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Column Handbook for Size Exclusion Chromatogruphy Edited by C h i-s a n W u International Specialty Products Wayne, New jersq 0kp A C A D E MIC PR E SS San Diego London Boston New York Sydney Tokyo Toronto - CONTENTS CONTRIBUTORS xix PREFACE xxi m CONTRIBUTIONS FROM SIZE EXCLUSION CHROMATOGRAPHY AND GEL-FILTRATION CHROMATOGRAPHY COLUMN MANUFACTURERS Preparation of Beaded Organic Polymers and Their Applications in Size Exclusion Chromatography MICHAEL J LU I Introduction II Basic Features of Conventional Suspension Polymerization A Droplet Formation B Droplet Stabilization C Particle Size and Pore Size III Preparation of Organic Polymeric Beads by Conventional Suspension Polymerization A Polystyrene/Polystyrene-Divinylbenzene B Polyacrylamide-Polymethacrylate C Polyvinyl Alcohol CONTENTS IV Special Polymeric Supports 10 A Templated Polymeric Supports 10 B Pore-Matrix Composite Supports 10 C Dual Functionality of Polymeric Supports 11 D Continuous Rods of Macroporous Polymeric Supports 14 E Porous Perfusion Particles 14 V Basic Features of Monosized Polymeric Particles Prepared by Activated Swelling and Polymerization 15 A Monosized Seed Particles 15 B Activated Seed Particles 16 C Swelling of the Activated Seed Particles with Monomers or a Mixture of Monomers 17 D Polymerization of Multistep Swelling Particles 11 E Preparation of Monosized Macroporous Particles 18 VI Application of Polymeric Beads as Size Exclusion Chromatography Supports A Polystyrene-Divinylbenzene-Based Resin 21 B Polyacrylamide- and Polymethacrylate-Based Gels 22 C Polyvinyl Alcohol-Based Gels 22 D Application of Monosized Polymeric Particles in Size Exclusion Chromatography 23 References 24 Size Exclusion for Analysis and Purification of Aqueous Macromolecules ALAN WILLIAMS AND LARS HAGEL I Introduction: Development of Size Exclusion Chromatography (SEC) 27 II Application Areas of SEC 29 A Determination of Molecular Size and Sample Purity 29 B High-Resolution Fractionation 35 C Desalting (Group Fractionation) 36 III Characteristics of Gel-Filtration/Size Exclusion Media 39 A Sephadex 39 B Sepharose C Sephacryl 44 D Superose 46 E Superdex 49 IV Columns for Packing SEC Media 54 A C Columns 55 B XK Columns 55 C SR Columns 56 D HR Columns 56 E INdEX Columns 57 F FineLINE Columns 58 G BPG Columns 59 CONTENTS H BioProcess Stainless-Steel Columns 59 I Stack Columns 59 V Preparation of the Size Exclusion Column 61 A Selection Criteria for Empty Columns 61 B General Packing Methodology 62 C Qualification of Column Packing 64 D Typical Results for Self-Packed Columns 66 VI Optimization of Gel-Filtration/Size Exclusion Separations A Selectivity 67 B Separating Volume 67 C Plate Height 69 D Column Length 69 E Sample Load 69 F Analytical Gel Filtration 70 G Preparative Gel Filtration 71 H Desalting 72 References 73 67 Zorbax Porous Silica Microsphere Columns for HighPerformance Size Exclusion Chromatography ROBERT T MOODY I Background Information 7.5 A Development of Silica Particles for High-Performance Size Exclusion Chromatography (HPSEC) 75 B Advantages and Disadvantages of Porous Silica Packings for HPSEC 76 C Manufacture of Zorbax Porous Silica Microspheres (PSM) 76 D Separation Mechanism 77 II Using and Selecting Zorbax PSM Columns and Mobile Phases 78 A Conducting HPSEC Experiments with Zorbax PSM Columns 78 B Selecting Columns and Mobile Phases for HPSEC 79 III HPSEC Troubleshooting 82 IV Applications 85 A General Applications 85 B Special Applications: Proteins 86 References 92 Bibliography 92 Size Exclusion Chromatography Using TSK-GEL Columns and Toyopearl Resins YOSHIO KATO, J KEVIN O’DONNELL, AND JON FISHER I Silica-Based TSK-GEL SW/SWxL Columns for Gel-Filtration Chromatography (GFC) 93 A Description 93 CONTENTS II III IV V VI B Conditions 94 C Applications 102 D Preparative TSK-GEL SW Columns 104 Polymer-Based TSK-GEL PW/PWxr Columns for Aqueous GFC 106 A Description 106 B Conditions 106 C Applications 118 Choosing TSK-GEL SW Type or TSK-GEL PW Type Columns for GFC 128 Cleaning and Rehydration of TSK-GEL Columns 134 A Cleaning Procedures 134 B Rehydration Procedure 135 TSK-GEL H Type Columns 135 A Introduction 135 B Product Offerings 136 C Limitation of Operation 138 D Separation Conditions 141 E Applications 145 Toyopearl Resins for Size Exclusion Chromatography 145 A Advantages of Toyopearl HW Resins 148 B Optimizing Separation Conditions 153 C Applications 154 References 157 American Polymer Standards Columns for Size Exclusion Chromatography JOHN E ARMONAS AND BRIAN H PEABODY I Introduction 159 II American Polymer Gel-Permeation/Size Exclusion Chromatography (GPC/SEC) Column Repacking 160 III GPC/SEC Styrene-Divinylbenzene Gel 163 IV Manufacture of GPUSEC Gels 163 A Discussion 163 B Experimental 164 C Optimizing the Formulation 166 D Classification of the GPC/SEC Gel 168 E Packing and Testing GPC/SEC Columns 168 V Conclusion 169 References 169 ix CONTENTS Shodex Columns for Size Exclusion Chromatography HIROSHI SUZUKI AND SADAO MORI I Introduction 171 II Gel Technology of Shodex Columns 171 III Shodex Organic GPC Series 172 A Column Characteristics 172 B Application Examples 189 IV Shodex OHpak SB-800HQ Series 193 A Column Characteristics 193 B Application Examples 193 C Preparative and Semipreparative Columns 197 V Shodex Asahipak GYGF Series 197 A Column Characteristics 97 B Application Examples: 198 C Preparative and Semipreparative Columns 200 VI Shodex Protein KW Series 204 A Column Characteristics 204 B Application Examples 205 C Preparative and Semipreparative Columns 210 VII Conclusion 210 Size Exclusion Chromatography on Fractogel EMD BioSEC LOTHAR R JACOB AND LOTHAR BRITSCH I II III IV V VI VII VIII IX X XI XII Introduction to Size Exclusion Chromatography (SEC) 219 Synthesis of Fractogel EMD BioSEC 220 Properties of Fractogel EMD BioSEC 221 Adequate Buffers 222 Recommended Columns for SEC 224 Packing the Column 226 A Preparing the Gel 228 B Packing a Superformance 600-l Column 228 C Packing of Preparative Columns 229 D Checking the Quality of the Packed Gel Bed 230 Recommended Flow Rates for SEC Columns 232 Loading Capacity 233 Determination of Molecular Mass of Proteins 235 Column and Gel Maintenance, Cleaning, Regeneration, and Storage Methods 237 Application Areas 241 Troubleshooting 246 References 247 X CONTENTS Size Exclusion High-Performance Liquid Chromatography of Small Solutes ANDREW J ALPERT I Overview of Size Exclusion Chromatography (SEC) of Small Solutes 249 II Introduction of PolyHYDROXYETHYL Aspartamide and the Effect of Chaotropes on the Fractionation Range 250 III Verification of SEC Mechanism for Small Solutes 254 A Effect of pH 255 B Effect of Salt 257 C Effect of Organic Solvents 258 D How Big Are Amino Acids? 259 E Superdex Peptide 260 IV Fractionation Range with PolyHEA as a Function of Pore Diameter 260 V Applications 265 A SEC of Peptides and Amino Acids 265 B SEC of Small Solutes Other Than Peptides 265 C Mass Spectrometry: On-Line Desalting 265 References 265 Design, Properties, and Testing of Polymer Standards Service Size Exclusion Chromatography (SEC) Columns and Optimization of SEC Separations P KILZ I Introduction 267 II Basis of SEC Column Design 268 A Major SEC Column Design Characteristics 269 III Review: SEC Sorbents and Requirements 270 IV SEC Column Selection Criteria 271 A Chromatographic Modes of Column Separation 272 B Selection of Pore Size and Separation Range 27.5 C Influence of Particle Size on Separation Eficiency 278 D Influence of Column Dimensions 281 E Influence of Operating Parameters 282 V Testing and Evaluation of SEC Columns 283 A Calculation of Plate Count 284 B Determination of Peak Asymmetry 286 C Calculation of Resolution 287 D Calculation of Efficiency 287 VI PSS Column Quality Control System 287 A Raw Materials 287 B Sorbent Manufacturing 288 C Testing of Sorbents 288 D Column Packing Process 289 XI CONTENTS VII Applications of PSS SEC Columns 291 A Organic SEC Applications 291 B Aqueous Applications 291 C Applications of PSS Polar Fluoro Gel Columns VIII Conclusion 298 References 303 295 IO SynChropak, Size Exclusion Columns KAREN M GOODING I Introduction 305 II Support Technology 306 A Physical Properties 306 B Chemical Properties 306 C Calibration 306 D Quality Assurance III Column Technology 10 A Hardware B Packing Techniques 12 C Quality Control 314 IV Operation 314 A Mobile Phase Selection 314 B Flow Rate 316 C Loading D Restrictions 318 V Applications 323 References 323 1 Waters Columns for Size Exclusion Chromatography UWE DIETER NEUE I Introduction 325 II Column Selection Principles 326 A Packing Type 326 B Pore Size Selection 328 C Particle Size Selection 332 D Narrow-Bore Columns 333 III Waters Products for Size Exclusion Chromatography 335 A The Styragel Line of Size Exclusion Columns 33.5 B The Ultrahydrogel Line of Size Exclusion Columns 343 C The Protein-Pak Line of Size Exclusion Columns 346 IV Summary 347 References 348 xii CONTENTS 12 Size Exclusion Chromatography Columns from Polymer Laboratories ELIZABETH MEEHAN I Introduction 349 II PLgel Organic Size Exclusion Chromatography (SEC) Columns A PLgel Individual Pore Size Columns 350 B PLgel Mixed Gel Columns 353 C PLgel Column Performance Testing 355 D Column Selection and Operation 3.55 III PL HFIPgel Columns 359 IV PL aquagel-OH Aqueous SEC Columns 360 V Column Hardware 363 A Narrow-Bore SEC Columns 364 B Preparative SEC Columns 365 VI Summary 366 References 366 13 Jordi Gel Columns for Size Exclusion Chromatography HOWARD JORDI I Introduction 367 II The Basic Polydivinylbenzene Packing 368 III Basic and Chemically Modified Polydivinylbenzene Packings 372 IV Modified GBR/PDVB Gel 376 V Discussion of Chromatographic Applications of Various Bonded and Nonbonded Phases 379 References 425 m CONTRIBUTIONS FROM USERS OF SIZE EXCLUSION CHROMATOGRAPHY AND GEL-PERMEATION CHROMATOGRAPHY 14 General Characterization of Gel-Permeation Chromatography Columns RUDOLPH BRUESSAU I Introduction 429 350 XIII CONTENTS II III IV V VI VII VIII IX Determination of the Number of Theoretical Plates 431 Asymmetry Factor 435 Separation Efficiency 435 Apparent Pore Size Distribution 437 Surface Interactions 438 Separation of the Oligomer Range from System Peaks 439 Column Dimensions 440 Conclusion 441 A Characterization of a GPC Column by the Manufacturer 441 B Testing a GPC Column before Using It 442 References 443 15 Interactive Properties of PolystyrenelDivinylbenzene and Divinylbenzene-Based Commercial Size Exclusion Chromatography Columns DUiAN BEREK I Introduction 445 II Experimental 448 A Columns 448 B Polymers 448 C Solvents 448 D Apparatus 449 E Data Evaluation 449 III Results 450 A Column No 450 B Column No 451 C Column No 453 D Column No 453 E Column No 453 F Column No 453 IV Discussion 4.55 References 457 16 Analytical and Preparative Columns for Aqueous Size Exclusion Chromatography of Polysaccharides ANTON HUBER AND WERNER PRAZNIK I Introduction 459 A Data Processing 460 B Broad Standard Size Exclusion Chromatography (SEC) Calibration 463 ... D Narrow-Bore Columns 333 III Waters Products for Size Exclusion Chromatography 335 A The Styragel Line of Size Exclusion Columns 33.5 B The Ultrahydrogel Line of Size Exclusion Columns 343 C... INdEX Columns 57 F FineLINE Columns 58 G BPG Columns 59 CONTENTS H BioProcess Stainless-Steel Columns 59 I Stack Columns 59 V Preparation of the Size Exclusion Column 61 A Selection Criteria for. .. Gel-Filtration /Size Exclusion Media 39 A Sephadex 39 B Sepharose C Sephacryl 44 D Superose 46 E Superdex 49 IV Columns for Packing SEC Media 54 A C Columns 55 B XK Columns 55 C SR Columns 56 D HR Columns