Methods in Lignin Chemistry

The structural complexity of lignin has continually challenged the in­ genuity of researchers to develop suitable methods for its charac­ terization prior to and following a wide variety of chemical, biologi­ cal, and physical treatments. Initially, activity along these lines was fueled by a desire to interpret technical delignification (Le. , pulping) processes in terms of accompanying structural changes in the lignin. Subsequently, increasingly wide ranging, in-depth investigations on the structure and reactivity of lignin exposed the inadequacy of many of the methods currently in use and underscored the ever-continuing need to develop new methods capable of solving the unique analytical problems associated with lignin. Characteristically, such methods should be selective, sensitive, suitable for quantitative measurements, and capable of being applied directly to, and without destruction of, the lignin or lignocellulose sample. One notable example of the head­ way being made in reaching this objective is the relatively recent devel­ opment and refinement of methods based on the use of sophisticated instrumentation, e. g. , lH_ and 13C-NMR spectroscopy. Although the utility of many of these and other recently developed methods de­ scribed in this book has yet to be fully and satisfactorily exploited, we believe that progress already made in this direction will continue and most likely accelerate. The decision to produce this book was prompted mainly by the acknowledged need for an up-to-date, single source compilation of lignin methodology. Hitherto, this need was, in part, satisfied by B. L.

1 Introduction.- 1 Introduction.- 1.1 General Structural Features of Lignin.- 1.2 Variations in Concentration and Composition of Lignin in Xylem.- 1.3 Isolation and Recovery of Lignin.- 1.4 Physical Properties of Lignin.- 1.5 Structure-Modifying Reactions.- 1.5.1 Modification of the Aromatic Ring.- 1.5.1.1 Electrophilic Substitution.- 1.5.1.2 Conversion of Aromatic Rings to Nonaromatic Cyclic Structures.- 1.5.1.3 Conversion of Cyclic to Acyclic Structures.- 1.5.1.4 Ring Coupling and Condensation Reactions.- 1.5.2 Modification of the Propanoid Side Chain.- 1.5.2.1 Cleavage of Ether Bonds.- 1.5.2.2 Cleavage of Carbon-Carbon Bonds.- 1.5.2.3 Substitution Reactions.- 1.5.2.4 Formation and Elimination of Multiple Bond Functionalities.- 1.5.3 Future Analytical Needs.- References.- 2 Detection and Determination.- 2.1 The Detection of Lignin.- 2.1.1 Introduction.- 2.1.2 Reagents for the Detection of Lignin.- 2.1.2.1 Aliphatic Compounds.- 2.1.2.2 Phenols and Aromatic Amines.- 2.1.2.3 Heterocyclic Compounds.- 2.1.2.4 Inorganic Reagents.- 2.1.2.5 Unclassified Reagents.- 2.1.3 Color-Forming Reaction Sequences.- 2.1.4 Procedures for the Detection of Lignin.- 2.1.4.1 Color Reaction with Phloroglucinol-Hydrochloric Acid.- 2.1.4.2 The Mäule Color Reaction.- 2.1.4.3 The Cross and Bevan Color Reaction.- 2.1.4.4 Color Reaction with Tosyl Chloride/Pyridine, p-Nitrosodimethylaniline/Potassium Cyanide.- 2.1.4.5 Color Reaction with Quinone Monochlorimide.- 2.1.4.6 Color Reaction with Potassium Nitrosodisulfonate.- 2.1.4.7 Color Reaction Based on the Formation of Nitrosophenols (Pearl-Benson Method).- References.- 2.2 The Determination of Lignin.- 2.2.1 Introduction.- 2.2.2 Direct Methods.- 2.2.2.1 Acid Hydrolysis of Wood and Pulp.- 2.2.2.2 Determination of Acid-Insoluble (Klason) Lignin in Wood and Pulp.- 2.2.2.3 Determination of Acid-Soluble Lignin in Wood and Pulp.- 2.2.2.4 Acid Hydrolysis of Annual Plants, Developing Wood, and Forage.- 2.2.2.5 Determination of Acid-Insoluble Lignin in Annual Plants, Developing Wood, and Forage.- 2.2.3 Indirect Methods.- 2.2.3.1 Spectrophotometric Methods.- 2.2.3.2 Determination of Lignin in Wood and Pulp by the Acetyl Bromide Method.- 2.2.3.3 Methods Based on Oxidant Consumption.- 2.2.3.4 Determination of the Kappa Number of Pulp.- 2.2.4 Determination of Lignin in Solution.- 2.2.4.1 Determination of Dissolved Lignin by the Modified Pearl-Benson (Nitrosation) Method.- References.- 3 Isolation and Purification.- 3.1 Wood.- 3.1.1 Introduction.- 3.1.2 Preferred Method for Isolation and Purification of Lignin.- 3.1.2.1 Isolation.- 3.1.2.2 Purification.- 3.1.3 Discussion.- 3.1.3.1 Modifications of and Comments on the Preferred Method.- 3.1.3.2 Milled Wood Lignin as a Representative of the Lignin in Wood.- 3.1.3.3 Milled Wood Lignin Compared with Brauns Lignin.- 3.1.3.4 Milled Wood Lignin Compared with Cellulolytic Enzyme Lignin.- References.- 3.2 Isolation of Lignin from Pulp.- 3.2.1 Introduction.- 3.2.2 Procedures.- 3.2.2.1 Preparation of Pulp Samples and Enzyme Solution.- 3.2.2.2 Isolation of Residual Lignins in Unbleached Pulps.- 3.2.2.3 Isolation of Residual Lignins from Semi-Bleached Pulps.- 3.2.2.4 Purification of the Residual Lignins.- 3.2.3 Concluding Remarks.- References.- 3.3 Commercial Spent Pulping Liquors.- 3.3.1 Introduction.- 3.3.2 Principle.- 3.3.2.1 Kraft (Sulfate) Lignin.- 3.3.2.2 Lignosulfonate (Sulfite Lignin).- 3.3.3 Methods.- 3.3.3.1 Isolation Procedure for Kraft Lignin.- 3.3.3.2 Isolation Procedure for Lignosulfonates.- 3.3.4 Composition of Isolated Lignin.- 3.3.4.1 Kraft Lignin Fractions.- 3.3.4.2 Lignosulfonate Fractions.- 3.3.5 Discussion.- References.- 4 Characterization in Solid State.- 4.1 Fourier Transform Infrared Spectroscopy.- 4.1.1 Principles and Instrumental Techniques.- 4.1.1.1 Introduction.- 4.1.1.2 Scope of FTIR Spectroscopic Applications.- 4.1.1.3 Description of a Standard FTIR Instrument.- 4.1.1.4 Advantages of FTIR Spectroscopy.- 4.1.2 Method.- 4.1.2.1 FTIR Spectroscopy in Practice.- 4.1.2.2 Procedures.- 4.1.3 Lignin Characterization in the Mid-Infrared Region (4000?500 cm?1).- 4.1.3.1 Band Assignment and Classification of Lignin IR Spectra.- 4.1.3.2 Influence of Carbonyl Groups.- 4.1.3.3 Mathematical Resolution of Spectra.- 4.1.3.4 Effect of Acetylation.- 4.1.3.5 Quantitative Evaluation.- 4.1.4 Lignin Spectra in the Near-Infrared Region (4000?10000 cm?1).- 4.1.5 Lignin Spectra in the Far-Infrared Region (50?500 cm?1).- 4.1.6 Concluding Remarks.- References.- 4.2 Ultraviolet Microscopy.- 4.2.1 Introduction.- 4.2.2 Information Obtained by UV Microscopy.- 4.2.2.1 Photomicrographs.- 4.2.2.2 Lignin Concentration.- 4.2.2.3 UV Absorption Spectra.- 4.2.3 Experimental Methods.- 4.2.3.1 Specimen Preparation.- 4.2.3.2 Photography.- 4.2.3.3 Quantitative Analysis.- 4.2.4 Concluding Remarks.- References.- 4.3 Interference Microscopy.- 4.3.1 Introduction.- 4.3.2 Principle.- 4.3.3 Method.- 4.3.3.1 Specimen Preparation.- 4.3.3.2 Measurement of Refractive Index.- 4.3.3.3 Calculation of Lignin Concentration from Refractive Index.- 4.3.3.4 Measurement of Porosity.- 4.3.3.5 Calculation of Lignin Concentration from Porosity.- 4.3.4 Discussion.- 4.3.4.1 Comparison with Other Techniques.- 4.3.4.2 Types of Specimen that Can be Examined.- 4.3.4.3 Accuracy and Precision.- References.- 4.4 Electron Microscopy.- 4.4.1 Introduction.- 4.4.2 Principles.- 4.4.2.1 Glossary.- 4.4.2.2 Principles of SEM-EDXA and TEM-EDXA.- 4.4.3 Methods.- 4.4.3.1 Specimen Thickness.- 4.4.3.2 Procedure for Bromination-EDXA.- 4.4.4 Lignin Distribution in Cell Walls.- 4.4.4.1 Softwoods.- 4.4.4.2 Hardwoods.- 4.4.4.2.1 Distribution of Lignin.- 4.4.4.2.2 Distribution of Guaiacyl and Syringyl Lignins.- 4.4.5 Comparisons of Bromination-EDXA with Other Methods.- References.- 4.5 Cross Polarization/Magic Angle Spinning Nuclear Magnetic Resonance (CP/MAS NMR) Spectroscopy.- 4.5.1 Introduction.- 4.5.2 Principle of CP/MAS NMR.- 4.5.2.1 Cross Polarization.- 4.5.2.2 Magic Angle Spinning.- 4.5.3 Description of Methods for CP/MAS NMR Spectroscopy.- 4.5.3.1 Choice of Spectrometer.- 4.5.3.2 Sample Preparation.- 4.5.3.3 Determination of Lignin in Wood and Pulp.- 4.5.3.4 Interrupted Decoupling.- 4.5.3.5 Estimation of Syringyl/Guaiacyl Ratios.- 4.5.3.6 Estimation of the Degree of Etherification.- 4.5.3.7 Resolution Enhancement.- 4.5.4 Discussion.- 4.5.5 Future Developments.- References.- 4.6 Raman Spectroscopy.- 4.6.1 Introduction.- 4.6.2 Principle.- 4.6.3 Method.- 4.6.3.1 Raman System.- 4.6.3.2 Scattering Geometries.- 4.6.3.3 Specimens and Sampling.- 4.6.4 Spectral Information.- 4.6.4.1 Identification of Native Lignin Features.- 4.6.4.2 Quantitative Analysis of Lignin in Wood.- 4.6.4.3 Orientation and Composition Studies of Lignin in Woody Tissue.- 4.6.5 Discussion.- 4.6.5.1 Comparison with Other Techniques.- 4.6.5.2 Future Development.- References.- 4.7 Pyrolysis-Gas Chromatography-Mass Spectrometry.- 4.7.1 Introduction.- 4.7.2 Principle and Methods of Analytical Pyrolysis.- 4.7.2.1 Combination of Pyrolysis with Other Instruments.- 4.7.2.2 Pyrolysis Techniques.- 4.7.2.2.1 Induction Heating (Curie-Point Pyrolysis).- 4.7.2.2.2 Resistance Heating.- 4.7.2.3 Sample Preparation, Size, Geometry, and Contamination: General Considerations.- 4.7.3 Gas Chromatography.- 4.7.3.1 Operating Conditions.- 4.7.4 Mass Spectrometry.- 4.7.4.1 Instrumentation.- 4.7.4.2 Ionization.- 4.7.4.3 Data Handling.- 4.7.5 Evaluation of Pyrograms and Spectra.- 4.7.6 Utility of the Method.- References.- 4.8 Thermal Analysis.- 4.8.1 Introduction.- 4.8.2 General Principles and Techniques of Thermal Analysis.- 4.8.2.1 Thermogravimetry.- 4.8.2.1.1 Apparatus.- 4.8.2.1.2 TG Curve.- 4.8.2.1.3 Factors Affecting TG Measurements.- 4.8.2.2 DTA and DSC.- 4.8.2.2.1 Apparatus.- 4.8.2.2.2 DTA and DSC Curves.- 4.8.2.2.3 Factors Affecting DTA and DSC Measurements.- 4.8.3 Applications of Thermal Analysis to Lignin.- 4.8.3.1 Kinetic Study of the Thermal Degradation of Lignin.- 4.8.3.2 Glass Transition of Lignin.- 4.8.3.3 Heat Capacity of Lignin.- 4.8.4 Conclusions.- References.- 5 Characterization in Solution: Spectroscopic Methods.- 5.1 Ultraviolet Spectrophotometry.- 5.1.1 Introduction.- 5.1.2 Principle of Measurement.- 5.1.3 Method.- 5.1.3.1 Solvent Selection.- 5.1.3.2 Preparation of Solutions for Spectral Measurements.- 5.1.3.3 Procedure for Quantitative Determination (Neutral Spectra).- 5.1.3.4 Procedure for Measurement of Ionization Difference Spectra.- 5.1.3.5 Second Derivative Photometry.- 5.1.4 Ultraviolet Absorption Characteristics of Lignins and Lignin-Related Model Compounds.- 5.1.4.1 Solvent Effect.- 5.1.4.2 Absorption Bands.- 5.1.4.3 Ionization Difference Spectra.- 5.1.4.4 NaBH4 Reduction Difference Spectra.- 5.1.4.5 Second Derivative Photometry.- 5.1.4.6 The Effect of Chemical Modification on Ultraviolet Absorption.- 5.1.5 Discussion.- References.- 5.2 Fourier Transform Infrared Spectroscopy.- 5.2.1 Introduction.- 5.2.2 Method.- 5.2.2.1 Transmission Cells.- 5.2.2.2 Attenuated Total Reflection (ATR).- 5.2.2.3 Circular Attenuated Total Reflection (CATR).- 5.2.3 Liquid State FTIR Spectroscopy of Lignins.- 5.2.3.1 Acetylated Lignins in Chloroform.- 5.2.3.2 Lignosulfonates in Water.- 5.2.3.3 Lignins in Alkaline Solutions.- 5.2.3.4 FTIR Spectra of Pulping Liquors.- References.- 5.3 Proton (1H) NMR Spectroscopy.- 5.3.1 Introduction.- 5.3.2 Description of Preferred Method.- 5.3.2.1 Acetylation.- 5.3.2.2 Purification of the Acetate Derivative.- 5.3.2.3 NMR Examination.- 5.3.3 Discussion.- 5.3.3.1 Comments on the Preferred Method.- 5.3.3.2 Alternative Conditions for the Recording of Spectra.- References.- 5.4 Carbon-13 Nuclear Magnetic Resonance Spectrometry.- 5.4.1 Introduction.- 5.4.2 Principles of Pulsed Fourier Transform 13C NMR.- 5.4.2.1 Glossary of Terms and Symbols.- 5.4.2.2 Basic Principles and Elements of an NMR Experiment.- 5.4.2.3 Pulsed NMR in the Rotating Frame of Reference.- 5.4.2.4 Relaxation Times.- 5.4.2.5 Acquisition of the Free Induction Decay (FID) and Fourier Transformation (FT) of the FID.- 5.4.2.6 Chemical Shifts.- 5.4.2.7 Spin-Spin Couplings: Scalar and Dipolar.- 5.4.2.8 Signal Intensities.- 5.4.2.9 One- and Two-Dimensional NMR Experiments.- 5.4.3 Experimental Procedures.- 5.4.3.1 Preparation of Samples.- 5.4.3.2 Spectrometer Specifications.- 5.4.3.3 Recording of 13C NMR Spectra: Summary of Acquisition Parameters.- 5.4.3.3.1 Routine Spectra.- 5.4.3.3.2 Spectra for Quantitative Analysis.- 5.4.3.3.3 DEPT (Distortionless Enhancement by Polarization Transfer) Spectra.- 5.4.3.4 Integration of NMR Signals.- 5.4.4 Results and Discussion.- 5.4.4.1 Qualitative Analysis.- 5.4.4.2 13C NMR DEPT Spectra.- 5.4.4.3 Quantitative Analysis.- 5.4.5 Concluding Remarks.- References.- 5.5 Electron Spin Resonance (ESR) Spectroscopy.- 5.5.1 Introduction.- 5.5.2 Principle of ESR.- 5.5.3 Spectral Parameters.- 5.5.3.1 The g-Value.- 5.5.3.2 Intensity.- 5.5.3.3 Line Shape.- 5.5.3.4 Hyperfine Structure.- 5.5.4 Methods.- 5.5.4.1 Sample Preparation.- 5.5.4.2 ESR Measurements.- 5.5.4.3 ESR Operation.- 5.5.5 ESR of Lignin.- 5.5.5.1 Detection of Mechanoradicals in Lignin.- 5.5.5.2 Detection of Free Radicals in Photoirradiated Lignin.- 5.5.5.3 Detection of Free Radicals in a Photoirradiated Lignin Model Compound.- 5.5.6 Discussion.- References.- 6 Characterization in Solution: Chemical Degradation Methods.- 6.1 Acidolysis.- 6.1.1 Introduction.- 6.1.2 Characterization of Lignins by Analysis of Low-Molecular Weight Acidolysis Products.- 6.1.3 Applications of the Acidolysis Method.- 6.1.4 Preferred Acidolysis Procedure.- 6.1.4.1 Preparation of the Acidolysis Reagent.- 6.1.4.2 Acidolysis Reaction.- 6.1.4.3 Work-Up of Acidolysis Mixture.- 6.1.4.4 Analysis of Acidolysis Products.- 6.1.5 Discussion of Procedure.- 6.1.6 Dimeric Acidolysis Products.- References.- 6.2 Nitrobenzene and Cupric Oxide Oxidations.- 6.2.1 Introduction.- 6.2.1.1 Nitrobenzene Oxidation.- 6.2.1.2 Cupric Oxide Oxidation.- 6.2.1.3 Nitrobenzene and Cupric Oxide Oxidations of Lignins in Grass Tissues.- 6.2.2 Experimental Procedures.- 6.2.2.1 Sample Preparation.- 6.2.2.2 Nitrobenzene Oxidation.- 6.2.2.3 Cupric Oxide Oxidation.- 6.2.2.4 Qualitative Determination of Oxidation Products.- 6.2.2.4.1 Gas Chromatography (GC).- 6.2.2.4.2 Gas Chromatography-Mass Spectrometry (GC-MS).- 6.2.2.5 Quantitative Determination of Oxidation Products.- 6.2.2.5.1 Gas Chromatography (GC).- 6.2.2.5.2 High Performance Liquid Chromatography (HPLC).- 6.2.2.6 Determination of p-Hydroxycinnamic Acid and Ferulic Acid Ester Units in Grass Lignins.- 6.2.2.7 Interpretation of Mass Spectra.- 6.2.3 Discussion of the Methods.- References.- 6.3 Chemical Degradation Methods: Permanganate Oxidation.- 6.3.1 Introduction.- 6.3.2 Experimental Procedure.- 6.3.2.1 Preparation of Samples.- 6.3.2.2 Alkylation.- 6.3.2.3 Oxidation with Potassium Permanganate.- 6.3.2.4 Oxidation with Hydrogen Peroxide.- 6.3.2.5 Esterification.- 6.3.2.6 Gas Chromatographic Analysis.- 6.3.3 Discussion of Method.- 6.3.3.1 Reaction Selectivity.- 6.3.3.2 Identification and Estimation of Structural Unit Types.- 6.3.3.3 Estimation of Phenolic Hydroxyl Groups.- References.- 6.4 Thioacidolysis.- 6.4.1 Introduction.- 6.4.2 Thioacidolysis Reactions of Lignin.- 6.4.3 Description of Method.- 6.4.3.1 Reagents.- 6.4.3.2 Treatment of Lignin with Thioacidolysis Reagent.- 6.4.3.3 GC Analysis of Monomeric Products.- 6.4.3.4 Quantitative Determination of the Main Monomeric Products.- 6.4.4 Discussion of Method.- 6.4.4.1 Sensitivity and Reproducibility.- 6.4.4.2 Identification of the Major Thioacidolysis Products.- 6.4.4.3 Quantitative Aspects of the Thioacidolysis Method.- References.- 6.5 Hydrogenolysis.- 6.5.1 Introduction.- 6.5.2 Proposed Sequence for Hydrogenolysis.- 6.5.3 Hydrogenolysis and Hydrogenation Reactions.- 6.5.3.1 Cleavage of Interunitary Linkages.- 6.5.3.2 Hydrogenation of Side Chain and Aromatic Ring.- 6.5.4 Factors Affecting Hydrogenolysis.- 6.5.4.1 Catalyst Activity.- 6.5.4.2 Catalyst Amount.- 6.5.4.3 Hydrogen Pressure.- 6.5.4.4 Solvent.- 6.5.5 Hydrogenolysis to Monomeric Products.- 6.5.6 Hydrogenolysis to Dimeric and Trimeric Products.- References.- 6.6 Nucleus Exchange Reaction.- 6.6.1 Introduction.- 6.6.2 Reaction Mechanism.- 6.6.2.1 Phenolation.- 6.6.2.2 Nucleus Exchange.- 6.6.2.3 Demethylation.- 6.6.2.4 The Response of Various Structural Units in Lignins to the NE Treatment.- 6.6.3 Description of the NE Method.- 6.6.3.1 Sample Preparation.- 6.6.3.2 Preparation of Reagent.- 6.6.3.3 Description of Procedure.- 6.6.3.4 Quantitative Analysis of Products by Gas Chromatography.- 6.6.4 Quantitative Determination of Noncondensed and Condensed Guaiacyl Nuclei in Softwood Protolignins.- 6.6.5 Quantitative Determination of Noncondensed and Condensed Phenyl Nuclei in Hardwood Protolignins.- 6.6.6 Discussion of Method.- 6.6.6.1 Confirmation of the Quantitative Nature of the NE Reaction.- 6.6.6.2 Use of the NE Method.- References.- 6.7 Ozonation.- 6.7.1 Introduction.- 6.7.2 Mechanism of Ozonation.- 6.7.3 Factors Involved in the Design of an Ozonation Procedure.- 6.7.3.1 Conditions Used for the Reaction with Ozone.- 6.7.3.2 Work-Up of Ozonation Product Mixtures.- 6.7.3.3 Procedures for Separation and Identification of Ozonation Products.- 6.7.3.4 Procedures for Quantitative Determination of Ozonation Products.- 6.7.4 Ozonation Procedure.- 6.7.4.1 Ozonation, Saponification, and Derivatization.- 6.7.4.2 Gas Chromatography and Gas Chromatography-Mass Spectrometry.- 6.7.5 Information on the Structure of the Lignin Side Chain.- 6.7.6 Conclusion.- References.- 7 Functional Group Analysis.- 7.1 Determination of Total and Aliphatic Hydroxyl Groups.- 7.1.1 Introduction.- 7.1.2 Determination of Total Hydroxyl Groups.- 7.1.2.1 Acetylation of the Lignin Preparation.- 7.1.2.2 Determination of Total O-Acetyl Groups.- 7.1.2.2.1 The Kuhn-Roth Procedure.- 7.1.2.2.2 The Modified Bethge-Lindström Procedure.- 7.1.2.3 Total Hydroxyl Content.- 7.1.2.4 Total Aliphatic Hydroxyl Content.- 7.1.3 Discuss...