Modern HPLC separations in theory and practice

Czech Chromatographic School

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Modern HPLC separations in theory and practice

Price: 155 € + VAT
840 Pages
ISBN 978-80-11-04472-5
Language: English
Published in June 2024 by Czech Chromatographic School
Authors: Lucie Nováková, Michal Douša, Petr Česla, Jiří Urban, and collective
Reviewers: Davy Guillarme, Deirdre Cabooter

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Book Content

1 HISTORY AND DEVELOPMENT OF LIQUID CHROMATOGRAPHY

1.1 FROM THE FIRST CHROMATOGRAPHIC SEPARATIONS TO HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

1.2 DEVELOPMENT OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY IN THE NEW MILLENNIUM

2 BASIC CONCEPTS OF CHROMATOGRAPHIC SEPARATION

2.1 DESCRIPTION OF RETENTION IN THE CHROMATOGRAPHIC SYSTEM

2.2 DESCRIPTION OF SELECTIVITY IN THE CHROMATOGRAPHIC SYSTEM

2.3 DESCRIPTION OF EFFICIENCY IN THE CHROMATOGRAPHIC SYSTEM

2.3.1 Shape of Chromatographic Peak
2.3.2 Description of the Symmetry of the Chromatographic Peak
2.3.3 Separation Efficiency of the Chromatographic System
2.3.4 Dynamic van Deemter Theory
2.3.5 Extra-Column Contributions

2.4 DESCRIPTION OF RESOLUTION

2.4.1 Chromatographic Resolution
2.4.2 Effect of Parameters in Resolution Equation on Chromatographic Separation

2.5 SEPARATIONS USING GRADIENT ELUTION

2.5.1 Chromatographic Separation Parameters in Gradient Elution
2.5.2 Practical Aspects and Benefits of Gradient Elution
2.5.3 Determining the Gradient Dwell Time

3 HPLC INSTRUMENTATION

3.1 BASIC DESCRIPTION OF A LIQUID CHROMATOGRAPH

3.2 MOBILE PHASE TRANSPORT

3.2.1 Mobile Phase Reservoirs
3.2.2 Mobile Phase Degassing
3.2.3 High-Pressure Pumps of Current HPLC Instrumentation
3.2.4 HPLC Pump Seal Wash
3.2.5 Creation of the Mobile Phase Gradient
3.2.6 Other Types of High-Pressure Pumps

3.3 SAMPLE INJECTION

3.3.1 Evolution of Methods for Sample Injection
3.3.2 Automatic Injection Devices - Autosamplers
3.3.3 Fixed-Loop Injection Systems
3.3.4 Flow-Through Needle Injection Systems
3.3.5 Autosampler Configurations
3.3.6 Sample Injection Containers - Vials

3.4 CHROMATOGRAPHIC COLUMNS FOR HPLC

3.4.1 Chromatographic Column Construction
3.4.2 Bioinert Column Hardware
3.4.3 Chromatographic Column Packing Characteristics
3.4.4 Column Thermostatting and Temperature Gradient

3.5 DETECTION IN HPLC

3.5.1 Detector Response
3.5.2 Noise and Drift
3.5.3 Spectrophotometric Detectors
3.5.4 Fluorescence Detectors
3.5.5 Chemiluminescence Detection
3.5.6 Electrochemical Detectors
3.5.7 Conductivity Detectors
3.5.8 Contactless Conductivity Detectors
3.5.9 Refractometric Detectors
3.5.10 Universal Aerosol Detectors
3.5.11 Mass Spectrometric Detection

3.6 DERIVATIZATION IN HPLC

3.6.1 Pre-Column Derivatization
3.6.2 Post-Column Derivatization

4 STATIONARY PHASES

4.1 PARAMETERS CHARACTERIZING STATIONARY PHASES

4.2 SILICA GEL

4.3 CHEMICALLY BONDED SILICA GEL BASED STATIONARY PHASES

4.3.1 Chemical Stability of Silica gel Stationary Phases
4.3.2 Stationary Phases with Chemically Bonded Alkyl Chains
4.3.3 Stationary Phases Modified with Aromatic Functional Groups
4.3.4 Fluorinated Stationary Phases
4.3.5 Brominated stationary phases
4.3.6 Chemically Bonded Stationary Phases with a Combined Ligand
4.3.7 Diol Stationary Phase
4.3.8 Stationary Phases with Aminopropyl and Cyanopropyl Groups
4.3.9 Free Silanol Groups and Related Issues
4.3.10 Silica Gel Stationary Phases for Separations with Aqueous Mobile Phases

4.4 METAL OXIDE-BASED STATIONARY PHASES

4.4.1 Aluminium Oxide
4.4.2 Zirconium Dioxide

4.5 STATIONARY PHASES BASED ON ORGANIC POLYMERS

4.6 STATIONARY PHASES FOR ION-EXCHANGE CHROMATOGRAPHY

4.6.1 Anion Exchangers
4.6.2 Cation Exchangers

4.7 HYBRID STATIONARY PHASES

4.7.1 Hybrid Stationary Phases with Ethylene Bridges
4.7.2 Hybrid Stationary Phases Prepared by Surface Modification
4.7.3 Hybrid Stationary Phases of Mixed-Mode Character

4.8 POROUS GRAPHITIC CARBON-BASED STATIONARY PHASES

4.9 MIXED-MODE STATIONARY PHASES

4.9.1 Bimodal Mixed-Mode Stationary Phases
4.9.2 Trimodal Stationary Phases
4.9.3 Hybrid Mixed-Mode Stationary Phases
4.9.4 Mixed-Mode Stationary Phases Based on Organic Polymer

5 CHROMATOGRAPHIC MODES

5.1 NORMAL-PHASE CHROMATOGRAPHY (NP-HPLC)

5.1.1 Principle of Retention in Normal-Phase Mode
5.1.2 Stationary Phases for Normal-Phase Chromatography
5.1.3 Mobile Phase and Eluotropic Series
5.1.4 Description of Retention in Normal-Phase Mode
5.1.5 Applications of Normal-Phase Chromatography

5.2 REVERSED-PHASE CHROMATOGRAPHY (RP-HPLC)

5.2.1 Principle of Retention in Reversed-Phase Mode
5.2.2 Stationary Phases in a Reversed-Phase Mode
5.2.3 Mobile Phases in Reversed-Phase Mode
5.2.4 Description of Retention in Reversed-Phase Mode
5.2.5 Controlling Retention in Reversed-Phase Mode
5.2.6 Non-Aqueous Reversed-Phase Mode
5.2.7 Applications of Reversed-Phase Chromatography

5.3 ION-EXCHANGE CHROMATOGRAPHY (IEC)

5.3.1 Principle of Retention in Ion-Exchange Chromatography
5.3.2 Stationary Phases for Ion-Exchange Chromatography
5.3.3 Mobile Phases in Ion-Exchange Chromatography
5.3.4 Applications of Ion-Exchange Chromatography

5.4 HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY (HILIC)

5.4.1 Principle of Retention in Hydrophilic Interaction Liquid Chromatography
5.4.2 Stationary Phases for Hydrophilic Interaction Chromatography
5.4.3 Mobile Phases in Hydrophilic Interaction Chromatography
5.4.4 Description of Retention in Hydrophilic Interaction Chromatography
5.4.5 Controlling Retention in Hydrophilic Interaction Chromatography
5.4.6 Sample Injection Solvent and Injection Volume in HILIC
5.4.7 Advantages and Drawbacks of the Hydrophilic Interaction Chromatography
5.4.8 Applications of Hydrophilic Interaction Chromatography

5.5 HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC)

5.5.1 Principle of Retention in Hydrophobic Interaction Chromatography
5.5.2 Stationary Phases for Hydrophobic Interaction Chromatography
5.5.3 Controlling Retention in Hydrophobic Interaction Chromatography
5.5.4 Applications of Hydrophobic Interaction Chromatography

5.6 MIXED-MODE CHROMATOGRAPHY

5.6.1 Principle of Retention in Mixed-Mode Chromatography
5.6.2 Stationary and Mobile Phases in Mixed-Mode Chromatography
5.6.3 Controlling Retention in Mixed-Mode Chromatography
5.6.4 Applications and Benefits of Mixed-Mode Chromatography

5.7 ION-PAIR CHROMATOGRAPHY

5.7.1 Principle of Retention Using Ion-Pair Chromatography
5.7.2 Applications of Ion-Pair Chromatography

5.8 AFFINITY CHROMATOGRAPHY

5.8.1 Principle of Retention in Affinity Chromatography
5.8.2 Description and Controlling Retention in Affinity Chromatography
5.8.3 Practical Implementation of Affinity Chromatography
5.8.4 Types of Support in Affinity Chromatography
5.8.5 Ligands in Affinity Chromatography
5.8.6 Applications of Affinity Chromatography

5.9 SIZE-EXCLUSION CHROMATOGRAPHY (SEC)

5.9.1 Principle of Elution in Size-Exclusion Chromatography
5.9.2 Stationary Phases in Size-Exclusion Chromatography
5.9.3 Mobile Phases in Size-Exclusion Chromatography
5.9.4 Applications of Size-Exclusion Chromatography

5.10 CHIRAL CHROMATOGRAPHY

5.10.1 Separation of Enantiomers
5.10.2 Principle of Chiral Separation
5.10.3 Types of Chiral Stationary Phases and Their Interactions
5.10.4 Separation Modes in Chiral Chromatography
5.10.5 Optimization of Chiral Separations
5.10.6 Polysaccharide Chiral Stationary Phases
5.10.7 Macrocyclic Antibiotics
5.10.8 Pirkle Chiral Stationary Phases
5.10.9 Cyclodextrin Chiral Stationary Phases
5.10.10 Chiral Stationary Phases Based on Cyclic Polyethers
5.10.11 Glycoprotein Chiral Stationary Phases
5.10.12 Chiral Separations Based on Ligand Exchange Reaction
5.10.13 Applications of Chiral Chromatography

6 CURRENT TRENDS IN HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

6.1 ULTRA-HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (UHPLC)

6.1.1 Separation Efficiency in Ultra-High-Performance Liquid Chromatography
6.1.2 Instrumentation in Ultra-High-Performance Liquid Chromatography
6.1.3 Stationary Phases for Ultra-High-Performance Liquid Chromatography
6.1.4 Method Transfer from HPLC to UHPLC
6.1.5 Applications and Benefits of Ultra-High-Performance Liquid Chromatography

6.2 POROUS SHELL PARTICLES

6.2.1 Characteristic of Porous Shell Particles
6.2.2 Separation Efficiency of Porous Shell Particles
6.2.3 Stationary Phases with Porous Shell Particles
6.2.4 Applications, Benefits, and Drawbacks of Porous Shell Particles

6.3 MONOLITHIC COLUMNS

6.3.1 Inorganic Monoliths Based on Silica Gel
6.3.2 Organic Polymer-Based Monoliths
6.3.3 Mixed-Mode Monoliths

6.4 HIGH-TEMPERATURE LIQUID CHROMATOGRAPHY (HTLC)

6.4.1 Description of Retention in High Temperature Liquid Chromatography
6.4.2 Separation Efficiency in High-Temperature Liquid Chromatography
6.4.3 Instrumentation in High-Temperature Liquid Chromatography
6.4.4 Applications and Benefits of High-Temperature Liquid Chromatography

6.5 SUPERCRITICAL FLUID CHROMATOGRAPHY

6.5.1 Supercritical Fluids as Mobile Phase
6.5.2 Separation Efficiency in Supercritical Fluid Chromatography
6.5.3 Instrumentation in Supercritical Fluid Chromatography
6.5.4 Stationary Phases in Supercritical Fluid Chromatography
6.5.5 Coupling Supercritical Fluid Chromatography with Mass Spectrometry Detection
6.5.6 Applications of Supercritical Fluid Chromatography

6.6 TWO-DIMENSIONAL LIQUID CHROMATOGRAPHY

6.6.1 Two-Dimensional Separation Concept
6.6.2 Experimental 2D LC Setup
6.6.3 Sample Characteristics and Selection of Separation Systems
6.6.4 Gradient Elution in Comprehensive 2D LC Analysis
6.6.5 Data Processing and Evaluation
6.6.6 Current Trends in 2D LC

6.7 MINIATURIZATION IN LIQUID CHROMATOGRAPHY

6.7.1 Micro- and Nano-Liquid Chromatography
6.7.2 Liquid Chromatography on Chip
6.7.3 3D Printing in Liquid Chromatography
6.7.4 Micro-Pillar Array Columns

7 MODERN HPLC PRACTICE

7.1 MOBILE PHASE PREPARATION AND USE

7.1.1 General Rules for the Mobile Phase Preparation
7.1.2 Mobile Phase Filtration
7.1.3 Mobile Phase Degassing
7.1.4 Mobile Phase Buffering

7.2 CHROMATOGRAPHIC COLUMN – CARE AND USE

7.2.1 General Rules of the Chromatographic Column Use
7.2.2 Installation of the Column and System Capillaries
7.2.3 Chromatographic Column Storage
7.2.4 Column Equilibration
7.2.5 Use of Guard Columns and Pre-column Filters
7.2.6 Column Regeneration

7.3 REGULAR MAINTENANCE OF HPLC SYSTEM

7.3.1 Mobile Phase Transport
7.3.2 HPLC Pump
7.3.3 HPLC Autosampler
7.3.4 Column Thermostat
7.3.5 HPLC Detectors

8 HPLC METHOD DEVELOPMENT AND OPTIMIZATION

8.1 PHYSICOCHEMICAL PROPERTIES OF THE ANALYTES AND METHOD OBJECTIVE

8.2 SELECTION OF DETECTION APPROACH

8.2.1 UV Detection conditions
8.2.2 Fluorescence Detection Conditions
8.2.3 Universal Aerosol Detection Conditions
8.2.4 Electrochemical Detection Conditions
8.2.5 Mass Spectrometry Detection Conditions

8.3 SELECTION OF CHROMATOGRAPHIC MODE

8.3.1 Low Molecular Weight Analytes
8.3.2 Sugars
8.3.3 Inorganic Ions – Analysis of Metals
8.3.4 Polymers
8.3.5 Peptides
8.3.6 Proteins
8.3.7 Nucleic Acids
8.3.8 Lipids

8.4 SELECTION OF STATIONARY AND MOBILE PHASES

8.4.1 Stationary Phases
8.4.2 Column Dimensions
8.4.3 Particle Size
8.4.4 Particle Pore Size
8.4.5 Mobile Phases and Elution Type

8.5 PRELIMINARY EXPERIMENTS

8.5.1 Selection of Initial Conditions and Optimization Strategy
8.5.2 Systematic Approach to Method Development

8.6 METHOD OPTIMIZATION

8.6.1 Optimization of the Mobile Phase pH in RP-HPLC
8.6.2 Gradient Program Optimization
8.6.3 Sample Injection Volume
8.6.4 Mobile Phase Flow Rate
8.6.5 Optimization of Separation Temperature
8.6.6 Calculation and Optimization of Analysis Time

8.7 SOLVING SEPARATION PROBLEMS IN METHOD DEVELOPMENT

8.8 SPECIFIC OPTIMIZATION APPROACHES

8.8.1 Sequential Methods
8.8.2 Simultaneous Methods
8.8.3 Chromatographic Optimization Software

9 SAMPLE PREPARATION FOR CHROMATOGRAPHIC ANALYSIS

9.1 CONVENTIONAL TECHNIQUES FOR SAMPLE PREPARATION

9.1.1 Direct Solvent Extraction
9.1.2 Protein Precipitation
9.1.3 Liquid-Liquid Extraction
9.1.4 Solid Phase Extraction
9.1.5 Additional Treatment of Extracts Obtained by Sample Preparation Methods

9.2 MODERN TRENDS IN SAMPLE PREPARATION TECHNIQUES

9.2.1 LLE-based Modern Sample Preparation Approaches: Microextractions
9.2.2 SPE-based Modern Sample Preparation Approaches: Microextractions
9.2.3 SPE-based Modern Sample Preparation Approaches: Automation
9.2.4 SPE-based Modern Sample Preparation Approaches: Selectivity
9.2.5 Miscellaneous Modern Sample Preparation Approaches

10 PREPARATIVE CHROMATOGRAPHY

10.1 PREPARATIVE HPLC

10.1.1 Instrumentation for Preparative HPLC
10.1.2 Development of Preparative HPLC Methods
10.1.3 Determination of Loading Capacity
10.1.4 Linear Scale-up
10.1.5 Operation Modes in Preparative HPLC for Higher Productivities
10.1.6 Displacement and Tag-along Effects
10.1.7 Multi-Column Preparative HPLC Systems

10.2 PREPARATIVE SFC

10.2.1 Multicolumn SFC systems

11 DATA ANALYSIS IN HPLC

11.1 QUALITATIVE ANALYSIS

11.2 QUANTITATIVE ANALYSIS

11.2.1 Calibration curve
11.2.2 External Standard Method
11.2.3 Internal Standard Method
11.2.4 Standard Addition Method
11.2.5 Internal Normalization Method

11.3 METABOLOMICS APPROACHES

12 METHOD VALIDATION IN HPLC

12.1 DEFINITIONS OF METROLOGICAL TERMS

12.2 VALIDATION PARAMETERS

12.3 PRACTICAL IMPLEMENTATION OF VALIDATION

12.3.1 Method Accuracy
12.3.2 Method Precision
12.3.3 Method Range and Linearity
12.3.4 Limit of Detection and Limit of Quantification
12.3.5 Method Selectivity
12.3.6 Method Robustness

12.4 PHARMACOPEIAL METHODS

12.5 SYSTEM SUITABILITY TEST

12.6 HPLC SYSTEM QUALIFICATION

13 HPLC TROUBLESHOOTING

13.1 BACK PRESSURE ISSUES

13.1.1 Increased System Pressure
13.1.2 Low Pressure
13.1.3 Unstable System Pressure

13.2 BASELINE ISSUES

13.2.1 Baseline Noise and Drift
13.2.2 Cyclic Fluctuating Baseline
13.2.3 Non-Cyclic Fluctuating Baseline

13.3 RETENTION TIME ISSUES

13.3.1 Fluctuating Retention Time
13.3.2 Increasing or Decreasing Retention Time
13.3.3 Change of the Retention Time by a Constant Value

13.4 PEAK SHAPE AND PEAK AREA PROBLEMS

13.4.1 Double Peak
13.4.2 Fronting Peak
13.4.3 Tailing Peak or Deformed Peak Shape
13.4.4 Negative peak
13.4.5 Flat-Top Peak
13.4.6 Decrease in Separation Efficiency
13.4.7 Peak Area Changes

13.5 CHROMATOGRAM PROBLEMS

13.5.1 More Peaks Than Expected
13.5.2 Fewer Peaks Than Expected
13.5.3 Uncommon Chromatogram Profile

14 LITERATURE REFERENCES

15 TABLES USEFUL IN HPLC DAILY PRACTICE

15.1 DYNAMIC VISCOSITY η OF ACETONITRILE/WATER AND METHANOL/WATER MIXTURES

15.2 MISCIBILITY OF ORGANIC SOLVENTS USED IN HPLC

15.3 ABSORPTION MAXIMA AND MOLAR EXTINCTION COEFFICIENTS

16 LIST OF ABBREVIATIONS

17 LIST OF SYMBOLS

CHROMATOGRAPHIC VENDORS’ PRESENTATION

Czech Chromatographic School: Modern HPLC separations in theory and practice - Book Partners

Authors’ short biographies

Lucie Nováková is a Full Professor at the Charles University, Faculty of Pharmacy in Hradec Králové, Department of Analytical Chemistry, the Czech Republic. Her research is focused on separation techniques, in liquid and supercritical phase, and their coupling to mass spectrometry. She is involved in a broad scope of research projects focused on pharmaceutical analysis, doping control, plant analysis, and bioanalytical methods. She extended her scientific experience during the fellowships at world-recognized universities, such as the University of Geneva and Vrije Universiteit Brussel, beyond others. She published over 155 peer-reviewed scientific articles with H-index of 41 and more than 5500 citations, one book, and nine book chapters. She is also widely involved in teaching and education activities, such as HPLC and SFC training courses, seminars, but also conference lectures and organization. Recently, she has become also a Vice-dean for External and International Relationships.

Czech Chromatographic School: Lucie Nováková (Charles University) and Michal Douša (Zentiva)

Michal Douša has been working in the pharmaceutical company Zentiva since 2007 as a head of the separation methods department. His research is focused on separation techniques, especially hydrophilic interaction liquid chromatography and chiral separation. He gained previous work experience at various workplaces in the field of HPLC analysis of water, soil, food,
and feed. He is the author of a book on HPLC theory and practice and one book chapter. He published over 65 peer-reviewed scientific articles withmore than 850 citations and H-index of 18. He is also involved in teaching and education activities, such as HPLC and validation training courses and seminars.

Petr Česla is an Associate professor of analytical chemistry, Head of the Department of analytical chemistry, and Vice-dean for Research and Creative Activities of the Faculty of Chemical Technology, University of Pardubice. In his research work, he focuses on the development of separation techniques in liquid phase, mainly liquid chromatography and capillary electrophoresis, with the special attention to the two-dimensional separations, optimization procedures, data processing, and coupling of LC and CE to mass spectrometry. He has been engaged in many research projects and acts as principal investigator of several projects of Czech Science Foundation. He is author of more than 60 papers in scientific journals with H-index of 18 and more than 1070 citations, co-author of one book and two book chapters.

Czech Chromatographic School: Petr Česla (University of Pardubice) and Jiří Urban (Masaryk University)

Jiří Urban received a Ph.D. in 2007 in the group of Pavel Jandera at the University of Pardubice, the Czech Republic, where he worked until 2016. During 2009 – 2011, he followed post-doctoral research at the University of California, Berkeley, USA, in the František Švec and Jean M. J. Fréchet group. In 2017 he moved to the Department of Chemistry, Masaryk University, Brno, Czech Republic, where he became an Associate Professor in 2018. In his research, he utilizes multifunctional polymer monoliths to develop new analytical methods applicable to metabolomics and proteomics. He also focuses on the design of new instrumental setups for two-dimensional liquid chromatography. He published over 51 peer-reviewed scientific articles with more than 1400 citations and H-index of 22.

Reviewers’ feedback

“It was an honor to serve as a reviewer for this comprehensive book on liquid chromatography, and I am grateful to the authors for the opportunity to contribute to such a valuable resource. The book provides an excellent balance of content for those new to the field and those with advanced expertise, making it a versatile and accessible guide for chromato- graphers at all levels. The clarity of the explanations, the quality of the figures (I cannot imagine the time it takes to draw all the figures contained within the book), coupled with the in-depth discussions, allow me to further expand my understanding of liquid chromatography in some points. This book, with its well-structured content and broad appeal, is sure to become a bestseller and make a significant contribution to the chromatography community. I am confident that readers, whether novice or experienced, will find immense value in its pages.”

Davy Guillarme

Czech Chromatographic School: Davy Guillarme (University of Geneva) and Deirdre Cabooter (KU Leuven)

“This book presents a very comprehensive overview of modern HPLC theory and practice that will be of great interest to anyone looking to immerse themselves into the technique. The book gives an overview of all aspects of liquid chromatography, from basic concepts of chromatography, covering all chromatographic modes, to novel trends in HPLC, such as 2D-LC and micro-pillar array columns, and a very useful chapter on troubleshooting. The many clear and informative illustrations make the book very practical and applicable. This is a must-have for anyone who is teaching or learning HPLC.”

Deirdre Cabooter