Multi-Dimensional Liquid Chromatography: Principles, Practice, and Applications

Cover
Half Title
Series Information
Title Page
Copyright Page
Table of Contents
Preface
Contributors
1 Introduction to Two-Dimensional Liquid Chromatography
	1.1 The Basic Concept of Multi-Dimensional Separation
	1.2 Scope of This Book
	1.3 Terminology
	1.4 Why Do We Need More Than One Dimension of Separation?
	1.5 Types and Modes of 2D-LC Separation
		1.5.1 Types of 2D Separation
			1.5.1.1 Serial Coupling of Columns Is Not Two-Dimensional Separation
			1.5.1.2 Offline vs. Online Separation
			1.5.1.3 2D-LC Separations With Discontinuous 1D Elution
		1.5.2 Modes of Online 2D-LC Separation
			1.5.2.1 Single Heartcut 2D-LC (LC-LC)
			1.5.2.2 Multiple Heartcut 2D-LC (mLC-LC)
			1.5.2.3 Selective Comprehensive 2D-LC (sLC×LC)
			1.5.2.1 Fully Comprehensive 2D-LC (LC×LC)
			1.6.1 2-For-1 Methods
	1.6 Scope of Application for 2D-LC in the Context of Analytical Chemistry
		1.6.2 Methods for Enhancing the Selectivity of 1D-LC
		1.6.3 Methods for Profiling Complex Materials
		1.6.4 Methods for Extraction of Chemical Information From Patterns in 2D Chromatograms
		1.6.5 Methods for Inherently Difficult Analyses
			1.6.5.1 Desalting And/or Solvent Exchange
			1.6.5.2 Separations of Analytes With a Large Range in Physicochemical Properties
			1.6.5.3 Separation of All the D-/L- Enantiomers of a Large Number of Amino Acids
	1.7 History of 2D-LC Development
		1.7.1 1970s–1980s: Introduction of Conceptual Frameworks for Multi-Dimensional Separations, and Instrumentation for …
		1.7.2 1990s: Automated LC×LC, and the Rise of LC×LC in Proteomics
		1.7.3 2000s: Development of a Solid Theoretical Foundation
		1.7.4 2010s: Reduction to Practice
	1.8 Opportunities and Challenges
		1.8.1 Challenges
		1.8.2 Opportunities
	1.9 Method Development Goals for 2D-LC
	1.10 Data Analysis
	1.11 Coupling LC With Other Separation Modes in a 2D Format
		1.11.1 Coupling With Capillary Electrophoresis
		1.11.2 Coupling With Supercritical Fluid Chromatography
		1.11.3 Coupling With Gas Chromatography
	References
2 Speed and Performance in Liquid Chromatography
	2.1 Introduction
	2.2 Overview of Aspects of Performance Optimization
	2.3 Impact of Column and Instrument Technologies On Separation Performance
		2.3.1 Smaller Particles and Higher Pressures
		2.3.2 Higher Temperatures
		2.3.3 Superficially Porous Particles (SPPs)
		2.3.4 Monolithic Columns
		2.3.5 Non-Porous Particles
	2.4 Approaches to Optimizing Speed and Performance
		2.4.1 One-Parameter Optimization
		2.4.2 Two-Parameter (Poppe) Optimization
			2.4.2.1 Introduction to Poppe Plots
			2.4.2.2 An Alternative Way to Compute Poppe Plots
			2.4.2.3 Long-Time and Short-Time Limits of LC as Revealed By the Poppe Plot
		2.4.3 Impact of System Parameters On Poppe Plots, the Speed and Limiting Plate Count in LC
			2.4.3.1 Impact of High Pressure On Speed of LC
			2.4.3.2 Impact of Particle Size On the Speed of LC
			2.4.3.3 Impact of Column Temperature On Speed of LC
			2.4.3.4 Effect of Column Parameters On Speed of LC
			2.4.3.5 Impact of Analyte Diffusion Coefficient On Speed of LC
			2.4.3.6 Limitations of Poppe Plots
		2.4.4 Three-Parameter (Knox-Saleem-Halasz) Optimization
			2.4.4.1 Implications of the Three-Parameter (K-S-H) Optimization
			2.4.4.2 Comparison of Pressure and Temperature Effects On Speed Based On Knox-Saleem-Halasz Optimization
	2.5 The Kinetic Plot – A Variant of the Poppe Plot
	2.6 Analysis Time and Resolution
		2.6.1 Optimum Retention Factor and Speed
	2.7 Comparison of Speed and Performance Predicted By Two-And Three-Parameter Optimization Methods
	2.8 Introduction to Peak Capacity
		2.8.1 Limitations of the Peak Capacity Concept
	2.9 Gradient Elution Reversed-Phase Liquid Chromatography
	2.10 Speed in Liquid Chromatography and Optimization of Peak Capacity
	2.11 Effect of the Gradient Compression Factor On the Peak Capacity
	2.12 Summary
	Appendix
	A.1 Relationships Between Plate Height and Mobile Phase Velocity
	A.2 Details Related to the “A” Term
	A.3 Details Related to the “B” Term
	A.4 Details Related to the “C” Terms
	A.5 Relationships Related to Mobile Phase Flow and Pressure Drop Across the Column
	A.6 COLUMN SELF-HEATING Due to Viscous Flow and Other Thermal Effects
	A.7 Estimation of Molecular Diffusion Coefficients in Liquids
	References
3 Theoretical Guiding Principles for Two-Dimensional Liquid Chromatography
	3.1 Introduction
	3.2 Two-Dimensional Peak Capacity
	3.3 Separation Orthogonality and Complementarity
	3.4 Undersampling
	3.5 Optimization of Two-Dimensional Peak Capacity
	3.6 Detection Sensitivity
	References
4 Instrumentation for Two-Dimensional Liquid Chromatography
	4.1 Challenges Unique to 2D-LC Separations
		4.1.1 Each 2D Separation Involves Many 2D Separations
		4.1.2 Requirement for Fast 2D Separations
		4.1.3 Injection Process
		4.1.4 Pressure Spiking at 1D Detector
	4.2 2D-LC System Control
		4.2.1 Native Software vs. Master Controller
		4.2.2 Precision of Module Control
	4.3 Pumps
		4.3.1 Gradient Delay Volume
		4.3.2 Pressure and Flow Rate Ranges
	4.4 Interface and Modulation
		4.4.1 Dual Loop Interfaces
			4.4.1.1 8-Port/2-Position Valves
			4.4.1.2 10-Port/2-Position Valves
			4.4.1.3 Dual 6-Port/2-Position Valves
		4.4.2 Multi-Loop Interfaces
			4.4.2.1 Multi-Loop Sampling With Serial 2D Analysis
			4.4.2.2 Multi-Loop Sampling With Multiple 2D Column Chemistries
			4.4.2.3 Multi-Loop Sampling With Parallel 2D Analysis, and Selective Comprehensive 2D Separation
		4.4.3 Other Interfaces
		4.4.4 Challenges Associated With Mobile Phase Mismatch in Multi-Dimensional Separations
		4.4.5 Interfaces for Solvent-Based Mitigation of Mobile Phase Mismatch
			4.4.5.1 Inline Dilution
			4.4.5.2 Fixed Solvent Modulation
			4.4.5.3 Active Solvent Modulation
			4.4.5.4 Inline Dilution Through Use of a Transfer Pump
			4.4.5.5 Implementation of Solvent-Based Modulation Approaches
		4.4.6 Interfaces for Sorbent-Based Modulation
			4.4.6.1 Implementation of Sorbent-Based Modulation Approaches
		4.4.7 Interfaces for Evaporation-Based Modulation
		4.4.8 Interfaces for Temperature-Based Modulation
		4.4.9 Approaches Based On Splitting the 1D Flow Prior to Modulation
		4.4.10 Approaches Based On Discontinuous 1D Elution
			4.4.10.1 Stop-Flow
			4.4.10.2 Pulsed-Elution
		4.4.11 LC – Transformation – LC
		4.4.12 Injection Loop Matching
		4.4.13 Injection Loop Filling
			4.4.13.1 Analyte Losses Due to Breakthrough
			4.4.13.2 First-In-First-Out vs. First-In-Last-Out Modes of Operation
			4.4.13.3 Partial Filling
		4.4.14 Use of Parallel 2D Columns
	4.5 Second Dimension Elution Schemes
	4.6 Extra-Column Peak Dispersion
		4.6.1 Dispersion Due to Injection Into the 2D Column
		4.6.2 Fast 2D Separations Produce Narrow Peaks
		4.6.3 Turbulent Flow in 2D Connecting Capillaries
	4.7 Detection
		4.7.1 Post-Column Flow Splitting
	4.8 Closing Remarks
	References
5 Selecting Separation Modes and Selectivities for Multi-Dimensional LC
	5.1 Introduction to the Thought Process of Choosing Separation Modes and Selectivities
		5.1.1 The Concept of Sample Dimensionality
		5.1.2 Potential for Mixed-Mode And/or Unintended Interactions
		5.1.2 Suppression of Retention Mechanisms
	5.2 Combining Selectivities
		5.2.1 Normal-Phase Liquid Chromatography
			5.2.1.1 Argentation (Silver-Ion) Normal-Phase Chromatography
			5.2.1.2 Hydrophilic-Interaction Liquid Chromatography (HILIC)
		5.2.2 Ion-Exchange Chromatography
		5.2.3 Size-Exclusion Chromatography and Hydrodynamic Chromatography
		5.2.4 Hydrophobic Interaction Chromatography
		5.2.5 Chiral Chromatography
		5.2.6 Affinity Chromatography
		5.2.7 Supercritical-Fluid Chromatography
	5.3 Choosing Reversed-Phase Selectivities
		5.3.1 Selecting an RP Column for One Dimension of a 2D-LC Separation
		5.3.2 Selecting RP Columns for Both Dimensions of a 2D-LC Separation
	5.4 Closing Remarks
	References
6 Method Development for Non-Comprehensive Two-Dimensional Liquid Chromatography
	6.1 Introduction
	6.2 General Thoughts On Method Development for Non-Comprehensive 2D-LC
	6.3 Focus of This Chapter
	6.4 Experimental Details
		6.4.1 First Dimension
		6.4.2 Second Dimension
		6.4.3 Multiple Heartcutting 2D-LC Interface
		6.4.4 Mass Spectrometry
	6.5 Influence of the Mobile Phase On Selectivity
		6.5.1 Solvent Types
		6.5.2 Temperature
		6.5.3 PH
	6.6 Influence of the Stationary Phase On Selectivity
	6.7 Optimizing Gradient Elution Conditions for the 2D Separation
	6.8 Active Solvent Modulation
		6.8.1 Choosing the Right ASM-f
		6.8.2 Using the Right Loop Size
	6.9 Using a Diverter Valve in Combination With MS
	6.10 Quantification in 2D-LC
		6.10.1 Precision
		6.10.2 Linearity
		6.10.3 Recovery
	6.11 Closing Remarks
	References
7 Method Development for Comprehensive Two-Dimensional Liquid Chromatography Separations
	7.1 Introductory Remarks
	7.2 A Generic Approach to Method Development
		7.2.1 Phase I – Sample Preparation, Sample Dimensionality, and Separation Modes
		7.2.2 Phase II – Physical Parameters
			7.2.2.1 Prioritizing Peak Capacity
			7.2.2.2 Prioritizing Detection Sensitivity
			7.2.2.3 Prioritizing Retention Repeatability
			7.2.2.4 Isocratic vs. Gradient Elution in the Second Dimension
		7.2.3 Phase III – Optimization
	7.3 Case Study #1 – Method Development for Small Molecule Dyes
		7.3.1 Phase I – Sample Preparation, Sample Dimensionality, and Separation Modes
			7.3.1.1 Sample Dimensionality
			7.3.1.2 Column Selection
			7.3.1.3 Analysis Time and Detection
		7.3.2 Phase II – Physical Parameters
		7.3.3 Phase III
	7.4 Case Study #2 – Method Development for Tryptic Peptides
		7.4.1 Phase I – Sample Preparation, Sample Dimensionality, and Separation Modes
		7.4.2 Phase II – Physical Parameters
			7.4.2.1 1D Parameters
			7.4.2.2 2D Parameters
			7.4.2.3 Modulation Parameters
		7.4.3 Phase III – Optimization
	7.5 Optimization
		7.5.1 Deciding Whether Or Not to Continue With Optimization
		7.5.2 Optimizing Stationary Phase Selection
		7.5.3 Optimization of Elution Conditions Using Retention Modeling
	7.6 Closing Remarks
	References
8 Data Analysis for Multi-Dimensional Liquid Chromatography
	8.1 Introduction
	8.2 Data Reformatting and Visualization
		8.2.1 Data Reformatting
		8.2.2 Visualization
		8.2.3 Correction for Wrap-Around
	8.3 Data Preprocessing – Goals and Techniques
		8.3.1 Noise Reduction
		8.3.2 Baseline Drift and Comprehensive Background Correction Approaches
			8.3.2.1 Penalized Least-Squares Approaches
			8.3.2.2 Local Minimum Values (LMV)
			8.3.2.3 Baseline Estimation and Denoising Using Sparsity (BEADS)
			8.3.2.4 Bayesian Approaches
			8.3.2.5 Background Correction Using Profile Spectra From Multi-Channel Detectors
			8.3.2.6 Dedicated Approaches Relevant to Comprehensive Two-Dimensional Chromatography
	8.4 Retention-Time Alignment
		8.4.1 Introduction
		8.4.2 Correlation-Optimized Warping
		8.4.3 Automatic Time-Shift Alignment
		8.4.4 Alignment Using Mass Spectra
	8.5 Peak Detection
		8.5.1 Classical Peak Detection
		8.5.2 Continuous Wavelet Transformation (CWT)
		8.5.3 Automatic Peak Detection and Background Drift Correction
		8.5.4 Comprehensive Two-Dimensional Approaches
			8.5.4.1 Two-Step Peak Detection Using Peak Clustering
			8.5.4.2 Watershed Algorithm
	8.6 Multi-Way Approaches
		8.6.1 Multivariate Curve Resolution-Alternating Least Squares
		8.6.2 PARAFAC and PARAFAC2
	8.7 Classification
	8.8 Summary
	References
9 Applications of Two-Dimensional Liquid Chromatography for Analysis of Synthetic Pharmaceutical Materials
	9.1 Preface
	9.2 Introduction
	9.3 Overview of Challenges Encountered in Pharmaceutical Analysis
	9.4 Coupling Non-MS Compatible LC Separations to MS (Figure 9.1A)
	9.5 Chiral Separation (Figure 9.1B)
		9.5.1 Achiral-Chiral 2D-LC to Separate Achiral Interferences From Chiral Compounds
		9.5.2 Compounds With Multiple Chiral Centers
		9.5.3 Integrated Method Development and Sample Analysis
	9.6 Peak Coelution, Peak Purity, and Stability Indicating Method Assessment (Figure 9.1C)
		9.6.1 Use of 2D-LC for Characterization of Synthetic Therapeutic Peptides
		9.6.2 Use of 2D-LC for Characterization of Therapeutic Oligonucleotides
		9.6.3 Other Examples of Different 2D-LC Modes Used to Resolve Coelution for Pharmaceutical Materials
		9.6.4 Column Screening in the Second Dimension to Identify Complementary Stationary Phases
		9.6.5 Use of Multiple Combinations of Separation Mechanisms to Solve Difficult Coelution Problems
	9.7 Resolving Sample Matrix Effects Using 2D-LC (Figure 9.1D)
		9.7.1 Drug Product Matrix Interference Removal
		9.7.2 Trace Analysis
	9.8 High Throughput Analysis
	9.9 Concluding Remarks and Outlook
	References
10 Application of Two-Dimensional Liquid Chromatography to Analysis of Biologics
	10.1 Introduction to Characterization of Biologics
	10.2 Characterization of Therapeutic Monoclonal Antibodies and Related Molecules
		10.2.1 Intact and Subunit Level
			10.2.1.1 Determination of Titer and Aggregation
			10.2.1.2 Determination of Charge Variants and Aggregation
			10.2.1.3 Determination of Charge Variants and Subunits
			10.2.1.4 Determination of Glycoforms and Subunits
			10.2.1.5 Measuring Bispecific Antibody Mispairing
		10.2.2 Peptide Level
		10.2.3 Online Digestion of MAbs
		10.2.4 Released Glycan and Glycopeptide Levels
	10.3 Characterization of Host-Cell Proteins
	10.4 Characterization of Antibody-Drug Conjugates (ADCs)
	10.5 Characterization of Excipients
	10.6 Closing Remarks
	References
11 Applications of Two-Dimensional Liquid Chromatography to Chemical Analysis in Academic Research and Industry
	11.1 2D-LC for Chemical Analysis
	11.2 Target Analysis – Small Molecules in Complex Matrices
	11.3 Characterization of Oligomeric/Isomeric Compounds: Surfactants, Lubricants, Oils, Chemical Process Intermediates, Lignin, Wastewater
	11.4 2D-LC for Polymer Analysis
		11.4.1 Techniques for Analysis of Synthetic Polymers
		11.4.2 Copolymer Analysis By 2D-LC
		11.4.3 Complex Polymer Mixtures
		11.4.4 High-Temperature and Temperature Gradient 2D-LC Methods
		11.4.5 Other Polymer Applications Involving Unconventional Combinations of Separation Mechanisms
	11.5 Outlook
	References
12 Applications of Two-Dimensional Liquid Chromatography to Natural Product and Food Analysis
	12.1 Introduction
	12.2 Major Food and Natural Product Components
		12.2.1 Carbohydrates
		12.2.2 Lipids
		12.2.3 Proteins
	12.3 Minor Food and Natural Product Components
		12.3.1 Polyphenols
			12.3.1.2 HILIC×RP Separations
			12.3.1.3 Other
		12.3.2 Saponins
		12.3.3 Carotenoids
		12.3.4 Miscellaneous
	12.4 Food Additives and Contaminants
	12.5 Conclusions and Future Outlook
	Notes
	References
13 Application of Two-Dimensional Liquid Chromatography to the Analysis of Chiral and Structurally Similar Molecules
	13.1 Introduction
	13.2 Enantioselective LC-LC
		13.2.1 Single Heartcut Achiral-Chiral 2D-LC
		13.2.2 Multiple Heartcut Achiral-Chiral 2D-LC for Metabolism Studies
		13.2.3 Multiple Heartcut Achiral-Chiral 2D-LC for Enantioselective Amino Acid Analysis
		13.2.4 Multiple Heartcut 3D-LC for Trace-Level Enantioselective Amino Acid Quantitation in Biological Samples
		13.2.5 Selective Comprehensive Achiral-Chiral 2D-LC for Enantioselective Analysis of All Proteinogenic Amino Acids
		13.2.6 Heartcut Chiral-Achiral and Chiral-Chiral 2D-LC
	13.3 Enantioselective LC×LC
		13.3.1 Chiral×Achiral 2D-LC
		13.3.2 Chiral×chiral 2D-LC
	13.4 Online Multi-Column 2D-LC Platform Technologies
	13.5 Enantioselective 2D Chromatography By LC-SFC
	13.6 Enantioselective 2D Chromatography By Microchip LC-LC
	13.7 Other Multi-Dimensional Isomer Separations With Chiral Columns
	13.8 Concluding Remarks
	Acknowledgments
	References
Glossary
Index