Methodology for Structural Analysis of Polysaccharides (eBook)

Dr. Steve W. Cui is a Senior Research Scientist at the Guelph Food Research Centre, Agriculture and Agri-Food Canada, Adjunct Professor at the University of Guelph, Department of Food Science. Dr. Cui’s expertise includes dietary fibre and dietary fibre analysis, structure and functional properties of bioactive carbohydrates and from agricultural products and natural hydrocolloids. He is expert on extraction, fractionation, analysis of natural polysaccharides, and elucidation of polysaccharide structures using methylation analysis, 2D NMR and mass spectroscopic techniques. He has been studying the bioactivity and structure-function relationship of polysaccharides by examining their conformation, rheological properties and functionality using light scattering and computer modelling approaches, and developing methodologies and process technologies to incorporate soluble dietary fibre into food products to exert health benefits.  Dr. Cui is the founder and Editor-in-Chief of the Bioactive Carbohydrates and Dietary Fibre, and Editor and Editorial Board Member of Food Hydrocolloids. He was Editor of Food Research International for 3 years (2009-2012) and served and a Director for CIFST during the same peroid. He is the Executive Director of the International Complex Carbohydrates Consortium (ICCC).  He authored and edited two books: “Polysaccharide Gums from Agricultural Products: Processing, Structures and Functionality (2000)” and “Food Carbohydrate: Chemistry, Physical Properties and Applications” (CRC Press, 2005).  Dr. Cui holds 8 patents and published over 170 peer-reviewed scientific papers and dozens of book chapters in the area of food carbohydrates.  Dr. Cui graduated from the Peking University, China with a B.Sc. degree in 1983, from Jiangnan University (Wuxi, China) with a M.Sc. degree in 1986, and from the University of Manitoba (Winnipeg, Manitoba) with a Ph.D. degree in 1993 specialized in food carbohydrates.Dr. Qingbin Guo is currently working as  an formal professor at Tianjin University of Science & Technology, China. He received his Ph.D. degree from University of Guelph  and  had postdoctoral experience with both University of Guelph (Canada) and Kansas State University (USA). His expertise includes carbohydrate analysis, molecular structure/conformation analysis, food dietary fibres and food extrusion for value added process. He has published over 30 peer reviewed papers in different well recognized journals and 4 book chapters in the area of food carbohydrate.

Contents 6
Abbreviations 9
1 Strategies for Structural Characterization of Polysaccharides 12
1.1 Complexity of Polysaccharide Molecules 12
1.1.1 Monosaccharide Composition 13
1.1.2 Glycosidic Bonds 14
1.1.3 Branching Position and the Degree of Substitution 14
1.1.4 Configurations (?-Form and ?-Form) 15
1.1.5 Special Functional Groups 15
1.1.6 Molecular Weight and Molecular Weight Distribution 16
1.2 Strategies for Structural Characterization of Polysaccharides 16
References 18
2 Polysaccharide Extraction and Fractionation 19
2.1 Extraction and Purification 19
2.2 Fractionation 21
2.2.1 Sequential Ammonium Sulphate Precipitation 21
2.2.2 Sequential Ethanol Precipitation 22
2.2.3 Column Separation 22
2.3 Purity Evaluation of Polysaccharide Samples Prior to Structural Analysis 26
References 26
3 Molecular Weight Distribution and Conformational Properties of Polysaccharides 28
3.1 Triple Detector HPSEC 29
3.1.1 Separation Principle of Size Exclusion Chromatography and Three Calibration Methods of Molecular Weight Determination 29
3.1.2 Conformational Properties of Polysaccharides by HPSEC 29
3.1.3 Branched Structure of Polysaccharides 31
3.2 Batch Mode Multi-angle Light Scattering 33
References 35
4 Monosaccharide Composition Analysis 37
4.1 Complete Hydrolysis 37
4.2 Separations and Detections 38
4.2.1 Anion-Exchange Chromatography 38
4.2.2 GC with Flame Ionization Detection 40
4.2.3 Reverse Phase HPLC Methods 41
4.2.4 Capillary Electrophoresis for Monosaccharide Composition Analysis 42
References 43
5 Partial Acid Hydrolysis and Molecular Degradation 45
5.1 Non-specific Degradation 45
5.1.1 Partial Acid Hydrolysis 45
5.1.2 Ultrasonication Degradation 46
5.2 Controlled Degradation 47
5.2.1 Enzymatic Degradation 47
5.3 Removal of Glycosides from Glycoproteins for Structure Determination 50
References 51
6 Linkage Pattern Analysis 52
6.1 Methylation Analysis 52
6.2 Acidic Polysaccharides Reduction Before Methylation Analysis 54
6.3 Strategies of Methylation Analysis for Glycoprotein or Other Uncommon Sugars 56
6.4 Smith Degradation (Goldstein, Hay, Lewis, & Smith, 1959, 1965)
6.4.1 Periodate Oxidation 57
6.4.2 Reduction and Acid Hydrolysis 57
References 58
7 1D & 2D and Solid-State NMR
7.1 Sample Preparation 59
7.2 1D NMR 60
7.3 2D NMR 61
7.3.1 COSY Spectrum 62
7.3.2 TOCSY Spectrum 63
7.3.3 HSQC Spectrum 64
7.3.4 Sequence Determination by NOESY and HMBC 65
7.4 Solid-State NMR 67
References 68
8 MALDI-TOF-MS for Polysaccharides Analysis 70
8.1 Introduction 70
8.2 Principles and Procedures (Cui, 2005a) 70
8.3 Applications 71
References 73
9 Fourier Transform Infrared Spectroscopy (FTIR) for Carbohydrate Analysis 74
References 76
10 Detailed Experimental Procedures 77
10.1 Total Sugar Analysis (Phenol-Sulfuric Acid Assay) 77
10.1.1 Sample Preparation 77
10.1.2 Testing Method 77
10.2 Uronic Acid Analysis 78
10.2.1 Sample Preparation 78
10.2.2 Testing Method 78
10.3 Column Separation for Polysaccharides from Flaxseed Hulls 78
10.4 Methylation Analysis 79
10.4.1 Reduction of Uronic Acids 79
10.4.2 Methylation Analysis 80
10.5 Controlled Degradation 81
10.5.1 Periodate Oxidation and Smith Degradation (Ai et al., 2016) 81
10.5.2 Partial Acid Hydrolysis 81
10.5.3 Trifluoromethanesulfonic Acid (TFMS) Hydrolysis 82
10.6 Sample Preparation and Experimental Procedure for NMR Analysis (Guo et al., 2015) 82
References 82
11 Summary 84
Reference 85