HPLC Made to Measure Edited by Stavros Kromidas 1293vch00.pmd 10.05.2006, 09:17 Related Titles S Kromidas Practical Problem Solving in HPLC 2000 ISBN 3-527-29842-8 S Kromidas More Practical Problem Solving in HPLC 2004 ISBN 3-527-31113-0 V R Meyer Practical High-Performance Liquid Chromatography 2004 ISBN 0-470-09378-1 P C Sadek Troubleshooting HPLC Systems A Bench Manual 2000 ISBN 0-471-17834-9 U D Neue HPLC Columns Theory, Technology, and Practice 1997 ISBN 0-471-19037-3 L R Snyder, J J Kirkland, J L Glajch Practical HPLC Method Development 1997 ISBN 0-471-00703-X 1293vch00.pmd 10.05.2006, 09:17 HPLC Made to Measure A Practical Handbook for Optimization Edited by Stavros Kromidas 1293vch00.pmd 10.05.2006, 09:17 The Editor Dr Stavros Kromidas Rosenstrasse 16 66125 Saarbrücken Germany All books published by Wiley-VCH are carefully produced Nevertheless, authors, editors, and publisher not warrant the information contained in these books, including this book, to be free of errors Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at © 2006 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim All rights reserved (including those of translation into other languages) No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers Registered names, trademarks, etc used in this book, even when not specifically marked as such, are not to be considered unprotected by law Printed in the Federal Republic of Germany Printed on acid-free paper Cover Design SCHULZ Grafik-Design, Fußgönheim Typesetting Manuela Treindl, Laaber Printing betz-druck GmbH, Darmstadt Binding J Schäffer GmbH, Grünstadt ISBN-13: 978-3-527-31377-8 ISBN-10: 3-527-31377-X 1293vch00.pmd 10.05.2006, 09:17 V Foreword HPLC has become the analytical method against which all others are measured and compared It is perhaps the most widely employed method of analysis of all those instrumental approaches that have ever been or are now in vogue Having been involved with HPLC for perhaps the past 35 years, since the early 1970s, I have seen the technique and field grow and prosper, academically and commercially It has become an incredible commercial success, and the cornerstone of many academic careers in analytical and other fields of chemistry Annual, dedicated meetings, as well as major parts of ACS, ASMS, AAPS and AAAS meetings, are routinely devoted to talks and discussions on or involving HPLC Though it has not quite displaced GC or flat-bed electrophoresis, it has surely been highly competitive for volatiles and biological macromolecules, respectively Indeed, one could argue that it is the very first technique that most analysts, biologists or biochemists would consider investigating and applying for virtually any class of analytes, regardless of molecular weight, size, volatility, ionic charges, polarity, hydrophobicity, or other physical or chemical properties HPLC has become a technique that can be applied to virtually any analyte or class of analytes, almost without regard to the properties thereof There are very few other analytical methods for which this can be claimed HPLC has truly become the “800 pound gorilla”, and it may be virtually impossible for any other technique to displace it from this niche in the analytical world, not even CEC or 2DE or multidimensional CE Why then another book dealing with this same topic? I have read some other texts by Stavros Kromidas, and was thus eager to preview this current one This text is really an edited book, though Stavros Kromidas has contributed several excellent chapters of his own The other contributions come from an international group of invited authors, mainly from the US, Canada, and Western Europe Virtually all of these individuals are well known in the HPLC community, such as Uwe Neue, Michael McBrien, Lloyd Snyder, John Dolan, Klaus Unger, and so forth Most, if not all, have been heavily involved in HPLC matters for decades, and have, in their own right, become well-regarded and recognized experts in their various fields The book is heavily practice and practicality oriented, in that it aims to help the readers become more knowledgeable and better adept at using various forms of and approaches in HPLC However, it is not a “Methods”-type text, such as those published by Humana Press; it is not just a compilation of practice-oriented HPLC methods for various analytes HPLC Made to Measure: A Practical Handbook for Optimization Edited by Stavros Kromidas Copyright © 2006 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim ISBN: 3-527-31377-X 1293vch00.pmd 10.05.2006, 09:20 VI Foreword Rather, the text is quite detailed, with scientific discussions and theory, lots of equations and principles, reference to a variety of practical software, and with an emphasis on understanding the fundamentals in each and every chapter This is not an introductory text; it is not meant as a text for a graduate Analytical Separations type course Rather, it is quite an advanced text, dealing with many recent and contemporary aspects of HPLC It deals with approaches for method optimization, currently available software and practices, chemometrics, principal component analysis, the selection of ideal stationary phases, and tools for column characterization and method optimization Of course, it deals extensively with reversed-phase HPLC, but it also covers many other areas, including GPC/SEC, affinity chromatography, chiral separations, microLC, nanoLC, and even microchip-based LC instrumentation and techniques It also deals with immunochromatographic methods, two-dimensional HPLC (MDLC), LC-MS, LC-NMR, and even how magic-angle spinning NMR spectroscopy can be used to better understand the selectivity of stationary phases in HPLC All in all, there are over two-dozen individual chapters, some authored by the same author(s), but most not It is to the Editor’s credit that he has not written most or even close to 50% of the total chapters, but rather that he has invited the most highly regarded and best-known authors, young and old, to contribute in areas of their unique expertise He has made an exceptionally good selection of such authors, each of whom has done an admirable job in their final writings and efforts This is not a book that you will pick up and read in a single sitting; that would appear impossible, even for those of us who have already devoted a major portion of our careers to researching and developing HPLC areas It is not an easy read; it is not a trivial text Rather, it is clearly an advanced, involved, and detailed text It is a book to be read slowly and carefully, because it contains an incredible amount of useful and important practical knowledge It also covers the very latest developments in HPLC, not just the fundamentals, but where the field stands today, and where it is going tomorrow It is a practical handbook for the optimization of HPLC and its ultimate application, but it is far from being just a handbook or “how to it” text It is really more of a summary of where HPLC stands today, what can be done with its various techniques and instrumentation, and what is important to know about its future developments and applications It is an incredibly useful and practical tome, collated by experts pooling their expertise, and it will make better chromatographers out of those of us who take up the book, study it carefully, and then apply its lessons to our own future needs It is not a simplistic methods development type book, though it does aim to help us optimize and improve our methods development approaches It is far more than “just” a Practical Handbook for Optimization, though the subtitle might make that suggestion It is my hope that those of you thinking of purchasing this particular, newer text on HPLC, and those who have already made this wise decision and are about to pursue the text itself, will benefit from these choices They were and are wise choices; now it is up to you to make the most of the book, which means not just reading the text and studying the figures and tables, but making every effort 1293vch00.pmd 10.05.2006, 09:20 Foreword possible to really understand what the authors are trying to impart to the readers This may require re-reading of the same chapter more than once; I did – actually several times, as these are not easy chapters or contributions However, in the long run, the time will be well spent and such efforts will be rewarded, for the book is truly a wealth of useful and practical information Obviously, I highly recommend the book to those contemplating purchase and study, for it is really one of the better texts to have come along in many years dealing with this, one of our very favorite subjects, HPLC January 2006 1293vch00.pmd Ira S Krull Associate Professor Department of Chemistry and Chemical Biology Northeastern University Boston, MA, USA 10.05.2006, 09:20 VII IX Preface The optimizing of practices and processes constitutes an essential prerequisite for long-term success The objective and the motives may be very different: selfpreservation among living things, “saving lives” among volunteers in Africa, maximizing profits among marketing strategists, new discoveries among scientists This principle is of course also valid in chemistry and in analytics This book deals exclusively with the subject of optimization in HPLC The aim is to examine this important aspect of HPLC from diverse perspectives First, we have set out the fundamental aspects, encompassing the principal considerations and background information At the same time, we have endeavored to present and discuss as many practical examples, ideas, and suggestions as possible for the everyday application of HPLC The implementation of concepts for rapid optimization should equally aid and support the planning of effective method development strategies as in daily practice at the laboratory bench The aim of the book is to contribute to purposeful, affordable, forward-looking method development and optimization in HPLC To this end, internationally renowned experts have offered their knowledge and experience I extend my sincere thanks to these colleagues I also thank Wiley-VCH, in particular Steffen Pauly, for their valued collaboration and good cooperation Saarbrücken, January 2006 Stavros Kromidas HPLC Made to Measure: A Practical Handbook for Optimization Edited by Stavros Kromidas Copyright © 2006 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim ISBN: 3-527-31377-X 1293vch00.pmd 10.05.2006, 09:20 XI Contents Foreword Preface V IX List of Contributors Structure of the Book XXV XXXI Fundamentals of Optimization 1.1 Principles of the Optimization of HPLC Illustrated by RP-Chromatography Stavros Kromidas 1.1.1 1.1.2 1.1.3 1.1.3.1 1.1.3.2 1.1.3.3 Before the First Steps of Optimization What Exactly Do We Mean By “Optimization”? Improvement of Resolution (“Separate Better”) Principal Possibilities for Improving Resolution What has the Greatest Effect on Resolution? 10 Which Sequence of Steps is Most Logical When Attempting an Optimization? 11 1.1.3.4 How to Change k, α, and N 17 1.1.3.4.1 Isocratic Mode 17 1.1.3.4.2 Gradient Mode 18 1.1.3.4.3 Acetonitrile or Methanol? 19 1.1.4 Testing of the Peak Homogeneity 22 1.1.5 Unknown Samples: “How Can I Start?”; Strategies and Concepts 1.1.5.1 The “Two Days Method” 36 1.1.5.2 “The 5-Step Model” 39 1.1.6 Shortening of the Run Time (“Faster Separation”) 48 1.1.7 Improvement of the Sensitivity (“To See More”, i.e Lowering of the Detection Limit) 48 HPLC Made to Measure: A Practical Handbook for Optimization Edited by Stavros Kromidas Copyright © 2006 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim ISBN: 3-527-31377-X 1293vch00.pmd 11 10.05.2006, 09:20 35 Subject Index – versus silanol activity 256 hydrophobicity parameter 305, 307, 312, 313, 318 hydroquinone 302 Hydrosphere C18 250 3-hydroxybenzoic acid, degrees of ionization 242 4-hydroxybenzoic acid, degrees of ionization 242 N-hydroxysuccinimide (NHS) activation 410 N-hydroxysuccinimide ester 516 hyper-cuboid 657 Hypercarb 37, 38, 152, 232, 251, 706 Hypersil Advance 38, 234, 706 Hypersil BDS 152, 179, 183, 236, 251, 706 Hypersil Beta Basic-18 325 Hypersil BetamaxNeutral 325 Hypersil C18 BDS 272 Hypersil Elite 706 Hypersil Elite C18 272 Hypersil ODS 152, 272, 611, 706 HyPURITY 251 HyPURITY Advance 152, 183, 251, 269 HyPURITY C18 152, 183, 233, 272, 325 i ibuprofen 73 – fast UPLC gradient separation 503 – gradient separation 65 ICH guidelines 140 IGF-1 512 iminodiacetic acid 406 immobilization – of soyabean trypsin inhibitor on Toyopearl AF Tresyl 650M 421 – site-directed 415 immobilization methods 409 immobilized metal-chelate affinity chromatography (IMAC) 406 – resins for 414 immobilized reagents 513 immunoaffinity column 520 immunoaffinity resin 407 immunoassays 511 immunochromatographic techniques 509 ff – nomenclature 511 immunoprecipitation (IP) 518 imprinted polymers 450 Inertsil 317 Inertsil C8 column 328 Inertsil columns 316 Inertsil ODS-2 152, 232, 238, 324, 706 Inertsil ODS-3 153, 238, 251, 272, 324, 329, 706 1293vch07.pmd 739 information-rich detectors 371 infrared spectroscopy 375 injection, delayed 67 instrumental variance 110 intelligent peak tracking 599 interaction chromatography 531 interferon 406 intermolecular interaction forces, relative strengths 430 internal standard 106 internal standard method 107 interparticle volume 384 interphase 299 interstitial volume 386 intrinsic molar volume 298 intrinsic viscosity 379 inversion-recovery pulse sequence 343 ion optics 544 – hysteresis 545 ion polarity 545 ion source – in LC/MS 544 – matrix stress of an 545 ion suppression 544, 545 – by trifluoroacilic acid 79 – in mass spectrometry 69 ion-exchange capacity – acidic 264 – total 264 ion-exchange chromatography (IEC), application of 676 ion-pairing agents 543 ionization 71 – influence of the organic solvent on 81 ionization modes 546 ionization principle 542 ionization process 541 IR absorption bands 375 isocratic elution in microchip-based LC 491 isocratic mode 17 isocratic separation – fast 501 – fully automated optimization 592 – of diuretics 85 – on a CN column 27 – on a Nucleosil C18 column 27 – on CN and C18 columns placed in series 28 – using short columns 501 isocratic separation, high-powered 500 isocratic separations 433 – with HPLC 499 isoelectric focusing 629 isohomovanillic acid, degrees of ionization 242 26.04.2006, 14:49 739 740 Subject Index isopropanol 26 – as modifier 38 – as solvent 351 – viscosity 350 8-isoprostane 512 isotachophoresis 492 j J’Sphere H80 325, 330 J’Sphere L80 325 J’Sphere M80 325 J’Sphere ODS 272 J’Sphere ODS JH 268 Jupiter 37, 153, 183, 233, 249, 251, 706 Jupiter C18 300A 272 k k-nearest-neighbor algorithm 703 k-means algorithm 703 Kamlet–Taft hydrogen-bond acceptor 298 Kamlet–Taft hydrogen-bond donor 298 Kamlet–Taft polarity parameter 298 kαN principle 17 Keppra 460 ketones 169 ketoprofen 311 – analysis of degraded 278 – as parameter B 314 kinases 406 Knox equation 570 Kromasil 153, 183, 251, 706 Kromasil 100-5C18 324 Kromasil C18 272 Kromasil C8 329 Kromasil-DMB 341 Kromasil-TBB 341 l L-classes 271 L1 phases 271 – comparison for the analysis of hydrophilic bases 275 lab-on-a-chip systems 487 labeling – fluorescent dyes 511 – radioactive isotopes 511 LaChrom 588 LaChromElite 588 Langmuir adsorption isotherm, modified 431 laser-induced fluorescence (LIF) 485 LC Packings Dionex 630 LC-FTIR interface 375 LC-NMR coupling 551 ff LC/MS 541 ff 1293vch07.pmd 740 – selectivity 545 LC/MS peaks 542 LCQdeca XP 633 lectin column 418 lectins 514 Levetiracetam 460 Lexapro 460 LiChrosorb 32, 153, 183, 706 LiChrosorb RP-selectB 303 LiChrospher 37, 38, 153, 233, 249, 251, 706 LiChrospher 100 RP-18 303 LiChrospher 100 RP-18e 303, 307 LiChrospher 100 RP-8 303, 307 LiChrospher 60 674 LiChrospher 60 RP-Select B 325 LiChrospher PAH 303 LiChrospher RP-18 272, 306 LiChrospher RP-18e 306 LiChrospher Select B 153, 183, 706 lidocaine – fast UPLC gradient separation 503 – gradient separation 65 ligand, arrangements at the particle surface 413 ligand concentration effect 419 ligand density 415 ligand immobilization 516 ligand utilization for different IMAC resins 415 ligand-exchange chromatography 451 ligand-exchange phases 451 light-scattering detection 377 limit of quantification 106 linear calibration function 134 – statistical comparison of 135 linear free energy relationship (LFER) 296, 297 linear free enthalpy relationship (LFER) – empirical, phase characterization 317 – mathematical determination of parameters 312 – parameters 307 – prediction of selectivity 308 – using solvation equation 301 – variation of eluent conditions 315 linear mixed-bed column, comparison of molar mass separation range 366 linear mixed-bed columns 366 – advantages and disadvantages of 365 linear regression 107, 110 linear-solvent-strength model 570 liquid adsorption chromatography (LAC) 371 liquid adsorption chromatography at the critical adsorption point (LACCC) 363 26.04.2006, 14:49 Subject Index liquid chromatography 691 – detectors for 371 – of polymers 371 liquid chromatography at the critical point of adsorption (LC-CC) 371 loading 266 loading capacity of enantiomer separation 455 loading factor 455 loading matrix 704 loading plot 281, 704 – in PCA 266 – obtained in mobile phase 287 – obtained in mobile phase 290 – of C18-phases type A 268 – of non-C18 phases 268 – of USP designed L1 phases 274 local optimum 602 log P value 241 loops 558 low-angle laser light scattering detector (LALLS) 377 Luna 153, 183, 706 Luna 251 Luna C18 272, 611 Luna C18(2) 37, 272, 325, 329 Luna Phenyl 249 lysergic acid diethyl amide 543 lysergic acid methyl propyl amide 543 lysine 406 lysozyme 398 – size-exclusion protein refolding of 399 lysozyme binding capacity 414 m Macro-Prep® 408 macrocyclic glycopeptide antibiotics 447 macrocyclic selectors 446 macromolecular materials, dectection 369 macromolecular samples, GPC analysis 359 magic triangle 362 magnetic field 551 magnetic field strength 552 main column 478 main effects 664 MALDI AnchorChip™ 639 MALDI fingerprint spectrum 636 MALDI PSD spectrum 636 MALDI-TOF-MS 640 MALDI-TOF-MS/MS analysis 637, 640 Mandel test 140 map of critical resolution 643 – one-dimensional 647 – two-dimensional 647 1293vch07.pmd 741 Mascot 637 mass spectrometer 484 – conditions and parameter 689 – in nanoLC 628 mass spectrometric analysis, full and fragmented 634 mass spectrometric method development 692 mass spectrometry conditions, optimization of 693 MassLynx 689 matrix blank 547 matrix effect 133, 136, 545 – evaluation by analyte infusion 548 – extended evaluation 547 – interfering 136 matrix spike 546 matrix-assisted laser desorption/ionization (MALDI) 628 McGowan characteristic volume 300 measurement uncertainty 107, 112, 113 – as criterion in optimization 120 mefenamic acid 327 – as parameter B 314 memory effect 341, 453, 544 mepivacaine 282 2-mercaptoethanol 395 metal chelate resin 414 metal complexation 451 MetaSil AQ 250 MetaSil Basic 250 methadone 282, 285 methanol 292, 307 – as organic solvent 81 – as solvent 351 – bandwidth of the selectivity 231 method development 567 – factorial 608 – for calibration 143 – for unknown samples 35 – in affinity chromatography 405 – multifactorial 609 – partly automated, in RP-HPLC 604 – PCA in 277 – strategy for 12 – strategy for partially automated 603 – two days method 36 – with ChromSword® 588 method development kit 249 method development systems for HPLC 605 ff method of least squares see linear regression method robustness – calculation of 651 26.04.2006, 14:49 741 742 Subject Index – quantification of 650 cis-4-methoxychalcone as parameter S 314 trans-4-methoxychalcone as parameter S 314 2-methoxyethanol gradient 520 methyl benzoate 302 methyl tert-butyl ether as solvent 350 methylamine, pK values 80 methylene group selectivity 255 methylparaben 302 methylphenylhydantoin, NOE cross-peak 343 micellar electrokinetic chromatography (MEKC) 491 micro-LC 467 – detectors 484 – packing materials 484 – sample loading strategies 478 – sensitivity and resolution 483 – system choice 469 – volume control 472 micro-LC pumps, guard column switching for 479 micro-SEC 383 micro-total analysis systems 487 microchip-based LC 487 ff – comparison of common techniques in 496 – formation of bubbles 492 – separation performance 490 microchips, liquid-phase for separations 487 microcoil-NMR 560 microcystins 512 – by RP chromatography 524 microfabricated chromatographic beds 489 MicroPro 469 microtitration plates (MTP) 520 middle polar phases 200 Millennium 588 miniaturization 56, 467 mixed-mode stationary phase 34 mobile phase – descriptors 298 – pH effect 73 – selection for HPLC 440 mobile phase pH 543 – correction factors for acidic buffer, acetonitrile 93 – optimization of 94 – troubleshooting 102 mobile phases – chromatographic test optimization 292 – influence of LFER 307 – pH of 255 moderately polar phases 249 modern multi-angle systems (MALLS) 379 modifier, organic 94 1293vch07.pmd 742 molar mass distribution 360, 370 – determination by GPC 371 molar mass-sensitive detector in GPC 377 molecular imprinting 518 molecular imprints (MIP) 523 molecular weight – apparent 372 – determination of 398 molecularly imprinted polymer (MIP) 518 moniliformin 512 monoclonal antibodies 510 monolithic silica columns 672 monolithic stationary phases in situ polymerized 489 monoliths 416 Monte Carlo optimization method 598 more-dimensional separation 492 morphine 282 MP Gel C18 611 MP-Gel 153, 232, 251, 706 MS-compatible buffer, capacity 75 MS-compatible pH control 79 MSFit 637 multi-angle laser light scattering detector (MALLS) 377 multi-angle light-scattering data analysis 379 multi-dimensional LC/MS, peptide mapping of biofluids 384 multidetector chromatogram of commercial compounds 692 multidimensional HPLC 670 – application of 675 – critical parameters of 682 – sample preparation 674 multidimensional separation, techniques for 672 multifactorial systematic method development 601 multilinear regression analysis for the derivation of CLND response factors 690 multiple detection, copolymer GPC analysis by 372 multivariate distances 703 multivariate linear regression 301 multivariate regression, descriptors for 300 n N-hydroxysuccinimide, IMAC resins nano-LC 467 – detectors 484 – guard column switching for 480 – packing materials 484 – sample loading strategies 478 26.04.2006, 14:49 417 Subject Index – sensitivity and resolution 483 – system choice 469 – volume control 472 nanoESI tip 633 nanoLC – columns for 629 – sample preparation for 628 – technique 629 nanoLC-ESI-MS/MS 638 nanoLC-MALDI-MS/MS 638 nanoLC-MS/MS – application 637 ff – in proteomics 627 ff α-naphthol 302 β-naphthol 302 α-naphthylamine 302 naproxen – fast UPLC gradient separation 503 – gradient separation 65 Nautilus C18 285 Nexium™ 456 nicotine 275, 282 nitrile phase 305 p-nitroaniline 302 nitroanilines – isomer separations 169 – separations of 172 nitrobenzene 302, 355 2-nitrobenzoic acid 313 – as parameter B 314 3-nitrobenzoic acid as parameter B 314 cis-4-nitrochalcone as parameter S 314 trans-4-nitrochalcone as parameter S 314 4-nitrophenol 687 p-nitrophenol 302 o-nitrotoluene 302 nitrotyrosine 512 nLC 469 NMR basics 551 NMR experiment, sample requirements 552 NMR measurements – continuous-flow 553 – probe designs 553 NMR probes for LC-NMR 553 13 C NMR spectroscopy 551 – developments 562 H NMR spectroscopy 551 – solution-state 336 NMR spectroscopy 553 H NMR spectrum – of tocopherols 556 – solid-state 337 2D NMR spectrum of estradiol 557 NMR time scale 339 1293vch07.pmd 743 N,N′-diallyl-l-tartardiamide bis(4-tert-butylbenzoate) 339 f Nonidet P40 394 norethisterone acetate, trNOE 342 norfloxacin, change in capacity factor 90 normal-phase HPLC 349 ff – applications 355 – mobile phases in 350 – troubleshooting 356 nortriptyline 282, 285, 316, 327 – as parameter C 314 Novapak 38, 153, 249, 251, 706 Novapak C18 272 NP separation on a mixed-mode 34 Nuclear Overhauser Effect Spectroscopy (NOESY) 552 nuclear-Overhauser-effect (NOE) – cross-peaks 340 – negative intramolecular cross-peaks 343 Nucleodex 446 Nucleodur 100-C18 Gravity 324 Nucleodur C18 Gravity 324 Nucleodur Gravity 37, 250, 251 Nucleosil 50 37, 38, 153, 250, 251, 706 Nucleosil 100 153, 250, 706 Nucleosil AB 153, 179, 183, 232, 285, 706 Nucleosil AB/HD 250 Nucleosil C18 27, 272, 276 Nucleosil C18 HD 272 Nucleosil C18 Pyramid 250 Nucleosil HD 153, 183, 233, 251, 706 Nucleosil HD/AB 38, 236 Nucleosil Nautilus 153, 183, 250, 251, 706 Nucleosil Protect 153, 183, 234, 706 o ochratoxin A 512 off-line affinity enrichment 513 off-line coupling 521 – advantages 520 off-line LC-MALDI-MS/MS coupling 635 off-line technique in 2D-HPLC 672 ofloxacin, change in capacity factor 90 oleic acid methyl ester, capillary-HPLC-NMR 559 omeprazole – analytical enantiomer separation of 457 – preparative enantiomer separation of 457 Omnispher C18 325 Omnisphere C18 268 on-column focusing 673 on-line 2D-HPLC, schematic overview of 677 on-line coupling 521 on-line HPLC-NMR 560 26.04.2006, 14:49 743 744 Subject Index on-line LC-ESI-MS/MS coupling 633 on-line sample preparation 674 on-line technique – continuous flow 672 – in 2D-HPLC 672 – interrupted flow 672 open-channel electrochromatography (OCEC) 488, 491 – of coumarin dyes 491 OpenLynx Diversity 689 OpenLynx® 686 optimization – goals – principles of ff – sequence of steps 11 – through enhanced efficiency 12 – through peak homogeneity 12 – through selectivity 12 organic acids, RP-phases 241 organic bases, selectivity 237 orthogonal chromatography see 2D chromatography orthogonal separation 330 – light version 35 orthogonal separation techniques, coupling 29 orthogonal tests 248 OVAT method 601 p packed-bed electrochromatography 488 packing materials in micro- and nano-LC 484 1D-PAGE, protein separation 627 2D-PAGE, protein separation 627 palmitoleic acid methyl ester, capillaryHPLC-NMR 559 papain 406 Pareto diagram 660 – standardized effects in 662 patulin 512 peak capacity 59, 60, 61, 671 – as a function of flow rate and gradient duration of cm columns 63 – as a function of flow rate for columns of equal ratio of column length to particle size 64 – as a function of gradient duration 62 – as a function of the gradient span 61 – in 2D chromatography 527 peak homogeneity 22 – proof with the helpf of an orthogonal column 31 – the 1/2 hour method 22 1293vch07.pmd 744 peak pair – critical, separation of 660 – separation under worst case combination of variables 665 peak tracking 610 peak width 59 peak-tracking module 570 PEEK 472 PEEK capillaries 475 PEEK-encased glass capillaries 477 PEEKSil 472 pentylbenzene 264 – retention factors 268 4-n-pentylbenzoic acid as parameter B 314 pepsin 406 peptide 450 – immobilization 415 – two-dimensional separation of 495 peptide mass fingerprint (PMF) 637 perfluorinated phases – PC1–PC2 score and loading plots 270 – PCA of 270 perfluoroctyl phases 271 perfluorohexyl phases – endcapped 271 – non-endcapped 271 perfluorophenyl (PFP) phases 267 perfluoropropyl phases 271 Perfluorpropyl ESI 271 periodate cleavage 516 perylene 237 pH 71, 315 – corrections based on the presence of organic solvent 93 – dependency of C-parameters on 318 – impact on the immobilization result 422 – of mobile phase 73 – optimization of 90, 94 – or organic mobile phase solvents 93 pH effect 419 pH meter, calibration 76 pH range 73 – effective 94 pH scale 76 pH selection, systematic approach 96 pH value – changes in the presence of an organic solvent 76 – of an ammonium hydrogencarbonate buffer 78 – of several buffers in acetonitrile/water mixture 77 – selecting the 89 ff pharmaceuticals by SMB 459 ff 26.04.2006, 14:49 Subject Index phase collapse 270 phase equivalency, use of PCA in the identification 271 phase parameter 312 phase ratio 297, 389, 429 phases – embedded 232 – of polymer type 232 – similar 232 – with a polymer layer 232 phenetole 355 phenol 275 – in neutral water/acetonitril solution 19 – in neutral water/methanol solution 19 – onto hydrophobic, endcapped column 32 – pKa values 73 phenolic activity 259 Phenonemex AQUA 269, 270 phenyl columns 323 phenylboronate 406 2-phenylethylbromide 355 phenylurea herbicides 512 phosphatases 406 phosphate buffers, pKa value 78 phosphoric acid 316 phosphotyrosine 512 photoionization 542 phthalic acid, degrees of ionization 242 PicoTips™ 633 PicoView™ 100 633 pillar arrays 489 – applicability of 494 pilot-scale column 455 Pirkle phases 444 pK value of several buffers for methanol/ water mixtures 76 pKa value – basis of prediction 92 – changes in the presence of an organic solvent 76 – look-up and prediction 91 ff Plackett–Burman experimental plan 659 ff plasminogen 406 plate count 60 plate number 176 – correlation between normed plate number and normed pressure 180 – effective – in DryLab 571 – influence on 179 – normed, of ethylbenzene 177 – theoretical Platinum C18 38, 153, 183, 233, 234, 251, 267, 269, 270, 272, 273, 611, 706 1293vch07.pmd 745 polar bases, selectivity 238 polar embedded phases – PC1–PC2 score and loading plots 269 – PCA in 269 polar interactions 171, 259 – in stationary RP-phase 156 polar phases 200, 249, 251 – as watcher 252 – suitability of 247 polar RP-phases, properties 234 polar selectivity 259 – extended 261 – versus hydrophobicity of the stationary phase 260 Polaris 37 Polaris Amid C18 250, 269 Polaris C18 A 250 Polaris C8-Ether 250 Polaris NH2 250 polarization of the nuclei 551 poly(methyl methacrylate) (PMMA), separation of 363 polyacrylamide, packings in SEC 389 polyaromatic hydrocarbons, planar and twisted 318 polyclonal antibodies 510 polydisperse macromolecules, distributions of properties 370 polyethylene glycol, spacer molecules 413 polymer-bonded PAH column 305 polymeric monoliths 492 polymers, synthetic and imprinted 450 polynomial retention models 589 polysaccharide phases 453 – pure and coated 440 polystyrene resins 408 polystyrene/divinylbenzene 515 polyvinylamine 343 pore diameter 249 pore size 364 pore volume 384, 386 Poros 514 porous monoliths 494 post-column affinity detection 513 post-column derivatization 487 post-source decay 635 potassium phosphate buffer 255 pre-column 629, 632 pre-saturation techniques 557 PRECISION C18 324 Precision C8 column 328 prediction interval 113 prednisolone – fast UPLC gradient separation 503 26.04.2006, 14:49 745 746 Subject Index – gradient separation 65 prednisone 313 – as parameter S 314 pressure-driven chromatographic separations 494 pressure-driven LC 488 Primesep™ 100 34 Primesep A 38, 251 principal component (PC) – classification into groups 269 – projection of objects onto 266 principal component analysis (PCA) 264 ff., 704 – autoscaled data 265 – evaluation of charomatographic supports 285 – for method development 277 – in identification of column/phase equivalency 271 – of perfluorinated phases 270 – of polar embedded, enhanced polar selectivity and aqua phases 269 ff – of the database of RP silica materials 267 – physicochemical properties 281 – stationary phase optimization by 278 probability network 664 probenecid, gradient separation 84 Probot 635 procainamide 275, 282 Prodigy 153, 183, 232, 706 Prodigy ODS 329 Prodigy ODS (3) 273, 325 Prodigy ODS2 273 ProFound™ 637 ProLab 469 prolintane as parameter C 314 ProntoSil 233 ProntoSil 120-C18 ace EPS 611 ProntoSil 300 38 ProntoSil ACE 154, 183, 251, 706 ProntoSil AQ 154, 233, 706 ProntoSil C18 154, 183, 706 ProntoSil C18-AQ 270 propranolol 237, 313 – as parameter C 314 propylbenzene, fast isocratic UPLC separation 501 propylparaben 302 prostaglandins 512 protein A 406, 514 – IMAC resins 417 protein aggregation, studied by gel filtration 398 protein analysis 637 1293vch07.pmd 746 protein, binding capacity 405 protein folding, studied by gel filtration 398 protein G 406, 514 protein phases 440 – immobilized 450 proteins 450 – effect of flow rate on the resolution of 391 proteome analysis, SEC in 400 proteomics 627 – analysis steps and methods 639 Proteomweaver 628 PS-PMMA, GPC-FTIR analysis 377 pseudo 3D stacked transfer injections 534 PSS SDV GPC columns, separation characteristics for organic eluents 365 pulsation damper 471 pulse repetition rate 555 pulsed Fourier-transform (PFT) 551 Purospher 37, 38, 154, 251, 303, 706 Purospher e 232 Purospher RP-18 325 Purospher RP-18e 273, 307 Purospher Star 154, 233, 706 Purospher STAR RP18e 325 Pursuit C18 250 pyridine 282, 302 – in neutral water/acetonitril solution 19 – in neutral water/methanol solution 19 – onto hydrophobic, endcapped column 32 – pK values 80 1-(2-pyrimidyl)piperazine · HCl 687 pyrrolidine, pKa value 78 q quinine 282 r racemate, separation of 432 radius of gyration 385 RAM pre-column 678 RAM-CIX-RP separation 680 RAM/CIX-RP separation of peptides 682 Reactive Blue, IMAC resins 417 rebinding 515 reciprocity concept 445 recovery function 133 – evaluation 133 reference matrix 134 reference phase 312 refolding buffer 399 refractive index 377 regression coefficients 300 – as a function of organic modifier 308 relative CLND response factors 698 26.04.2006, 14:49 Subject Index relative deviation 106 relative percentage deviation 112 relative standard deviation 680 relaxation 552 replacement columns 330 reproducibility 85 – or retention times 86 ReproSil 233 ReproSil AQ 32, 154, 233, 251, 706 ReproSil ODS 706 ReproSil ODS 154, 183 residual chart, calibration function and uncertainty 121, 123, 128 residual standard deviation 113 resin – activation methods 409 – NHS-activation 410 – preparation of an activated 409 resin design in affinity chromatography 405 resolution – as function of gradient run time 644 – as function of gradient slope 648 – as function of pH value 647 – as function of temperature 645 – critical 644 – dependence – effect of flow rate 391 – effect of injection volume of neutral compounds 25 – effect of injection volume of polar compounds 25 – effect of sampling rate 23 – examples of improvement – greatest effect on 10 – improvement ff – improvement by increasing efficiency 16 – in enantiomeric separation 429 – in micro- and nano-LC 483 – in UPLC 499 – optimization 670 – possibilities for improving 8, 18 – representation in dependence on the stationary phases 619 – representation with SynergiPOLAR RP 620 resolution map 97, 569 – in DryLab 571 Resolve 154, 169, 183, 249, 706 Resolve C18 267, 273 Resource®/Source® 408 response factor of comonomer 372 Restek Ultra C18 325 restricted access materials (RAM) – columns 674 1293vch07.pmd 747 – principle of 675 – synthesis 674 restricted access, SEC columns based on 400 retention 71 – as a function of eluent pH 316 – as a function of pH 89 – dependence, of a zwitterionic sample on the pH of the mobile phase 73 – differences under gradient conditions as a function of pH 79 – influence of the samples on 79 – of acid and base as a function of pH 95 – of acids 80 – of bases 80 – of zwitterions 80 retention and separation factor in neutral and acidic methanol/acetonitrile phosphate buffers 710 retention factor 6, 7, 59, 60, 176, 297, 299, 334, 429 – change as a function of pH for norfloxacin, fleroxacin, and ofloxacin 90 – correlation between retention and separation 181 – dependence, of an acidic and a basic analyte on the pH value of the mobile phase 72 – deprotonated form of the analyte 72 – expressiveness 175 – for chrysene/perylene in methanol 162 – for tricyclic anti-depressants in acetonitrile/acidic phosphate buffers 170 – in acidic/alkaline methanol/acetonitrile phosphate buffers 707, 708 – in alkaline methanol/phosphate buffers 717 – in methanol/acetonitrile eluents 711 – in neutral and alkaline methanol/ acetonitrile phosphate buffers 709 – influence on 179 – method development with ChromSword® 589 – of isobutyl/isopropyl ketones 174 – protonated form of the analyte 72 retention mechanism 334 retention times in NP-HPLC 356 rhodamine as label 511 Ristocecin A 447 robustness – in analytical RP-HPLC 643 ff – settings of the influencing parameters for the test of 656 robustness criteria, determination of 647 robustness parameter 649 26.04.2006, 14:49 747 748 Subject Index robustness test 654 – by statistical experimental design 652 – choice of the correct influencing variables 655 – influencing variables 654 – influencing variables of an HPLC method 661 rotating frame overhauser effect spectroscopy 552 RP chromatography – application of 676 – retention according to the model of interphase and solvophobic effects 299 RP mechanism, ideal 157 RP separation on a mixed-mode 34 RP silica materials, PCA of the databasis 267 RP-chromatography – CMD approaches in 569 – economics 48 – factorial viewpoint 601 – optimization steps – pH and selectivity in 71 – potentially influences on separation 603 – robustness 643 ff RP-columns – comparison 151 – polar interactions 156 – properties 152 ff – selection of commercial 151 RP-phases – criteria for comparing 174 – examples of hydrophobic and polar 183 – for different classes of substances 237 – hexagons of the hydrophobic and polar type 228 – polar 234 – polar and hydrophobic 233 – polar and hydrophobic versions based on the same silica gel 186 – ranking according decreasing selectivity 184 ff – ranking according their surface homogeneity 189, 190 – selectivity hexagons 205 – similarity of 195 RP-phases I, types of 251 RP-phases II, types of 251 run time, shortening of 48 ruthenium complexes 450 s salbutamol 275 sample buffer 521 sample preparation in gel filtration 1293vch07.pmd 748 394 sample preparation variance 110 sample rate 26 sample type 80 sample viscosity in SEC 395 sample volume in SEC 395 sampling period 22 saturation transfer difference (STD) NMR 347 SB-100 317 SB-90 317 score matrix 704 score plot 704 – in PCA 266 – of C18-phases type A 268 – of non-C18 phases 268 – of USP designed L1 phases 274 – with chromatographic parameters 286 – with physico-chemical properties of several substances 235 – with test compounds 291 scouting 603 SCX-RAM column 402 – separation profile 402 SCX-RAM exclusion limit 401 SCX-RAM silica particle 400 SDS-PAGE, protein separation 627 segmented gradient 576 selecting valve 36 selectivity 71 – aromatic 157 – comparison of different column types 326 – hydrophobic 182 – of mixtures in phosphate buffer/ acetonitrile 20 – of mixtures in phosphate buffer/methanol 20 – of mixtures in water/acetonitrile 21 – of mixtures in water/methanol 21 – of RP-phases 157 – of the mobile phase 254 – polar and hydrophobic RP-phases for the separation of polar and apolar compounds 239 ff – prediction with LFER data 308 – reproducibility 261 – steric 157, 187 – theoretical 309 selectivity chart 258, 259 selectivity factor see separation factor – expressiveness 175 selectivity feature – for RP-phases I 251 – for RP-phases II 251 selectivity hexagons 205 26.04.2006, 14:49 Subject Index – aromatic acids in unbuffered eluent/acidic phosphate buffer 221 ff – different aromatic compounds in methanol/water 207 ff – neutral and acidic aromatic compounds in unbuffered eluent/acidic phosphate buffer 214 ff selectivity map 196 – application 200 selectivity map 1, hydrophobic/polar selectivity 197 selectivity map 2, hydrophobic/polar selectivity 199 selectivity plot 200 ff – LiChrospher/Superspher 203 – Reprosil/Prontosil 203 – Symmetry C18/Purospher 201 f – Symmetry Shield C18/XTerra 204 – Zorbax ODS/Hypersil BDS 204 selectivity triangle 350 – of organic solvents 351 selector-to-analyte ratio 346 selectors for enantiomer separation 433 Selectosil C18 276 sensitivity – improvement of 48 – in micro-LC/nano-LC 467 SepaBeads FP-HP 413 separation – complex sample mixtures 669 ff – electro-driven 489 – of a critical pair of peaks 660 – of a pharmaceutical sample on an original column and replacement columns 330 – of 4-hydroxy-/3-hydroxybenzoic acid on a polar and on a hydrophobic RP-phase 244 – of ionizable compounds 85 – of nitroaniline isomers 169 – of phthalic/terephthalic acids on a polar and on a hydrophobic RP-phase 245 – of steroids on several RP materials 168 – of tricyclic anti-depressants 169 – of weak and strong aromatic acids on various RP-phases 246 – one-dimensional 670 – optimization with ChromSword® 589 – possible influences in RP-HPLC 603 – single-stage 670 2D separation – advantages 527 – by combination of HPLC with GPC 527 – of non ionic tensides 539 separation column, pressure of 633 separation factor 6, 567 1293vch07.pmd 749 – biplot of 714 – correlation between retention and separation 181 – for chrysene/perylene in methanol 162 – for tricyclic anti-depressants in acetonitrile/acidic phosphate buffers 170 – in acetonitrile/water 718, 725 – in acetonitrile/water eluents 713 – in acidic acetonitrile/phosphate buffer 719, 726 – in acidic methanol/phosphate buffer 720, 723 – in acidic/alkaline methanol/acetonitrile phosphate buffers 707 f – in alkaline acetonitrile/phosphate buffer 727 – in alkaline methanol/phosphate buffer 716, 724 – in all methanol-containing eluents 721 – in methanol/acetonitrile eluents 711 – in methanol/water 722 – in neutral and acidic methanol/ acetonitrile phosphate buffers 710 – in neutral and alkaline methanol/ acetonitrile phosphate buffers 709 – influence on 179 – of 4-hydroxy-/3-hydroxybenzoic acid in methanol/phosphate buffers 243 – of ethylbenzene/fluorenone on some commercial RP-phases 160 – of ethylbenzene/toluene on some commercial RP-phases 158 – of isobutyl/isopropyl ketones 174 – of methanol/water on some commercial RP-phases 158 separation performance in microchip-based LC 490 separation power of the multidimensional HPLC 678 separation selectivity 329, 567 separation value for o-ter/tri and EB/T in 80/20 (v/v) methanol/water 164 Sephacryl 390 Sephadex 383, 390, 408 Sepharose 408 SEQUEST™ 637, 639 serine proteases 406 shape selectivity see steric selectivity signal suppression, measuring 69 signal-to-noise ratio 106, 551 significant F-test 112 silanes 151 silanol activity 255, 286 26.04.2006, 14:49 749 750 Subject Index – standard deviation 258 – versus hydrophobicity of the stationary phase 256 ff silanol dissociation 315 silanol groups, interaction 171 silanol masking capacity 287 silanols – acidic 350 – end-cap unreacted 323 – free 350 – geminal and vicinal 350 silanophilicity 181, 248 silanophilicity/hydrophobicity 181 silica 237 – as NP stationary phase 349 – as stationary phases 255 – gradient separation 355 silica gel 515 – alternatives to 355 – as matrix 151 – polar and hydrophobic versions 186 silica matrix, separation data 186 silica surface – cavity 299 – polar groups on 350 SilicaRod 706 similar phases 232 similarity 703 simulated moving bed (SMB) chromatography 458 – applications 431 – principles of 458 single porosity 366 single-linkage algorithm 703 single-quadrupole Waters ZQ 692 size-exclusion chromatography (SEC) see also gel filtration; gel-peremeation chromatography (GPC) – application of 675 – calibration curve 385 – column selection and flow rate 388 ff – comparison with other HPLC variants 387 – theory 384 sleeves 475 SMT 232, 251, 706 SMT OD 37, 154, 183, 236 sodium azide, markers for SEC columns 385 sodium deoxycholate, detergent in SEC 394 sodium dodecyl sulfate 518 software for automatic method development 608 solenoidal micro flow probe 553 solid-phase extraction 492, 513 1293vch07.pmd 750 solid-phase-extraction NMR 558 solubility 86 solute descriptors, determination of LFER 310 solute parameters, experimental determination 311 solute–carbamate interactions 306 solute–column interactions 322 solvation equation 300, 321 – in LFER 301 solvation parameter 318 solvatochromic descriptors 301 solvatochromic effects 300 solvent properties 351 solvent standard 546 solvent strength of binary mixture 353 solvent suppression 559 solvent suppression technique 557 solvents – basic, localizing 352 – miscibility of 354 – non-basic, localizing 352 – non-localizing 352 – without matrix 133 solvophobic retention model 300 Sorbex process 458 soya bean trypsin inhibitor, immobilization of 420 spacer – polymeric 412, 413 – with a single ligand attached 412 spacer molecules 414 specific CLND response factor 690 Spherisorb ODS-1 38, 154, 183, 234, 251, 267, 273, 706 Spherisorb ODS-2 38, 154, 183, 251, 273, 706 spiking prove 136 spiking experiment, calibration and uncertainty 138 spin-lattice relaxation time 552 spin-spin relaxation time 552 split system – at high pressure 469 – at high pressure level 471 – regulated 469, 470 – simple 469 splitting system 55 stainless steel 472 stainless steel capillaries 475 standard addition method 137 standard deviation 106, 108, 113 standard free energy 297 staphylococcus aureus V8 protease 406 26.04.2006, 14:49 Subject Index star block copolymer – 2D analysis 537 – gradient HPLC analysis 536 – SEC separation 536 star-branched copolymer, 3D surface representation 536 Statgraphics Plus 657 stationary phase 26 – characterization parameter 265 – optimization 278 – physico-chemical parameters 303 – silanol activity versus hydrophobicity 256 ff stationary phase cavity 299 stationary phase characterization – with C-parameters 317 – with empirical LFER parameters 317 stationary phases – characterization 264 – characterization and selection 296 – comparability on the basis of LFER data 301 – derived from tartaric acid 444 – for enantiomer separation 433 – for micro-LC and nano-LC 469 – in microchip-based LC 492 – polar selectivity versus hydrophobicity 260 – reproducibility of a stationary phase 262 – selection of 611 – silanol activity 255 – tailor-made 492 – test method 262 – with a low hydrophobicity 258 – with high silanol activity 258 – with metal atoms 450 – with strongest hydrophobicity 258 statistical experimental design 652 step gradients 520, 673 steric parameter 318 steric selectivity 157, 187, 237, 248, 264 – for small molecules 33 steric selectivity parameter 311 steroid contaminants, chromatograms of 614 steroids 237, 512 trans-stilbene oxide 452 stopped-flow measurements 557 streptavidin 406, 514 Student’s t-factor 107, 109, 113, 114 styrene–divinylbenzene co-polymers 490 styrene/butadiene star block copolymer, 2D result 537 styrene/MMA block copolymer, molar mass and composition determination 374 1293vch07.pmd 751 subtraction model of reversed-phase column selectivity 323 successive RP chromatogram 681 sudan red 7B 355 sugars, WAC separation 524 sulfonamides 260 – pK values 80 sulfonic acids, pK values 80 SunFire 249 Supelcogel TRP 154 Supelcosil ABZ Plus 154, 183, 234, 250, 251, 285, 706 supercritical fluid chromatographic (SFC) conditions, method development 463 supercritical fluids 462 Superdex 390 Superose 390 Superspher 154, 706 Superspher 100 RP-18e 325 Superspher RP18e 273 Superspher Select B 154, 183, 706 Suplex pkb 100 267, 269 surface covering in micro- and nano-LC 484 surface homogeneity 188 – of RP-phases 189, 190 3D surface plot 534 susceptibility distortions 337 suspended-state high-resolution magicangle spinning NMR spectroscopy see suspended-state HR/MAS NMR spectroscopy suspended-state HR/MAS NMR spectroscopy 334 ff – cross-peaks 342 – rotor set-up 337 – spectrum of a chromatographic sorbent suspended in [D4]methanol 338 ff – T1 measurements on binaphtyl-2,2′-diol 344 – T1 relaxation measurements 343 ff – trNOESY spectrum of binaphthyl-2,2′-diol mixtur 345 Switchos™ 630 SWXL columns 390 symmetric Gaussian distribution 697 Symmetry 37, 232, 316, 317, 706 Symmetry 300 38 Symmetry C18 82, 154, 183, 236, 249, 273, 325 Symmetry Shield 169, 249, 251, 305, 706 Symmetry Shield C18 155, 183, 306 Symmetry Shield phases 306 Symmetry Shield RP-18 82, 273 26.04.2006, 14:49 751 752 Subject Index Symmetry Shield RP-C18 303, 307 Symmetry Shield RP-C8 303, 307 Synchropak RP-C18 303 Synchropak RP-C4 303 Synergi Fusion-RP 38, 249 Synergi Hydro-RP 270, 273 Synergi Max-RP 38, 155, 183, 236, 249, 251, 325, 706 Synergi Polar-RP 37, 155, 183, 234, 249, 251, 269, 270, 613, 618, 706 – resolution as a function of column temperature and gradient run time 620 – reversed phase 615 syringe pumps, double 469 ff t T-2 512 T1 relaxation 343 tailing peaks 68 Targa C18 324 target 635 Teicoplanin 447 temperature, influence on peak homogeneity 28 terbinafine – degradation profile of 500 – high-powered isocratic UPLC separation 500 terbutaline 275 terephthalic acid, degrees of ionization 242 o-terphenyl, separation in methanol/water 167 tert-butylcarbamoyl)quinine selector, HR/MAS 1H NMR spectrum of 337 test compounds – chromatographic properties 284 – for chromatographic test optimization 291 – groups 283 – physicochemical properties 281 test kits 522 Tetralon 460 theophylline 687 theoretical plate height 61 ThermoElectron 633 THF 26 – as modifier 38 thin-layer chromatography for use in HPLC trials 352 thin-layer separation 29 thiopyridine 406 thiourea, fast isocratic UPLC separation 501 three-level design 655 thyroglobulin, markers for SEC columns 385 time constant 68 1293vch07.pmd 752 time-of-flight (TOF) 628 tocopherol, continuous-flow measurements 555 toluene 302, 327 – fast isocratic UPLC separation 501 – separation in methanol/water 167 o-toluidine 302 Toyopearl 650M 414 Toyopearl Epoxy HW70 EC 413 ToyoScreen columns 418 2D transfer injection options 530 transferred nuclear Overhauser effect (trNOE) 340 transients 551 trap column 519 tresyl activation 411, 516 triamterene, gradient separation 84 triazine herbicides 512 trichothecenes 512 tricyclic antidepressants 255 – selectivity 238 triethylamine – as solvents 351 – pKa value 78 trifluoroacetic acid 85 – as buffer 79 trimethylamine, pK values 80 2,2,7-trimethylguanosine 512 triphenylene, separation in methanol/water 167 triphenylene/terphenylene, separation values 164 triple detection 380 tris(carboxy)ethylenediamine 406 Trisacryl® 408 Triton X-100 394 Tröger’s base 452 true affinity chromatography 511 see also weak affinity chromatography Tryptic peptides 628 tryptophan 398 TSK 155, 706 TSK-GEL SWXL columns 390 TSK-Gel, Toyopearl® 408 Tween 20 515 two days method 36 two-level design 655 u uBondpak 273 Ultimate™ 630 Ultra Flow 514 ultra-performance liquid chromatography (UPLC) 498 ff 26.04.2006, 14:49 Subject Index – fast gradient separation 503 – high-powered gradient separation 503 – separation performance 499 Ultracarb ODS 268 Ultracarb ODS(30) 267, 273 Ultrasep ES 155, 232, 233, 251, 706 Ultrasep ES RP18e Pharm 611 Ultrasphere ODS 324 United States Pharmacopoeia (USP) 271 universal calibration 379 universal column 220, 248 Universal Oil Products (UOP) 458 uracil – in acidic acetonitrile/phosphate buffer 19 – in acidic methanol/phosphate buffer 19 – in neutral water/acetonitril solution 19 – in neutral water/methanol solution 19 – onto hydrophobic, endcapped column 32 – separation in methanol/water 167 USP L1 phases – chromatographic properties 272 ff – PC1–PC2 and PC1–PC3 score and loading plots 274 UV detection 691 UV/Vis detector 484 v van Deemter curve 499 van Deemter equation 60, 176 vancomycin 447 variance heterogeneity 114 variance quotient 109, 111 variance ratio 111 variances, homogeneous 125 variation coefficient, constant 122 virial coefficient, second 377 viscometry detection 379 vitamin A metabolites, elution orders 334 ff vitamin B12 512 void time 428 void volume, detection of 632 volumetric flow rates 456 VYDAC 155, 233, 611, 706 w Wakosil II 5C18AR 325 Waters CapLC 469 weak affinity chromatography see also true affinity chromatography weak affinity chromatography (WAC) 511 – examples 523 weighted calibration, weighting 1/s2 applies standard deviations proportional to concentration 130, 131 1293vch07.pmd 753 weighting with concentrations ‘x’ applies 1/x2 with absolute units 132 weighting exponent 110 weighting factor 111 Whelk-O phase 445 working range 105, 109, 114 x XTerra 38, 81, 155, 183, 249, 618, 706 XTerra MS 155, 183, 233, 249, 251, 706 XTerra MS C18 85, 273, 325 XTerra RP18 273, 611, 613 – reversed-phase 616 y YMC 15 317 YMC 16 317 YMC AQ 233, 250, 706 YMC Hydrosphere 611 YMC J’Sphere ODS-H80 611 YMC ODS AQ 155 YMC ODS-AQ 270, 273 YMC Pro 251 YMC Pro C18 37, 155, 183, 250, 273, 611, 706 YMC Pro C4 611 YMC Pro C8 325, 611 YMCbasic 250, 325 z Z-cell 630, 633 zearalenone 512 zero quantum 340 ZirChrom 37, 38 ZirChrom-MS 251 Zoloft 460 Zorbax Bonus RP 155, 169, 183, 234, 250, 251, 706 Zorbax Bonus/Chromolith Performance 26 Zorbax Eclipse XDB 250 Zorbax Eclipse XDB-C18 273, 324 Zorbax Extend C18 38, 155, 183, 233, 236, 250, 251, 273, 285, 324, 706 Zorbax ODS 155, 182, 183, 233, 250, 251, 706 Zorbax phases SB-100, non-endcapped 317 Zorbax Rx C18 273, 324 Zorbax SB 300 38 Zorbax SB 300 C8 303 Zorbax SB 300 CN 303 Zorbax SB AQ 250, 269, 270 Zorbax SB C18 155, 273, 706 Zorbax SB C18/300 250 Zorbax SB C8 37, 155, 183, 234, 250, 691, 706 zwitterion 72 26.04.2006, 14:49 753 [...]... Optimization means more than merely the “correct” choice of method parameters Efforts to obtain as much information as possible, or at least the necessary information, are also a part of optimization In this context, the evaluation of chromatographic data and calibration take on a special significance These topics are dealt with in Chapter 1.5 (Hans-Joachim Kuss) and Chapter 1.6 (Stefan Schömer) Part 2 Characteristics... International GmbH Scientific Instruments Hilpertstrasse 2 0A 64295 Darmstadt Germany Roberto Biancardi Solvay Solexis SpA Viale Lombardia, 20 20021 Bollate (MI) Italy Diane M Diehl Waters Corporation, CAT 34 Maple Street Milford, MA 01757 USA John W Dolan BASi Northwest Laboratory 3138 NE Rivergate Building 301C McMinnville, OR 97128 USA HPLC Made to Measure: A Practical Handbook for Optimization Edited... Liquid Chromatography (LC) 488 Open-Channel Electrochromatography (OCEC) 488 Packed-Bed Electrochromatography 488 Microfabricated Chromatographic Beds (Pillar Arrays) 489 In Situ Polymerized Monolithic Stationary Phases 489 Optimization and Possibilities 490 Separation Performance 490 Isocratic and Gradient Elution 491 Tailor -Made Stationary Phases 492 Sample Pretreatment and More-Dimensional Separations... Optimization Automation can generally lead to the elimination of errors and to time saving Meanwhile, fully automated computer-aided method development and semiautomated optimization in HPLC have reached a remarkable level of maturity and sophistication Through several real examples, Lloyd R Snyder (Chapter 4.1) and Sergey Galushko (Chapter 4.2) describe the possibilities offered by the software packages DryLab®... Alden Waters Corporation, CRD 34 Maple Street Milford, MA 01757 USA Hans Bilke Sandoz GmbH Biochemiestrasse 10 6250 Kundl Austria Yung-Fong Cheng Cubist Pharmaceuticals 65 Hayden Ave Lexington, MA 02421 USA Maristella Colombo Oncology – Analytical Chemistry Nerviano Medical Sciences Via le Pasteur, 10 20014 Nerviano (MI) Italy Mario Arangio CarboGen AG Schachenallee 29 5001 Aarau Switzerland Wolf-Dieter... questions prior to commencing the development of a method or the optimization of a given separation • What do I want? In other words, what is the true intention of the separation? • What do I have? That is to say, what relevant information about the analytical purpose and the samples is available? • How should I do it? Do I have all what I need, and is what I want to do really possible? At first glance, these... Euerby, Cinzia Stella), and LC-MS (Friedrich Mandel, Katrin Markus) The reader may benefit from the different descriptions of the topics and from the individual evaluations of the authors 1293vch00.pmd 33 10.05.2006, 09:21 XXXIII 1 1 Fundamentals of Optimization HPLC Made to Measure: A Practical Handbook for Optimization Edited by Stavros Kromidas Copyright © 2006 WILEY-VCH Verlag GmbH & Co KGaA, Weinheim... optimal specificity? • Must the relative standard deviation Srel be no higher than 2%? What loss of qua- lity would be incurred if Srel were to be 2.5%? Is there actually a correlation between the cost of the analysis and real improvement in the quality of the product? HPLC Made to Measure: A Practical Handbook for Optimization Edited by Stavros Kromidas Copyright © 2006 WILEY-VCH Verlag GmbH & Co KGaA,... quickly calculate the pKa value of the known main component in the sample with appropriate software (see Chapter 1.4)? Has a colleague in a neighboring department worked in the past with similar compounds and might therefore be able to provide valuable insights? As far as possible, all means of communication with colleagues should be pursued to gather information At times it may be helpful not to make this... of Chiral Additives to the Mobile Phase in HPLC and Capillary Electrophoresis 461 2.6.8 Determination of Enantiomeric Purity Through the Formation of Diastereomers 462 2.6.9 Indirect Enantiomer Separation on a Preparative Scale 462 2.6.10 Enantiomer Separations Under Supercritical Fluid Chromatographic (SFC) Conditions 462 2.6.11 New Chiral Stationary Phases and Information Management Software 463