Gradient HPLC for Practitioners

Stavros Kromidas, PhD, works as an independent consultant for analytical chemistry, based in Saarbrucken (Germany). For more than 20 years he has regularly held lectures and training courses on HPLC, and has authored numerous articles and several books on various aspects of chromatography. He is the founder of NOVIA GmbH, a provider of professional training and consulting in analytical chemistry, and served as its CEO until 2001.

• Preface ixThe Structure of the Book xiNotes on Contributors xiiiList of Contributors xviiPart 1 Principles of Gradient Elution 11 Aspects of Gradient Optimization 3Stavros Kromidas (translated from German by Steve Ross)1.1 Introduction 31.2 Special Features of the Gradient 31.3 Some Chromatographic Definitions and Formulas 51.4 Detection Limit, Peak Capacity, Resolution – Possibilities for Gradient Optimization 81.4.1 Detection Limit 81.4.2 Peak Capacity and Resolution 91.5 Gradient “Myths” 141.6 Examples for the Optimization of Gradient Runs: Sufficient Resolution in an Adequate Time 151.7 Gradient Aphorisms 382 Instrumental Influences on the Quality and Performance of Gradient Methods and Their Transfer Between Different HPLC Devices 41Frank Steiner2.1 Technical Implementation of the Gradient Elution and the Respective Characteristics 412.1.1 Low-Pressure and High-Pressure Gradient Proportioning – Two Fundamentally Different Technical Principles 412.1.2 The Role of the Mixing Device in HPG and LPG Systems 422.1.3 The Operational Principle of Mixing Devices and the Systematic Characterization of Their Effectiveness 472.1.4 Effects of Volume Contraction when Mixing Water and Organic Solvents in Gradient Instruments 582.1.5 Effects of Minimum Leak Rates of Pump Heads in Sensitive Applications and HPG Synchronization Techniques to Correct Them 632.2 The Determination and Significance of the Gradient Delay Volume of the System 652.2.1 The Determination of the GDV and its Dependence on the Specific Operation Conditions of the System 662.2.2 The Influence of GDV on the Chromatographic Results 762.2.3 Possibilities of the User to Influence System GDV and its Impact on Chromatography 772.3 The Transfer of Gradient Methods Between Different HPLC Systems 802.3.1 Practical Tips for Dealing with Deviating GDVs and Possible Countermeasures 802.3.2 The Relevance of the Pressure Dependence of the GDV in Method Transfer 822.3.3 Effect of a too High Elution Strength of the Sample Solvent in the Presence of Weakly Eluting Solvent at the Gradient Start 852.4 Influence of Fluctuations of the Eluent Composition on the Quality of the Detection 872.4.1 Influence of a Reference Channel on the Baseline in Diode Array Detectors 882.4.2 The Special Challenge in Methods with UV-Absorbing Retained Additives in the Mobile Phase such as TFA 902.5 Other Kinds of Practical Application of Gradient Systems in HPLC 952.5.1 Alternative and Combined Gradient Modes in HPLC 962.5.2 Advantages in the Implementation of Isocratic Methods with Gradient Instruments 972.5.3 Use of Gradient Systems in Method Development and Method Optimization 983 Optimization of a Reversed-Phase Gradient Separation Using EXCEL 103Hans-Joachim KussPart 2 Specifics of the Gradient in Different Elution Modes 1114 Gradient Elution of Ionic Compounds 113Joachim Weiss4.1 Introduction 1134.2 Theoretical Aspects 1144.3 Gradient Types in Ion Chromatography 1164.4 Choice of Eluent 1194.4.1 Possibilities for Optimizing Concentration Gradients 1254.5 Gradient Elution of Anions on Anion Exchangers 1264.6 Gradient Elution of Cations on Cation Exchangers 1364.6.1 pH Gradients for the Separation of Monoclonal Antibodies 1444.7 Gradient Elution of Anions and Cations on Mixed-Mode Stationary Phases 1485 The Gradient in Biochromatography 161Oliver Genz5.1 Biomolecules 1615.2 Biochromatography 1615.3 The Gradient in Biochromatography 1625.3.1 A Gradient you Should Definitely Avoid ... 1635.4 Gradients for Different Biochromatographic Techniques 1645.4.1 Gel-Filtration, Size-Exclusion Chromatography (SEC) 1645.4.2 Ion Exchange Chromatography (IEX) 1645.4.3 Hydrophobic Interaction Chromatography (HIC) 1685.4.4 Reversed-Phase Chromatography of Biomolecules 1705.4.5 Affinity Chromatography (AC) 1715.5 Summary 1736 Specifications of Gradients in Hydrophilic Interaction Liquid Chromatography (HILIC) 175Thomas Letzel7 Specifications of Gradients in Supercritical Fluid Chromatography 183Stefan Bieber and Thomas Letzel7.1 Types of Gradients in SFC 1837.1.1 Mobile Phase Gradients 1837.1.2 Pressure Gradients 1847.1.3 Temperature Gradients 1857.2 Effects of gradients 1858 Aspects of Gradient Elution in LC-MS Analysis 189Markus M. Martin8.1 Role and Importance of Gradient Elution for LC-MS 1898.2 Technical Aspects of Gradient Elution in LC-MS Analysis 1928.2.1 Technical Impact of the LC System: System Dispersion, Gradient Proportioning Precision, and How they Affect MS Results 1928.2.2 Technical Impacts of a Mass Spectrometer: LC Gradients and Signal Generation in the MS 1988.2.3 Quantitation in Mass Spectrometry Within a Gradient Separation: Matrix Effects and How to Address Them 2048.2.4 MS Workload Balancing in Gradient Elution – Column Equilibration as a Throughput Bottleneck 2068.2.5 Gradient Delay, Flow Rate, and Column Dimension – How Far Can we Get With Downsizing of Gradient Separations in LC-MS? 2088.3 Summary 2118.4 Abbreviations 2119 Additional Tools for Method Development: Flow and Temperature Gradients 215Egidijus Machtejevas9.1 Introduction 2159.2 Temperature Gradients 2159.3 Flow Gradients 2169.4 Combination of Flow and Temperature Gradients 2179.5 Case Example 2179.6 Conclusions 219Index 223