Introduction to Modern Liquid Chromatography, Third Edition part 4 pdf

xxviii CONTENTS 17.3.5 Module Substitution, 820 17.3.6 Put It Back, 821 17.4 Common Symptoms of HPLC Problems, 821 17.4.1 Leaks, 822 17.4.1.1 Pre-pump Leaks, 822 17.4.1.2 Pump Leaks, 823 17.4.1.3 High-Pressure Leaks, 825 17.4.1.4 Autosampler Leaks, 825 17.4.1.5 Column Leaks, 828 17.4.1.6 Detector Leaks, 828 17.4.2 Abnormal Pressure, 830 17.4.2.1 Pressure Too High, 831 17.4.2.2 Pressure Too Low, 832 17.4.2.3 Pressure Too Variable, 833 17.4.3 Variation in Retention Time, 833 17.4.3.1 Flow-Rate Problems, 834 17.4.3.2 Column-Size Problems, 834 17.4.3.3 Mobile-Phase Problems, 834 17.4.3.4 Stationary-Phase Problems, 835 17.4.3.5 Temperature Problems, 836 17.4.3.6 Retention-Problem Symptoms, 836 17.4.4 Peak Area, 838 17.4.4.1 Peak Area Too Large, 839 17.4.4.2 Peak Area Too Small, 840 17.4.4.3 Peak Area Too Variable, 840 17.4.5 Other Problems Associated with the Chromatogram, 841 17.4.5.1 Baseline Drift Problems, 841 17.4.5.2 Baseline Noise Problems, 844 17.4.5.3 Peak Shape Problems, 847 17.4.6 Interpretation of System Performance Tests, 856 17.4.6.1 Interpretation of Gradient Performance Tests, 857 17.4.6.2 Interpretation of Additional System Tests, 864 17.5 Troubleshooting Tables, 865 References, 876 APPENDIX I. PROPERTIES OF HPLC SOLVENTS 879 I.1 Solvent-Detector Compatibility, 879 I.1.1 UV Detection, 879 I.1.2 RI Detection, 881 CONTENTS xxix I.1.3 MS Detection, 881 I.2 Solvent Polarity and Selectivity, 882 I.3 Solvent Safety, 885 References, 886 APPENDIX II. PREPARING BUFFERED MOBILE PHASES 887 II.1 Sequence of Operations, 887 II.2 Recipes for Some Commonly Used Buffers, 888 Reference, 890 Index 891 PREFACE H igh-performance liquid chromatography (HPLC) is today the premier technique for chemical analysis and related applications, with an ability to separate, analyze, and/or purify virtually any sample. The second edition of this book appeared in 1979, and for tens of thousands of readers it eventually became their choice of an HPLC reference book. The remarkable staying power of the second edition (with significant sales into the first decade of the present century) can be attributed to certain features which continue to be true for the present book. First, all three editions have been closely tied to short courses presented by the three authors over the past four decades, to an audience of more than 10,000 industrial, governmental, and academic chromatographers. Teaching allows different approaches to a subject to be tried and evaluated, and a pragmatic emphasis is essential when dealing with practicing chromatographers as students. Second, all three editions have tried to combine practical suggestions (‘‘how to?’’) with a theoretical background (‘‘why?’’). Both theory and practice continue to be emphasized so that the reader can better understand and evaluate the various recommendations presented here. Finally, each of the three authors has been an active participant in HPLC research, development, and/or routine application throughout most of their careers. Since the preparation of the second edition in 1979, there have been major improvements in columns and equipment, as well as numerous advances in (1) our understanding of HPLC separation, (2) our ability to solve problems that were troublesome in the past, and (3) the application of HPLC for new kinds of samples. Whereas six different HPLC procedures received comparable attention in the second edition, today reversed-phase chromatography (RPC) accounts for about 80% of all HPLC applications—and therefore receives major (but not exclusive) attention in the present edition. Over the past three decades the use of HPLC for biological samples, enantiomeric (chiral) separations, and sample purification has expanded enormously, accompanied by a much better understanding of these and other HPLC applications. Commercial HPLC columns continue to be improved, and many new kinds of columns have been introduced for specific applications, as well as for faster, trouble-free operation. Prior to 1990, HPLC method development was an uncertain process—often requiring several months for the acceptable separation of a sample. xxxi xxxii PREFACE Since then it has become possible to greatly accelerate method development, espe- cially with the help of appropriate software. At the same time HPLC practice is increasingly carried out in a regulatory environment that can slow the release of a final method. These various advances and changes in the way HPLC is carried out have mandated major changes in the present edition. The organization of the present book, while similar to that of the second edition, has been significantly modified in light of subsequent research and experience. Chapter 1 provides a general background for HPLC, with a summary of how its use compares with other modern separation techniques. Chapter 1 also reviews some of the history of HPLC. Chapter 2 develops the basis of HPLC separation and the general effects of different experimental conditions. Chapters 3 and 4 deal with equipment and detection, respectively. In 1979 the detector was still the weak link in the use of HPLC, but today the widespread use of diode-array UV and mass-spectrometric detection—as well as the availability of several special-purpose detectors—has largely addressed this problem. Chapter 5 deals with the column: the ‘‘heart’’ of the HPLC system. In 1979, numerous problems were associated with the column: peak tailing—especially for basic samples, column instability at elevated temperatures or extremes in mobile-phase pH, and batch-to-batch column variability; today these problems are much less common. We also now know a good deal about how performance varies among different columns, allowing a better choice of column for specific applications. Finally, improvements in the column are largely responsible for our current ability to carry out ultra-fast separations (run times of a few minutes or less) and to better separate mixtures that contain hundreds or even thousands of components. Chapter 6, which deals with the reversed-phase separation of non-ionic samples, extends the discussion of Chapter 2 for these important HPLC appli- cations. A similar treatment for normal-phase chromatography (NPC) is given in Chapter 8, including special attention to hydrophilic interaction liquid chromatog- raphy (HILIC). In Chapter 7 the separation of ionized or ionizable samples is treated, whether by RPC, ion-pair chromatography, or ion-exchange chromatogra- phy. Gradient elution is introduced in Chapter 9 for small-molecule samples, and as an essential prerequisite for the separation of large biomolecules in Chapter 13; two-dimensional separation—another technique of growing importance—is also discussed. Chapter 10 covers the use of computer-facilitated method development (computer simulation). Other important, general topics are covered in Chapters 11 (Qualitative and Quantitative Analysis) and 12 (Method Validation). Chapter 13 introduces the separation of large molecules, including both biological and synthetic polymers. HPLC procedures that are uniquely useful for these separations are emphasized: reversed-phase, ion-exchange, and size-exclusion, as well as related two-dimensional separations. Chapter 14 (Enantiomer Separations) marks a decisive shift in approach, as the resolution of enantiomers requires columns and conditions that are sample-specific—unlike most of the HPLC applications described in earlier chapters. Chapter 15 deals with preparative separations (‘‘prep-LC’’), where much larger sample weights are introduced to the column. The big change since 1979 for prep-LC is that we now have a much better understanding of how such separations vary with conditions, in turn making method development much more systematic and efficient. Chapter 16 (Sample Preparation) provides a comprehensive PREFACE xxxiii coverage of this important supplement to HPLC separation. As in the case of other HPLC-related topics, the past 30 years have seen numerous developments that today make sample preparation a routine addition to many HPLC procedures. Finally, Chapter 17 deals with HPLC troubleshooting. Despite all our advances in equipment, columns, materials, technique, and understanding, trouble-free HPLC operation is still not guaranteed. Fortunately, our ability to anticipate, diagnose, and solve HPLC problems is now more informed and systematic. One of our three authors (JWD) has been especially active in this area. Different readers will use this book in different ways. An experienced worker may wish to explore topics of his or her choice, or find an answer to specific problems. For this audience, the Index may be the best starting place. Beginning readers might first skim Chapters 1 through 7, followed by 9 through 10, all of which emphasize reversed-phase HPLC. The latter sequence is similar to the core of the basic HPLC short courses developed by the authors. After this introduction, the reader can jump to chapters or sections of special interest. Other readers may wish to begin with topics of interest from the Contents pages at the front of the book or at the beginning of individual chapters. The present book has been organized with these various options in mind. This third edition is highly cross-referenced, so as to allow the reader to follow up on topics of special interest, or to clarify questions that may arise during reading. Because extensive cross-referencing represents a potential distraction, in most cases it is recommended that the reader simply ignore (or defer) these invitations to jump to other parts of the book. Some chapters include sections that are more advanced, detailed, and of less immediate interest; these sections are in each case clearly identified by an introductory advisory in italics, so that they can be bypassed at the option of the reader. We have also taken pains to provide definitions for all symbols used in this book (Glossary section), along with a comprehensive and detailed index. Finally, attention should be drawn to a ‘‘best practices’’ entry in the Index, which summarizes various recommendations for both method development and routine use. We very much appreciate the participation of eight collaborators in the preparation of the present book: Peter Schoenmakers (Sections 9.3.10, 13.10), Mike Swartz (Chapter 12), Tim Wehr (Sections 13.1–13.8), Carl Scandella (Section 13.9), Wolfgang Lindner, Michael L ¨ ammerhofer, and Norbert Maier (Chapter 14), Geoff Cox (Chapter 15), and Ron Majors (Chapter 16). Their affiliations are as follows: Peter Schoenmakers University of Amsterdam Mike Swartz Synomics Pharma Tim Wehr BioRad Corp. Carl Scandella Carl Scandella Consulting (4404 91st Avenue NE Bellevue, WA 98004) Wolfgang Lindner, Michael L ¨ ammerhofer, and Norbert Maier University of Vienna Geoff Cox Chiral Technologies Ron Majors Agilent Technologies xxxiv PREFACE We also are indebted to the following reviewers of various parts of the book: Peter Carr, Tom Chambers, Geoff Cox, Roy Eksteen, John Fetzer, Dick Henry, Vladimir Ioffe, Pavel Jandera, Peter Johnson, Tom Jupille, Ron Majors, Dan Marchand, David McCalley, Imre Molnar, Tom Mourey, Uwe Neue, Ravi Ravichandran, Karen Russo, Carl Scandella, Peter Schoenmakers, and Loren Wrisley. However, the authors accept responsibility for any errors or other shortcomings in this book. L LOYD R. SNYDER J. J. (JACK)KIRKLAND JOHN W. DOLAN Orinda, CA Wilmington, DE Amity, OR GLOSSARY OF SYMBOLS AND ABBREVIATIONS T his section is divided into ‘‘frequently used’’ and ‘‘less-frequently used’’ sym- bols.’’ Most symbols of interest will be included in ‘‘frequently used symbols’’. Equations that define a particular symbol are listed with that symbol; for example, ‘‘Equation 2.18’’ refers to Equation (2.18) in Chapter 2. The units for all symbols used in this book are indicated. Where IUPAC definitions or symbols differ from those used in this book, we have indicated the corresponding IUPAC term (from ASDLID 009921), for example, t M instead of t 0 . FREQUENTLY USED SYMBOLS AND ABBREVIATIONS A the ‘‘weak’’ component in a binary-solvent mobile phase (A/B); in RPC, the A-solvent is water or aqueous buffer; also, ‘‘type-A’’ silica (older, more acidic silica) ACN acetonitrile B (%B) the ‘‘strong’’ component (and its %-volume) in a binary-solvent mobile phase (A/B); in RPC, the B-solvent is an organic, such as acetonitrile; also, ‘‘type-B’’ silica (newer, less acidic silica; Section 5.2.2.2) CSP chiral stationary-phase CV coefficient of variation (equivalent to %-relative standard deviation); also, column volumes (Section 13.9) C 8 ,C 18 Reversed-phase column-packing designations, indicating length of alkyl ligand bonded to the particle d c column inner diameter (mm) d p column-packing particle-diameter (μm) F mobile-phase flow rate (mL/min) xxxv xxxvi GLOSSARY OF SYMBOLS AND ABBREVIATIONS H column plate height (equal to L/N); see also ‘‘less-frequently used symbols’’ below HIC hydrophobic interaction chromatography HILIC hydrophilic interaction chromatography i.d. column or tubing inner diameter (mm) IEC ion-exchange chromatography IPC ion-pair chromatography k retention factor (same as capacity factor k  ); equal to (t R /t 0 ) − 1 k ∗ gradient retention factor; Equation (9.5) L column length (mm) LC-MS liquid chromatography–mass spectrometry LC-MS/MS LC-MS with a triple-quadrupole mass spectrometer M molecular weight (Da) MeOH methanol MS mass spectrometry N column plate number; Equation (2.9) n c ‘‘equivalent’’ peak capacity, usually referred to as ‘‘conditional’’ or ‘‘sample’’ peak capacity NPC normal-phase chromatography P pressure drop across the column (psi); bar or atmospheres = 14.7psi; megaPascal (MPa) = 10 bar = 147 psi; also, partition coefficient (Section 6.2) PC peak capacity; Equation (2.30), Figure 2.26a (isocratic); Equation 9.20, Figure 9.20 (gradient) pK a logarithm of the acidity constant for an acid or base; Equations (7.2), (7.2a) R F solute fractional migration in TLC; Equation (8.6), Figure 8.8 RI refractive index RPC reversed-phase chromatography R s resolution; Equation (2.23) S slope of plots of log k versus φ(d log k/dφ); Equation (2.26) SEC size-exclusion chromatography SPE solid-phase extraction T temperature ( o C) t D dwell time (min); equal V D /F TFA trifluoroacetic acid t G gradient time (min); Figure 9.10 GLOSSARY OF SYMBOLS AND ABBREVIATIONS xxxvii t 0 column dead-time (min); also the retention time of a non-retained solute;equaltoV m /F; Equations (2.4a), (2.7) T-P touching-peak; Figure 15.9b t R retention time (min); Equation (2.5) type-A older, more acidic silica (Section 5.2.2.2) type-B newer, less acidic silica (Section 5.2.2.2) UV ultraviolet absorption V D equipment dwell volume; Section 9.2.2.4 V m column ‘‘dead-volume’’; volume of the mobile phase within a column (mL); Equation (2.7a) W baseline peak width W; Figure 2.10a w s column saturation capacity (g) w x weight of solute injected (g) α separation factor; Equation (2.24a) φ change in φ during a gradient; Figure 9.2g ε mobile-phase solvent strength in NPC; Equations (8.2), (8.5); also, dielectric constant ε 0 value of ε (in NPC) for a pure solvent φ volume-fraction of the B-solvent (equal to 0.01 × %B) φ ∗ value of φ during gradient elution for a solute, when the band reaches the column midpoint ν reduced velocity; Equation (2.18a) η mobile-phase viscosity (cP) LESS-FREQUENTLY USED (OR LESS-COMMONLY UNDERSTOOD) SYMBOLS AND ABBREVIATIONS A absorbance A column hydrogen-bond acidity; Equation (5.3) AAPS American Society of Pharmaceutical Scientists AIQ analytical instrument qualification (or validation) AMT analytical method transfer AOAC Association of Official Analytical Chemists APCI atmospheric pressure chemical ionization API active pharmaceutical ingredient (also atmospheric pressure ionization) . Retention-Problem Symptoms, 836 17 .4. 4 Peak Area, 838 17 .4. 4.1 Peak Area Too Large, 839 17 .4. 4.2 Peak Area Too Small, 840 17 .4. 4.3 Peak Area Too Variable, 840 17 .4. 5 Other Problems Associated with the Chromatogram,. Column Leaks, 828 17 .4. 1.6 Detector Leaks, 828 17 .4. 2 Abnormal Pressure, 830 17 .4. 2.1 Pressure Too High, 831 17 .4. 2.2 Pressure Too Low, 832 17 .4. 2.3 Pressure Too Variable, 833 17 .4. 3 Variation in. 833 17 .4. 3.1 Flow-Rate Problems, 8 34 17 .4. 3.2 Column-Size Problems, 8 34 17 .4. 3.3 Mobile-Phase Problems, 8 34 17 .4. 3 .4 Stationary-Phase Problems, 835 17 .4. 3.5 Temperature Problems, 836 17 .4. 3.6