High-Speed Countercurrent Chromatography

Edited by

YOICHIRO ITO

Laboratory of Biophysical Chemistry National Institutes of Health

Bethesda, Maryland

WALTER D. CONWAY

Department of Pharmaceutics SUNY Buffalo

Buffalo, New York

A WILEY-INTERSCIENCE PUBLICATION

JOHN WILEY & SONS

New York / Chichester / Brisbane / Toronto / Singapore

High-Speed Countercurrent Chromatography

Edited by

YOICHIRO ITO

Laboratory of Biophysical Chemistry National Institutes of Health

Bethesda, Maryland

WALTER D. CONWAY

Department of Pharmaceutics SUNY Buffalo

Buffalo, New York

A WILEY-INTERSCIENCE PUBLICATION

JOHN WILEY & SONS

New York / Chichester / Brisbane / Toronto / Singapore

CONTENTS

PREFACE xvii CUMULATIVE LISTING OF VOLUMES IN SERIES xix

INSTRUMENTATION

CHAPTER 1 PRINCIPLE, APPARATUS, AND METHODOLOGY OF HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY 3 Yoichiro Ito

1.1. Introduction 3 1.2. Principle of High-Speed Countercurrent

Chromatography 5 1.2.1. Two-Phase Distribution in a Rotating

Coil in Unit Gravity 5 1.2.2. Flow-Through Coil Planet Centrifuge

Free of Rotary Seals 11 1.2.3. Mechanism of High-Speed

Countercurrent Chrmatography 13 1.3. High-Speed Countercurrent Chromatograph

Instrumentation 16 1.3.1. Multilayer Coil Planet Centrifuge (Type J) 16 1.3.2. Cross-Axis Synchronous Flow-Through

Coil Planet Centrifuge 22 1.4. Phase Distribution Diagrams 26

1.4.1. Retention of Stationary Phase in Type J Coil Planet Centrifuge 28

1.4.2. Retention of Stationary Phase in Cross-Axis Coil Planet Centrifuges 31

1.4.3. SettlingTime 34 1.5. General Methodology of High-Speed

Countercurrent Chromatography 36 1.5.1. Partition Coefficient 36 1.5.2. Preparation of Sample Solution 39

vn

Vlll CONTENTS

1.5.3. Elution 1.5.4. Detection

References

40 41 42

CHAPTER 2 ANALYTICAL HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY Daniel E. Schaufelberger

2.1. Introduction 2.2. Development of Analytical High-Speed

Countercurrent Chromatography 2.3. Theory 2.4. Practical Considerations

2.4.1. Instrumentation 2.4.2. Solvent Systems 2.4.3. Handling 2.4.4. Detection

2.5. Applications 2.5.1. Methods Development 2.5.2. Measurement of Partition Coefficients 2.5.3. Natural Products Isolation

2.6. Discussion References

45

45

45 46 51 51 56 56 57 60 60 62 63 66 68

SPECIAL TECHNIQUES

CHAPTER 3 HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY/MASS SPECTROMETRY 73 Hisao Oka

3.1. Introduction 73 3.2. Previous Interfacing of HSCCC With

Thermospray Mass Spectrometry 75 3.3. Direct Interfacing of HSCCC With Frit Electron

Ionization, Chemical Ionization, and Fast Atom Bombardment Mass Spectrometry 75

3.4. HSCCC/FritEIMSoflndoleAuxins 78 3.5. HSCCC/Frit CIMS of Mycinamicins 80 3.6. HSCCC/Frit FABMSof Colistin Complex 85 3.7. Conclusion 90 References 90

CONTENTS IX

CHAPTER 5

CHAPTER 4 DUAL COUNTERCURRENT CHROMATOGRAPHY 93 Y. W. Lee

4.1. Introduction 93 4.2. Principles and Mechanism 94 4.3. Methods and Apparatus 95 4.4. Applications 97 4.5. Conclusion 104 References 104

FOAM COUNTERCURRENT CHROMATOGRAPHY OF BACITRACIN COMPLEX 107 Hisao Oka

5.1. Introduction 107 5.2. Apparatus of Foam Countercurrent

Chromatography 108 5.3. Previous Foam Countercurrent

Chromatography Work 109 5.4. Separation of Bacitracin Components With

Nitrogen and Additive-Free Water 110 5.5. Continuous Enrichment and Stripping of

Bacitracin Components 117 5.6. Conclusion 118 References 120

CHAPTER 6 pH-PEAK-FOCUSING AND pH-ZONE-REFINING COUNTERCURRENT CHROMATOGRAPHY 121 Yoichiro Ito

6.1. Introduction 121 6.2. pH-Peak-Focusing Countercurrent

Chromatography 121 6.2.1. Research for the Causative Agent

Sharpening the BrAcT3 Peak 121 Chemohydrodynamic Mechanism of pH-Peak-Focusing 126 Advanced Peak-Focusing Technique 136 Conclusion 140

6.3. pH-Zone-Refining Countercurrent Chromatography 141

6.2.2.

6.2.3. 6.2.4.

X CONTENTS

6.3.1. Characteristic Features of pH-Zone-RefiningCCC

6.3.2. Chemohydrodynamic Mechanism of pH-Zone-Refining CCC

6.3.3. Simple Mathematical Model of pH-Zone-Refining CCC

6.3.4. Displacement pH-Zone-Refining CCC 6.3.5. pH-Zone-Refining CCC versus

Displacement Chromatography 6.3.6. Applications of pH-Zone-Refining

CCC 6.3.7. Conclusion

References

141

143

147 155

158

163 173 174

APPLICATIONS

CHAPTER 7 HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY OF NATURAL PRODUCTS 179 Marc Maillard, Andrew Marston, and Kurt Hostettmann

7.1. Introduction 7.1.1. Instruments 7.1.2. Solvent Systems

7.2. Preparative Applications 7.2.1. Flavonoids 7.2.2. Xanthones 7.2.3. Tannins 7.2.4. Lignans 7.2.5. Monoterpene Glycosides 7.2.6. Saponins 7.2.7. Alkaloids 7.2.8. Carotenoids 7.2.9. Gingerols 7.2.10. Polyacetylenic Alcohols 7.2.11. Marine Natural Products 7.2.12. Antibiotics

7.3. Analytical Applications 7.3.1. Alkaloids 7.3.2. Anthraquinones 7.3.3. Coumarins 7.3.4. Flavonoids

179 181 184 189 190 195 195 198 200 202 203 204 204 205 206 210 211 212 215 215 215

CONTENTS XI

7.3.5. Lignans 216 7.3.6. Macrolides 216 7.3.7. Triterpenoids 217

7.4. Conclusions 218 References 218

HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY ON MEDICINAL HERBS 225 Tian- You Zhang

8.1. Introduction 225 8.2. The Present Status of High-Speed

Countercurrent Chromatography on Isolation of Medicinal Herbs 226

8.3. Separations of Substances Prepared From Medicinal Herbs by the Horizontal Flow-Through Coil Planet Centrifuge 227 8.3.1. Apparatus and Experimental

Procedures 227 8.3.2. Separations of a Group of Similar

Substances 228 8.3.3. Studies on the Preparative Capability of

CPC and HPLC in the Separation of Polar Compounds 231

8.4. Separation of Alkaloids, Hydroxyanthraquinones, and Flavonoids by Analytical HSCCC 236 8.4.1. Apparatus and Experimental

Procedures 236 8.4.2. Separation of Alkaloids Extracted from

Stephania tetranda S. Moore 236 8.4.3. Separation of Hydroxyanthraquinone

Derivatives Extracted from Rhubarb 240 8.4.4. Rapid Separation of Flavonoids

Extracted from Sea Buckthorn (Hippophae rhamnoides) 242

8.5. Separations of Flavonoids and Alkaloids by Multilayer Coil Separator and Extractor 245 8.5.1. Apparatus and Experimental

Procedures 245

CONTENTS

8.5.2. Separation of Daphne genkwa Flavonoids 247

8.5.3. Separation of Flavonoids from Crude Extracts of Sea Buckthorn (Hippophae rhamnoides) 249

8.5.4. Separation of Alkaloids from Anisodus tangulicus (Maxin) Pasch 250

8.6. Semipreparative Separation of Alkaloids by GS-10A HSCCC 252 8.6.1. Apparatus and Experimental

Procedures 252 8.6.2. Separation of Alkaloids from

Sophora flavescens Ait and Datura mete L. 252

8.6.3. Separation of Alkaloids from Cephalotaxusfortunei Hook F. 255

8.6.4. Separation of Alkaloids from Senecio fuberi Hemsl 256

8.6.5. Separation of Vincamine and Vincine 259 8.7. Conclusion 262 References 263

CHAPTER 9 ISOLATION OF MARINE NATURAL PRODUCTS BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY 265 Nancy L. Fregeau and Kenneth L. Rinehart

9.1. Introduction 265 9.2. Alternative Separation Techniques 267 9.3. Countercurrent Chromatography 268 9.4. Examples of Marine Natural Product Isolations

Using High-Speed Countercurrent Chromatography 268 9.4.1. Macrolides 269 9.4.2. Polyethers 273 9.4.3. Other Acetate-Derived Metabolites 275 9.4.4. Terpenes and Steroids 276 9.4.5. Peptides 278 9.4.6. Nitrogen-Containing Heterocycles 280

9.5. Conclusion 296 References 297

CONTENTS Xll l

CHAPTER 10 SEPARATION OF COMPLICATED ANTIBIOTICS BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY 301 Ken-Ichi Harada

10.1. Introduction 301 10.2. Selected Antibiotics 302 10.3. Operation of HSCCC 304 10.4. Establishment of HPLC for SVD and BC 304 10.5. Selection of Proper Two-Phase Solvent

System 307 10.6. Separation of SVD and BC Components by

HSCCC 314 10.7. Comparison of Elution Behavior of SVD and

BC between HPLC and HSCCC 314 10.8. Conclusion 317 References 318

CHAPTER 11 N-BROMOACETYL-3,3 5-TRIIODO-L-THYRONINE AND JV-BROMOACETYL-L-THYROXINE: SYNTHESIS AND CHARACTERIZATION 321 Hans J. Cahnmann

11.1. 11.2.

11.3.

11.4.

11.5. Refe

Introduction Synthesis 11.2.1. Synthesis of BrAcT3

11.2.2. Synthesis of BrAcT4

Purification 11.3.1. High-Speed Countercurrent

Chromatography 11.3.2. Solvents 11.3.3. Elution 11.3.4. Eluate Scanning 11.3.5. Storage of HSCCC-Purified BrAcT3

and BrAcT4

Verification of Identity and Purity 11.4.1. HPLC 11.4.2. TLC 11.4.3. Absorption Spectra in the Near UV Summary

rences

321 322 324 325 326

326 328 330 330

330 331 331 331 333 334 335

XIV CONTENTS

CHAPTER 12 SEPARATION AND PURIFICATION OF DYES BY CONVENTIONAL HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY AND pH-ZONE-REFINING COUNTERCURRENT CHROMATOGRAPHY 337 Adrian Weisz

12.1. Introduction 337 12.2. Instrumentation 338 12.3. Conventional HSCCC Separations 338

12.3.1. Sulforhodamine B 338 12.3.2. Tetrabromotetrachlorofluorescein and

Phloxine B 340 12.3.3. Complementary Use of HSCCC and

Preparative HPLC in the Separation of a Synthetic Mixture of Dyes 342

12.4. pH-Zone-Refining Countercurrent Chromatography 347 12.4.1. Introduction 347 12.4.2. Principle 347 12.4.3. Preliminary Requirements for pH-

Zone-Refining CCC 350 12.4.4. Preparative Separations of Brominated

and Chlorinated Fluoresceins 352 12.4.5. Preparative Separations of Iodinated

Fluoresceins 375 12.5. Conclusions 381 References 382

CHAPTER 13 SEPARATION OF PROTEINS BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY 385 Yoichi Shibusawa

13.1. Introduction 385 13.2. Apparatus 386 13.3. Polymer Phase Systems for Protein Separation 391 13.4. Countercurrent Chromatography

Fractionation of Proteins 391 13.4.1. PEG-Potassium Phosphate Systems 391 13.4.2. PEG-Dextran Polymer Phase System 404

13.5. Conclusion 412 References 413

CONTENTS XV

CHAPTER 14 SEPARATION OF RARE EARTH AND CERTAIN INORGANIC ELEMENTS BY HIGH­SPEED COUNTERCURRENT CHROMATOGRAPHY 415 Eiichi Kitazume

14.1. Introduction 415 14.2. Separation of Rare Earth Elements by High-

Speed Countercurrent Chromatography 416 14.2.1. Description of the Instrument 416 14.2.2. Preparation of Two-Phase Solvent and

Sample Solutions 419 14.2.3. Separation Procedure 419 14.2.4. Measurement of Partition Coefficients

KTand KE 419 14.2.5. Partition Efficiencies 422 14.2.6. Gradient Elution of Fourteen Rare

Earth Elements 426 14.3. Analytical Capability of High-Speed

Countercurrent Chromatography for Rare Earth Elements 427 14.3.1. Reproducibility of Chromatogram 428 14.3.2. Quantitative Determination of Cerium

Impurities in Erbium Chloride 430 14.4. Separation of Other Inorganic Elements by

High-Speed Countercurrent Chromatography 430 14.4.1. Separation of Ortho-and

Pyrophosphate Ions 431 14.4.2. Separation of Cesium and Strontium 432 14.4.3. Separation of Zirconium and Hafnium 432 14.4.4. Separation of Cadmium and Zinc 435 14.4.5. Separation of Nickel, Cobalt,

Magnesium, and Copper 435 14.4.6. Separation of Copper, Cadmium, and

Manganese 437 14.4.7. Separation of Iron(II) and Iron(III) 438 14.4.8. Preconcentration of Trace Elements

from Certain Rock Macrocomponents 438 References 441

INDEX 445