Table of Contents

Cover

Preface

The Structure of the Book

Notes on Contributors

List of Contributors

Part 1: Principles of Gradient Elution

Chapter 1: Aspects of Gradient Optimization
1. 1.1 Introduction
2. 1.2 Special Features of the Gradient
3. 1.3 Some Chromatographic Definitions and Formulas
4. 1.4 Detection Limit, Peak Capacity, Resolution – Possibilities for Gradient Optimization
5. 1.5 Gradient “Myths”
6. 1.6 Examples for the Optimization of Gradient Runs: Sufficient Resolution in an Adequate Time
7. 1.7 Gradient Aphorisms
8. References
Chapter 2: Instrumental Influences on the Quality and Performance of Gradient Methods and Their Transfer Between Different HPLC Devices
1. 2.1 Technical Implementation of the Gradient Elution and the Respective Characteristics
2. 2.2 The Determination and Significance of the Gradient Delay Volume of the System
3. 2.3 The Transfer of Gradient Methods Between Different HPLC Systems
4. 2.4 Influence of Fluctuations of the Eluent Composition on the Quality of the Detection
5. 2.5 Other Kinds of Practical Application of Gradient Systems in HPLC
6. References
Chapter 3: Optimization of a Reversed-Phase Gradient Separation Using EXCEL
1. References

Part 2: Specifics of the Gradient in Different Elution Modes

Chapter 4: Gradient Elution of Ionic Compounds
1. 4.1 Introduction
2. 4.2 Theoretical Aspects
3. 4.3 Gradient Types in Ion Chromatography
4. 4.4 Choice of Eluent
5. 4.5 Gradient Elution of Anions on Anion Exchangers
6. 4.6 Gradient Elution of Cations on Cation Exchangers
7. 4.7 Gradient Elution of Anions and Cations on Mixed-Mode Stationary Phases
8. References
Chapter 5: The Gradient in Biochromatography
1. 5.1 Biomolecules
2. 5.2 Biochromatography
3. 5.3 The Gradient in Biochromatography
4. 5.4 Gradients for Different Biochromatographic Techniques
5. 5.5 Summary
6. References
Chapter 6: Specifications of Gradients in Hydrophilic Interaction Liquid Chromatography (HILIC)
1. Mechanistic Notes on the use of Gradients in HILIC
2. Types of Gradients in HILIC
3. Solvent Gradients
4. Salt Gradients
5. Temperature Gradients
6. pH Gradient
7. Effects of Gradients
8. References
Chapter 7: Specifications of Gradients in Supercritical Fluid Chromatography
1. 7.1 Types of Gradients in SFC
2. 7.2 Effects of gradients
3. References
Chapter 8: Aspects of Gradient Elution in LC-MS Analysis
1. 8.1 Role and Importance of Gradient Elution for LC-MS
2. 8.2 Technical Aspects of Gradient Elution in LC-MS Analysis
3. 8.3 Summary
4. 8.4 Abbreviations
5. References
Chapter 9: Additional Tools for Method Development: Flow and Temperature Gradients
1. 9.1 Introduction
2. 9.2 Temperature Gradients
3. 9.3 Flow Gradients
4. 9.4 Combination of Flow and Temperature Gradients
5. 9.5 Case Example
6. 9.6 Conclusions
7. References

Index

End User License Agreement

List of Illustrations

Chapter 1

Figure 1.1 Influence of the flow rate. XBridge Shield 150 × 4.6 mm, 5 μm. (a) 0–...
Figure 1.2 Influence of the flow rate. (a) 0.6 ml/min, 10 °C, (b) 1 ml/min, 10 °...
Figure 1.3 Influence of the flow rate. XBridge Shield, 150 × 4.6 mm, 5 μm. (a) 5...
Figure 1.4 The gradient duration required. Zorbax SB C8, 150 × 4.6 mm, 5 μm, 40–...
Figure 1.5 Effect of initial % B. XBridge Shield, 150 × 4.6 mm, 5 μm. (a) 0–100%...
Figure 1.6 Regarding the starting conditions with a small number of peaks. Gemin...
Figure 1.7 Effect of initial % B and slope. XBridge Shield, 150 × 4.6 mm, 5 μm. ...
Figure 1.8 Influence of initial % B and slope. Symmetry C18 150 × 4.6 mm, 5 μm. ...
Figure 1.9 Effect of initial % B and gradient duration on elution order and reso...
Figure 1.10 Influence of column length. (a) Synergi Fusion RP, 20 × 4.6 mm, 2 μm...
Figure 1.11 Influence of gradient duration, Synergi MAX RP 20 × 4 mm, 2 μm. (a) ...
Figure 1.12 Gradient run at 35 °C (a) and 15 °C (b) on LUNA Omega PS. The larges...
Figure 1.13 Gradient run at 35 °C (a) and 15 °C (b) on Primesep C. A change of e...
Figure 1.14 Gradient run on Cortecs C18 on two apparatus with different dwell vo...
Figure 1.15 Gradient run on Poroshell EC 120-C18 on two apparatus with different...
Figure 1.16 Gradient run on Cortecs Phenyl on two apparatus with different dwell...
Figure 1.17 Gradient run on Atlantis T3 on two apparatus with different dwell vo...
Figure 1.18 Gradient separation on Primesep C on two apparatus with different dw...
Figure 1.19 Different volumes of mixing chambers may have a different influence ...
Figure 1.20 Mixers of different volumes: (a) 400 μl; (b) 10 μl, in a high-pressu...

Chapter 2

Figure 2.1 Schematic representation of the working principle of (a) HPG pumps (o...
Figure 2.2 Illustration of the resulting waviness of the composition of the mobi...
Figure 2.3 Schematic representation of the technical concept of the SpinFlowTM m...
Figure 2.4 Programmed sinusoidal composition pattern generated with an HPG pump ...
Figure 2.5 Programmed sinusoidal composition pattern with an HPG pump with 13 di...
Figure 2.6 Residual pulsation amplitude as a function of the residence volume of...
Figure 2.7 Representation of the damping effect as a function of the logarithmic...
Figure 2.8 Curves of volume contraction when mixing water with acetonitrile and ...
Figure 2.9 Diagram (a) shows the programmed pump parameters of a simple linear g...
Figure 2.10 Overlay of nine runs of an amino acid method with UV detection after...
Figure 2.11 Application of the marker-pulse method for GDV determination by dire...
Figure 2.12 Example of atypical Dolan test result. The round edges in the part o...
Figure 2.13 (a) Two different evaluation methods described by Dolan for the dete...
Figure 2.14 Flow (a) and pressure (b) dependence of the GDV determined by the Do...
Figure 2.15 Influence of the GDV change in a USP-based method for the determinat...
Figure 2.16 Experimental Dolan test curves recorded with different Spin Flow mix...
Figure 2.17 Scheme to illustrate the influence of changes in the idle volume set...
Figure 2.18 Practical measures to transfer the acetaminophen method (Figure 2.16...
Figure 2.19 Painkiller application to demonstrate a GDV adjustment by changing t...
Figure 2.20 Transfer of a pesticide analysis gradient method from Agilent 1260 t...
Figure 2.21 Influence of the injection volume on the peak shape of 4-aminophenol...
Figure 2.22 Influence of different precolumn mixing efficiency in different HPLC...
Figure 2.23 Example for generation of baseline ripples in the UV range due to us...
Figure 2.24 Amplitude comparison of baseline ripples with TFA to demonstrate the...
Figure 2.25 Comparison of the detector baseline with two different mixer volumes...
Figure 2.26 Residual ripple amplitude in a TFA gradient with and without a colum...
Figure 2.27 Comparison of absolute base line waviness from isocratic mixing of 1...
Figure 2.28 Schematic representation of an automated method development system e...

Chapter 3

Figure 3.1 Chroma to gram using a 15 × 0.46 cm 5 μm XBridge Shield column with 3...
Figure 3.2 Similar to Fig. 3.1 at 40% ACN.
Figure 3.3 Retention times on the y-axis for the three chromatograms at 30% ACN ...
Figure 3.4 Diagram of ln(k) against % B with conditions according to Figure 3.1.
Figure 3.5 Simulated chromatogram at 47% ACN according to the LSS model with whi...
Figure 3.6 The Excel solver minimizes the sum of deviation squares (SAQ) for tol...
Figure 3.7 Bent lines fitted with the Excel solver using the Neue model.
Figure 3.8 Prediction of a chromatogram according to Snyder’s LSS model.
Figure 3.9 Prediction of a chromatogram with the same conditions as in Figure 3....

Chapter 4

Figure 4.1 Comparison between classical anion exchange and anion exchange on met...
Figure 4.2 Comparison of isocratic and capacity gradient separations of inorgani...
Figure 4.3 Schematic illustration of a cartridge for a contamination-free electr...
Figure 4.4 Separation of inorganic anions with an electrolytically generated KOH...
Figure 4.5 Representation of log(Vms − Vd)/Vd as a function of log R for various...
Figure 4.6 Separation of standard inorganic anions and oxyhalides on Dionex IonP...
Figure 4.7 Gradient elution of inorganic and organic anions with an electrolytic...
Figure 4.8 High-resolution separation of inorganic and organic anions on Dionex ...
Figure 4.9 Gradient elution of inorganic and organic acids in beer. Separator co...
Figure 4.10 Gradient elution of hydrolysate amino acids and O-phosphorylated ami...
Figure 4.11 Gradient elution of mannose-7-isomers on a pellicular anion exchange...
Figure 4.12 HPAE-PAD analysis of inulin. Separator column: Dionex CarboPac PA200...
Figure 4.13 Gradient elution of an oligonucleotide mixture such as d (AC)10–11 2...
Figure 4.14 Capacity gradient analysis of linear polyphosphates. Separator colum...
Figure 4.15 Isocratic elution of inorganic cations and ethyl amines in compariso...
Figure 4.16 Gradient elution of lanthanides. Separator column: Dionex IonPac CS5...
Figure 4.17 (a) Gradient elution of inorganic cations and petrochemically releva...
Figure 4.18 Gradient elution of inorganic cations and biogenic amines. Separator...
Figure 4.19 Gradient elution of inorganic cations, various diamines, paraquat, a...
Figure 4.20 Gradient elution of a monoclonal antibody with an MES/NaCl eluent. S...
Figure 4.21 Optimization of the analysis time for the separation of monoclonal a...
Figure 4.22 High-resolution separation of mAb variants on Dionex MAbPac SCX-10 u...
Figure 4.23 Comparison between the programmed and measured pH value during a lin...
Figure 4.24 Example of a linear pH gradient based on a zwitterionic Good buffer ...
Figure 4.25 Separation of a protein standard with a pH gradient based on phospha...
Figure 4.26 Optimization of the separation of mAb charge variants with a linear ...
Figure 4.27 Gradient elution of basic, neutral, and acidic pharmaceutically rele...
Figure 4.28 Gradient elution of native N-glycans from bovine fetuin with charged...
Figure 4.29 Gradient elution of labeled N-glycans from bovine fetuin with fluore...
Figure 4.30 Simultaneous gradient separation of pharmaceutically relevant counte...
Figure 4.31 Gradient elution of acidic and basic APIs in a pharmaceutical formul...
Figure 4.32 Gradient elution of mono- and multivalent pharmaceutical counterions...

Chapter 5

Figure 5.1 Typical SEC run (also known as gel filtration). No gradient run is po...
Figure 5.2 Typical ion exchange run (CIX) with different phases. 1: Equilibratio...
Figure 5.3 Typical IEX run with a step gradient [2].
Figure 5.4 Salt characteristics in terms of binding strength for the preparation...
Figure 5.5 Typical chromatofocusing run [2].
Figure 5.6 Typical HIC run with a linear gradient with decreasing salt concentra...
Figure 5.7 HIC separation can also be performed with step gradients [2].
Figure 5.8 The chaotropic character of the ions increases from left to right. Sa...
Figure 5.9 Typical reversed-phase chromatography (RPC) run for the separation of...
Figure 5.10 Typical affinity chromatography separation [2].

Chapter 6

Figure 6.1 Typical HILIC gradient showing gradient and content of the mobile pha...
Figure 6.2 HILIC separation of 2-aminobenzoic acid (1), 2,3-dihydroxybenzoic aci...

Chapter 7

Figure 7.1 UV-chromatogram of a SFC separation of caffeine (A), theophylline (B)...
Figure 7.2 Separation influencing parameters in SFC. Interactions of parameters ...

Chapter 8

Figure 8.1 RP retention diagram for a small molecule and a protein.
Figure 8.2 Signal intensity of Leu-enkephalin dissolved in various common LC-MS ...
Figure 8.3 (a) Fluidic scheme of a LC-MS system with inverse gradient and dual g...
Figure 8.4 Base peak chromatograms of a UHPLC separation of the diuretics Amilor...
Figure 8.5 Normalized response factors and signal-to-noise ratios with and witho...
Figure 8.6 Reduction of re-equilibration time and throughput enhancement by usin...

Chapter 9

Figure 9.1 Effect of gradient flow on Purospher STAR separation. Dark – constant...
Figure 9.2 Effect of flow rate gradient and flow rate and temperature gradient o...
Figure 9.3 Effect of flow rate gradient and flow rate and temperature gradient o...

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The Structure of the Book

The book consists of two parts: Part 1 provides the basic information on the gradient technique, while Part 2 presents the specifics of the gradient in different modes and separation techniques.

Part 1: The Principles of Gradient Elution

In Chapter 1 (Aspects of Gradient Optimization) Stavros Kromidas discusses in a compact fashion what is important in gradient optimization and presents simple “to-do” rules. Frank Steiner explains in Chapter 2 (Instrumental Influences on the Quality and Performance of Gradient Methods and Their Transfer Between Different HPLC Devices) to what extent even the smallest differences between HPLC systems can strongly influence chromatography. Part 1 ends with Chapter 3 by Hans-Joachim Kuss (Optimization of a Reversed-Phase Gradient Separation Using EXCEL), which shows one way to predict gradients using EXCEL.

Part 2: The Specifics of the Gradient in Different Separation Modes

Chapters 4 and 5 deal with the separation of ionic or ionizable components. In Chapter 4 (Gradient Elution of Ionic Compounds) Joachim Weiss deals with both the separation of small molecules such as inorganic ions and the separation of large molecules such as monoclonal antibodies and shows the specifics of pH and salt gradients. In Chapter 5 (The Gradient in Biochromatography) Oliver Genz deals with the different separation modes in biochromatography, which should also be noted here in particular for gradient runs. In Chapter 6 (Specifications of Gradients in Hydrophilic Interaction Liquid Chromatography (HILIC)) Thomas Letzel discusses all applicable gradients in HILIC, including temperature gradients. In Chapter 7 (Specifications of Gradients in Supercritical Fluid Chromatography), Stefan Bieber and Thomas Letzel present the three possibilities of gradient elution in SFC in condensed form. In Chapter 8 (Aspects of Gradient Elution in LC-MS Analysis) Markus Martin deals in detail with gradients in LC-MS coupling. Here, instrumental aspects of the LC and MS parts as well as the problem of quantification of gradients are discussed. Finally, in Chapter 9, Egidijus Machtejevas describes some rare gradient modes (Additional Tools for Method Development: Flow and Temperature Gradients).

The book does not have to be read linearly. The individual chapters have been written in such a way that they represent completed stand-alone modules – “jumping” between them is possible at any time. We have tried to do justice to the character of the book as a reference work. Let the reader benefit from this.

Notes on Contributors

Stavros Kromidas

Stavros Kromidas studied Chemistry in Saarbruecken, Germany, completing his PhD thesis on the development of new optically active phases for HPLC. He subsequently worked as a Sales Manager for Waters, when he founded 1989 NOVIA GmbH, an independent consultancy company for analytical chemistry. Since 2001, he has been working as a consultant and has given lectures and training courses on HPLC and Validation. Stavros Kromidas has authored, coauthored and edited numerous books on HPLC, validation, and quality in analytical chemistry.

Joachim P. Weiss

After his graduation in Chemistry in 1979 from the Technical University of Berlin, Germany, he worked in the field of Liquid and Gas Chromatography at the Hahn-Meitner Institute in Berlin and received his PhD in Analytical Chemistry in 1982 from the Technical University of Berlin. Weiss habilitated in Analytical Chemistry at the Leopold-Franzens University in 2002. He currently holds the position of International Technical Director for Dionex Products within the Chromatography and Mass Spectrometry Division (CMD) of Thermo Fisher Scientific, located in Dreieich (Germany). Dr. Weiss is recognized as an international expert in Analytical Chemistry (especially in the field of Liquid/Ion Chromatography). The 4th edition of his Handbook of Ion Chromatography was published in 2016.

Markus M. Martin

Markus M. Martin works as Manager, Product Management UHPLC Systems at Thermo Fisher Scientific in Germering (Germany). He joined the former Dionex Corporation, now part of Thermo Fisher Scientific, in 2010 as Solutions Manager for LC/MS, being responsible for UHPLC and LC/MS solutions marketing. He received his Doctorate in Analytical Chemistry from the Saarland University in Saarbruecken, Germany, in 2004 for capillary electrophoresis investigations on polyelectrolytes in the research group of Prof. Heinz Engelhardt. Before his Thermo Fisher Scientific engagement, he worked as Analytical Lab Head at Sanofi-Aventis and as a Research Fellow at the Saarland University; his scientific work has been focused on UHPLC, HPLC-MS, CE, and CE-MS techniques as well as integrated sample preparation.

Thomas Letzel

Thomas Letzel is a habilitated analytical chemist with almost 20 years of experience in the field of analytical screening techniques using LC and GC with mass spectrometric detection. He is Head of the Analytical Research Group at the Chair of Urban Water Systems Engineering at the Technical University of Munich (TUM), Germany. He holds a Diploma and PhD in Chemistry and the license to teach analytical and bioanalytical chemistry from TUM. Currently, the key aspects in his research cover technological, analytical-methodological, and analytical-chemical properties and can be applied in water and wastewater analysis as well as in other relevant environmental matrices, such as food analysis, beverage and plant extract analysis, among others. A special focus of his work is on chemical analysis with simultaneous functionality analysis using mass spectrometric detection. He is the author and coauthor of more than 150 journal papers, book contributions, conference proceedings, and four books.

Stefan Bieber

Stefan Bieber studied Pharmaceutical Bioprocessing Engineering at the Technical University of Munich, Germany. He received his PhD at the Chair of Urban Water Systems Engineering, where he investigated the occurrence of trace organic compounds in the aquatic environment and evaluated innovative separation techniques. Since 2018, he has been Director of AFIN-TS GmbH. His research focuses on the basics of SFC separations, aiming to achieve a better understanding of this technique and to improve the applicability of SFC.

Frank Steiner

Frank Steiner heads the marketing application lab in the HPLC organization of Thermo Fisher Scientific and serves as a Scientific Advisor for HPLC. In this function he coordinates scientific collaborations with external partners to advance UHPLC technologies and applications. Frank received his PhD degree in Chemistry in 1995 from Prof. Dr. Dr. Heinz Engelhardt at the Saarland University in Saarbruecken, Germany, working on the development of stationary phases for IC. He then became a postdoctoral research fellow at the CEA, Saclay in France focusing on elementary and isotopic analysis by IC and IC-ICP/MS in 1996. Frank returned to Saarland University in 1997 to conduct research on electro-driven separation (nonaqueous CE and CEC), LC purification, and MS coupling technologies and became an Assistant Professor in 2003. In 2005, Frank joined Dionex Softron GmbH in Germering, Germany, now a part of Thermo Fisher Scientific and held different roles in marketing as product manager, manager of LC hardware marketing, and manager of solutions marketing before he became a Scientific Advisor. Frank played a significant role in developing and launching the UltiMate 3000 UHPLC systems and solutions, as well as the new Vanquish UHPLC platform.

Oliver Genz

Oliver Genz studied Biology and Chemistry in Krefeld, Mainz and Freiburg (Germany). He worked for about 10 years at Pharmacia Biotech (today GE Healthcare) in sales, technical support, and the application lab and was responsible for running international training courses in theory and hands-on-training in analytical, preparative and process chromatography. After that he spent many years in sales, marketing, and technical support for chromatography instrumentation and stationary phases for preparative and process scale at YMC, Grace Davison (today GRACE) and Labomatic. He is the author of several publications related to preparative- and process-scale chromatography. Since 2000, he has been a freelance consultant for preparative- and process-scale chromatography and downstream processing with separation technologies.

Hans-Joachim Kuss

After studying Chemistry in Karlsruhe (Germany), he graduated in the field of Spectroscopy (PhD). He was engaged in HPLC, GC, and GCMS for 34 years at the University of Munich. Hans-Joachim has held some hundreds of courses on chromatography and implementation of weighted regression, prediction of gradients, and integration problems in EXCEL.

Egidijus Machtejevas

Egidijus studied Organic Chemistry and Biotechnology at Kaunas University of Technology, Lithuania. He completed his PhD in Analytical Chemistry (dissertation title “Design of chiral adsorbents and enantioseparations by means of HPLC”) in 2001. From 2001, he worked as a post-doc with Prof. Klaus Unger at Mainz University, Germany. He joined the R&D Department at Merck KGaA in Darmstadt, Germany in 2008 and worked on applications of silica monolithic columns. Currently, he is a global chromatography specialist. Egidijus Machtejevas has more than 20 scientific papers and ten book chapters to his name and his major research focuses include multidimensional liquid chromatography, proteomics, and the development of monolithic stationary phases for chromatography.

List of Contributors

Stefan Bieber

AFIN-TS GmbH

Am Mittleren Moos 48

86167 Augsburg

Germany

Oliver Genz

Bioprocess Chromatography

Consulting

In den Schliermatten 19

79219 Staufen im Breisgau

Germany

Hans-Joachim Kuss

Neubibergerstr. 54

85640 Putzbrunn

Germany

Stavros Kromidas

Breslauer Str. 3

66440 Blieskastel

Germany

Thomas Letzel

TU München

LS Siedlungswasserwirtschaft

Am Coulombwall 3

85748 Garching

Germany

Egidijus Machtejevas

Merck KGaA

Frankfurter Str. 250, D042/208

64293 Darmstadt

Germany

Markus Martin

Thermo Fisher Scientific

Dornierstraße 4

82110 Germering

Germany

Frank Steiner

Thermo Fisher Scientific

Dornierstr. 4

82110 Germering

Germany

Joachim Weiß

Thermo Fisher Scientific

Im Steingrund 4–6

63303 Dreieich

Germany

Gradient HPLC for Practitioners

RP, LC-MS, Ion Analytics, Biochromatography, SFC, HILIC

Preface

The Structure of the Book

Part 1: The Principles of Gradient Elution

Part 2: The Specifics of the Gradient in Different Separation Modes

Notes on Contributors

List of Contributors

Part 1
Principles of Gradient Elution