic relationship. Chemical Society Reviews, 1994. 23(6): p. 429-438. 85 25. I.M. Ganiev, E.S. Suvorkina, and N.N. Kabal'nova, Reaction of chlorine dioxide with phenol. Russian Chemical Bulletin, 2003. 52(5): p. 1123-1128. 26. C. Hansch, A. Leo, and R.W. Taft, A survey of Hammett substituent constants and resonance and field parameters. Chemical Reviews, 1991. 91(2): p. 165-195. 27. L.R Hammett, The effect of structure upon the reactions of organic compounds -benzene derivatives. Journal of the American Chemical Society, 1937. 59: p. 96-103. 28. S.V. Jovanovic, M . Tosic, and M.G. Simic, Use of the Hammett correlation and o~+ for calculation of one-electron redox potentials of antioxidants. Journal of Physical Chemistry, 1991. 95(26): p. 10824-10827. 29. N. Mita, S. Tawaki, H. Uyama, and S. Kobayashi, Structural control in enzymatic oxidative polymerization of phenols with varying the solvent and substituent nature. Chemistry Letters, 2002. 3: p. 402-403. 30. N. Mita, S. Tavaki, and S. Kobayashi, Enzymatic oxidative polymerization of phenols in an aqueous solution in the presence of a catalytic amount of cyclodextrin. Macromolecular Bioscience, 2002. 2(3): p. 127-130. 31. Y. Kim, H. Uyama, and S. Kobayashi, Peroxidase-catalyzed oxidative polymerization of phenol with a nonionic polymer surfactant template in water. Macromolecular Bioscience, 2004. 4: p. 497-502. 32. O.A. Kholdeeva, A.V. Golovin, R.I. Maksimovskaya, and I.V. Kozhevnikov, Oxidation of 2,3,6-trimethylphenol in the presence of molybdovanadophosphoric heteropoly acids. Journal of Molecular Catalysis, 1992. 75(3): p. 235-244. 86 3. Polyoxometalate (POM) Oxidation of L ignin Model Compounds A version of this chapter has been accepted for publication. Kim, Y.S., Chang, H.-M. and Kadla, J. (2007) Polyoxometalate (POM) Oxidation of Lignin Model Compounds, Holzforschung. 3.1 I n t r o d u c t i o n Due to environmental concerns, there is increasing interest in using polyoxometalates (POMs) for the delignification of wood pulp [1-3]. POMs are environmentally benign and relatively high selective alternatives to traditional bleaching reagents such as elemental chlorine. Recently, POMs have been shown to efficiently and selectively remove lignin from wood pulps without severely degrading carbohydrates [1-41-Lignin model compounds have widely been used to enhance the understanding of the reactions involved in the delignification of pulps. Detailed mechanistic studies of the POM ([SiVWio04o]5") oxidation of guaiacyl and syringyl P-O-4 dimer lignin model compounds have been reported by Weinstock et al. [5]. They found the POM oxidation of phenolic compounds involves an electron-transfer mechanism and generation of a phenoxy radical intermediate, which is either further oxidized by a second POM or reacts with another radical intermediate to give dimeric products. Similarly, Evtuguin et al. [6] studied the catalytic reactions of heteropolyanions [ P M 0 7 V 5 O 4 0 ] 8 " under aerobic conditions. They proposed a reaction mechanism involving a one-electron oxidation of the lignin aromatic group by the V v of the catalyst to generate cyclohexadienyl cation type structures, followed by hydrolytic cleavage. Although pulp bleaching experiments have been performed on a laboratory scale and 88 a number of studies on the reactions of POMs with phenolic lignin model compounds have been reported, the oxidative mechanisms of lignin model compounds by POMs are only partially understood. In order to further develop and optimize the process, it is necessary to obtain a detailed understanding of the reaction kinetics and mechanisms of the POM delignification process. In this paper, we report the reactions of POM ([SiVWnO40]5") with various lignin model compounds, specifically, the effect of lignin model compound structure on the reaction pathway and kinetics. 3 .2 E x p e r i m e n t a l 3 .2.1 M a t e r i a l s K5[SiVWii04o]-12H20 (POM) was provided from the USDA Forest Products Laboratory (Madison WI, U.S.) [1]. CDCI3, DMSO-d6, and acetone-d6 were purchased from Cambridge Isotope Laboratories. Boron trifluoride-methanol (25 %, BF3-mefhanol) complex, N,0-bis(trimethylsilyl)acetamide, 3,5-dimethoxyl-4-hydroxylacetphenone, 4-hydroxylacetophenone, 4-hydroxyl-3-methoxylacetophenone, mercuric chloride, sodium bisulfate, sodium methoxide (25 wt %) and pyridine were purchased from Sigma-Aldrich. Magnesium sulfate, sodium acetate, sodium chloride, silica gel (column chromatography grade, 60 A, 70 - 230 mesh), anhydrous potassium carbonate, potassium ferricyanide, zinc metal, acetic acid, acetone, chloroform, concentrated hydrochloric acid, ethanol, 89 methanol, methylene chloride, and petroleum ether were purchased from Fisher Scientific. Bromine and sodium borohydride were purchased from Acros Chemicals. Al l chemicals and solvents were used as received. The lignin model compounds ( F i g u r e 3.1): 4-acetyl-2-methoxylphenol (6), 4-acetyl-2,6-dimethoxylphenol (7) and 2-methoxyl-4-methyl-phenol (8) were purchased from Sigma-Aldrich and used as received. l-(4-hydroxylphenyl)-ethanol (1), 1-(4-hydroxyl-3-methoxylphenyl)-ethanol (2), l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol (3), l-(4-hydroxyl-3-methoxylphenyl)-ethyl methyl ether (4), l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethyl methyl ether (5), 4-ethyl-2-methoxylphenol (9), 3,3'-dimethoxyl-5,5'-dimethyl-biphenyl-2,2'-diol (10), and 4-[2-hydroxyl-2-(4-hydroxyl-3-methoxylphenyl)ethoxyl]-3-methoxyl-benzoic acid (11) were synthesized as described below. 4-[2-hydroxyl-2-(4-hydroxyl-3,5-dimethoxylphenyl)ethoxyl]-3-methoxyl-benzoic acid (12), and 4-[2-hydroxyl-2-(4-hydroxyl-3,5-dimethoxylphenyl)ethoxyl]-3,5-dimethoxyl-benzoic acid (13) were provided from the Dept. of Wood and Paper Science at North Carolina State University (Raleigh NC, U.S.A.). NaHS0 4»Si0 2 and amalgamated zinc were prepared as detailed below. 90 1: RT = H, R 2 = H 2: R, = H, R 2 = OCH 3 3: Ri = O C H 3 , R 2 = O C H 3 4: RT = H, R 2 = OCH 3 5: Ri = 0CH 3 , R2 = O C H 3 6: Rr = H, R 2 = OCH3 7: R, = OCH3, R 2 = OCH3 8: R = CH 3 9: R = CH 2 CH 3 10 11:R1 = H, R 2 = OCH 3 , R 3 = H, R 4 = OCH 3 12: R1 = OCH3, R 2 = OCH3, R 3 = H, R 4 = OCH 3 1 3 ^ = 0 0 ^ , R 2 = OCH 3 , R 3 = OCH 3 , R 4 = OCH 3 Figure 3.1 Lignin model compounds studied. NaHS04»Si02 (silica gel-supported sodium bisulfate) Silica gel-supported sodium bisulfate (NaHSCVSiC^) was prepared according to Ramu et al. [7]. Accordingly, 4.14 g (0.03 mmol) of NaHSCu was dissolved in 20 mL of H 2 0 followed by the addition of lOg of Si0 2 (column chromatography grade, 60 A, 70 -230 mesh). The mixture was gently heated until most of the water evaporated. The catalyst was further oven-dried at 130 °C prior to use. 9 1 Amalgamated Zinc Amalgamated zinc was prepared according to Brewster [8]. Zinc (5 g) was amalgamated in a reaction flask by covering it with a solution of mercuric chloride (0.1 g) in 15 mL of water with occasionally agitation over a 30 min period. The solution was poured off and the amalgamated zinc was rinsed once with water. 3.2.2 Syntheses of Lignin Model Compounds l-(4-Hydroxylphenyl)-ethanol (1) l-(4-Hydroxylphenyl)-ethanol 1 was synthesized according to Yokoyama et al. [9]. Accordingly, 4 equivalents of NaBfL (1.1 g, 29.2 mmol) were added to a stirring solution of 4-hydroxylacetophenone (1 g, 7.3 mmol) in 50 mL of EtOH/H20 (3:1, v/v) at room temperature and left overnight. The reaction mixture was neutralized by purging with CO2 and subsequently extracted with CH2CI2 (3 x 50 mL). The organic layer was dried over anhydrous MgSCu, filtered and evaporated under reduced pressure to give l-(4-hydroxylphenyl)-ethanol 1, a pure white solid, in high yield. 'H-NMR: (acetone^) 5 1.36(3H, d, CH 3), 3.97(1H, d, OH), 4.77(1H, q, CH), 6.77(2H, d, J=8.6, ArH), 7.20(2H, d, J=8.6, ArH), 8.14(1H, s, ArOH); 1 3C-NMR: (Aceton-d6) 5 25.30, 68.79, 114.75, 126.50, 138.16, 156.19; m/z (low resolution) EI, 138(M+, 14), 123(100), 95(87), 77(85), 65(13). The assignments are in agreement with literature data [10]. 92 l - ( 4 - H y d r o x y l - 3 - m e t h o x y l p h e n y l ) - e t h a n o l (2) l-(4-Hydroxyl-3-methoxylphenyl)-ethanol 2 was synthesized by reacting 4 equivalents of NaBH 4 (1.8 g, 48.1 mmol) with 4-hydroxyl-3-methoxylacetophenone (2 g, 12.0 mmol) as outline for 1. 'H-NMR: (acetone-d6) 5 1.37(3H, d, CH 3), 3.85(3H, s, ArOCH3), 3.97(1H, d, OH), 4.77(1H, q, CH), 6.75(1H, d, J=3.0, 8.2, ArH), 6.80(1H, dd, J=2.1, 8.2, ArH), 7.01(1H, d, J= 8.2, ArH), 7.35(1H, s, ArOH); 1 3C-NMR: (Aceton-d6) 5 25.39, 55.29, 68.97, 108.98, 114.39, 117.85, 138.92, 145.35, 147.17; m/z (low resolution) EI, 168(M+, 51), 153(100), 125(62), 93(58), 65(45), 51(9). The assignments are in agreement with literature data [11]. l - ( 4 - H y d r o x y l - 3 , 5 - d i m e t h o x y l p h e n y l ) - e t h a n o l (3) l-(4-Hydroxyl-3,5-dimethoxylphenyl)-ethanol 3 was synthesized by reacting 4 equivalents of NaBH 4 (0.8 g, 20.4 mmol) with 3,5-dimethoxyl-4-hydroxylacetphenone (1 g, 5.1 mmol) as outline for 1. 'H-NMR: (acetone-d6) 5 1.37(3H, d, CH 3), 3.82(6H, s, ArOCH3), 3.98(1H, d, OH), 4.76(1H, q, CH), 6.68(2H, s, ArH), 6.97(1H, s, ArOH); 1 3 C-NMR: (Aceton-de) 5 25.44, 55.68, 69.22, 102.88, 134.72, 137.95, 147.57; m/z (low resolution) EI, 198(M+, 100), 183(78), 155(83), 123(81), 95(54), 77(61), 65(11), 51(8). The assignments are in agreement with literature data [12]. l - ( 4 - H y d r o x y l - 3 - m e t h o x y l p h e n y l ) - e t h y l m e t h y l e the r (4) l-(4-Hydroxyl-3-methoxylphenyl)-ethyl methyl ether 4 was synthesized according 93 to Ramu et al. [7]. Accordingly, 3 equivalents of MeOH (0.22 mL, 5.3 mmol) were added to a stirring solution of l-(4-hydroxyl-3-methoxylphenyl)-ethanol 2 (0.3 g, 1.8 mmol) in 10 mL of CH 2C1 2, followed by 100 mg of NaHS0 4 'Si0 2 (silica gel-supported sodium bisulfate). The mixture was stirred at room temperature for 90 min and monitored by thin layer chromatography (TLC, elution CH2Cl2/Me2CO, 1:1, v/v). The reaction mixture was then filtered, and the solvent was removed under reduced pressure to give 1 -(4-hydroxyl-3-methoxylphenyl)-ethyl methyl ether 4, a pure white solid, in high yield. 'H-NMR: (acetone-d6) 5 1.3313H, d, CH 3), 3.14(3H, s, OCH3), 3.86(3H, s, ArOCH 3), 4.18(1H, q, CH), 6.74(1H, dd, J=2.0, 8.1, ArH), 6.79(1H, d, J=8.1, ArH), 6.92(1H, d, J= 2.0, ArH), 7.44(1H, s, ArOH); 1 3C-NMR: (Aceton-d6) 5 23.37, 55.24, 55.32, 79.07, 109.35, 114.57, 118.95, 135.29, 145.89, 147.49; m/z (low resolution) EI, 182(M+, 12), 167(100), 151(28), 107(8), 91(11), 77(6). l - ( 4 - H y d r o x y l - 3 , 5 - d i m e t h o x y l p h e n y l ) - e t h y l m e t h y l e t h e r (5) l-(4-Hydroxyl-3,5-dimethoxylphenyl)-ethyl methyl ether 5 was synthesized using the same preparation as for 4; 3 equivalents of MeOH (0.2 mL, 7.9 mmol) were added to a stirring solution of l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol 3 (0.2 g, 1.0 mmol) in 10 mL of C H 2 C 1 2 . A pure bright orange oil product was obtained in high yield. 'H-NMR: (acetone-d6) 5 1.33(3H, d, CH 3), 3.15(3H, s, OCH3), 3.84(6H, s, AxOCH3), 4.17(1H, q, CH), 6.61(2H, s, ArH), 7.07(1H, s, ArOH); 1 3C-NMR: (Aceton-d6) 5 23.48, 55.34, 55.73, 94 79.40, 103.52, 134.34, 135.20, 147.82; m/z (low resolution) EI, 212(M+, 27), 197(100), 182(26), 167(6), 91(9). 4-Ethyl-2-methoxylphenol (9) 4-Ethyl-2-methoxylphenol 9 was synthesized according to Brewster [8]. 4-Acetyl-2-methoxylphenol 6 (2 g, 12.0 mmol) was dissolved in acetic acid (5 mL) and carefully added to an amalgamated zinc solution (5g amalgamated zinc in 10 mL acetic acid and 10 mL concentrated HCI). The reaction mixture was refluxed for 2 days. At the end of the reaction 20 mL of a 20 % NaCl solution was added and the mixture was diluted with petroleum ether (30 mL). The organic phase was separated, and the aqueous layer was extracted with petroleum ether (2 x 30 mL). The combined organic phases were dried over anhydrous MgSCu, filtered and evaporated under reduced pressure to give 4-ethyl-2-methoxylphenol 9. A pure bright orange oil product was obtained in high yield. 'H-NMR: (acetone-d6) 5 1.18(3H, t, CH 3), 2.51(2H, q, CH 2), 3.83(3H, s, ArOCH3), 6.63(3H, m, ArH), 7.23(1H, s, ArOH); 1 3C-NMR: (CDC13) 5 15.98, 28.61 55.87, 110.61, 114.29, 120.32, 136.32, 143.58, 146.43; m/z (low resolution) EI, 152(M+, 100), 137(98), 122(8), 91(11), 65(4). The assignments are in agreement with literature data [12]. 3,3'-Dimethoxyl-5,5'-dimethyl-biphenyl-2,2'-diol (10) 3,3'-Dimethoxyl-5,5'-dimethyl-biphenyl-2,2'-diol 10 was synthesized according to Haynes et al. [13]. A solution of K3[Fe(CN)6] (36 g, 0.28 mol) in 250 mL of deionized 95 H2O was added dropwise to a vigorously stirring mixture of creosol (7.5 g, 54.3 mmol) and sodium acetate (15.25 g, 0.19 mol) in 550 mL of deionized H2O using an addition funnel at room temperature. After 3 h a cream-colored solid formed. The reaction mixture was acidified to pH 2 with concentrated HCI, and extracted with CHCI3 (3 x 250 mL). The organic layers combined and dried over anhydrous MgSCM, filtered and evaporated under reduced pressure to give crude white crystals. Recrystallization from ether/petroleum ether (1:10, v/v) gave pure 3,3'-dimethoxyl-5,5'-dimethyl-biphenyl-2,2'-diol 10 (yield 8.3 g, 56 %). 'H-NMR: (acetone-d6) 5 2.29(6H, s, CH 3), 3.86(6H, s, ArOCH3), 6.68(2H, d, J=2.0, ArH), 6.79(2H, d, J=2.0, ArH), 7.28(1H, s, ArOH); 1 3 C-NMR: (Aceton-d6) 5 20.19, 55.48, 111.41, 123.56, 125.52, 128.21, 141.51, 147.69; m/z (low resolution) EI, 274(M+, 100), 259(12), 241(25), 227(33), 213(17), 199(34), 171(9), 143(8), 128(13), 115(11), 91(5), 76(4). The assignments are in agreement with literature data [14]. 4^2-Hydroxyl-2-(4-hydroxyl-3-methoxylphenyl)ethoxyl]-3-methoxyl-benzoic acid (11) 4-[2-Hydroxyl-2-(4-hydroxyl-3-methoxylphenyl)ethoxyl]-3-methoxyl-benzoic acid 11 was synthesized according to Chen et al. [15] (scheme 3.1). First, 4-acetyl-2-methoxyl-phenyl acetic acid ester A was prepared by acetylating 2 g (12.0 mmol) of 4-hydroxyl-3-methoxylacetophenone in 20 mL of pyridine with 8 mL of acetic anhydride 96 overnight at room temperature. The reaction mixture was poured into 200 mL of deionized H 2 O and extracted with CHCI3 (3 x 50 mL). The organic layer was dried over anhydrous MgSC>4, filtered, and evaporated under reduced pressure to give a pure colorless oil; 4-acetyl-2-methoxyl-phenyl acetic acid ester A. 'H-NMR: (CDC13) 5 2.36(3H, s, ArOCOCH3), 2.62(3H, s, COCH3), 3.91(3H, s, ArOCH3), 7.13(1H, d, J=8.1, ArH), 7.56(1H, dd, J=2.0, 8.1, ArH), 7.62(1H, d, J=2.0, ArH). Bromine (0.37 mL, 7 mmol) in 10 mL of CHCI3 was slowly added over 3 h to a stirring reaction mixture of 1.5 g (7.2 mmol) of A in 20 mL of CHCI3 at 5 - 10 °C. The reaction mixture was then poured into 100 mL of H 2 O at 0 °C and washed with deionized H20 (2 x 50 mL), saturated NaHC03 (3 x 50 mL), and deionized H20 (3 x 50 mL). The organic layer was dried over anhydrous MgSC^, filtered, and evaporated under reduced pressure. The crude white solid was recrystallized from a mixture of ethyl acetate/hexane (3:7, v/v) to give pure 4-(2-bromo-acetyl)-2-methoxyl-phenyl acetic acid ester A l (yield 1.5 g , 74 %) [16]. 'H-NMR: (CDC13) 5 2.36(3H, s, ArOCOCH3), 3.92(3H, s, ArOCH3), 4.45(2H, s, CH2), 7.16(1H, d, J=8.2, ArH), 7.59(1H, dd, J=1.8, 8.2, ArH), 7.64(1H, d, J=1.8, ArH). The assignments are in agreement with literature data [15]. 4-Hydroxyl-3-methoxyl-benzoic acid methyl ester B was prepared by refluxing 2 g (11.9 mmol) of 4-hydroxyl-3-methoxyLbenzoic acid in 25 mL (24 mmol) of BF3-MeOH complex for 9 h. The mixture was poured into 200 mL of saturated NaHC03 solution and 97 extracted with CH2CI2 (3 x 50 mL). The combined organic layers were dried over anhydrous MgSCu, filtered, and evaporated under reduced pressure. A pure brown-colored oil, 4-hydroxyl-3-methoxyl-benzoic acid methyl ester B, was obtained in high yield. 'H-NMR: (CDC13) 5 3.91(3H, s, ArOCH3), 3.96(3H, s, ArCOOCH 3), 6.07(1H, s, ArOH), 6.94(2H, d, J=8.1, ArH), 7.56(1H, d, J=2.1, ArH), 7.64(1H, dd, J=2.1, 8.1, ArH). The assignments are in agreement with literature data [16]. 3-Methoxyl-4-(4'-acetoxy-3'-methxoyl-|3-oxophenethoxy)benzoic acid methyl ester (C) was prepared by refluxing A l (1.5 g, 5.2 mmol) and B (1.8 g, 10 mmol) with 3 g of anhydrous K 2 C 0 3 in 50 mL of acetone for 90 min. The reaction mixture was then cooled, filtered to remove the inorganic salts, and the solvent was removed under reduced pressure. The residue was then dissolved in 150 mL of CHC13 and the solution was washed with 50 mL of 0.5 M NaOH, followed by 100 mL of deionized H 2 0 . The organic phase was dried over anhydrous MgS04, filtered and the solvent removed under reduced pressure. The crude white solid product was recrystallized from acetone-MeOH to give pure 3-methoxyl-4-(4'-acetoxy-3'-methxoyl-f3-oxophenethoxy)benzoic acid methyl ester C (yield 2.4 g , 77 %). 'H-NMR: (acetone-d6) 5 2.30(3H, s, ArOCOCH3), 3.85(3H, s, ArCOOH3), 3.92(3H, s, ArOCH3), 3.93(3H, s, ArOCH3), 5.66(2H, d, CH 2), 7.03(1 H, d, J=8.8, ArH), 7.25(1H, d, J=8.8 ArH), 7.57(2H, m, ArH), 7.73(2H, m, ArH). The assignments are in agreement with literature data [15]. 98 to the reaction temperature. For compounds 10 - 13, 200 uL of ethanol were added to the buffer solution to dissolve the lignin model compounds. The temperature range studied was 20 - 50 ± 1 °C. Reactions were initiated by injecting 1 mL of a stock POM solution (2.25 mmol L"1). The concentrations of lignin model compound and POM were 0.10 mmol L"1 and 0.25 mmol L ' 1 , respectively. Reaction kinetics were spectrophotometrically recorded by monitoring the absorbance of the reduced POM at 520 nm (e = 610 M^cm"1). Data were recorded at 0.1 -5s intervals for 10 to 120 min, depending on the experiment. All reactions were run in triplicate, and rate constants are reported as average values. Figure 3.2 Schematic representation of the stainless steel UV-Vis reaction cell. The cell was equipped with two 1 cm thick quartz windows, a void volume of 10 cm3 and a 4 cm UV path-length. 102 3.2.4 Reaction Conditions for Product Analysis In a typical experiment the lignin model compound (~ 0.25 mmol) was dissolved in ethanol (1 mL) and added via a syringe to a sealed reaction flask containing POM (- 0.50 mmol) dissolved in 0.2 M sodium acetate buffer (50 mL, pH 5.0) under argon. For compounds, 1, 6 and 7 the reaction mixture was stirred for 8 h at 60 °C. For compound 2 the reaction mixture was stirred for 8 h at 25 °C, while for compounds 3, 5, 10 and 13 the reactions were reacted for 1 h at 25 °C. The analysis of intermediate reaction products was performed by sampling 1 mL of the reaction solution at set time intervals during the course of reaction. The sample was quickly, extracted three times with 1 mL of chloroform/acetone 2:1 (v/v), followed by the addition of an internal standard, p-nitrobenzaldehyde. The organic layers were collected, dried (MgS04), concentrated under reduced pressure, and used for GC analysis. For detailed product analysis the reaction mixture was acidified to pH 2 with concentrated HC1 and diluted with a 2:1 (v/v) mixture of chloroform and acetone (70 mL). The organic phase was separated, and the aqueous layer was extracted two more times with 2:1 (v/v) chloroform/acetone (2 x 70 mL). The organic phases were combined, dried over anhydrous MgS04, filtered, and the solvent removed under reduced pressure. In the case of 6 the precipitated product was collected by filtration using a Nylon membrane filter (0.45 pm, 47 mm) and freeze-dried. 103 A portion of the crude product mixture was then analyzed by GC-MS; one sample (-1 mL) was directly analyzed, while the second sample (~ 1 mL) was first silylated by reacting with N,0-bis(trimethylsilyl)acetamide (200 pL) in pyridine (0.5 mL) at room temperature for 24 h. The remaining reaction product mixture was separated, if possible, by thin layer chromatography (eluent: 2 - 10 % acetone in CHCI3) and analyzed by NMR. 3.2.5 Analytical Methods Gas chromatography-mass spectroscopy (GC-MS) analyses were conducted on a ThermoFinnigan TraceGC and PolarisQ ion trap mass spectrometer using a J&W Scientific Inc. DB-5 column (30 m x 0.32 mm x 0.25 um). The injection temperature was set to 200 °C, the transfer temperature was set to 200 °C, and the ion source was set to 300 °C. Helium flow was 1 mL min"1. After a 5 min solvent delay at 70 °C, the oven temperature was increased at 5 °C min"1 to 280 °C, then held for 5 min before being cooled down to 70 °C. Mass spectra were recorded from m/z = 50 to 650 at 0.58 s scan"1 with an electron ionization of 70 eV. When available, commercial/synthesized samples of the identified products were used to verify chromatographic retention time and spectral data. ' H and 13C-nuclear magnetic resonance (NMR) analyses of the isolated products were conducted on a Bruker AVANCE 300 MHz NMR spectrometer at 300 K using 104 CDC13, acetone-d6 or DMSO-d 6 as the solvent. Chemical shifts were referenced to tetramethylsilane (TMS; 0.0 ppm). 3 .2 .6 I d e n t i f i c a t i o n o f R e a c t i o n P r o d u c t s 4 - A c e t y l - 2 , 6 - d i m e t h o x y l p h e n o l (7): m/z (low resolution) EI, 196(M+, 60), 181(100), 153(20), 138(4), 123(4), 93(4), 65(7). A commercial sample (Aldrich) was used to compare the chromatographic retention time and spectral data. S ^ ' - D i m e t h o x y l - S ^ ' - d i m e t h y l - b i p h e n y l ^ ^ ' - d i o l (10). m/z (low resolution) EI, 274(M+, 100), 259(12), 241(25), 227(33), 213(17), 199(34), 171(9), 143(8), 128(13), 115(11), 91(5), 76(4). The assignments are in agreement with literature data [14]. 4- ( l - H y d r o x y I - e t h y l ) - [ l , 2 ] b e n z o q u i n o n e (14): m/z (low resolution) EI, 152(M+, 2), 137(100), 121(26), 91(26), 77(11), 65(9). The assignments are in agreement with literature data [18]. 5- ( l - H y d r o x y l - e t h y l ) - 3 - m e t h o x y l - [ l , 2 ] b e n z o q u i n o n e (15): m/z (low resolution) EI, 182(M+, 62), 167(100), 139(23), 124(3), 65(7). 2 , 6 - D i m e t h o x y l - [ l , 4 ] b e n z o q u i n o n e (16): m/z (low resolution) EI, 168(M+, 21), 140(42), 112(72), 97(25), 69(100), 52(16); 'H-NMR: (CDC13) 5 3.84(6H, s, ArOCH3), 5.87(2H, s, ArH); 1 3C-NMR: (CDC13) 5 56.48, 107.42, 157.32, 176.67, 186.84. The assignments are in agreement with literature data [5]. 105 2 ,6 -D imethoxy l -4 - v iny l -pheno l (17): m/z (low resolution) 180(M+, 100), 165(55), 137(35), 119(11), 91(40), 77(10), 65(8). The assignments are in agreement with literature date [12]. 4 -Hyd roxy l - 3 ,5 - d ime thoxy l - benzo i c a c i d (18): m/z (low resolution) 198(M+, 100), 183(37), 153(7), 137(8), 127(16), 109(21), 93(6), 81(7), 65(7), 53(7). A commercial sample (Aldrich) was used to compare the chromatographic retention time and spectral data. 5 ,5 ' -B is - ( l -hydroxy l -e thy l ) -b ipheny l -2 ,2 ' - d io l (19): m/z (low resolution) trimethyl silyl ether 562(M+, 13), 547(100), 473(7), 385(14), 311(3), 73(11). 5,5 ' -B is - ( l -hydroxy l -e thy l ) -3 ,3 ' -d imethoxy l -b ipheny l -2 ,2 ' -d io l (20): m/z (low resolution) trimethyl silyl ether 622(M+, 31), 607(100), 533(7), 445(33), 357(9), 73(16). l - [6 ,2 ' -D ihydroxy l -5 ' - ( l -hyd roxy l -e thy l ) -5 ,3 ' - d ime thoxy l -b ipheny l -3 - y l ] -e thanone (21): m/z (low resolution) trimethyl silyl ether 548(M+, 5), 533(15), 460(40), 445(100), 429(21), 416(8), 371(17), 343(14), 117(19), 73(22). l , l ' - ( 6 , 6 ' -D i hyd roxy l - 5 , 5 ' - d ime thoxy l - l , l ' - b i pheny l - 3 , 3 ' - d i y l ) d i e t hanone (22): 'H-NMR: (DMSO-d6) 5 2.51*(6H, s, ArCOCH3), 3.91(6H, s, ArOCH 3), 7.46QH, d, J=2.0 ArH), 7.48(1H, d, J=2.0 ArH); 1 3C-NMR: (CDC13) 8 26.75, 56.45, 110.13, 124.95, 125.74, 128.31, 147.94, 149.65, 196.62. The assignments are in agreement with literature data [19]. * overlapped with DMSO-d 6 NMR solvent. 106 2 , 6 - D i m e t h o x y l - 4 , 8 - d i m e t h y l - d i b e n z o f u r a n - l - o l (23): m/z (low resolution) 272(M+, 100), 257(45), 240(55), 229(20), 197(10), 185(7), 169(6), 141(5), 115(5). 4 - H y d r o x y l m e t h y l - 2 , 6 - d i m e t h o x y l - 8 - m e t h y l - d i b e n z o f u r a n - l - o l (24): m/z (low resolution) 288(M+, 100), 274(5), 260(32), 241(20), 227(33), 199(27), 189(9), 172(6), 128(8), 115(4), 91(2). l - H y d r o x y l - 2 , 6 - d i m e t h o x y l - 8 - m e t h y l - d i b e n z o f u r a n - 4 - m e t h y l es te r a c e t i c a c i d (25): m/z (low resolution) 330(M+, 100), 287(57), 272(16), 257(11), 229(5), 204(3), 157(3), 142(2), 111(2), 75(2). 4 - [ 2 - ( 4 - H y d r o x y l - 3 , 5 - d i m e t h o x y l - p h e n y l ) - 2 - o x o - e t h o x y ] - 3 , 5 - d i m e t h o x y l - b e n z o i c a c i d (26): 'H-NMR: (DMSO-d6) 5 3.79(6H, s, ArOCH3), 3.82(6H, s, ArOCH 3), 5.26(2H, s, ArCOCH2), 7.25(2H, s, ArH), 7.27(2H, s, ArH); 1 3C-NMR: (DMSO-d6) 5 56.49, 56.60, 74.50, 106.38, 107.11, 125.27, 126.26, 140.32, 141.74, 148.11, 152.63, 167.36, 192.97. The assignments are in agreement with literature data [15]. 3 , 3 ' - D i m e t h o x y l - 5 ' - ( 2 - m e t h o x y l - 4 - m e t h y l - p h e n o x y m e t h y l ) - 5 - m e t h y l - b i p h e n y l - 2 , 2 ' -d i o l (27): m/z (low resolution) 410(M+, 60), 258(100), 242(18), 227(8), 199(4), 128(2). The assignments are in agreement with literature data [20]. 107 26 27 Figure 3.3 Reaction products identified from the POM oxidation of 1-3, 5-8, 10, and 13. 3.3 Results and Discussion 3.3.1 Kinetics The initial rates of formation of the reduced POM were measured by UV-VIS spectroscopy. The reaction order of both the POM and selected lignin model compounds (1, 2, 3, and 11) was calculated according to equation 2.3 (Chapter 2). The concentration 108 of POM was varied from 0.03 to 3.1 mmol L"1 at a constant lignin model compound concentration of 1.9 mmol L"' for 1 and 0.08 mmol L"1 for 2, 3 and 11. Then, the concentration of lignin model compound was varied from 0.13 to 5.1 mmol L"' with a constant POM concentration of 0.25 mmol L" 1. Plots of the initial rates against lignin model compound or POM concentration produced straight lines that passed through the origin (Appendix). This indicates first-order kinetics with respect to both lignin model compounds and POM. Similarly, first-order kinetics were shown from plots of log[v] versus log [POM0X] or logfsubstrate], Figure 3.4a and 3.4b, respectively [21]. Overall, the PCM oxidation of these lignin model compounds is a second order reaction, and the kinetic rate expression can be written as; dt[POM r H] = 2k[POM o x ] [Substrate] (3.1) However, the initial rate method only samples a few initial data points. In complex reaction systems, the products may participate in the reaction and affect the reaction rate. To avoid this, the rate law should be fit to the data throughout the entire reaction. Accordingly, nonlinear regression analysis was used to confirm the overall second order reaction rate of this system. An explicit equation (equation 3.2) was derived by integrating the effective second-order rate law (see Appendix) [22]. 109 Figure 3.4 Plots of a) log[initial rate] versus log[POM] or b) log[model compounds] at 45 °C (1), 25 °C (2 and 3), and 40 °C (11), in sodium acetate buffer (I = 0.2 M , pH 5.0) under argon. 1 (l-(4-hydroxylphenyl)-ethanol), 2 (l-(4-hydroxyl-3-methoxylphenyl)-ethanol), 3 (l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol) and 11 (4-[2-hydroxyl-2-(4-hydroxyl-3-methoxylphenyl)ethoxyl]-3-methoxyl-benzoic acid). 1 1 0 A, =A 0 +4e [POM]0 [Substrate] 0 -[POM]Q [Substrate] 0 [POM]o exp k [ S u b s t r a t e l _ [ f ° ^ k I 2 (3.2) The second-order rate constant, k was determined by fitting the absorbance versus time data to e q u a t i o n 3.2. The sum of the squares of the deviation was minimized by varying either k or e (extinction coefficient) within less than 0.5 % of the experimental value. F i g u r e 3.5 illustrates the fit (red line) of the experimental data to e q u a t i o n 3.2. Reaction time (min) F i g u r e 3.5 Absorbance-time plot for the reaction of POM (SiVW u0 4o 5") with 3 (l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol) at 40 °C in sodium acetate buffer (I = 0.2 M , pH 5.0) under argon. The open circles represent the experimental data points and the red curve represents the calculated data using e q u a t i o n 3 .2 . The sum of the squares of the standardized residuals was 0.2 x 10"4. I l l T a b l e 3.1 lists the second-order rate constants, k, calculated using the initial rate law and nonlinear regression methods. The values obtained by both methods are in good agreement with each other, and provide additional support for the second order reaction, e q u a t i o n 3 .1 . T a b l e 3.1 Second-order rate constants (k) calculated by either the initial rate law or the nonlinear regression equation. [Substrate] = 0.1 mmol L" 1, [SiVWnCuo5] = 0.25 mmol L"1 in sodium acetate buffer (I = 0.2 M , pH 5.0) under argon. Second order rate constant (M'V1) Temp. Compound — Initial rate Nonlinear regression (°C) 1 0.51 + 0.05 - 50 2 31.3 + 0.5 28.4 + 0.4 40 3 161 + 5 160 + 3 40 4 11.2 + 0.2 - 40 5 84.3 ± 1.8 81.7 + 1.6 40 6 2.95 + 0.07 - 50 7 1.19 + 0.07 - 40 8 59.0 + 0.5 51.0 ± 0.2 40 9 45.4 + 0.8 46.6 ± 0.6 40 10 272 + 10 277 + 12 20 11 4.67 + 0.24 - 30 12 51.6 + 1.1 50.7 + 1.8 30 13 52.9 + 1.3 59.5 + 1.5 30 112 3.3.2 Reactions of p-Hydroxylphenyl, Guaiacyl and Syringyl Lignin Model Compounds 1-(4-HydroxyIphenyl)-ethanol 1, l-(4-hydroxyl-3-methoxylphenyl)-ethanol 2, and l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol 3 were reacted with POM in sodium acetate buffer (pH 5.0) under argon. The reaction rates were very much dependent on the structure of the lignin model compound. The syringyl type model compound 3 reacted more rapidly with POM than the guaiacyl- (2) and p-hydroxylphenyl-type (1) lignin model compounds (Figure 3.6). Figure 3.6 Absorbance-time plots for the reaction of POM (SiVWuO^ 5) with compounds 1, 2, and 3. Reaction conditions: [model compounds 1-3] = 0.1 mmol L"1 and [SiVWuCUo5-] = 0.25 mmol L"1 in sodium acetate buffer (I = 0.2 M , pH 5.0) under argon at 50 °C (1) and 40 °C (2 and 3). 1 (l-(4-hydroxylphenyl)-ethanol), 2 (l-(4-hydroxyl-3-methoxylphenyl)-ethanol) and 3 (l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol). 113 It is well accepted that electron-transfer reactions involving phenols are highly dependent on the nature of the substituent groups on the aromatic ring [5, 23, 24]. Electron donating groups accelerate reaction rates whereas electron withdrawing groups retard reaction rates. Relationships such as the Hammett equation have been extensively used to relate rates and equilibria for many reactions with substituents para-and/or meta-to the phenolic hydroxyl group [25]. Ortho-substitutents are usually omitted as they can impart steric effects, depending on the size of substituent, in addition to electronic effects [25, 26]. In the case of lignin model compounds ortho-substituted methoxyl groups have been shown to form intra-molecular hydrogen bonds with the adjacent phenolic hydroxyl group; no difference exists in the dissociation constants of 2-methoxylphenol and 2,6-dimethoxylphenol derivatives [27]. However, the addition of methoxyl groups at the C-2 and C-6 positions of the phenolic aromatic ring dramatically increased the reactivity with POM; the observed rate constants were found to increase in the following order 1 < 2 < 3. This suggests that the reaction rates could be dependent on the ability of the ortho-substituted methoxyl group(s) to help stabilize the phenoxy radical intermediate via a conjugative resonance derealization as illustrated in Scheme 3.2. The same effect was observed in the reaction of P O M with 4 and 5 (Figure 3.7). The syringyl model compound 5 reacted more rapidly with P O M as compared to the guaiacyl model compound 4. 1 1 4 o o o R = H, O C H 3 Scheme 3.2 Possible conjugative delocalized resonance structures of the phenoxy radical intermediate. 0.7 Reaction time (min) Figure 3.7 Absorbance-time plots for the reaction of P O M (S iVWn0 4 o 5 ~) with compounds 4 and 5. Reaction conditions: [model compounds 4 and 5] = 0.1 mmol L" ' and [ S i V W n 0 4 o 5 " ] = 0.25 mmol L " 1 in sodium acetate buffer (I = 0.2 M , p H 5.0) under argon at 40 °C. 4 (l-(4-hydroxyl-3-methoxylphenyl)-ethyl methyl ether) and 5 ( l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethyl methyl ether). Product analysis revealed the formation of several oxidation and oxidative coupling reaction products. In the reaction of 1, 4-(l-hydroxyl-ethyl)-[l,2]benzoquinone (14) and 115 5,5'-bis-(l-hydroxyl-ethyl)-biphenyl-2,2'-diol (19), were detected, while in the reaction of 2, 5,5'-bis-(l-hydroxyl-ethyl)-3,3'-dimethoxyl-biphenyl-2,2'-diol (20) and l-[6,2'-dihydroxyl-5'-(l-hydroxyl-ethyl)-5,3'-dimethoxyl-biphenyl-3:-yl]-ethanone (21) were identified (Scheme 3.3). 19 :R = H 20: R = OCH3 Scheme 3.3 Possible reaction mechanisms for POM (SiVWuO^5") oxidation of 1 (l-(4-hydroxylphenyl)-ethanol) and 2 (l-(4-hydroxyl-3-methoxylphenyl)-ethanol). 116 In the reactions of 1 and 2 with POM, the initial electron-transfer reaction leads to the formation of a phenoxy radical intermediate. The phenoxy radical either reacts with a second phenoxy radical to form the dimeric products 19 and 20, or undergoes a second electron-transfer reaction with P O M to the corresponding cation intermediate and ultimately product 14. The reaction product 20 can further react with P O M to give the a-ketone product, 21. The reaction mechanism likely involves the same sequential single electron transfer process as that occurring in the oxidation of 3 to 7 as outlined in Scheme 3.4. Again, the first electron-transfer involves the phenolic moiety and phenoxy radical formation, however, the second electron-transfer likely involves the benzyl C-H bond to give the a-keto product. (The bond-dissociation energy of benzyl C-H in toluene (88 kcal/mol) is quite similar to that of phenolic O-H (86 kcal/mol) [28].) In the reaction of 3, the primary products detected were 4-acetyl-2,6-dimethoxylphenol (7) and 2,6-dimethoxyl-[l,4]benzoquinone (16), along with as a small amount of o-benzoquinone, 5-(l-hydroxyl-ethyl)-3-methoxyl-[l,2]benzoquinone (15). The formation of both 15 and 16 involve consecutive electron-transfer reactions to the corresponding resonance stabilized carbocation, followed by nucleophilic attack of water and subsequent substituent elimination [5]. 117 7 (23%) Scheme 3.4 Possible reaction mechanisms for the POM (SiVWnO^5") oxidation of 3 (1-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol). 118 3.3.3 Reactions of Guaiacyl Lignin Model Compounds with Different para-Position Substituent Groups The effect of para-substituents on the reactivity of lignin-based moieties with P O M was studied using several guaiacyl-based lignin model compounds; l-(4-hydroxyl-3-methoxylphenyl)-ethanol (2), l-(4-hydroxyl-3-methoxylphenyl)-ethyl methyl ether (4), 4-acetyl-2-methoxylphenol (6) and 4-ethyl-2-methoxylphenol (9). Figure 3.8 illustrates the relative reactivities of the various lignin model compounds. The observed rate constants are included in Table 3.1. Reaction time (min) Figure 3.8 Absorbance-time plots for the reaction of P O M (SiVWi,0 4 o 5 ~) with compounds 2, 4, 6 and 9. Reaction conditions: [model compounds 2, 4, 6 and 9] =0.1 mmol L ' 1 and [ S i V W u 0 4 o 5 " ] = 0.25 mmol L" 1 in sodium acetate buffer (I = 0.2 M , p H 5.0) under argon at 50 °C (6) and 40 °C (2, 4 and 9). 2 (l-(4-hydroxyl-3-methoxylphenyl)-ethanol), 4 (l-(4-hydroxyl-3-methoxylphenyl)-ethyl methyl ether), 6 (4-acetyl-2-methoxylphenol) and 9 (4-ethyl-2-methoxylphenol). 119 The observed rate constants increased in the following order 6 < 4 < 2 < 9. This trend indicates that the rates are sensitive to the nature of the substituent groups at the para-position of the guaiacyl ring. This is consistent with the electron density of the aromatic ring having a significant role in the first electron-transfer reaction. In the case of the oxygenated compounds 2 and 4, the substituents act as inductive electron-withdrawing groups, while in 6 the substituent acts as a resonance conjugated electron-withdrawing group. These substituents decrease the electron density of the aromatic system, and thereby the rates of electron transfer. By contrast, the ethyl substituent in 9 acts as a weak inductive electron-donating group, and activates the aromatic ring towards electron abstraction and oxidation. At the same time, the alpha methylene group acts to stabilize the forming phenoxy radical through hyperconjugation. Product analysis of the POM reactions with 2 and 6 reveal the major products were 5,5'-bis-(l-hydroxyl-ethyl)-3,3'-dimethoxyl-biphenyl-2,2'-diol (20) and l,l'-(6,6'-dihydroxyl-5,5'-dimethoxyl-l,l'-biphenyl-3,3'-diyl)diethanone (22), respectively. These dimeric products likely arise from radical coupling reaction between two phenoxy radical intermediates as outlined in Scheme 3.3. 120 3.3.4 Reactions of Syringyl Lignin Model Compounds with Different para Position Substituent Groups The effect of syringyl-based lignin model structure on the reactivity with POM was studied using l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol (3), l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethyl methyl ether (5), and 4-acetyl-2,6-dimethoxylphenol (7). As with the guaiacyl compounds (2, 4 and 6), the relative reactivities of the syringyl compounds was a-hydroxyl compound 3 > a-methyl ether compound 5 > a-carbonyl compound 7 (Figure 3.9). 0.7 0 1 2 3 4 5 Reaction time (min) Figure 3.9 Absorbance-time plots for the reaction of POM (S1VW11O40 5 ) with compounds 3, 5 and 7. Reaction conditions: [model compounds 3, 5 and 7] = 0.1 mmol L" 1 and [SiVWn04o5"] = 0.25 mmol L"1 in sodium acetate buffer (I = 0.2 M, pH 5.0) under argon at 40 °C. 3 (l-(44iydroxyl-3,5-dimethoxylphenyl)-ethanol), 5 (l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethyl methyl ether) and 7 (4-acetyl-2,6-dimethoxylphenol). 121 Unlike the guaiacyl counterparts, the syringyl-based compounds cannot undergo oxidative coupling reactions. The C-2 and C-6 position of the phenols are substituted with methoxyl groups. In the reaction of 3, the primary oxidation products were 4-acetyl-2,6-dimethoxylphenol (7 - 23 % yield) and 2,6-dimethoxyl-[l,4]benzoquinone (16 - 21 % yield), along with .a small amount of orthoquinone, 5-(l-hydroxyl-efhyl)-3-methoxyl -[l,2]benzoquinone (15 ~ 2 % yield). As illustrated in Scheme 3.3, products 15 and 16 likely form via nucleophilic attack of water to the cationic carbon positions of the cyclohexadienone intermediate formed after two consecutive one electron-transfer reactions. On the other hand, 7 may be formed via a second electron-transfer from the benzyl C-H bond to give a quinone-methide intermediate (Scheme 3.3). Although 16 could arise from further POM oxidation of 7, we do not believe this to be the case as the reaction rate of 7 under these conditions is extremely low (Figure 3.9, Table 3.1). As with 3 the primary reaction products from compound 5 were 7 (16 % yield) and 16 (15 % yield). Scheme 3.5 outlines a possible reaction mechanism leading to products 7 and 16. In the case of 7, a small amount of 2,6-dimethoxyl-[l,4]benzoquinone (16) was detected by GC-MS. In addition, a bright red extract was observed in the POM oxidation of the syringyl model compounds, 3, 5, and 7, which based on the earlier reports of Weinstock et al. [5] may be o-naphthaquinone type products. 122 o 16 (15%) Scheme 3.5 Possible reaction mechanism for POM (S1VW11O40 5") oxidation of 5 (l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethyl methyl ether). 3.3.5 R e a c t i o n s o f A l k y l a t e d G u a i a c y l a n d 5-5 D i m e r L i g n i n M o d e l C o m p o u n d s Figure 3.10 shows the relative reactivities of 4-methyl-2-methoxyl-phenol (8), 4-ethyl-2-methoxyl-phenol (9) and 3,3'-dimethoxyl-5,5'-dimethyl-biphenyl-2,2'-diol (10) with POM in sodium acetate buffer (pH 5.0) under argon. It is evident that in comparison to 8 and 9 the reaction of 10 with POM is extremely fast. In fact 10 exhibited the fastest reaction rate among all the lignin model compounds studied (Table 3.1). It is significant to note that dimeric structures such as 10 are abundantly present in both native and residual lignin, but have reportedly low reactivity towards other typical oxidative conditions [29, 30]. 123 0.7 Reaction time (min) F i g u r e 3.10 Absorbance-time plots for the reaction of POM (SiVWuO^ 5) with compounds 8, 9 and 10. Reaction conditions: [model compounds 8 - 10] = 0.1 mmol L"1 and [SiVWiiO4 0 5"] = 0.25 mmol L " ! in sodium acetate buffer (I = 0.2 M , pH 5.0) under argon at 20 °C. 8 (4-methyl-2-methoxyl-phenol), 9 (4-ethyl-2-methoxyl-phenol) and 10 (3,3'-dimethoxyl-5,5'-dimethyl-biphenyl-2,2'-diol). Analogous to methoxyl groups, ortho-phenol substitutes are inductive electron withdrawing and resonance electron donating substituent groups. In Chapter 2 a Hammett plot of the oxidation reaction kinetics for a series of substituted phenols revealed a relatively large negative reaction constant (p = - 4.7), indicating electron donating groups strongly facilitate the reaction. Furthermore, that study found the reaction system involves the formation of an electron-deficient radical intermediate as the rate-determining step. Therefore, the ortho-phenol substitute appears to not only increase the electron density of the phenolic ring, promoting oxidation, but also dramatically stabilizes 124 the phenoxy radical intermediate through resonance stabilization and derealization of the intermediate radical throughout the second aromatic ring. Comparison of the reaction rates of 8 and 9 reveals a slight increase in reactivity for 8 as compared to 9 (Figure 3.10). From Table 3.1 the second order rate constants are ~ 51 and 47 M"'s~', respectively. The slightly faster reaction rate of 8 is likely the result of the increased resonance stabilization of the phenoxy radical through increased hyperconjugation. Radical stabilization involves the interaction of the higher energy SOMO (singly occupied p orbital) and the lower energy HOMO (highest occupied molecular orbital, the C-H bonding orbital localized on the methylene group) and the formation of two new orbitals, one lower in energy than the HOMO and one higher in energy than the SOMO. A critical factor in this phenomenon is the correct geometry of the C-H bonding orbital with the p orbital; the C-H bonding orbital must eclipse the p orbital. Statistically and energetically, the methyl substituent will have increased hyperconjugation as compared to higher order alkyl chains; i.e. more readily form the required eclipsed geometry (more C-H bonds and a less bulky group). In the reaction of 8, the primary oxidation product was oxidadve coupling and the formation of 3,3'-dimethoxyl-5,5'-dimethyl-biphenyl-2,2'-diol (10), along with a trace amount of 3,3'-dimethoxyl-5'-(2-methoxyl-4-methyl-phenoxymethyl)-5-methyl-biphenyl-2,2'-diol (27). The reaction presumably involves an electron-transfer reaction to give a 125 phenoxy radical intermediate, followed by radical coupling. Subsequently, product 10 may undergo further POM oxidation and formation of a resonance stabilized phenoxy radical intermediate. This very stable intermediate can then undergo a second oxidation, this time involving the benzylic hydrogen followed by intra-radical coupling to give the product, 2,6-dimethoxyl-4,8-dimethyl-dibenzofuran-l-ol (23). In the presence of excess POM 23 can undergo further oxidation reactions leading to products such as 4-hydroxylmethyl-2,6-dimethoxyl-8-methyl-dibenzofuran-l-ol (24) and l-hydroxyl-2,6-dimethoxyl-8-methyl-dibenzofuran-4-methyl ester acetic acid (25) as outlined in Scheme 3.6. The reaction of POM with 10 produced trace amounts of numerous unidentified reaction products. The proposed products 23-25, could not be isolated and confirmed by NMR, nor are they reported anywhere in the literature. Therefore, the reaction mechanisms presented in Scheme 3.6 are just proposed mechanisms, and require further investigation. 126 OH 25 Scheme 3.6 Possible reaction mechanism for P O M ( S i V W i i O 4 0 5 " ) oxidation of 8 (2-methoxyl-4-methyl-phenol) and 10 (3,3'-dimethoxyl-5,5'-dimethyl-biphenyl-2,2'-diol). 127 3.3.6 Reac t ions of £ - 0 - 4 L i g n i n M o d e l C o m p o u n d s F i g u r e 3.11 shows the relative reaction rates of a guaiacyl-guaiacyl B-0-4 (11), syringyl-guaiacyl J3-0-4 (12), and syringyl-syringyl [3-0-4 (13) model compound with POM in sodium acetate buffer (pH 5.0) at 40 °C under argon. The syringyl-based phenolic compounds 12 and 13 were much more reactive towards POM oxidation than compound 11. This is consistent with the observed reactivity of simple syringyl- and guaiacyl-based lignin model compounds (Sec t i on 3.3.2). The initial reactivities of 12 and 13 are identical ( F i g u r e 3.11), but deviate slightly at higher POM conversion, with 13 having a higher reaction rate than 12. The slight increase in reactivity observed after 3 minutes for 1 3 as compared to 12 may be due to the influence of reaction products on the observed rate. It is likely that the syringyl fragments from 13 react faster than the guaiacyl fragments from 12. This observation provides further evidence that caution should be taken when utilizing initial rate methods to study the reaction kinetics of complex reaction systems. To simplify the reaction system for product analysis, we analyzed the syringyl-syringyl {3-0-4 model compound 13. As per the reaction with the simple syringyl lignin model compound 3 the major POM reaction product with 13 was the a-hydroxyl oxidation product 4-[2-(4-hydroxyl-3,5-dimethoxyl-phenyl)-ethoxyl]-3,5-dimethoxyl-benzoic acid (26 - 30 % yield). 128 0 3 , 6 9 12 15 Reaction time (min) Figure 3.11 Absorbance-time plots for the reaction of POM (SiVWnO^5") with compounds 11, 12 and 13. Reaction conditions: [model compounds 11 - 13] =0.1 mmol L"1 and [SiVW,,04o5"] = 0.25 mmol L"1 in sodium acetate buffer (1 = 0.2 M , pH 5.0) under argon at 30 °C. 11 (4-[2-hydroxyl-2-(4-hydroxyl-3-methoxylphenyl)ethoxyl]-3-methoxyl-benzoic acid), 12 (4-[2-hydroxyl-2-(4-hydroxyl-3,5-dimefhoxylphenyl)ethoxyl]-3-methoxyl-benzoic acid) and 13 (4-[2-hydroxyl-2-(4-hydroxyl-3,5-dimethoxylphenyl)ethoxyl]-3,5-dimethoxyl-benzoic acid). As per 3, the reaction mechanism likely involves first the formation of the phenoxy radical intermediate followed by a second oxidation involving the benzyl C-H bond [5]. GC-MS analysis of the reaction system also revealed the formation of a small amount of 4-acetyl-2,6-dimethoxylphenol (7), 2,6-dimethoxyl-4-vinyl-phenol (17) and 4-hydroxyl-3,5-dimethoxyl-benzoic acid (18) (Scheme 3.7). 129 OH OH OH 17 7 18 Scheme 3.7 Products detected from the POM (SiVWn04o5") oxidation of 13 (4-[2-hydroxyl-2-(4-hydroxyl-3,5-dimethoxylphenyl)ethoxyl]-3,5-dimethoxyl-benzoic acid) at 25 °C. 3.4 Conclusions Lignin is a complex biomacromolecule formed by dehydrogenative radical coupling of hydroxylphenyl-, guaiacyl- and syringyl-based precursors. As a result, lignins contain numerous structural moieties and functional groups. Therefore, to better understand the delignification reactions during POM ([SiVWuO^]5-) oxidation under anaerobic conditions we investigated the reactions of several monomeric and dimeric lignin model compounds. As was found for the simple phenols in Chapter 2, the lignin model compound reactions are overall second order reactions; first-order in lignin model-130 compound and POM. A dramatic increase in reactivity with POM was observed when methoxyl groups were introduced ortho to the phenolic hydroxyl group. The observed rate constants were found to increase in the following order hydroxylphenyl < guaiacyl < syringyl lignin model compounds. The observed increase in reaction rate is likely the result of the ortho-substituted methoxyl group increasing the electron density (reactivity) of the phenolic ring, in combination with stabilization of the phenoxy radical intermediate through resonance stabilization and derealization. The reactivity of POM with guaiacyl and syringyl model compounds having different para-substituents on the aromatic ring showed a strong dependence on the nature of the substituent. Inductive and/or resonance-conjugated electron-withdrawing groups retarded the rate of POM oxidation, whereas inductive electron-donating groups accelerated oxidation; the rate of electron transfer is significantly dependent upon the electron density of the phenolic ring. Multiple electron-transfer reactions could take place depending on the model compound structure and reaction conditions. Analysis of the POM oxidation products obtained from syringyl lignin model compounds and a syringyl-syringyl 0-0-4 lignin dimer compound revealed a second electron-transfer occurs at the benzylic proton resulting in the formation of a-carbonyl groups. The POM oxidation of the 5-5' dimer lignin model compound was extremely fast. In 131 fact, it had the fastest reaction rate of the model compounds studied. This is a significant finding, as 5-5'-type moieties are generally regarded as difficult structural units to remove during delignification. 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Polyoxometalate (POM) Oxidation of Mi l led Wood Lignin ( M W L ) 3 3 A version of this chapter has been submitted for publication. Kim, Y.S., Chang, H.-M. and Kadla, J.K. (2007) Polyoxometalate (POM) Oxidation of Milled Wood Lignin (MWL), Journal of Wood Chemistry and Technolody. 136 4 .1 I n t r o d u c t i o n Polyoxometalates (POMs) are a growing class of delignification reagents. They are environmentally benign alternatives to conventional bleaching chemicals that are well suited for total chlorine free (TCF) bleach plant processes [1-3]. POMs have been reported to efficiently and selectively remove lignin from pulps under both aerobic and anaerobic conditions [1-4]. Several investigations into the mechanisms of POM oxidation of lignin model compounds have been reported [1, 2, 5-7], and some pulp bleaching experiments have been performed [1, 2, 4, 8]. However, very little information is available on the effect of POM oxidation on the chemical structure of the lignin macromolecule at the molecular level. Detailed information on the mechanisms of oxidation of macromolecular lignin would significantly contribute to a more comprehensive understanding of the chemistry of POM oxidation, and help improve process operation. In particular information is lacking regarding both fundamental mechanisms of homogeneous oxidation as well as the effects of interactions between POMs and polymeric (insoluble) lignin in wood-cell walls. In this paper, we report the oxidation of a milled wood lignin by oc-[SiVWio04o]5", the dominant oxidatively active species in the prototypical industrially relevant POM system, Na5 (+i.9)[SiVi (_ o.i)MoWio(+o i)] [9]. In this paper we report on the chemical structural changes of MWL after POM oxidation using elemental and methoxyl analyses, gel permeation 137 chromatography (GPC) and spectroscopic techniques, including Fourier transform infrared (FT-IR) spectroscopy, *H and 1 3 C nuclear magnetic resonance (NMR) spectroscopy and two-dimensional heteronuclear single quantum coherence (HSQC) NMR spectroscopy. 4.2 E x p e r i m e n t a l 4.2.1 M a t e r i a l s Air-dried Lodgepole pine (Pinus contorta) was milled in a Wiley mill using a 40-mesh screen. The coarsely milled wood was extracted with acetone for 48 h using a Soxhlet apparatus. After extraction, the wood was air-dried and stored in a 40 °C vacuum oven and left at least 1 week prior to use. K5[SiVWn04o]12H20 (POM) was provided from the USDA Forest Products Laboratory (Madison WI, U.S.) [1]. CDC13 and DMSO-d(, were purchased from Cambridge Isotope Laboratories. Anhydrous ethyl ether, 1,2-dichlorethane, 1,4-dioxane, tetrahydrofuran and pyridine were purchased from Sigma-Aldrich. Acetic acid and ethanol were purchased from Fisher Scientific. All chemicals and solvents were used as received. 4.2.2 P l a n e t a r y B a l l M i l l i n g M e t h o d The dried Wiley wood was ground using a planetary ball mill (Retsch PM 200) with 138 two 50 mL zirconium oxide jars. Each jar contained 10 g of extractive-free Wiley wood and 6 zirconium oxide balls (20 mm diameter). Samples were ground under an argon atmosphere at 650 rpm for 15 h. To prevent overheating and thermal changes to the wood, the samples were milled for 30 min intervals, between which the samples were allowed to cool for 30 min. After milling, the milled wood was dried in a 40 °C vacuum oven for at least 1 week prior to use. 4.2.3 M i l l e d W o o d L i g n i n ( M W L ) I s o l a t i o n MWL was isolated according to the method of Bjorkman [10, 11]. Accordingly, 19 g of milled wood were put into a 500 mL capped centrifuge bottle and 200 mL of dioxane/water (96:4, v/v) was added. The solution was shaken at room temperature for 24 h under a nitrogen atmosphere. The mixture was centrifuged at 8000 rpm for 15 min and the supernatant collected. The remaining solid was resuspended in 200 mL of dioxane/water (96:4, v/v) and the above procedure repeated. The combined supernatants from centrifugation were concentrated and added dropwise to deionized water and then freeze-dried for 3 days using a VirTis EX freeze dryer. The resulting crude MWL (3.2 g) was stored in a 40 °C vacuum oven for 1 week prior to purification. The crude MWL (3 g) was dissolved in 60 mL of 90 % acetic acid. The acetic acid solution was then added dropwise with stirring to 660 mL of deionized water in order to 139 precipitate the lignin. The precipitated lignin was centrifuged, freeze-dried and then stored in a 40 °C vacuum oven. The dried lignin was dissolved in 60 mL of 1,2-dichloroethane/ethanol (2:1, v/v) and centrifuged to remove any insoluble material. The resulting supernatant was added dropwise to 690 mL of anhydrous ethyl ether to precipitate the purified lignin. After centrifugation, the pellet was washed three times with fresh petroleum ether and dried in a 40 °C vacuum oven for 1 week. The MWL isolation procedure is summarized in Figure 4.1. Lodgepole pine Wiley milling Acetone extraction Wiley wood Milling by a planetary ball-mill 15 hours Milled wood Extraction with dioxane/water and purified according to Bjorkman Soluble Milled wood lignin (MWL) Figure 4.1 Preparation of milled wood and milled wood lignin (MWL). 140 4.2.4 POM Treatment of MWL The reaction of POM with MWL was performed by first weighing 150 mg of MWL into a septum-sealed serum bottle containing 20 mL of degassed sodium acetate buffer (0.2 M , pH 5.0). All of the air inside the bottle was displaced by purging repeatedly with argon and 10 mL of degassed POM stock solution were added to the sealed bottle. The final concentration of MWL and POM was 0.5 wt. % and 0.05 M , respectively. The bottle was quickly immersed in an oil-bath maintained at 90 °C with constant stirring. After 1 h of reaction, the bottle was rapidly cooled by immersion in an ice-water bath. The lignin was then filtered, washed with water and freeze-dried. 4.2.5 Acetylation of MWL MWL and POM-treated MWL (POM-MWL) samples were acetylated for physical and chemical analysis. Acetylation was performed using 100 mg of purified lignin dissolved in 4 mL of pyridine/acetic anhydride (1:1, v/v), and the mixture then being stirred for 24 h at room temperature. The reaction solution was added dropwise, with stirring, to 100 mL of ice-water. The precipitated lignin was collected by filtration through a Nylon membrane (0.45 pm, 47 mm), washed with ice-water and freeze-dried using a VirTis EX freeze dryer. This procedure was repeated to ensure complete acetylation of the samples. 141 4.2.6 Determination of Lignin content in MWL The lignin content of the purified MWL and extractive-free Wiley wood was determined using the Klason method (TAPPI Method T249 cm-85). Acid-soluble lignin was quantified by UV-Vis spectroscopy at 205 nm according to TAPPI Useful Method UM250. Al l samples were measured in duplicate. 4.2.7 Gel Permeation Chromatography (GPC) and Fourier Transform Infrared (FT-IR) analysis The molecular mass distribution of the acetylated lignin samples were determined by GPC (Agilent 1100, UV and RI detectors) using styragel columns (Styragel HR 4 and HR 2) at 35 "C, THF as the eluting solvent (0.5 mL min"1) and UV detection at 280 nm. The lignin concentration was 1 mg mL'1 and the injection volume was 75 uL. The GPC system was calibrated using standard polystyrene samples with molecular weights ranging between 580 and 1,800,000 Daltons. Fourier transform infrared (FT-IR) analysis was performed using a Perkin-Elmer Spectrum One FT-IR equipped with attenuated total reflectance (ATR) attachment. A total of 128 scans per sample were acquired at a spectral resolution of 4.0 cm"1. FT-IR spectra were directly obtained using oven-dried lignin samples. The FT-IR spectra were further analyzed using Peak Fit software (SPSS Inc., 142 Chicago, IL). Deconvolution was performed using the Gaussian peak shape and a full width at half-maximum (fwhm) of 20 - 30 cm"1; compared to ensure good resolution of peaks without overfitting. The number of peaks was analyzed using the second-derivative spectra prior to deconvolution. All peaks were fit until regression values (r ) were greater than 0.995 [12]. 4.2.8 Nuclear Magnetic Resonance (NMR) analyses Table 4.1 lists the experimental parameters used in the respective NMR experiments. Table 4.1 Conditions for NMR experiments. Temperature Acquisition Relaxation Pulse Number of Experiment (°C) Time (s) Delay (s) Width Scans 'H 25 1.3 7.0 90° 128 , 3 C 40 1.4 1.7 90° 20,000 HSQC 25 0.12 1.0 90° 512 4.2.8.1 Quantitative lU NMR spectroscopy Quantitative *H NMR spectroscopy was performed on a Bruker Avance 300 MHz spectrometer equipped with a BBI probe at 40 °C. Forty mg of acetylated lignin was accurately weighed, dissolved in 0.75 mL of CDCI3 and filtered prior to NMR analysis. 143 Quantification of the relative amounts of the various functional groups was performed according to Adler et al. [13]; the integral of the aromatic proton region (7.2 - 6.4 ppm) was set equal to 2.7 and 2.6 for the MWL and POM-MWL, respectively. These values are based on 2 protons associated with C-2 and C-6, as well as 0.6 and 0.5 protons for the C-5 of MWL and POM-MWL, as well as a small contribution from certain vinyl proton (-0.1). The number of C-5 protons (0.6 and 0.5) was calculated based on the degree of condensation determined from 1 3 C NMR (39 and 46 per 100 aromatic rings for MWL and POM-MWL, respectively). 4.2.8.2 1H- 1 3C two-dimensional Heteronuclear Single Quantum Coherence (HSQC) NMR spectroscopy HSQC spectroscopy was performed on a Bruker Advance 300 MHz spectrometer equipped with a BBI probe using 40 mg of acetylated lignin in 0.75 mL of CDCI3. The solution was filtered prior to NMR analysis. 4.2.8.3 Quantitative 1 3 C NMR spectroscopy Quantitative 1 3 C NMR spectroscopy was performed on a Bruker Avance 300 MHz equipped with a BBO probe using 60 mg of acetylated lignin in 0.25 mL of DMSO-d6. The sample solutions were filtered prior to NMR analysis in a Shigemi tube. Relaxation 144 was facilitated by the addition of 10 uL of a chromium acetoacetonate solution (final concentration 10 mM) [14]. 4.3 Results and Discussion Table 4.2 reports the yield and lignin content of the extractive-free wood and purified MWL. The yield of extracted MWL was 26 % with a 91 % Klason lignin content after purification. The yield of POM-MWL was approximately 90 % after POM reaction. Table 4.2 Yield and Klason lignin content of the Lodgepole pine and purified MWL. Extracted lignin yield Klason lignin content (% + 4.2%) (%) soluble insoluble Total Extractive-free Wiley wood 0.5 25.7 26.5 Purified MWL 26.0a 0.2 90.6 90.8 a extracted lignin yield of purified MWL was calculated based on total lignin content in mountain pine determined by Klason lignin content as referenced 100 %. 4.3.1 Structural Analyses of MWL and POM-MWL using NMR Spectroscopy NMR spectroscopy has been extensively used to investigate the structure of lignin [14, 15]. NMR provides a comprehensive view of the entire lignin macromolecule. Quantitative ! H NMR spectra were obtained for both acetylated MWL and POM-MWL 145 (Figure 4.2). The relative amounts of the various functional groups was determined by setting the integral of the aromatic proton region (7.2 - 6.4 ppm) equal to 2.7 and 2.6 for acetylated MWL and POM-MWL, respectively [13]. Table 4.3 lists the 'H NMR signal assignments and their relative abundance for the MWL and POM-MWL samples. Comparison of the MWL before and after POM oxidation reveals significant changes in the MWL occur; specifically, cleavage of a-O-4 and P-O-4 linkages and lignin oxidation. There is an increase in the phenolic hydroxyl group content from 17 to 23 per 100 aromatic rings for the acetylated MWL and acetylated POM-MWL, respectively. There is also a decrease in the aliphatic hydroxyl content from 101 to 99 per 100 aromatic rings MWL and POM-MWL, respectively. The generation of new phenolic hydroxyl groups is likely arising from the cleavage of a-O-4 and [3-0-4 linkages (Schemes 3.4 and 3.5), which is evident from large decrease in the relative amount of benzylic protons (5.7 and 1.5 per 100 aromatic rings for MWL and POM-MWL, respectively), and the increase in the aromatic protons associated with the C-2 and C-6 position of the aromatic ring in phenolic oc-CO moieties (1.3 per 100 aromatic rings for POM-MWL). 146 Figure 4.2 Quantitative 'H NMR spectra for a) acetylated MWL and b) acetylated POM-MWL. ^Unknown signals or impurities. 147 Table 4.3 Signal assignment in the ] H NMR spectra of acetylated MWL and POM-MWL samples [16-19]. Peak Chemical shift Spectral region MWL POM-MWL label range (ppm) assignment (per 100 Ar) (per 100 Ar) 1 9 . 9 - 9 . 8 Ar-CHO (M) 0.3 0.4 2 9 . 8 - 9 . 5 Ar-CHO (L) 0.8 0.9 3 8 . 2 - 7 . 9 Ar-H 2 o r H 6 in D, R - 1.3 4 7 . 5 - 7 . 4 Ar-H in benzaldehyde and H-a in L 3.3 4.4 5 6 . 2 - 5 . 9 H-a in A , B , C, G and H-p in K 29 17 6 5 . 7 - 5 . 4 H-a in E 20 25 7 4 . 8 - 4 . 6 H-P in A , B , C, G and H- a in K 31 26 8 4 . 6 - 4 . 4 H -Y in A , E 39 47 9 4 . 4 - 4 . 2 H-y in several structures 47 53 10 4 . 1 - 3 . 4 Methoxyl groups 100* 94* 11 2 . 7 - 2 . 6 Benzylic protons in several structures 5.7 1.5 12 2 . 4 - 2 . 2 Aromatic OH (acetylated) 17* 2 3 * 13 2 . 2 - 1 . 7 Aliphatic OH (acetylated) 101* 99 * 14 1 . 6 - 1 . 5 Unknown - 1.0 15 1 .4 - 1.2 Hydrocarbon (contaminant) • 13 25 - Absent or too small for interpretation (see Figure 4 for substructure identity). * methoxyl groups and acetyl methyl groups contain 3H per group. In order to obtain detailed information on the abundance of each inter-unit linkage, quantitative l 3 C NMR spectroscopy was also performed for both acetylated and non-acetylated MWL and POM-MWL (Figure 4.3). 1 3 C NMR spectroscopy can be used to quantitatively estimate the abundance of each inter-unit linkage in the lignin macromolecule [14, 17, 20]. However, there still exists some overlap in the spectra, making estimations of the structural formations difficult [17]. Unfortunately, the non-148 acetylated POM-MWL did not completely dissolve at concentrations suitable for NMR analysis. This may be the result of significant chemical structural changes of the MWL after POM treatment. Quantitative carbon for this POM-MWL did not yield a spectrum with sufficient resolution to provide any useful data. As a result, only comparison between the acetylated MWL and POM-MWL are presented. Ac-MWL Ac-POM-MWL 1 ' 2 ' l' — i — . — i — i — i — . — i — . — i — . — , — 200 180 160 140 120 100 80 60 40 20 ppm Figure 4.3 Quantitative 1 3 C NMR spectra for acetylated MWL and acetylated POM-MWL. 149 The 1 3 C NMR spectra can be separated into different structural regions and integrated to obtain structural information (Tables 4.4 and 4.5 and Figure 4.3). For the estimation of the amounts of inter-unit linkages and functional groups, the aromatic region from 162 to 103 ppm was set equal to an integration of 6.12 [14, 21]. This value represents the six aromatic carbons plus a contribution of 0.12 per 100 aromatic units from the side-chain of coniferyl alcohol and coniferyl aldehyde structures [22]. 150 Table 4.4 Signal assignment in the 1 3 C NMR spectrum of acetylated MWL and POM-MWL samples [14, 15, 21]. Peak Chemical shift Spectral region MWL POM-MWL label range (ppm) assignment (per lOOAr) (per lOOAr) 1' 196-193 CO in a-CO/p-O-4 (D), L 3 6 2' 193-191 Ar-CHO (M) 1 2 3' 182-180 C-4 in I 1 1 4' 171.3-170.1 Primary aliphatic OH 73 69 5' 170.1 - 169.3 Secondary aliphatic OH 31 29 6' 169.3-168 Phenolic OH, conjugated COOR 21 25 7' 162-160 C-4 in etherified p-hydroxyl- - 8 ' phenyl-units 8' 162- 148.5 All C-3 (except E and p- 106 93 hydroxylphenyl-uni ts), C-5 in S, C-a in L, C-5 in I, C-4 in conjugated CO/COOR etherified and p-hydroxylphenyl-. units 9' 144.5 - 142.5 C-3 in E, C-4 in conjugated Si, 8 7 unknown 10' 87.8-86 C-a in E 10 9 11' 85.3 - 84.7 C-a in F, G 10 8 12' 83.2-81.5 •C-p inG 4 4 13' 77 - 72.5 C-a in A, H, P, carbohydrates 42 30 14' 58-54 OMe, C-l and C-P in I 108 98 15' 50.7 - 49 C-P in H, E 12 10 16' 34 - 33.4 C-a in J 1 1 17' 32.5-31.5 C-a inQ 1 1 Clusters 125 - 103 C A r -H 261 255 90-58 Alk-O- 215 196 90-77 Alk-O-Ar, a-O-Alk 69 64 77-65 y -OAlk, OH s e c 63 58 65-58 80 74 - Absent or too small for interpretation (see Figure 4.4 for substructure identity). 151 Table 4.5 Quantification of inter-unit linkages and functional groups in M W L and POM-M W L via quantitative l 3 C N M R [14]. M W L POM-MWL Structure Calculation (per 100 Ar) (per 100 Ar) P-0-4A (77 - 72.5 ppm) - H 40 29 Spirodienone (I) (182- 180 ppm) 1 1 Phenylcoumaran (E) (87.8 - 86 ppm) 10 9 Dibenzodioxocin (G)B (85.3 - 84.7 ppm) 10 8 (3-1 (H) (50.7 - 49 ppm) - E 2 1 Methoxyl groups (58 - 54 ppm) - H 106 97 Phenolic hydroxyl groups ' H N M R 17 23 Degree of condensation 3 - (125-103 ppm) 39 46 a overestimated because P + 3 (sugar) were not included. b overestimated because F was not included (see Figure 4.4 for substructure identity). 152 S T R = H R = C H O , C H = C H - C H O , C O O H Figure 4.4 Lignin substructures (from Capanema et al. 2004) [14]. 153 4.3.2 Functional Groups (Methoxyl, Hydroxyl and Carbonyl Groups) The estimated amount of inter-unit linkages and functional groups determined for the MWL and POM-MWL via quantitative l 3 C NMR are listed in Tables 4.4 and 4.5. The methoxyl content of the acetylated MWL and acetylated POM-MWL was determined by integrating the methoxyl peak region at 58 - 54 ppm to be 106 and 97 per 100 aromatic rings, respectively (Table 4.5). These values are comparable to that obtained from 'H NMR (100 and 94, respectively). The small decrease in methoxyl content between the two lignins is consistent with the demethoxylation products detected from the lignin model compound reactions (Chapter 3). In the reaction of l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol, demethoxylation and 1,2-benzoquinone formation was observed (Chapter 3). The detected ortho-quinone products (Scheme 3.4) indicate the initially formed phenoxy radical intermediates undergo a second electron transfer with POM followed by nucleophilic attack of water and subsequent hydrolysis of the intermediate. I 3 C NMR analysis of the acetylated MWL and acetylated POM-MWL was also used to determine the amount of aliphatic (OHa]jPh) and phenolic (OHPhen) hydroxyl groups. The aliphatic hydroxyl group content was 104 and 98 per 100 aromatic rings for the acetylated MWL and acetylated POM-MWL, respectively. These values are comparable to those determined by quantitative 'H NMR, 101 and 99 per 100 aromatic rings 154 respectively (Table 4.3). Similarly, the phenolic hydroxyl group content was 21 and 25 ( 1 3C NMR) and 17 and 23 ('H NMR) per 100 aromatic rings for the acetylated MWL and acetylated POM-MWL, respectively. The increase in phenolic hydroxyl content in after POM oxidation was not anticipated, as the lignin model compound studies showed the phenolic moieties react quickly with POM, the relative rate depending on the substituted groups on the aromatic ring. However, as discussed above, the slight increase in the phenolic hydroxyl content in the acetylated POM-MWL is likely the result of degradation of a-O-4 and P-O-4 linkages and the liberation of new free phenolic hydroxyl lignin moieties. Comparison of the carbonyl region (196 - 191 ppm) of the quantitative 1 3 C NMR spectra (Figure 4.3) for the acetylated MWL and acetylated POM-MWL confirms oxidation of the MWL during the reaction with POM. Integration of this region indicates the number of carbonyl structures doubles from 4 to 8 per 100 aromatic rings (Table 4.4). Of these, aldehyde groups such as in vanillin (M) constitute 1 and 2 per 100 aromatic rings for MWL and POM-MWL, respectively. This is slightly higher than the numbers estimated from quantitative ! H NMR (Table 4.3), which are 1.1 and 1.3 per 100 aromatic rings, respectively. Similarly, carbonyl groups such as those found in coniferaldehyde (L) and a-CO/P-O-4 (D) moieties were calculated to be 3 and 6 per 100 aromatic rings for MWL and POM-MWL respectively, (Table 4.4). The increased levels of oxidized carbon 155 structures in the POM-MWL are supported by the model compound studies in Chapter 3. The major reactions of oc-hydroxyl or oc-alkoxyl monomeric and |3-0-4 dimeric lignin model compounds were sequential electron-transfer reactions and carbonyl group formation. In fact, the predominate degradation product detected from the POM oxidation of (4-[2-hydroxyl-2-(4-hydroxyl-3,5-dimethoxylphenyl)ethoxyl]-3,5-dimethoxyl-benzoic acid), an a-OH/|3-0-4 model compound was 4-[2-(4-hydroxyl-3,5-dimethoxyl-phenyl)-2-oxo-ethoxyl]-3,5-dimethoxyl-benzoic acid, the corresponding a-CO/0-O-4 compound (Chapter 3). Furthermore, it is likely that the POM oxidation of MWL involves the degradation of other inter-unit linkages and the formation of smaller lignin moieties with carbonyl groups. 4.3.3 /3-0-4 Inter-unit Linkage The major inter-unit linkage in lignin is the 0-0-4 bond which accounts for approximately 48 % and 60 % of the inter-unit linkages in softwood and hardwood lignins, respectively [23]. Comparison of the amount of 0-0-4 linkages of MWL before and after POM oxidation reveals substantial differences: 40 and 29 per 100 aromatic rings for MWL and POM-MWL, respectively. Even though the amount of 0-0-4 is overestimated (Table 4.5) [14], a large decrease in the amount of 0-0-4 linkages in the POM-MWL indicates that most likely, 0-0-4 inter-unit linkages had reacted with POM. 156 In addition, as shown in Figure 4.3 two strong peaks around 120.3 and 130.6 ppm were observed in the quantitative 1 3 C NMR spectrum of the POM-MWL. In the acetylated HSQC spectrum, Figure 4.5 B, the two peaks occur at 5C/5H 130.6/7.9 and 120.3/8.1, and can be assigned to the aromatic carbons associated with the C-2 and C-6 position of the aromatic ring in phenolic OC-CO moieties. The increase in these peaks in the POM-MWL somewhat explains the observed increase in carbonyl group content. Interestingly, the increase in amount of the carbonyl groups after POM oxidation is only 4 per 100 aromatic rings. This is substantially less than the detected decrease in the amount of {3-0-4 inter-unit linkages, 11 per 100 aromatic rings. This may be due to some of the oxidized MWL fragments arising from POM oxidation of the fi-O-4 inter-unit linkages being soluble in the reaction media; approximately 10 wt % MWL was lost after POM oxidation. 157 Figure 4.5 'H- 1 3 C HSQC spectra of a) acetylated MWL and b) acetylated POM-MWL. Peak labels correspond to Figure 4.4. The yellow color indicates methoxyl groups [17]. 158 4.3.4 A r o m a t i c C a r b o n s a n d D e g r e e o f C o n d e n s a t i o n The aromatic region of the quantitative 1 3 C NMR spectrum can be divided into oxygenated aromatic carbons (162 - 141 ppm), condensed aromatic carbons (141 - 125 ppm), and protonated aromatic carbons (125 - 103 ppm). The degree of condensation was calculated using the protonated aromatic region (125 - 103 ppm) due to some ambiguity in the condensed aromatic carbon region (141 - 125 ppm region), which has overlapping vinylic carbons from coniferyl alcohol (Q) moieties and the {3-carbon in coniferaldehyde (M) [21]. The protonated aromatic region consists of methine carbons (C-2 and C-6) and any protonated C-5 carbons, which are not expected to contain overlapping signals. Softwood lignin primarily consists of guaiacyl phenylpropanoid units that contain a methoxyl and a phenolic hydroxyl group at C-3 and C-4, respectively. Thus, an uncondensed softwood lignin will contain three protonated aromatic carbons per aromatic ring. The degree of condensation is calculated by subtracting the integral of this region (125 - 103 ppm) from 3. Integration values obtained for the MWL and POM-MWL indicate a degree of condensation of 39 and 46 per 100 aromatic rings, respectively (Table 4.5). However, the degree of condensation of the POM-MWL may be slightly overestimated as the signal observed at 120.3 ppm can be assigned to the aromatic ortho carbons of a-CO side-chain containing moieties (discussed above). Regardless, these results suggest either an enrichment of. the condensed units due to preferential 159 degradation of other non-condensed linkages (e.g. P-O-4) or the formation of condensed lignin moieties through radical coupling reactions. In the model compound studies (Chapters 2 and 3), POM oxidation of lignin model compounds such as phenol, cresol and acetovanillone, led to dimeric and even oligomeric products. Furthermore, the reaction of condensed biphenol-type compounds was extremely fast towards POM. Therefore, the increased degree of condensation may reflect the fact that condensed structures were formed via radical coupling reactions during POM oxidation. The oxygenated aliphatic region can also provide more detailed structural information about the inter-unit linkages in the lignin macromolecule [17]. HSQC NMR spectra were used to determine the unobstructed chemical shifts for important inter-unit linkages in lignin such as (3-0-4, [3-0, (3-5, and dibenzodioxocin (Figure 4.5). Both spectra are very similar, as expected based on the subtle differences determined in the amounts of inter-unit linkages (Table 4.5). 4.3.5 Other Lignin Structures One lignin structure that was observed to change dramatically after POM oxidation was the amount of C-4 in p-hydroxylphenyl units (peak 7 in Table 4.4 and Ac-POM-MWL in Figure 4.3). Although this signal was too small to integrate in the MWL 1 3 C NMR spectrum, it was prevalent in the acetylated POM-MWL spectrum at 8 per 100 160 aromatic rings. This apparent enrichment of p-hydroxylphenyl lignin moieties is consistent with the decreased reaction rates of the simple p-hydroxylphenyl model compounds with POM (Chapter 3). The second order reaction rate constant for 1 -(4-hydroxylphenyl)-ethanol was almost two orders of magnitude lower that that of l-(4-hydroxyl-3-methoxylphenyl)-ethanol), 0.47 M'V 1 versus 31.4 M'V 1 , respectively. 4.3.6 F T - I R A n a l y s e s o f M W L a n d P O M - M W L Figure 4.6 shows the FT-IR spectra of the MWL before and after oxidation by POM. The identified peaks and relative absorbance are listed in Table 4.6. 10" 1 3 . . POM-MWL r A11"1.2"/! i" I S " J V I 2" . A 3 " 4 / 1 10" 13" / UT- n 12" A J ' Im"! f] MWL r . 6" Fe- I \ I Y \ 2" t yv 3" 4000 3600 3200 2800 2400 2000 1600 1200 800 Wavenumber (cm"1) Figure 4.6 FT-IR spectra of MWL and POM-MWL. 161 . Relative differences between the MWL and POM-MWL spectra can be seen in the band envelopes associated with the hydroxyl stretching region ( V O H ~ 3700 - 3100 cm"1) and the carbonyl stretching region (Vc=o - 1650 - 1850 cm"1). The other major IR absorption bands, e.g., aromatic C-H out-of-plane deformation (guaiacyl-type) region at 1140 - 800 cm"1, and aromatic C=C bond stretching region at 1600 - 1500 cm"1, do not appear to differ significantly between the MWL and POM-MWL (Table 4.6) [24]. Table 4.6 Signal assignment in the FT-IR spectra of MWL and POM-MWL [24]. Peak Band position Spectral MWL POM-MWL label (cm1) assignment Absorbance 1" 3400* O-H stretching 0.37 0.46 2" 2934 C-H stretching 0.18 0.22 3" 2833 C-H stretching 0.09 0.09 4" 1720 C=0 stretching (unconjugated) 0.11 0.14 5" 1662 C=0 stretching 0.21 0.28 6" 1602 aromatic skeletal vibration + C=0 0.41 0.37 stretching 7" 1507 aromatic skeletal vibration 0.73 0.70 8" 1454 C-H deformation (methyl and 0.50 0.49 methylene) 9" 1418 C-H in-plane deformation with 0.48 0.44 aromatic ring stretching 10" 1265 C-0 of the guaiacyl ring 0.86 0.85 11" 1215 C-C + C-0 stretch 0.64 0.64 12" 1134 aromatic C-H in-plane deformation 0.69 0.68 in the guaiacyl ring 13" 1028 aromatic C-H in-plane deformation 0.77 0.74 band position represented by the absorbance at the highest point of the band envelope. 162 Figure 4.7 Deconvoluted FT-IR spectra of the hydroxyl stretching region ( V 0 H ) of a) MWL and b) POM-MWL. *value in parenthesis indicates IR band area. The change in intensity of the hydroxyl stretching region between the MWL and POM-MWL is consistent with the increased phenolic hydroxyl contents determined by quantitative *H and l 3 C NMR. To better understand the complex hydroxyl stretching 163 region, deconvolution was performed using Peak Fit software. The deconvoluted hydroxyl stretching regions of the MWL and POM-MWL are shown in Figure 4.7. Analysis of the second derivative spectra for both samples detected 6 - 7 band centres (see Appendix). Application of Gausssian peak shape assumptions and fwhm of 30 cm"', and minimized potential overfitting of the data, was found to correspond well with previous lignin and lignin model compound results [12]. The number of bands and band centers of the MWL and POM-MWL does not vary significantly, with the MWL having 6 and the POM-MWL having 7. Both lignin samples had 5 bands in common, in which 4 of the 5 bands increased in the relative intensity in the POM-MWL spectrum, specifically, those associated with free hydroxyl groups >3600 cm"1 and involving biphenol hydroxyl groups <3300 cm"1 [12]. The intensity (area) of the bands at -3617 cm"1 and 3228cm"1 were - 70 % and -50 % greater, respectively, in the POM-MWL than in the MWL. Differences are also apparent in the bands associated with intra- and intermolecular (dimeric [12]) hydrogen bond formation at -3500 cm"1, the POM-MWL having an approximately 44 % increase over the MWL. The increase in both free and intra- and intermolecular hydrogen bonding is consistent with the NMR results and formation of phenolic moieties from cleavage of ot-O-4 and P-O-4 linkages. Likewise, the increased band intensities at <3300 cm"1 in the POM-MWL, which involve biphenol moieties, are in agreement with the increased degree of condensation measured by 1 3 C 164 NMR, 46 and 39 per 100 aromatic rings of POM-MWL and MWL, respectively. This agrees with the lignin model studies (Chapter 3), where the predominant products detected in the oxidation of guaiacyl model compounds with POM were dimers (discussed above). In addition to hydroxyl stretching, apparent differences can be seen between the MWL and POM-MWL bands associated with the carbonyl stretching region (1650-1850 cm"1). Figure 4.8 shows the deconvoluted carbonyl stretching region of the MWL and POM-MWL. Three distinct stretching bands were detected at 1720, 1662 and 1602 cm"1, corresponding to unconjugated, conjugated and aromatic skeletal vibrations plus carbonyl stretching, respectively [24]. It is clearly evident that the largest change occurs in the band associated with the conjugated carbonyl structures at ~ 1662 cm"1, where a nearly 78 % increase in the absorbance of this band occurs. This is in agreement with 1 3 C NMR analysis and the oxidation of a-OH groups in the |3-0-4 inter-unit linkages to a-CO groups. In fact, in the reaction of 0-0-4 lignin model compounds (Chapter 3), the major oxidation pathways involved the formation of a-carbonyl groups via sequential electron-transfer reactions. 165 a) 0.5 r M W L 1602 b) 1850 1800 1750 1700 1650 1600 1550 Wavenumber (cm 1) Figure 4.8 Deconvoluted FT-IR spectra of the carbonyl stretching region (Vc=o) of a) MWL and b) POM-MWL. *value in parenthesis indicates IR band area. 4.3.7 Molecular Mass Distributions The molecular mass distributions for the MWL before and after POM oxidation are shown in Figure 4.9. The POM-MWL clearly has a slightly broader molecular mass distribution and the presence of higher molecular mass material as compared to the MWL 166 sample. This is expected based on the change in P-O-4 content and the degree of condensation estimated by quantitative I3C NMR. Thus, in addition to the oxidative degradation of the lignin, most likely involving the (3-0-4 inter-unit linkages, substantial oxidative coupling of the generated phenoxy radical intermediates occurs. However, it is not known if the residual lignin in pulp would undergo the same condensation reactions and molecular mass distribution increases. It is likely that the condensed lignin structures observed during the POM oxidation of MWL may not occur to as significant a degree with pulp, as the generated lignin radicals will be immobilized within the cell wall structure, and potentially precluded from undergoing coupling reactions. MW (Da) 100000 10000 1000 100 MWL 15 20 25 30 35 40 45 50 Elution time (min) Figure 4.9 Molecular weight distributions of acetylated MWL and acetylated POM-MWL. 167 4.4 Conclusions Changes to the chemical structure of MWL as a result of POM oxidation were investigated using wet chemistry and spectroscopic techniques. Quantitative 'H and 1 3 C NMR showed that POM oxidation of lignin results in the degradation of {3-0-4 inter-unit linkages. POM oxidation leads to side chain oxidation and/or degradation of {3-0-4 inter-unit linkages with concurrent formation of carbonyl structures and phenolic hydroxyl groups. FT-IR analysis also showed an increase in the carbonyl stretching region (Vc=o ~ 1650 - 1850 cm"1) associated with para-substituted aryl ketones, along with an increase in the hydroxyl stretching region ( V O H ~ 3700 - 3100 cm"1), which is consistent with results estimated by quantitative and 1 3 C NMR. These results further support that POM oxidative degradation likely involves a-OH/{3-0-4 inter-unit linkages. In addition to oxidative degradation of inter-unit ether linkages, quantitative 1 3 C NMR analysis showed an increase in the degree of condensation. GPC analysis revealed a change in the relative molecular mass distribution after POM oxidation. The elution profile was much broader and showed the presence of higher relative molecular mass materials. Likewise, deconvolution of the hydroxyl stretching region of the FTIR spectra of POM-MWL and MWL confirmed the presence of increased condensed lignin structural moieties. A substantial increase in the bands associated with intermolecular hydrogen bonding involving biphenol-type structures at <3300 cm"1 was evident. 168 Together, these results support the findings from previous lignin model compounds wherein radical coupling reactions and the formation of dimeric and oligomeric products dominate. However, it is anticipated that the same extent of radical coupling and increase in degree of condensation may not occur in the POM bleaching of pulp due to the immobilization of lignin radicals within the cell wall structure. 169 4.5 R e f e r e n c e s 1. I.A.Weinstock, R.H. Atalla, R.S. Reiner, M.A. Moen, K.E. Hammel, C.J. Houtman, and C L . Hill, A new environmentally benign technology and approach to bleaching kraft pulp - polyoxometalates for selective delignification and waste mineralization. New Journal of Chemistry, 1996. 20(2): p. 269-275. 2. I.A .Weinstock, R.H. Atalla, R.S. Reiner, M.A. Moen, K.E. Hammel, CJ . Houtman, C L . Hill, and M.K. Harrup, A new environmentally benign technology for transforming wood pulp into paper - engineering polyoxometalates as catalysts for multiple processes. Journal of Molecular Catalysis A-Chemical, 1997. 116(1-2): p. 59-84. 3. D.V. Evtuguin and C R Neto, New polyoxometalate promoted method of oxygen delignification. Holzforschung, 1997. 51(4): p. 338-342. 4. D.V. Evtuguin, C R Neto, and J.D.P. De Jesus, Bleaching of kraft pulp by oxygen in the presence of polyoxometalates. Journal of Pulp and Paper Science, 1998. 24(4): p. 133-140. 5. I.A. Weinstock, K.E. Hammel, M.A. Moen, L.L. Landucci, S. Ralph, C E . Sullivan, and R.S. Reiner, Selective transition-metal catalysis of oxygen delignification using water-soluble salts of polyoxometalate (POM) anions. Part II. reactions of a-[SiVWn04o] 5 " with phenolic lignin-model compounds. Holzforschung, 1998. 52(3): p. 311-318. 6. T. Yokoyama, H.-M. Chang, R.S. Reiner, R.H. Atalla, I.A. Weinstock, and J.F. Kadla, Polyoxometalate oxidation of nonphenolic lignin subunits in water: effect of substrate structure on reaction kinetics. Holzforschung, 2004. 58(2): p. 116-121. 7. D.V. Evtuguin, CP. Neto, H. Carapuca, and J. Soares, Lignin degradation in oxygen delignification catalyzed by [PM07V5O40] 8 " polyanion. Part II. study on lignin monomeric model compounds. Holzforschung, 2000. 54(5): p. 511-518. 8. A. Gaspar, D.V. Evtuguin, and CP. Neto, Oxygen bleaching of kraft pulp catalyzed by Mn(III)-substituted polyoxometalates. Applied Catalysis A-General, 2003. 239(1-2): p. 157-168. 9. I.A. Weinstock, E.M.G. Barbuzzi, M.W. Wemple, J.J. Cowan, R.S. Reiner, D.M. Sonnen, R.A. Heintz, J.S. Bond, and C L . Hill, Equilibrating metal-oxide cluster ensembles for oxidation reactions using oxygen in water. Nature, 2001. 414(6860): p. 191-195. 10. A. Bjorkman, A. Studies on finely divided wood. Part I. extraction of lignin with neutral solvents. Svensk Papperstidning-Nordisk Cellulosa 1956, 59(13): p. 477-485. 11. J.R.Obst and T.K. Kirk, Isolation of Lignin. In Methods in Enzymology; W.A. 170 Wood, S.T. Kellogg, Eds.; Academic Press: San Diego, CA 1988: p. 3-12. 12. S. Kubo and J.F. Kadla, Hydrogen bonding in lignin: a Fourier transform infrared model compound study. Biomacromolecules, 2005. 6(5): p. 2815-2821. 13. E. Adler, G. Brunow, and K. Lundquist, Investigation of the acid-catalyzed alkylation of lignins by means of NMR spectroscopic methods. Holzforschung, 1987. 41(4): p. 199-207. 14. E.A. Capanema, M.Y. Balakshin, and J.F. Kadla, A comprehensive approach for quantitative lignin characterization by NMR spectroscopy. Journal of Agricultural and Food Chemistry, 2004. 52(7): p. 1850-1860. 15. D. Robert, Carbon 13 nuclear magnetic resonance spectroscopy, In Methods in Lignin Chemistry; CW. Dence, Ed.; Springer-Verlag, Berlin: New York, 1992: p. 250-273. 16. L.M. Zhang and G. Gellerstedt, NMR observation of a new lignin structure, a spiro-dienone. Chemical Communications, 2001. 24: p. 993-1003. 17. J. Ralph, C. Lapierre, J.M. Marita, H. Kim, F.C. Lu, R.D. Hatfield, S. Ralph, C. Chappie, R. Franke, M.R. Hemm, J. Van Doorsselaere, R.R. Sederoff, D.M. O'Malley, J.T. Scott, J.J. MacKay, N. Yahiaoui, A .M. Boudet, M . Pean, G. Pilate, L. Jouanin, and W. Boerjan, Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR. Phytochemistry, 2001. 57(6): p. 993-1003. 18. K. Lundquist, NMR studies of lignins 2. interpretation of the ! H NMR spectrum of acetylated birth. Acta Chemica Scandinavica B, 1979. 33: p. 27-30. 19. K. Lundquist, NMR studies of lignins 3. *H NMR spectroscopic data for lignin model compounds. Acta Chemica Scandinavica B, 1979. 33: p. 418-420. 20. F.C. Lu and J. Ralph, Non-degradative dissolution and acetylation of ball-milled plant cell walls: high-resolution solution-state NMR. Plant Journal, 2003. 35(4): p. 535-544. 21. K.M. Holtman, H.-M. Chang, H. Jameel, and J.F. Kadla, Quantitative C-13 NMR characterization of milled wood lignins isolated by different milling techniques. Journal of Wood Chemistry and Technology, 2006. 26(1): p. 21-34. 22. C.-L. Chen, Characterization of MWL and dehydrogenative polymerization from monolignols by carbon 13 NMR spectroscopy. In Lignin and Lignan Biosynthesis; S. Sarkanen, Ed,; American Chemical Society, Washington, DC, 1996: p. 255-275. 23. E. Adler, Lignin Chemistry - Past, Present and Future. Wood Science and Technology, 1977. 11(3): p. 169-218. 24. K.V. Sarkanen and C.H. Ludwig, Spectroscopic characterization of lignins. In Lignins : occurrence, formation, structure and reactions; K.V. Sarkanen, C.H. Ludwig, Eds.; Wiley-Interscience, New York, 1971: p. 241-293. 171 5 . Conclusions and Future Work 5.1 Conc lus i ons In this study the oxidations of lignin and various lignin model systems by a-[ S i V W u 0 4 o ] 5 \ the dominant oxidatively active species in the prototypical industrially relevant POM system, Na5(+].9)[SiVi(-o.i)MoWio(+o.i)04o] were investigated. In the first study, the reaction kinetics and mechanisms of a-[SiVWn04o] 5 " oxidation of a series of substituted phenols were studied. It was found that the overall reaction rates were second-order, and that the reaction mechanism was the same for all compounds in the series of phenols. The rate of the reaction was highly dependent on the nature of the substituent group: electron donating groups (EDG) accelerated reaction rates whereas electron withdrawing groups (EWG) retarded reaction rates. Furthermore, the rate-determining step appears to involve an electron-transfer from a neutral substrate to POM. In addition to phenol oxidation, oxidative polymerization was observed at high POM concentrations. This is likely the result of subsequent electron transfer reactions of the initial oxidized and oxidatively coupled dimeric products. In the second study, several typical monomeric and dimeric lignin model compounds were studied. Again, kinetic studies showed the reactions to be second-order, first order with respect to both model compound and POM. A dramatic increase in reactivity was observed with the additional of methoxyl and/or phenol substitutents at the C-3 and/or C-172 5 positions on the phenolic aromatic ring. The rate of reaction increased in the order: hydroxylphenyl < guaiacyl < syringyl. This is most likely due to the increase in electron density of the phenolic ring, promoting oxidation and dramatically stabilizing the phenoxy radical through resonance stabilization and derealization. Of the various lignin model compounds investigated, the 5-5' dimer reacted the fastest with POM. This is quite significant, since it is known that dimeric structures are abundantly present in both native and residual lignin, but have reportedly low reactivity towards other typical oxidative systems. Unfortunately, the reaction products could not be separated and isolated to confirm their identity by NMR. Therefore, the mechanism of POM oxidation of 5-5' dimer is still not clear, and further investigation on the identification of the reaction products is required. As with the simple phenols, the observed reaction rates were dependent on the nature of the substituents at the para-position of the guaiacyl and syringyl model compounds. Again, inductive or resonance donating groups and withdrawing groups, which can increase or decrease the electron density on the aromatic ring, dramatically affected the reaction rates with POM. It is apparent that the reaction rates are highly dependent upon the first electron-transfer from the phenolic moieties. In the final study, the reaction of POM with an isolated MWL was studied. 'H and 1 3 C NMR showed that POM oxidation of MWL results in the degradation of £5-0-4 inter-173 unit linkages. The degradation of (3-0-4 inter-unit linkages was accompanied by the formation of carbonyl structures and an increase in phenolic hydroxyl groups. FT-IR analysis showed an increase in the carbonyl stretching region associated with para-substituted aryl ketones, along with an increase in the hydroxyl stretching region. These results are consistent with the reaction mechanism observed for the POM oxidation of simple P-O-4 lignin model compounds, wherein the formation of a-carbonyl moieties and cleavage of P-O-4 inter-unit linkages were detected. 5 . 2 Future Work Preliminary results revealed that a-[SiVWu04o]5\ one of the active species present in a prototypical industrially relevant POM system, abbreviated Na5 ( + i .9 ) [SiVi ( . o.i)MoWio(+o.i)04o], had very low reactivity toward non-phenolic lignin model compounds. This is unfortunate, because non-phenolic groups represent 40 - 55 % of the inter-units linkages in native lignin. Moreover, it was shown that non-phenolic lignin model compounds were degraded by the Na5(+i.9)[SiVi(.0.i)MoWio(+o.i)04o] POM complex (dominant oxidatively active species present being Na5[SiVWn04o] and Na5[SiVMoWio040]), albeit at high temperatures (>100 °C). Therefore, it may be that the other active species, Na5[SiVMoWio04n] may have higher reactivity toward lignin, and more specifically non-phenolic lignin moieties. Future work could involve detailed 174 analysis of the reaction of this POM species with lignins and lignin models. By better understanding the chemistry (kinetics and mechanisms) of this POM species, it may be possible to better design and optimize the equilibrated POM ensemble. As mentioned in the chapter 3, the POM reaction with the 5,5'-dimer (3,3'-dimethoxyl-5,5'-dimethyl-biphenyl-2,2'-diol) was extremely fast. This is quite interesting because dimeric structures are abundantly present in both native and residual lignin, but have reported low reactivity towards other typical oxidative conditions. Consequently, the POM oxidation of the various dimeric lignin model compounds requires further investigation in order to get the detailed kinetics and mechanisms. In addition to simple variation in side-chain substituents, mixed phenolic/non-phenolic dimers and higher order oligomeric models, such as trimers and tetramers that possess both 5-5' and (3-0-4 inter-unit linkages, would be of particular interest. Finally, POM oxidation of MWL revealed an increase in the degree of condensation, likely the result of radical coupling reactions. It is speculated that such radical coupling reactions would not occur to such an extent in wood pulp, as the lignin is immobilized within the cell wall. Therefore, analysis of the chemical structure of residual lignin isolated from pulp before and after POM oxidation may provide more understanding of the POM bleaching of pulp. 175 6. Appendix 6.1 Reaction Order Determination by Initial Rate Law in Chapter 2. o i >— 1 . , I 0 0.5 1 1.5 2 2.5 ln[POM]+7 b) 1 2 3 4 ln[Phenol]+6 Figure 6.1 Plot of ln[initial rate] versus a) ln[POM] and b) ln[5] at 25°C in sodium acetate buffer (I = 0.2 M , pH 5.0). 5 (phenol). 176 6.2 R e a c t i o n O r d e r D e t e r m i n a t i o n b y I n i t i a l R a t e L a w i n C h a p t e r 3. 4 [POM], mM Figure 6.2 Plot of initial rate versus [POM] at 45 °C (1), 25 °C (2 and 3), and 40 °C (11), in sodium acetate buffer (I = 0.2 M, pH 5.0) under argon. 1 (l-(4-hydroxylphenyl)-ethanol), 2 (l-(4-hydroxyl-3-methoxylphenyl)-ethanol), 3 (l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol) and 11 (4-[2-hydroxyl-2-(4-hydroxyl-3-methoxylphenyl)ethoxyl]-3-methoxyl-benzoic acid). 1 7 7 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 [model compound], mM Figure 6.3 Plot of initial rate versus [model compound] at 45 °C (1), 25 °C (2 and 3), and 40 °C (11), in sodium acetate buffer (I = 0.2 M , pH 5.0) under argon. 1 (l-(4-hydroxylphenyl)-ethanol), 2 (l-(4-hydroxyl-3-methoxylphenyl)-ethanol), 3 (l-(4-hydroxyl-3,5-dimethoxylphenyl)-ethanol) and 11 (4-[2-hydroxyl-2-(4-hydroxyl-3-methoxylphenyl)ethoxyl]-3-methoxyl-benzoic acid). 178 6.3 Deviation of Equation 3.2. The 2:1 stoichiometry can be expressed by a second-order rate law: 2A + B^2C + D + 2H+ (6.1) and d[A] _ d[C] dt dt = k[A][B] (6.2) Where [ P O M o x ] = A , [lignin model compound] = B , [POM r e c i ] = C and [product] = D . If the concentration of A has been replaced by the initial concentration, A = [A]o - 2x, the concentration of B = [B]a - x and C = 2x, then Equation 6.2 can be expressed as: 1 dx -— = k([A]0-2x)([B]o-x) (6.3) Rearranging equation 6.3, equations 6.4 - 6.6 can be easily obtained. 1 ^ _ dx 179 \k(2mo-[A]o)\dt= r_?^—r d x 2 h J) M I - 9 . r * [A] o -2x * [ B ] 0 - J C (6.6) Integration of e q u a t i o n 6.6 and rearranging give e q u a t i o n 6.7. \k{[B\-[A]o)t = In [ f ] ° * + I n ^ 2 [A]„-2x [fi]o (6.7) Reintroduction of the concentration A gives e q u a t i o n 6.8. ;*(2[B] o-[A]) o=ln Wo Mo • + -2 ,4 + ln (6.8) Rearrangement as function of time of the concentration of A gives e q u a t i o n 6.9. A(t) = Mo exp 2 (6.9) C(t) = B 0 -B t , e q u a t i o n 6.9 was transformed into e q u a t i o n 6.10. 180 A'(t) = [B]„ 1*1 [Al, exp 1 2 (6.10) If Equation 6.10 was transformed into equation " A = eel" where A ' = absorbance, A 0 = initial absorbance, 1 = pathelength (4cm), and C = equation 6.10, Equation 6.11 for the formation of reduced form of P O M can be obtained, which is the same as equation 3.2. A\t) = A+Ae\ [BL Wo [Bl, ~ [Bh [Al, exp (6.11) 6.4 Methoxyl Content Analysis Methoxyl content analysis was performed according to Viebock and Schwappach [1] in a modified Zeisel method [2]. The sample (~ 50 mg) was weighed in a gelatin capsule which was placed in the reaction flask. The trap was half filled with a water suspension of red phosphorus (0.06 g in 100 mL) . 20 m L of bromine solution (5 m L of bromine in 145 m L of potassium acetate solution) were added to the receiver. The potassium acetate solution was prepared as fol lowing: 100 g of anhydrous potassium acetate was dissolved in a solution of 900 m L of glacial acetic acid and 100 m L of acetic anhydride. Propionic anhydride (2 mL) , hydroiodic acid (6 mL) and a few small boil ing chips 181 were then added to the 10 mL reaction flask and the joint was sealed with a few drops of hydriodic acid. Nitrogen was passed through the apparatus at a rate of about 2 bubbles per second using a glass tip of ~1 mm diameter. The flask was then immersed in an oil bath maintained at about 150 °C for 40 min. 10 mL of sodium acetate (2.4 N) were placed in a 500 mL Erlenmeyer flask and the contents of the receiver were washed into the flask with 150 mL of deionized water. Formic acid (90 %) was then added dropwise, while stirring, until the brown color of the bromine disappeared and then 6 more drops were added. After 3 min, 3 g of potassium iodide and 15 mL of 10% sulfuric acid (w/w) were added and the mixture was titrated with sodium thiosulfate (0.1 N) with addition of 1 % starch solution near the end point. A blank determination was carried out with a gelatin capsule and propionic anhydride in the reaction flask. Calculation of methoxyl content: (A - B)N x 0.00517 x 100 % methoxyl = — moisture - free sample in g Where A = titration for the sample (mL), B = titration for the blank, and N = normality of the thiosulfate solution. 182 6.5 K l a s o n L i g n i n C o n t e n t A n a l y s i s The lignin content of the purified MWL and extractive-free Wiley wood was determined using the Klason method (TAPPI Method T249 cm-85). The sample (about 200 mg of Wiley wood or 40 mg of lignin) was weighed accurately in a reaction flask. 3 mL of 72 % sulfuric acid (w/w) were added to the reaction flask and the mixture was mixed well with a stirring rod (approximately 1 min) at every 10 min for 2 h. After 2 h, the mixture was transferred into a septa-sealed bottle with 112 mL of deionized water to produce an acid concentration of 3 %. The mixture was then autoclaved at 121 °C for 1 h and allowed to stand overnight on the bench in order to allow the insoluble material (brown precipitate) to settle down. The cooled reaction solution was filtered through pre-weighed sintered glass crucibles. About 10 mL of filtrate were moved into a sealed vial for acid soluble lignin content. The remaining solids in the bottle were recovered by washing with deionized water into a sintered glass crucible. These solids were washed with 200 mL of warm deionized water. The crucible was then oven-dried at 105 °C and left overnight. Acid-insoluble lignin was determined from the weight of the dried crucible. Calculation of acid insoluble lignin content: 183 % acid insoluble lignin = x 100 W Where W c = oven-dried weight of acid insoluble lignin (g) and W = oven-dried weight of sample (g). Acid-soluble lignin was quantified by UV-Vis spectroscopy at 205 nm according to the TAPPI Useful Method UM250. The acid solution obtained from the filtrate was measured using a 3 % sulfuric acid as a blank. If the absorbance was greater than 0.7, the filtrate was further diluted to obtain an absorbance within 0.2 - 0.7. Calculation of acid soluble lignin content: C(glmL) = % acid soluble lignin = xlOO lOOOxW Where A = absorbance, V = total volume of filtrate and W = oven-dried weight of sample (g)-184 6.6 NMR Spectrum of Non-Acetylated MWL NMR peak assignments (Table 6.1) and related lignin structures (Figure 4.4) are shown according to Capanema et al. [3] and the references there in. 7 —I . | . | . , , , , , , , , , , , , , 180 160 140 120 100 80 60 40 20 ppm Figure 6.4 Quantitative 13C NMR spectrum for non- acetylated MWL. 185 Table 6.1 Signal assignment in the 13C NMR spectrum of non-acetylated MWL sample [3-5]. Peak Chemical shift label range (ppm) Spectral region assignment 1 196-193 CO in a-CO/p-0-4(D),L 2 193-191 Ar-CHO(M) 3 182-180 C-4 in I 4 168-166 Conjugated COOR 5 157-151 C-3 in T,, C-3,5 in Si, C-a in L, C-3,6 in I, C-4 conjugated 6 144.5 - 142.5 C-3 in E, C-4 in Tn, C-4 in conjugated Si, unknown 7 5 8 - 5 4 OMe, C-P in H, C - l in I and C-y in R 8 5 4 - 5 2 C-P in E and F 9 3 5 - 3 4 C-P in Q, C-a in J 10 32.5-31.5 C-a in Q 186 6.7 Analysis of the Second Derivative Spectra of Hydroxyl and Carbonyl Stretching regions of MWL and POM-MWL Figure 6.5 FT-IR spectra (upper) and second-derivative spectra (lower) of the hydroxyl stretching region of a) M W L and b) P O M - M W L . *the number of peaks was determined from the second-derivative spectra; local minima with values that tend to be above or near zero indicate hidden peaks. 187 a) Figure 6.6 F T - I R spectra (upper) and second-derivative spectra (lower) of the carbonyl stretching region in a) M W L and b) P O M - M W L . *the number of peaks was determined from the second-derivative spectra; local minima with values that tend to be above or near zero indicate hidden peaks. 6.8 Visible Spectra Absorbance-Time Data (in attached CD) 188 6.9 R e f e r e n c e s 1. B.L. Browning, Acetyl and methoxyl groups In Methods in Wood Chemistry; B.L. Browning, Ed.; Appleton, Wisconsin, 1967: p. 660-664. 2. S.Y. Lin and CW. Dence, Determination of methoxyl content by Viebock and Schwappach procedure In Methods in Lignin Chemistry; C.-L. Chen, Ed.; Springer-Verlag , Berlin: New York, 1992: p. 467-472. 3. E.A. Capanema, M.Y. Balakshin, and J.F. Kadla, A comprehensive approach for quantitative lignin characterization by NMR spectroscopy. Journal of Agricultural and Food Chemistry, 2004. 52(7): p. 1850-1860. 4. D. Robert, Carbon 13 nuclear magnetic resonance spectroscopy, In Methods in Lignin Chemistry; CW. Dence, Ed.; Springer-Verlag, Berlin: New York, 1992: p. 250-273. 5. K .M. Holtman, H.-M. Chang, H. Jameel, and J.F. Kadla, Quantitative C-13 NMR characterization of milled wood lignins isolated by different milling techniques. Journal of Wood Chemistry and Technology, 2006. 26(1): p. 21-34. 189 

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