The HPLC Solvent Guide SECOND EDITION PAUL C. SADEKTHE HPLC SOLVENT GUIDE. SECOND EDITION PAUL C. SADEK WILEY- INTERSCIENCE A John Wiley & Sons, Inc., Publication‘This book is printed on acid-free paper. @) Copyright © 2002 by John Wiley and Sons, Inc., New York, All rights reserved, Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form ot by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except as permitted under Sections 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA (01923, (978) 750-8400, fax (978) 750-4744. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 605 Third Avenue, New York, NY 10158-0012, (212) 850-601, fax (212) 850-6008, E-Mail: PERMREQ@WILEY.COM. For ordering and customer service, call 1-800-CALL-WILEY. Library of Congress Cataloging-in-Publication Data CIP data is available ISBN 0-471-41 138-8 Printed in the United States of America 10 9.8 7 6 5 45 21 CONTENTS Preface Introduction Abbreviations, Acronyms, and Symbols xvii Chapter 1 Physical and Chemical Solvent Properties J Ll UV Cutoff, 2 1.2 Absorbance Versus Wavelength Curves, 3 1.3. Reversed-Phase Solvents, 5 1.3.1 Mobile Phases and Mobile Phase Modifiers, 9 1.4 Normal-Phase Solvents, 11 1.5 System Peaks, 14 1.6 Lot-to-lot Solvent Variability, 16 1.7. Viscosity, 19 1.8 Miscibility and Solubility, 22 1.9 Buffers and Other Mobile Phase Additives, 25 1.10 Volatility, 29 Lu Instability, Reactivity, and Denaturants, 31 LILI Ethers, 32 1.11.2. Chlorinated Alkanes, 34 1.11.3 Ethanol, 36 1.11.4 Water, 36 1.11.5. Acetone,, 38 1.12 Sample Stability in Solvents, 39 1.13 Mobile Phase Interaction with Stationary Phase, 40 1.14 Inherent Contaminants, 41 1.15 Solvent Effects on Fluorescence, 42 1,16 Particulates and Solvent Filtering, 43 1.17 Manufacturers and Testing Protocols, 44vi CONTENTS Chapter 2 Method Optimization 45 21 ears a 24 pe) 2.6 Eluotropic Series and Solvent Strength Parameters, 46 2.1.1 Eluotropic Series, 46 2.1.2. Solvent Strength Parameters, 49 2.1.3 Mathematical Representation of Solvent Strength, 52 Triangulation and Other Algorithmic Methods, 53 Scout Gradients, 58 Window Diagrams, 60 Brute-force Method, 60 Other Optimization Techniques and Retention Parameters, 61 Chapter 3. Method Validation and Ongoing Performance Evaluation 63 ce 7. 33 Qualification Process, 64 3.1.1 Instrument Qualification, 64 3.1.2. Design Qualification, 65 3.1.3 Installation Qualification, 65 3.1.4 Operational Qualification, 66 3.1.5. Performance Qualification, 66 Method Validation, 67 3.2.1 Accuracy, 68 3.2.2 Precision, 68 3.2.3. Linearity, 69 3.2.4 Linear and Working Ranges, 71 3.2.5 Specificity, 72 3.2.6 Detection and Quantitation Limit, 72 3.2.7 Robustness, 73 3.2.8 Ruggedness, 73 System Suitability, 74 Chapter 4 Alcohols 15 41 42 4.3 Impurities, 81 General Analytes, 82 4.2.1 Simple Substituted Benzene Analytes, 82 4.2.2. Other Organic Compounds, 89 4.23 Organometallic Compounds and Metal-Ligand Complexes, 93 4.2.4 Summary, 96 Environmentally Important Analytes, 96 43.1 PAHS, Substituted PAHs, and Related Analytes, 96 43.2. Nitrated and Chlorinated Nonpesticide /Herbicide Poifutant Analytes, 100 43.3 Pesticides, Herbicide, and Fungicides, 103 43.3.1 Pesticides, 103 43.3.2 Herbicides, 108 4.3.3.3 Fungicides, 111 43.4 Summary, 113 44 aa 46 47 CONTENTS — vii Industrial and Polymer Analytes, 113 4.4.1 Surfactant and Additive Analytes, (13 4.4.2 Polymers and Polymer Additives, 115 443 Sunscreen Agents, 118 44.4 Dyes, [20 4.4.5 Other Industrial Analytes, 123 44.6 Summary, 124 Biological Analytes, 124 4.5.1 Carboxylic Acid Analytes, 124 4.5.2 Basic Amine Analytes, 130 : 45.3 Aflatoxin, Mycotoxins, and Other Toxic Analytes, 133 4.5.4 Vitamins and Related Analytes, 139 4.5.4.1 Water-soluble Vitamins and Related Compounds, 139 4.5.4.2 Fat-soluble Vitamins and Related Compounds, 140 4.5.5 Terpenoids, Flavonoids, and Related Compounds, 148 4.5.5.1 Terpenoids, 148 4.5.5.2. Flavanoids, 151 4.5.5.3. Caffeine and Related Compounds, 153 4.5.5.4 Other Compounds, 155 4.5.6 Analytes Derived from Oils and Fats, 169 45.7 Nucleotides, Nucleosides, and Related Analytes, 171 45.8 Other Analytes, 174 45.9 Summary, 178 Amino Acid, Peptide, and Protein Analytes, 178 4.6.1 Amino Acid Analytes, 178 46.2 Peptide Analytes, 180 4.6.3 Protein Analytes, 181 4.6.4 Summary, 184 Pharmaceutical Analytes, 184 4.7.1 Drug Surveys and Screening Procedures, 184 4.7.2. Retention Mechanisms for Drug Compounds, 186 4.7.3. NSAIDs and Analgesic Drugs, 187 4.7.4 Antibiotic Drugs, 189 4.7.5 Anticancer Drugs, 192 4.7.6 Antiepileptic Drugs, 195 4.7.7 Steroidal Drugs, 196 4.78 Anthelmintics, 198 4.7.9 Illicit Drugs, 199 4.7.10 Antihistamines, 201 4.7.11 Anti-HIV Drugs, 202 4.7.12 Antianxiety and Antipsychotic Drugs, 202 4.7.13 Other Drug Analytes, 204 4.7.14 Summary, 213vii. CONTENTS Chapter 5 Alkanes and Alkyl Aromaties 214 5.1 Les 5.3 54 == 5.6 5.7 Impurities, 216 General Analytes, 221 5.2.1 Simple Substituted Aromatic Analytes, 221 5.2.2. Organometallics and Metal-Ligand Complexes, 226 5.2.3. Summary, 227 Environmentally Important Analytes, 227 5.3.1 PAHs, Substituted PAHs, and Related Analytes, 227 5.3.2. Nitrated and Chlorinated Nonpesticide /herbicide Pollutant Analytes, 230 5.3.3 Pesticide, Herbicide, and Fungicide Analytes, 231 5.3.4 Summary, 233 Industrial and Polymer Analytes, 233 5.4.1 Surfactant and Additive Analytes, 233 5.4.2. Other Polymeric Analytes, 235 5.4.3 Fullerenes, 236 5.44 Summary, 237 Biological Analytes, 238 5.5.1 Carboxylic Acid Analytes, 238 5.5.2 Vitamins and Related Analytes, 239 5.5.3 Terpenoids, Flavonoids, Steroids, and Related Compounds, 242 5.5.4 Analytes Derived from Oils and Fats, 244 5.5.5 Other Analytes, 248 55,6 Summary, 250 ‘Amino Acid and Peptide Analytes, 250 5.6.1 Summary, 250 Pharmaceutical Analytes, 252 5.1.1 Cardiac Glycosides, 252 5.7.2. NSAIDs and Analgesic Drugs, 253 5.7.3 Benzodiazepines, 253 5.7.4 Other Analytes, 254 5.15 Summary, 257 Chapter 6 Chlorinated Alkanes and Chlorinated Benzenes 258 6.1 6.2 63 Impurities, 260 General Analytes, 266 6.2.1 Simple Substituted Benzene Analytes, 266 6.2.2 Organometallics and MetalLigand Complexes, 269 6.23 Summary, 269 Environmentally Important Analytes, 269 6.3.1 PAHs, Substituted PAHs, and Related Analytes, 269 6.3.2. Nitrated and Chlorinated Nonpesticide/herbicide Analytes, 271 63.3 Pesticide and Herbicide Analytes, 271 63.4 Summary, 272 64 6.5 6.6 67 CONTENTS ix Industrial and Polymer Analytes, 272 6.4.1 Surfactant and Additive Analytes, 272 6.4.2 Polymeric Analytes, 273 643 Fullerenes, 274 6.4.4 Summary, 275 Biological Analytes, 275 6.5.1 Carboxylic Acid Analytes, 275 6.5.2. Vitamins and Related Analytes, 275 6.5.3 Analytes from Fats and Oils, 277 6.5.4 Other Analytes, 278 6.5.5 Summary, 280 ‘Amino Acid and Peptide Analytes, 280 Pharmaceutical Analytes, 281 Chapter 7 Ethers 285 | 72 re 14 15 = 7 Impurities, 287 General Analytes, 288 7.2.1 Simple Substituted Benzenes and Related Analytes, 288 Environmentally Important Analytes, 291 73.1 PAHs, Substituted PAHs, and Related Analytes, 291 73.2. Pesticide and Herbicide Residue Analytes, 292 Industrial and Polymer Analytes, 294 7.4.1 Surfactant and Additive Analytes, 294 7.4.2. Polymeric Analytes, 294 74,3 Fullerenes and Other Industrial Analytes, 296 Biological Analytes, 297 7.5.1 Carboxylic Acid Analytes, 297 7.5.2. Vitamins and Related Analytes, 298 7.5.3 Terpenoids, Flavonoids, Steroids, and Related Analytes, 300 7.5.4 Analytes Derived from Oils and Fats, 302 75.5 Other Analytes, 303 ‘Amino Acid and Peptide Analytes, 305 Pharmaceutical Analytes, 305 7.7.1 Summary, 311 Chapter 8 Ketones and Esters 312 8.1 es te) 8.4 BS 8.6 Impurities, 314 General Analytes, 316 Environmentally Important Analytes, 318 Industrial and Polymer Analytes, 319 Biological Analytes, 320 Pharmaceutical Analytes, 325x CONTENTS Chapter 9 Nitriles and Nitrogenous Solvents 327 ie 9.2 93 94 ae 9.6 Impurities, 328 General Analytes, 329 9.2.1 General Sample Solvent Considerations, 329 9.2.2 Simple Substituted Hydrocarbons and Benzene Analyte Retention Studies, 332 9.2.3 Other Compounds, 337 9.2.4 Organometallic and Metal-Ligand Complexes, 341 9.2.5 Summary, 343 Environmentally Important Analytes, 343 9.3.1 Substitutes Benzenes and Related Analytes, 343 PAHs, Substituted PAHs, and Related Analytes, 344 Nitro-, Nitroso-, and Chlorinated Nonpesticide/herbicide Pollutant Analytes, 348 9.3.4 Pesticide, Herbicide, and Fungicide Analytes, 350 9.3.4.1 Pesticides, 350 9.3.4.2 Herbicides, 363 9.3.4.3 Fungicides, 368 9.3.4 Summary, 369 Industrial and Polymer Analytes, 370 9.4.1 Surfactant and Additive Analytes, 370 9.4.2 Polymeric Analytes, 373 9.4.3 Dyes and Related Analytes, 375 9.44 Other Industrial Analytes, 378 9.4.5. Personal Care and Cosmetic Analytes, 379 9.4.6 Summary, 380 Biological Analytes, 381 9.5.1 Carboxylic Acid Analytes, 381 9.5.2 Basic Amine Analytes, 387 9.5.3 Aflatoxins, Mycotoxins, and Other Toxic Analytes, 395 9.5.4 Vitamins and Related Analytes, 401 9.5.4.1 Water-soluble Vitamins and Related Compounds, 401 9.5.4.2 Fat-Soluble Vitamins and Related Compounds, 402 9.5.5 Terpenoids, Flavonoids, and Other Naturally Occurring Analytes, 405 9.5.5.1 Terpenoids, 405 9.5.5.2. Flavanoids and Related Compounds, 408 9.5.5.3 Alkaloids and Related Compounds, 418 9.5.5.4 Fats, Oils, and Related Analytes, 425 9.5.6 Nucleotides, Nucleosides, and Related Analytes, 428 9.5.7 Sugars and Related Analytes, 430 9.5.8 Other Analytes, 433 9.5.9 Summary, 450 ‘Amino Acid, Peptide, and Protein Analytes, 450 Be ee CONTENTS xi 9.6.1 Amino Acid Analytes, 450 9.6.2 Peptide Analytes, 452 9.6.3 Protein Analytes, 457 9.6.4 Summary, 459 Pharmaceutical Analytes, 459 9.7.1 Drug Surveys and Screening Procedures, 459 9.7.2. NSAIDs and Analgesic Drugs, 460 9.7.3. Antibiotic Drugs, 464 9.7.4 Anticancer Drugs, 476 9.7.5. Antiepileptic Drugs, 482 9.7.6 Anthelminties, 485 9.7.7 Illicit and Related Drugs, 487 9.7.8 Antihistamines, 490 9.7.9 Antiretroviral Drugs, 492 9.7.10 Antidepressants, 496 9.7.11 Antibacterial Drugs, 499 9.7.12 Anesthetics, 503 9.7.13 Immunosuppressants, 504 9.7.14 Analgesics, 505 9.7.15 Antihypercholesterolemic and Antihyperlipidemic Drugs, 506 9.7.16 Antihypertension Drugs, 507 9.7.17 Antimalarials, 511 9.7.18 Steroids, 512 9.7.19 Antipsychotics, 515 9.7.20 Other Drug Analytes, 518 Summary, 527 Chapter 10 Water, Dimethyl Sulfoxide, and Common Acidic Modifiers 531 10.1 10.2 10.3 10.4 10.5 10.6 General Considerations and Impurities, 531 10.1.2 Solvent Preparation, 532 Industrial Analytes, 535 Biological Analytes, 538 10.3.1 Carboxylic Acid Analytes, 539 10.3.2 Basic Amine Analytes, 540 10.3.3 Toxins, 541 10.3.4 Vitamins, $42 10.3.5 Nucleotides, Nucleosides, Amino Acids, and Peptides, 543 10.3.6 Terpenoids, Flavonoids, and Related Analytes, 545 Pharmaceutical Analytes, 546 Summary, 547 Dimethyl Sulfoxide, 549 References 552 Index 607PREFACE HPLC separations are a fundamental analytical tool for the vast majority of testing laboratories. Over the last few years HPLC has expanded in two important ways. First, it become even more prevalent in the pharmaceutical, nutraceutical (natural product), and protein/peptide characterization areas. Second, along with this, mass spectrometry has become more widespread as a detector of choice. The second edition has been updated to reflect these advances. In addition, the original 1123 references have been replaced, where possible, by newer citations and the reference base has been increased to over 1600. The original Chapter 1 has been reorganized into three chapters; the first still deals with solvent properties. The second expands the topics of method optimization somewhat, and the third deals briefly with method/system validation and operating parameters. The most striking change is the addition of chemical structures. This was done to give the analyst a ready reference to the chemicals cited in a separation and their similarities /differences. Obviously, presenting the structures for all compounds was not feasible (and not necessary). For example, simple substituted benzenes, common acids like acetic, benzoic, etc., and base structures such as pentane (and 1-pentanol) are not included. Great effort was made in trying to verify structures and to check and recheck their accuracy of depiction. However, with the size of the database and the paucity of available independent structure verification (i.e., second sources), there may be some errors. ‘As with the first edition, my hope is that the information contained in this volume will significantly decrease the time and effort for the analyst and in terms of both deriving final methods and avoiding the numerous potential problems associated with reaching that method. Paul C. Sadek Grand Rapids, MIThis book serves two separate and important functions for the chromatographer: practical and operational. The first three chapters deal with the operational aspects of solvents. They contain information regarding solvents and solvent classes, method optimization techniques, and the definition and use of method validation proto- cols/system suitability parameters. These chapters describe solvents from a practical use-oriented point of view. Here the physical and chemical properties of numerous solvents are discussed with respect to their impact on the chromatographic system. A clear understanding of the implications presented by these properties will save the chromatographer considerable time and effort. The method optimization chapter reviews five commonly used techniques for assisting bench chromatographers in method optimization or development: eluo- tropic series and solvent strength, triangulation, “scout gradient,” window diagrams, and brute force. Detailed examples and expianations are presented so that these concepts can readily be put to use. Theory is not discussed. : Chapter 3 deals with validation issues (e.g., determination of whether or not a method produces the correct results [accuracy], reproducibly [precision and robust- ness}, and from analyst to analyst and laboratory to laboratory [ruggedness]) as well as ongoing system suitability parameters (e.g., those variables that are monitored and recorded that ensure that the system is functioning properly during any given analysis). In total, these first three chapters embody the “use” aspect of solvents and chromatographic systems. The last seven chapters (Chapters 4 to 10) present documented real-world chromatographic uses for each solvent class on an individual basis: alcohols, ketones and esters, nitriles and nitrogenous, ethers, and so on. These chapters contain details regarding individual separations so that they may be implemented directly or used in conjunction with the method optimization chapter to modify existing methods in order to meet the needs of new and specific separations.ABBREVIATIONS, ACRONYMS, AND SYMBOLS a Selectivity factor [= (kj/#4)] APCL Atmospheric pressure chemical ionization AU Absorbance unit BHT 2,6-di-t-butyl-4-methylphenol c Butyl-bonded phase Cs Octyl-bonded phase Cig Octadecyl-bonded phase CG Alkyl chain carbons in length CN Cyanopropyl-bonded phase DMF Dimethylformamide DMSO, Dimethyl sulfoxide e Eluotropic strength EC Electrochemical EDTA Ethylenediaminetetraacetic acid ELSD Evaporative light scattering detector em Emission wavelength used in fluorescence detection x Excitation wavelength used in fluorescence detection FAB Fast atom bombardment fg Femtogram (10-'S g) fmol Femtomole (10-5 mol) FMOC 9-Fluoromethy|chloroformate FTIR Fourier transform infrared GPC Gel permeation chromatography HPLC High-performance liquid chromatography Icp Inductively coupled plasmaxviii IPA K LC mg mL mM mmol He. HL uM umol MiBE nmol ABBREVIATIONS, ACRONYMS, AND SYMBOLS Isopropyl alcohol, 2-propanol Capacity factor [= (te ~ fo)/to)] Wavelength Liquid chromatography Molar, molarity (mol/L) Milligram (10° g) Milliliter (10-3 L) Millimolar (10—> mol/L) Millimole (10-3 mol) Microgram (10~* g) Microliter (10-° L) Micromolar (10~* mol/L) Micromole (10- mol) Methyl t-butyl ether Molecular weight Mass spectrometry. Nanogram (107° g) Nanomole (107° mol) Normal phase o-Phthalaldehyde Solvent strength parameter Picogram (10-}? g) Picomole (10-"? mol) Polyaromatic hydrocarbon Refractive index Reversed phase Sodium dodecy] sulfate Signai-to-noise ratio Solute retention time Void volume of system Triethylamine Trifluoroacetic acid Tetrahydrofuran Ultraviolet THE HPLC SOLVENT GUIDE SECOND EDITION