Methods in Lignin Chemistry

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 Discussion of the Methods.- 7.1.3.1 Precision and Accuracy of the Methods.- 7.1.3.2 In-House as Opposed to Commercial O-Acetyl Analysis.- References.- 7.2 Determination of Phenolic Hydroxyl Groups.- 7.2.1 Significance of the Analysis.- 7.2.2 Survey of Analytical Approaches.- 7.2.3 Description of Procedures.- 7.2.3.1 Aminolysis.- 7.2.3.1.1 Instrumentation and Chemicals.- 7.2.3.1.2 Calibration of Internal Standard.- 7.2.3.1.3 Procedure.- 7.2.3.2 Periodate Oxidation.- 7.2.3.2.1 Instrumentation and Chemicals.- 7.2.3.2.2 Calibration of Internal Standard.- 7.2.3.2.3 Procedure.- 7.2.3.2.4 Calculation.- 7.2.4 Discussion of Methods.- References.- 7.3 Determination of Ethylenic Groups.- 7.3.1 Ethylenic Structures in Lignin.- 7.3.2 Survey of Methods.- 7.3.3 Determination of Total Cinnamaldehyde and Cinnamyl Alcohol Units.- 7.3.3.1 Pre-Reduction of Carbonyl Groups with Lithium Aluminum Hydride.- 7.3.3.2 Catalytic Hydrogenation.- 7.3.3.3 Measurement of Hydrogenation Difference Spectrum.- 7.3.4 Determination of Stilbenoid Units.- 7.3.4.1 Pre-Reduction of Carbonyl Groups with Lithium Aluminum Hydride (LiAlH4).- 7.3.4.2 Measurement of Ionization Difference Spectrum.- 7.3.5 Discussion of Procedures.- References.- 7.4 Determination of Carbonyl Groups.- 7.4.1 Introduction.- 7.4.2 Proposed Methods for the Determination of Carbonyl Groups.- 7.4.3 Determination of Total Carbonyl Content of Lignins by Reaction with Hydroxylamine Hydrochloride.- 7.4.4 Determination of Conjugated Carbonyl Groups by UV Spectroscopy.- 7.4.4.1 Preparation of Lignin Sample Stock Solution.- 7.4.4.2 UV Spectrum of Lignin in Alkaline Solution.- 7.4.4.3 UV Spectrum of Sodium Borohydride-Reduced Lignin in Alkaline Solution.- 7.4.4.4 Reduction Difference (??r) Spectrum.- 7.4.4.5 Calculation of the Conjugated Carbonyl Contents.- 7.4.5 Discussion of the Methods.- 7.4.5.1 Comparison of the Hydroxylamine Hydrochloride and Borohydride Methods.- 7.4.5.2 Accuracy of the Procedure for Determining Conjugated Carbonyl Groups.- References.- 7.5 Determination of Carboxyl Groups.- 7.5.1 Introduction.- 7.5.2 Determination of Carboxyl Groups by Nonaqueous Potentiometric Titration.- 7.5.2.1 Instrumentation and Chemicals.- 7.5.2.2 Standardization of Titrant.- 7.5.2.3 Procedure for Carboxyl Group Determination.- 7.5.2.4 Calculation.- 7.5.3 Discussion of Method.- References.- 7.6 Determination of Methoxyl Groups.- 7.6.1 Introduction.- 7.6.2 Sample Preparation.- 7.6.3 Determination of Methoxyl Content by the Vieböck Schwappach Procedure.- 7.6.4 Discussion of Method.- References.- 7.7 Determination of Sulfonate Groups and Total Sulfur.- 7.7.1 Significance of the Analysis.- 7.7.2 Survey of Available Methods.- 7.7.3 Sulfonate Group Determination.- 7.7.3.1 Principle of Conductometric Titration.- 7.7.3.2 Description of Conductometric Titration of Pulp.- 7.7.4 Total Sulfur Determination.- 7.7.4.1 Principle of Combustion/Ion Chromatography.- 7.7.4.2 Description of Combustion/Ion Chromatography.- 7.7.5 Discussion of Methods.- References.- 8 Determination of Molecular Weight, Size, and Distribution.- 8.1 Gel Permeation Chromatography.- 8.1.1 Introduction.- 8.1.1.1 Chromatography of Lignins on Sephadex Gels.- 8.1.1.2 Modified Sephadex Gels.- 8.1.2 High-Performance Size Exclusion Chromatography (HPSEC).- 8.1.3 Experimental.- 8.1.3.1 Equipment.- 8.1.3.2 Sample Preparation.- 8.1.3.3 Acetylation.- 8.1.3.4 Methylation.- 8.1.4 Discussion of Method.- 8.1.4.1 Problems Encountered in Gel Permeation Chromatography.- 8.1.4.2 HPSEC on Styragel.- References.- 8.2 Light Scattering.- 8.2.1 Introduction.- 8.2.2 Principle.- 8.2.2.1 Optical Anisotropy Correction.- 8.2.2.2 Absorbance Correction.- 8.2.2.3 Influence of Fluorescence.- 8.2.3 Method.- 8.2.3.1 Instrument Description.- 8.2.3.2 Procedure.- 8.2.3.2.1 Solvents and Solutions.- 8.2.3.2.2 Measurements.- 8.2.3.2.3 Determination of Rayleigh Factors.- 8.2.3.2.4 Specific Retractive Index Increment, dn/dc.- 8.2.3.3 Data Processing.- 8.2.4 Discussion.- 8.2.4.1 Clarification of Solvents and Solutions.- 8.2.4.2 Influence of Fluorescence.- 8.2.4.3 Influence of Anisotropy.- 8.2.4.4 Typical Analytical Data.- 8.2.4.5 Accuracy of the Method.- References.- 8.3 Vapor Pressure Osmometry.- 8.3.1 Introduction.- 8.3.2 Principle.- 8.3.3 Method.- 8.3.3.1 Instrument Description.- 8.3.3.2 Procedure.- 8.3.3.2.1 Selection of Solvents.- 8.3.3.2.2 Measurement Steps.- 8.3.3.3 Evaluation of Results.- 8.3.3.3.1 Calibration Factor.- 8.3.3.3.2 Number-Average Molecular Weight.- 8.3.4 Discussion.- 8.3.4.1 Drop Size Effects.- 8.3.4.2 Response Time.- 8.3.4.3 Purity of Solvents and Lignin.- 8.3.4.4 Constancy of the Calibration Factor.- 8.3.4.5 Typical Analytical Data.- References.- 8.4 Ultrafiltration.- 8.4.1 Introduction.- 8.4.2 Principle.- 8.4.3 Method.- 8.4.3.1 UF Equipment and Membranes.- 8.4.3.2 Preparation of Feed Solution.- 8.4.3.3 Procedure.- 8.4.3.4 Data Presentation.- 8.4.4 Discussion of Method.- References.- 9 Separation of Identification of Low-Molecular Weight Fragments and Model Compounds.- 9.1 Gas Chromatography-Mass Spectrometry (GC-MS).- 9.1.1 Introduction.- 9.1.2 Sample Preparation.- 9.1.2.1 Extraction of Mixtures of Chemical Degradation Products.- 9.1.2.2 Extraction of Spent Pulping Liquors and Bleaching Effluents.- 9.1.2.3 Derivatization.- 9.1.2.4 Preparation of Sample Solution.- 9.1.3 GC-MS Analysis.- 9.1.3.1 Selection of Capillary Column for GC Analysis.- 9.1.3.2 Selection of Optimum Conditions for GC Operation.- 9.1.3.3 Selection of Most Suitable Mode for MS Operation.- 9.1.3.4 GC-MS Operation.- 9.1.3.5 Retrieval and Editing of Mass Spectral Data.- 9.1.4 Discussion of Method.- 9.1.4.1 Validity of Structural Assignments.- 9.1.4.2 Mass Spectra Quality.- 9.1.4.3 Interpretation of Mass Spectra.- References.- 9.2 High Performance Liquid Chromatography (HPLC).- 9.2.1 Significance of the Analysis.- 9.2.2 Survey of Available Methods.- 9.2.3 Principle of the Preferred Method.- 9.2.3.1 Adsorption Chromatography.- 9.2.3.2 Reversed Phase Chromatography.- 9.2.3.3 Chromatographic Theory.- 9.2.4 Description of the Preferred Method.- 9.2.4.1 Mobile Phase and Pump Considerations.- 9.2.4.2 HPLC Column: Attachment, Equilibration, and Performance Checks.- 9.2.4.3 E/Z Monolignols - Separation and Calibration.- 9.2.5 Discussion of Methods.- 9.2.5.1 Monomers.- 9.2.5.2 Dimers.- 9.2.5.3 Trimers and Higher Oligomers (Lignin Model Compounds).- References.