Introduction to Modern
`Liquid Chromatography
`
`Second Edition
`
`L. R. SNYDER
`Technicon Instruments Corporation
`Research & Development Division
`Tarrytown, New York
`
`J. J. KIRKLAND
`E. I. du Pont de Nemours & Company Central
`Research & Development Department
`Wilmington, Delaware
`
`A Wiley-Interscience Publication
`JOHN WILEY & SONS, INC.
`New York • Chichester • Brisbane • Toronto
`
`CUREVAC EX2004
`Page 1 of 402
`
`

`

`Copyright © 1979 by John Wiley & Sons, Inc.
`
`All rights reserved. Published simultaneously in Canada.
`
`Reproduction or translation of any part of this work
`beyond that permitted by Sections 107 or 108 of the 1976
`United States Copyright Act without the permission of the
`copyright owner is unlawful. Requests for permission or
`further information should be addressed to the Permissions
`Department, John Wiley & Sons, Inc.
`
`Library of Congress Cataloging in Publication Data:
`Snyder, Lloyd R.
`Introduction to modern liquid chromatography.
`
`"A Wiley-Interscience publication."
`Includes bibliographies and index.
`1. Liquid chromatography. I. Kirkland, Joseph Jack,
`joint author. II Title.
`
`QD79.0454S58 1979 544'.924 79-4537
`ISBN 0-471-03822-9
`
`Printed in the United States of America
`
`10 9 8 7 6 5 4 3 2 1
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`Page 2 of 402
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`

`And thus the native hue of resolution
`Is sicklied o'er with the pale cast of
`thought.
`
`Shakespeare Hamlet,
`Scene I, Act 3
`
`Page 3 of 402
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`

`PREFACE TO THE FIRST EDITION
`
`This book is about modern liquid chromatography. By this we mean automated,
`high-pressure liquid chromatography in columns, with a capability for the high-
`resolution separation of a wide range of sample types, within times of a few
`minutes to perhaps an hour. Modern liquid chromatography (LC) is now about five
`years old. By early 1969 it was possible to purchase equipment and high perform-
`ance column packings which together largely bridged the gap between classical
`liquid chromatography and gas chromatography. Since that time there has been a
`flurry of activity on the part of companies that supply equipment and materials for
`LC. Within the past few years there have been further major advances in the theory
`and practice of LC. Finally, numerous applications of modern LC to a wide range
`of problems are now being reported. The technique has reached the point where the
`average chromatographer can achieve - by yesterday's standards - consistently
`spectacular results.
`To get the most out of modem LC, some care is required in choosing the right
`technique, selecting the best separation conditions, and using the proper equip-
`ment to best advantage. In short, the practical worker must know what he is doing.
`Moreover, his knowledge must be a balance of theory and experience; it must
`include both principles and practice. Unfortunately, there has been a tendency for
`those in chromatography to stress either the theoretical or the "practical" side of
`the subject. Also, the theory of chromatography - and of modern LC - has often
`been represented as highly complex, with its application to real separation
`problems either not obvious, or impossibly tedious. We think there is a better
`approach.
`An effective presentation of what modern LC is all about must be a blend of
`practical details plus down-to-earth theory. This conviction led us in 1971 to
`develop the American Chemical Society short course "Modern Liquid Chromato-
`graphy. " Within the next two years we presented the course to about 800 students,
`with a highly enthusiastic response. The course itself continued to evolve during
`this time, largely in response to the questions and comments of the students. By
`
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`viii
`Preface to the First Edition
`late 1972 it appeared worthwhile to reduce our approach to textbook form, and the
`present book is the result.
`Our goal for this book was to retain the essential elements of the short course,
`and to add certain materials that could not be included in a two-day series of
`lectures. We did not hope to present everything of conceivable interest to LC in a
`book of this size, yet we were determined not to slight any area of significant
`practical importance. Where compromise between these two objectives eventually
`proved necessary, it has been handled by referencing other sources. The book was
`written to be self-sufficient in terms of the needs of the average professional or
`technician who plans to work with modern LC. We believe this book will prove
`useful in most laboratories where modern LC is practiced.
`
`L. R. SNYDER
`
` J. J. KIRKLAND
`
`October 1973
`
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`PREFACE TO THE SECOND EDITION
`
`For several years liquid chromatography (LC) with a performance fully com-
`parable to that of gas chromatography has been available as a routine laboratory
`technique. In 1973, when the first edition of this book was completed, the
`advantages of modern LC were just coming to the attention of a wide audience. At
`that time there were many published examples of LC applications for a variety of
`sample types, and equipment and related materials were available to solve most
`practical problems. However, several additional techniques and instrument
`improvements remained to be developed. As a result, many LC applications
`proved challenging - and occasionally unworkable, even in the hands of experi-
`enced workers.
`Six years later, in 1979, we see great changes and advances in the practical
`application of LC. The ubiquitous detector problem has been largely solved with
`the advent of spectrophotometer detectors operating down to 190 nm, making
`possible the sensitive detection of almost any compound type. Increased use of
`fluorescence and electrochemical detectors, plus off-line and on-line derivatiza--
`tion, has further pushed detection problems into the background, even for trace
`analyses of complex samples such as blood, food, and soil. Recent developments
`in microprocessor-controlled instrumentation also have produced greatly
`improved equipment performance, to permit more convenient, versatile, and
`precise LC separations.
`Only in the past four years has the tremendous potential of small-particle,
`reverse-phase LC been exploited, particularly when augmented with gradient
`elution and special methods such as ion-pair formation. As a result, previously
`difficult or impossible separations of such compounds as polar dyes or basic drugs
`and their metabolites are now more or less routine. Also, much less time is
`required to develop the average LC application; successful separations within the
`first few tries are now common.
`The exponential improvement in column packings that occurred between 1968
`and 1973 has leveled off in the past four years, and most experts now agree that we
`ix
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`Preface to the Second Edition
`x
`are approaching a fundamental limit to further major increase in column efficiency
`or plate numbers. However, there has been continuing emphasis by manufacturers
`on reliability and reproducibility of both packings and packed columns. Con-
`sequently, today the user can expect closer agreement between packings and
`columns from different lots, both in terms of sample retention and column
`efficiency characteristics. During the past four years, packings for the separation
`of large, water-soluble molecules such as proteins were finally reported, and it
`seems likely that these and other useful new packings will become commercially
`available in the near future.
`Problem areas such as band tailing, trace analysis, preparative separation, and
`so on, have received considerable attention in the past five years, and these
`particular problems or applications can now be approached in a relatively systema-
`tic fashion. The further development and application of special "tricks" or
`techniques such as column switching continues to add greatly to the potential of
`LC and to enable the easier handling of problem samples. The increasing volume
`of certain LC testing, particularly in the areas of quality control, process control,
`and clinical chemistry, has made full automation of these assays necessary -
`which has led to a number of important developments and the commercial
`availability of automated peripheral equipment for sampling, sample pretreat-
`ment, and so on.
`Finally, superb examples of the application of LC in virtually every industry and
`technical area now abound, providing encouragement to the would-be practi-
`tioner, and specific examples for the chromatographer with a given job to do.
`Although this second edition has been almost completely rewritten, our
`approach closely follows that brought to the preparation of the first edition. Aside
`from including new developments that have occurred over the past five years, we
`have expanded our treatment of many practical areas, so that the reader will have
`less need to chase down references to work described elsewhere. We have also
`intentionally added a large number of actual chromatograms of "real" samples,
`both because such examples are readily available and because we feel that in this
`case "one picture is worth a thousand words." Inevitably, all of this has meant a
`larger and more expensive book - for which we apologize. The final format and
`relative emphasis on certain areas is the result of experience gained from two
`American Chemical Society short courses in modern liquid chromatography - the
`original basic course and the problem-solving course introduced in 1974. These
`two courses have now been presented in over 50 sessions to more than 2000
`students. Thus, the present book has benefited greatly from the questions and
`inputs of both beginning and experienced liquid chromatographers.
`In conclusion, we want to express our appreciation to a number of people for
`their help in creating this second edition. Specifically, we wish to thank several of
`our co-workers and friends who reviewed the orginal manuscript for many helpful
`corrections and modifications, especially Dr. Eli Grushka of the Hebrew Univer-
`
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`Preface to the Second Edition xi
`sity, Jerusalem (Israel); Drs. Pedro A. Rodriguez, Thomas S. Turan, C. Grant
`Birch, Mark D. Seymour, and William J. Kozarek, all of the Proctor & Gamble
`Company, Cincinnati, Ohio; Dr. Dennis L. Saunders of the Union Oil Research
`Center, Brea, California; and Drs. John W. Dolan and J. Russel Gant of
`Technicon. Dr. Dolan has also contributed Section 17.2 on Sample Cleanup. We
`are also grateful to Mrs. Patricia C. Lyons of Du Pont for extensive assistance with
`the typing of the manuscript. Finally, we gratefully acknowledge the considerable
`support of Du Pont and Technicon in many different ways.
`
`L. R. S. J.
`J. K.
`
`July 1979
`
`Page 8 of 402
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`

`

`CONTENTS
`
`1 Introduction, 1
`1.1 Liquid versus Gas Chromatography, 2
`1.2 Modern versus Traditional LC Procedures, 3
`1.3 How Did Modem LC Arise?, 8
`1.4 The Literature of LC, 9
`1.5
`About the Book, 12
`References, 13
`Bibliography, 13
`
`2 Basic Concepts and Control of Separation, 15
`2.1 The Chromatographic Process, 16
`2.2 Retention in LC, 22
`2.3 Band Broadening, 27
`2.4 Resolution and Separation Time, 34
`2.5
`The Control of Separation, 37
`References, 81
`Bibliography, 82
`
`3 Equipment, 83
`3.1
`Introduction, 84
`3.2
`Extracolumn Effects, 86
`3.3 Mobile-Phase Reservoirs, 88
`3.4 Solvent Pumping (Metering) Systems, 90
`3.5 Equipment for Gradient Elution, 103
`
`XIII
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`xiv
`
`Contents
`
`3.6 Sample Injectors, 110
`3.7 Miscellaneous Hardware, 117
`3.8
`Integrated and Specialized Instruments, 119
`3.9 Laboratory Safety, 123
`References, 123
`Bibliography, 124
`
`4 Detectors, 125
`4.1
`Introduction, 126
`4.2 Detector Characteristics, 127
`4.3 UV-Visible Photometers and Spectrophotometers, 130
`4.4 Differential Refractometers, 140
`4.5 Fluorometers, 145
`4.6
`Infrared Photometers, 147
`4.7 Electrochemical (Amperometric) Detectors, 153
`4.8 Radioactivity Detectors, 158
`4.9 Conductivity Detectors, 161
`4.10 Summary of the Characteristics of Most-Used Detectors, 161
`4.11 Other Detectors, 165
`References, 165
`Bibliography, 166
`
`5 The Column, 168
`5.1
`Introduction, 169
`5.2 Characteristics and Use of Different Column Packings, 173
`5.3 Available Column Packings, 183
`5.4 Column Packing Methods, 202
`5.5 Column Evaluation and Specifications, 218
`5.6 Column Techniques, 225
`5.7 Reduced Parameters and Limiting Column Performance, 234
`References, 243 Bibliography, 245
`
`6 Solvents, 246
`6.1
`Introduction, 247
`6.2 Physical Properties, 251
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`Contents
`6.3
`
`Intermolecular Interactions Between Sample and
`Mobile-Phase Molecules, 255
`Solvent Strength and "Polarity," 257
`6.4
`Solvent Selectivity, 260
`6.5
`Purification of LC Solvents, 265
`6.6
`References, 267
`Bibliography, 268
`
`xv
`
`7 Bonded-Phase Chromatography, 269
`7.1
`Introduction, 270
`7.2
`Preparation and Properties of Bonded-Phase Packings, 272
`7.3 Mobile-Phase Effects, 281
`7.4 Other Separation Variables, 289
`7.5
`Special Problems, 294
`7.6 Applications, 301
`7.7
`The Design of a BPC Separation, 316
`References, 319
`Bibliography, 321
`
`8 Liquid-Liquid Chromatography, 323
`8.1
`Introduction, 323
`8.2 Essential Features of LLC, 325
`8.3 Column Packings, 327
`8.4 The Partitioning Phases, 332
`8.5 Other Separation Variables, 336
`8.6
`Special Problems, 336
`8.7 Applications, 338
`8.8
`The Design of an LLC Separation, 342
`References, 347
`Bibliography, 348
`
`9 Liquid-Solid Chromatography, 349
`9.1
`Introduction, 351
`9.2 Column Packings, 361
`9.3 Mobile Phases, 365
`9.4 Other Separation Variables, 389
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`xvi
`
`Contents
`
`Special Problems, 391
`9.5
`9.6 Applications, 398
`9.7
`The Design of an LSC Separation, 405
`References, 407
`Bibliography, 409
`
`10 Ion-Exchange Chromatography, 410
`10.1
`Introduction, 410
`10.2 Column Packings, 414
`10.3 Mobile Phases, 419
`10.4 Other Separation Variables, 426
`10.5
`Special Problems, 427
`10.6 Applications, 429
`10.7
`The Design of an Ion-Exchange Separation, 445
`References, 450
`Bibliography, 452
`
`11 Ion-Pair Chromatography, 453
`11.1
`Introduction, 454
`11.2 Column Packings, 457
`11.3
`Partitioning Phases, 458
`11.4 Other Separation Variables, 470
`11.5
`Special Problems, 471
`11.6 Applications, 473
`11.7
`The Design of an Ion-Pair Separation, 477
`References, 481
`Bibliography, 482
`
`12 Size-Exclusion Chromatography, 483
`12.1
`Introduction, 484
`12.2 Column Packings, 487
`12.3 Mobile Phases, 500
`12.4 Other Separation Variables, 503
`12.5 Molecular-Weight Calibration, 509
`12.6 Recycle Chromatography, 519
`12.7
`Problems, 522
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`xvii
`
`Contents
`12.8 Applications, 525
`12.9
`The Design of an SEC Separation, 534
`References, 538
`Bibliography, 540
`
`13 Quantitative and Trace Analysis, 541
`13.1
`Introduction, 542
`13.2
`Peak-Size Measurement, 545
`13.3
`Calibration Methods, 549
`13.4
`Selection of Calibration Method, 556
`13.5
`Trace Analysis, 560
`References, 573
`
`14 Qualitative Analysis, 575
`14.1 Retention Data for Sample Characterization, 576
`14.2 Qualitative Analysis of Sample Bands from
`Analytical-Scale LC Separations, 589
`On-Line Spectroscopic Analysis of LC Peaks, 603
`14.3
`References, 612
`Bibliography, 614
`
`15 Preparative Liquid Chromatography, 615
`15.1
`Introduction, 615
`15.2
`Separation Strategy, 617
`15.3
`Experimental Conditions, 623
`15.4 Operating Variables, 641
`15.5 Applications, 647
`15.6 A Preparative Separation Example, 654
`References, 661
`
`16 Gradient Elution and Related Procedures, 662
`16.1 Gradient Elution, 663
`16.2 Column Switching and Stationary-Phase
`Programming, 694
`Flow Programming, 712
`16.3
`16.4 Temperature Programming, 715
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`xviii
`
`Contents
`
`16.5 Practical Comparison of Various Programming
`and Column-Switching Procedures, 715
`References, 717
`Bibliography, 718
`
`17 Sample Pretreatment and Reaction Detectors, 720
`17.1
`Introduction, 720
`17.2
`Sample Cleanup, 722
`17.3
`Sample Derivatization, 731
`17.4 Reaction Detectors, 740
`17.5 Automation of Sample Pretreatment, 746
`References, 748
`Bibliography, 750
`
`18 Selecting and Developing One of the LC Methods, 752
`18.1
`Introduction, 753
`18.2 Developing a Particular Separation, 762
`18.3
`Special Applications, 776
`References, 779
`
`19 Troubleshooting the Separation, 781
`19.1
`Troubleshooting the Equipment, 782
`19.2
`Separation Artifacts, 791
`19.3
`Troubleshooting Analytical Errors, 813
`References, 823
`
`Appendix I
`Suppliers of LC Equipment, Accessories, and Columns, 824
`
`Appendix II
`Miscellaneous Tables Used by Workers in LC, 833
`II. 1 The Gaussian or Error Function, 833
`II.2 Reduced Plate-Height Data for "Good" Columns, 836
`II.3 Viscosity of Solvent Mixtures, 836
`II.4 Particle Size Expressed as Mesh Size, 838
`References, 839
`
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`

`

`Contents
`
`List of Symbols, 840 List
`
`of Abbreviations, 844
`
`Index, 847
`
`xix
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`

`

`ONE
`
`INTRODUCTION
`
`1.1 Liquid versus Gas Chromatography
`1.2 Modern versus Traditional LC Procedures
`1.3 How Did Modern LC Arise?
`1.4 The Literature of LC
`1.5 About the Book
`References
`Bibliography
`
`2
`3
`8
`9
`12
`13
`13
`
`Over the past 40 years the practice of chromatography has witnessed a continu-
`ing growth in almost every respect: the number of chromatographers, the
`amount of published work, the variety and complexity of samples being sepa-
`rated, separation speed and convenience, and so on. However, this growth curve
`has not moved smoothly upward from year to year. Rather the history of
`chromatography is, one of periodic upward spurts that have followed some major
`innovation: partition and paper chromatography in the 1940s, gas and thin-
`layer chromatography in the 1950s, and the various gel or size-exclusion
`methods in the early 1960s. A few years later it was possible to foresee still
`another of those major developments that would revolutionize the practice of
`chromatography: a technique that we call modern liquid chromatography.
`What do we mean by "modern liquid chromatography"? Liquid chromato-
`graphy (LC) refers to any chromatographic procedure in which the moving phase
`is a liquid, in contrast to the moving gas phase of gas chromatography. Tradi-
`tional column chromatography (whether adsorption, partition, or ion-exchange),
`thin-layer and paper chromatography, and modern LC are each examples of
`liquid chromatography. The difference between modern LC and these older
`procedures involves improvements in equipment, materials, technique, and the
`
`1
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`

`Introduction
`2
`application of theory. In terms of results, modern LC offers major advantages in
`convenience, accuracy, speed, and the ability to carry out difficult separations.
`To appreciate the unique value of modern LC it will help to draw two compari-
`sons:
`• Liquid versus gas chromatography.
`• Modern versus traditional LC procedures.
`
`1.1 LIQUID VERSUS GAS CHROMATOGRAPHY
`
`The tremendous ability of gas chromatography (GC) to separate and analyze
`complex mixtures is now widely appreciated. Compared to previous chromato-
`graphic methods, GC provided separations that were both faster and better.
`Moreover, automatic equipment for GC was soon available for convenient, un-
`attended operation. However, many samples simply cannot be handled by GC.
`Either they are insufficiently volatile and cannot pass through the column, or
`they are thermally unstable and decompose under the conditions of separation.
`It has been estimated that only 20% of known organic compounds can be satis-
`factorily separated by GC, without prior chemical modification of the sample.
`LC, on the other hand, is not limited by sample volatility or thermal stability.
`Thus, LC is ideally suited for the separation of macromolecules and ionic species
`of biomedical interest, labile natural products, and a wide variety of other high-
`molecular-weight and/or less stable compounds, such as the following:
`Proteins
`Polysaccharides
`Synthetic polymers
`Nucleic acids
`Plant pigments
`Surfactants
`Amino acids
`Polar lipids
`Pharmaceuticals
`Dyes
`Explosives
`Plant and animal metabolites
`Liquid chromatography enjoys certain other advantages with respect to GC.
`Very difficult separations are often more readily achieved by liquid than by gas
`chromatography, because of
`• Two chromatographic phases in LC for selective interaction with sample
`molecules, versus only one in GC.
`• A greater variety of unique column packings (stationary phases) in. LC.
`• Lower separation temperatures in LC.
`Chromatographic separation is the result of specific interactions between sample
`molecules and the stationary and moving phases. These interactions are essen-
`tially absent in the moving gas phase of GC, but they are present in the liquid
`phase of LC - thus providing an additional variable for controlling and improving
`
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`3
`1.2 Modern versus Traditional LC Procedures
`separation. A greater variety of fundamentally different stationary phases have
`been found useful in LC, which again allows a wider variation of these selective
`interactions and greater possibilities for separation. Finally, chromatographic
`separation is generally enhanced as the temperature is lowered, because inter-
`molecular interactions then become more effective. This favors procedures such
`as LC that are usually carried out at ambient temperature.
`Liquid chromatography also offers a number of unique detectors that have so
`far found limited application in GC:
`• Colorimeters combined with color-forming reactions of separated sample
`components.
`• Amperometric (electrochemical) detectors.
`• Refractive index detectors.
`• UV-visible absorption and fluorescent detectors.
`Although GC detectors are generally more sensitive and also provide unique se-
`lectivity for many sample types, in many applications the available LC detectors
`show to advantage. That is, LC detectors are favored for some samples, whereas
`GC detectors are better for others.
`A final advantage of liquid versus gas chromatography is the relative ease of
`sample recovery. Separated fractions are easily collected in LC, simply by
`placing an open vessel at the end of the column. Recovery is quantitative and
`separated sample components are readily isolated, for identification by supple-
`mentary techniques or other use. The recovery of separated sample bands in GC
`is also possible but is generally less convenient and quantitative.
`
`1.2 MODERN VERSUS TRADITIONAL LC PROCEDURES
`
`Consider now the differences between modern LC and classical column or open-
`bed chromatography. These three general procedures are illustrated in Figure
`1.1. In classical LC a column is often used only once, then discarded. Therefore,
`packing a column (step 1 of Figure 1.1, "bed preparation") has to be repeated
`for each separation, and this represents a significant expense of both manpower
`and material. Sample application in classical LC (step 2), if done correctly, re-
`quires some skill and time on the part of the operator. Solvent flow in classical
`LC (step 3) is achieved by gravity feeding of the column, and individual sample
`fractions are collected manually. Since typical separations require several hours
`in classical LC, this is a tedious, time-consuming operation. Detection and quan-
`titation (step 4) are achieved by the manual analysis of individual fractions.
`Normally, many fractions are collected, and their processing requires much time
`and effort. Finally, the results of the separation are recorded in the form of a
`chromatogram: a bar graph of sample concentration versus fraction number.
`
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`

`4
`
`Introduction
`
`Figure 1.1 Different forms of liquid chromatography
`
`The advent of paper chromatography in the 1940s and thin-layer chromato-
`graphy (TLC) in the 1950s greatly simplified the practice of analytical liquid
`chromatography. This is also illustrated in Figure 1.1. Bed preparation in TLC
`or paper chromatography (step 1) is much cheaper and simpler than in classical
`LC. The paper or adsorbent-covered plates can be purchased in ready-to-use
`form at nominal expense. Sample application is achieved rather easily, and sol-
`vent flow is accomplished by placing the spotted paper or plate into a closed
`vessel with a small amount of solvent. Solvent flow up the paper or plate pro-
`ceeds by capillary action, without the need for operator intervention. Finally,
`detection and quantitation can be achieved by spraying the dried paper or plate
`
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`

`5
`1.2 Modern versus Traditional LC Procedures
`with some chromogenic reactant to provide a visible spot for each separated
`sample component.
`The techniques of paper and thin-layer chromatography greatly simplified
`liquid chromatography and made it much more convenient. A further advantage,
`particularly for TLC, was that the resulting separations were much better than
`in classical LC and required much less time-typically 30-60 min rather than
`several hours. However, certain limitations were still apparent in these open-bed
`methods:
`• Difficult quantitation and marginal reproducibility, unless special precautions
`are taken.
`• Difficult automation.
`• Longer separation times and poorer separation than in GC.
`• Limited capacity for preparative separation (maximum sample sizes of a few
`milligrams).
`Despite these limitations, TLC and paper chromatography became the tech-
`niques of choice for carrying out most LC separations.
`Let us look now at modern LC. Closed, reusable columns are employed (step
`1, Figure 1.1), so that hundreds of individual separations can be carried out on a
`given column. Since the cost of an individual column can be prorated over a
`large number of samples, it is possible to use more expensive column packings
`for high performance and to spend more time on the careful packing of a
`column for best results. Precise sample injection (step 2) is achieved easily and
`rapidly in modern LC, using either syringe injection or a sample valve. Solvent
`flow (step 3) is provided by high-pressure pumps. This has a decided advantage:
`controlled, rapid flow of solvent through relatively impermeable columns. Con-
`trolled flow results in more reproducible operation, which means greater accu-
`racy and precision in LC analysis. High-pressure operation leads to better, faster
`separation, as is shown in Chapter 2. Detection and quantitation in modern LC
`are achieved with continuous detectors of various types. These yield a final
`chromatogram without intervention by the operator. The result is an accurate
`record of the separation with minimum effort.
`Repetitive separation by modern LC can be reduced to a simple sample injec-
`tion and final data reduction, although the column and/or solvent may require
`change for each new application. From this it should be obvious that modern
`LC is considerably more convenient and less operator dependent than either
`classical LC or TLC. The greater reproducibility and continuous, quantitative
`detection in modern LC also lead to higher accuracy and precision in both quali-
`tative and quantitative analysis. As discussed in Chapter 13, quantitative analysis
`by modern LC can achieve a precision of better than ±0.5% (1 standard devi-
`ation or S.D.). Finally, preparative LC separations of multigram quantities of
`sample are now proving relatively straightforward.
`
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`

`6
`
`Introduction
`
`Figure 1.2 Rapid LC separation of aromatic hydrocarbons. Peaks 1, 2 are CH2C12 and
`CHCl3, peak 3 is benzene, and peaks 4-15 are polycyclic aromatic hydrocarbons, ending
`with dibenzanthracene (peak 15). Column, 6.5 × 0.4 cm of porous silica (4.4 ,(cid:151)m); mobile
`phase, n-pentance; ambient; velocity 0.93 cm/sec, (cid:39)P = 1060 psi; UV, 254 nm; 3-50 (cid:151)g each
`compound. Reprinted from (8) with permission.
`
`Modern LC also provides a major advance over the older LC methods in speed
`and separation power. In fact, LC now rivals GC in this respect. Figure 1.2 shows
`an example of the speed of modern LC: the separation of 15 aromatic hydrocar-
`bons in 1 min., using a small-particle silica column. Figure 1.3 shows the separa-
`tion of a urine sample into over 100 peaks in less than half an hour, using a
`small-particle reverse-phase column. Modern LC also features a number of new
`column packings that provide separations that were previously impossible.
`Figure 1.4 shows a chromatogram for a synthetic polymer sample, providing a
`rapid determination of the molecular-weight distribution of this polymer. Simi-
`lar determinations by classical, physical methods required literally months of
`work, as compared to the 10 min. for the assay of Figure 1.4. Most important,
`all these advantages of modern LC are now routinely available with commercial
`LC equipment and supplies.
`What we have called modern LC has been referred to by other names: high-
`performance or high-pressure LC (HPLC), high-speed LC, and simply liquid
`chromatography (LC). In the present book we refer to modern liquid chromato-
`
`Page 21 of 402
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`

`

`Figure 1.3 LC separation of acidified urine extract by reverse-phase small-particle column.
`Column, 25 × 0.46 cm LiChrosorb ODS (bonded-phase porous silica) (5 (cid:151)m); mobile phase,
`gradient elution from 0.1 M phosphate in water (pH = 2.1) to 40 %v acetonitrile; temp.,
`70°; flowrate, 2.0 ml/min; detector, UV, 280 nm; sample, 10 (cid:151)l injection from a 10-x con-
`centrated urine extract. Reprinted from (9) with permission.
`
`7
`
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`8
`
`Introduction
`
`Figure 1.4 Rapid LC separation of cellulose hemi-formal for determination of molecular
`weight distribution (size exclusion chromatography). Columns, four 10 × 0.79 cm (in series):
`PSM 50S, 800 S, 1500 S and 4000 S; mobile phase, dimethyl sulfoxide; temp., 23°;
`flowrate, 1.0 ml/min (2000 psi); detector, refractive index, 5 X 10-5 RI units full-scale.
`Reprinted from (10) with permission.
`
`graphy in columns as LC. Where high-pressure operation is to be contrasted with
`low-pressure LC, we use the term HPLC to define the usual technique that em-
`ploys high pressure.
`Recently, an improved version of TLC has been introduced (1), and referred
`to as high-performance TLC (HP-TLC). It has been implied that this new tech-
`nique will displace modern LC from many of its present applications. This seems
`to us an overoptimistic assessment of the potential of HP-TLC (e.g., la, Ib). How-
`ever, TLC itself has proved to be a complementary technique that can be used
`effectively in conjunction with LC (see Chapter 9), and any improvement in
`HP-TLC will only increase the value of TLC in these applications.
`
`1.3 HOW DID MODERN LC ARISE?
`
`Modern LC is based on developments in several areas: equipment, special
`columns and column packings, and theory. High-pressure, low-dead-volume
`equipment with sensitive detectors plays a vital role. The new column packings
`that have been developed specifically for modern LC are also essential for rapid,
`high-performance separations. Theory has played a much more important role
`in the development of modern LC than for preceding chromatographic innova-
`tions. Fortunately, the theory of LC is not very different from that of GC, and
`a good understanding of the fundamentals of GC had evolved by the early 1960s
`
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`9
`1.4 The Literature of LC
`[see, e.g., (2)]. This GC theory was readily extended to include LC, and this in
`turn led directly to the development of the first high-performance column pack-
`ings for LC and the design of the first modern LC units. A proper understanding
`of how the different separation variables are selected for optimum performance
`is particularly important in modern LC; theory has been most useful in providing
`the necessary insights.
`The potential advantages of modern LC first came to the attention of a wide
`audience in early 1969, when a special session on LC was organized as part of
`the Fifth International Symposium on Advances in Chromatography (3). How-
`ever, modern LC had its beginnings in the late 1950s, with the introduction of
`automated amino acid analysis by Spackman, Stein, and Moore (4). This was
`followed by the pioneering work of Hamilton (5) and Giddings (2) on the funda-
`mental theory of high-performance LC in columns, the work of C. D. Scott at
`Oak Ridge on high-pressure ion-exchange chromatography, and the introduction
`of gel permeation chromatography by J. C. Moore (6) and Waters Associates in
`the mid-1960s. At this point a number of workers began active research into
`what was to become modern LC, and their combined efforts [see (5) for a
`review] led to the 1969 breakthrough. Since 1969 tremendous activity has been
`aimed at the development of better equipment and columns and further im-
`provements in our understanding of modern LC. These developments have now
`leveled off somewhat, so that it seems possible to write an account of LC that
`should remain useful and up-to-date for some time to come.
`
`1.4 THE LITERATURE OF LC
`
`The literature of LC extends back into the 1930s. Some of this info