Method Development in Analytical HPLC

1st Edition - October 22, 2024
Imprint: Elsevier
Authors: Serban C. Moldoveanu, Victor David
Language: English
Paperback ISBN:
9 7 8 - 0 - 4 4 3 - 2 9 8 4 9 - 3
eBook ISBN:
9 7 8 - 0 - 4 4 3 - 2 9 8 5 0 - 9

Method Development in Analytical HPLC presents the essential information for understanding the process of developing an HPLC method of analysis. It includes foundational informati… Read more

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Method Development in Analytical HPLC presents the essential information for understanding the process of developing an HPLC method of analysis. It includes foundational information related to HPLC, as well as discussion of sample types, the properties of analytes and matrices in the samples, and sample preparation. The core of the book describes the best ways for approaching method development in various types of HPLC and the criteria for method optimization and validation.

This book provides clear guidance for adopting analytical methods from the literature and describes the development of original methods with selection of the suitable type of HPLC, of specific columns, mobile phase, and detection techniques with an emphasis on the use of mass spectrometry for detection, as well as optimization and validation of the chosen analytical method. The book includes useful details on method development for specific types of chromatography such as RP-HPLC, HILIC, ion exchange, size exclusion, and chiral.

Method Development in Analytical HPLC also includes information about green chemistry in analytical methods, computer assisted method development, and other key contemporary aspects of the subject.

Title of Book
Cover image
Title page
Table of Contents
Copyright
Preface
1. Introductory concepts related to a chemical analysis
1.1 Workflow in a chemical analysis
Key points
1.2 Basic information about sampling and preservation of samples
Types of sampling
Procedures for gas sampling
Procedures for sampling of liquids
Procedures of sampling for solids
Sampling of mixed-phases materials
Sampling of composite materials
Sampling of biological materials
On-line sampling coupled with HPLC analysis
Handling and storage of samples
Key points
1.3 Collection of information and planning for developing a method of analysis
Collection of information
Planning and collection of necessary resources
Collection of chemicals and certified reference materials
Key point
1.4 Short review of methods used as core analytical procedures
Wet chemistry
Optical spectroscopy based on light absorption
Optical spectroscopy based on light emission
Optical nonspectroscopic techniques
X-ray spectroscopy
Mass spectrometry
Electrochemical methods
Nuclear magnetic resonance
Radiochemical methods
Thermal methods of analysis
Other nonhyphenated analytical techniques
Chromatographic methods of analysis
Other methods of analysis which are including an independent separation step
Key points
1.5 Introductory information regarding HPLC
Qualitative analysis based on HPLC
Quantitative analysis based on HPLC
Key points
1.6 Decision to use an HPLC analytical method
Comparison of HPLC with several nonhyphenated analytical methods
Comparison of HPLC with other hyphenated analytical methods
The goals of developing an HPLC analytical method
Key points
1.7 Introduction to data processing in HPLC analysis
Average and standard deviation
Propagation of errors
Verification if the errors are random or systematic
Comparison of two measuring procedures regarding accuracy
Comparison of two procedures regarding precision
Least square regression
Elimination of suspect experimental data
Other mathematical procedures used in data processing
Key points
2. Retention and elution processes in high-performance liquid chromatography
2.1 Equilibrium types in HPLC
Partition equilibrium model
Adsorption equilibrium model
Equilibrium model involving ionic molecules
Equilibrium model in size exclusion processes
Other types of equilibrium models
Key points
2.2 Intermolecular interactions involved in HPLC separations
Ionic interactions or charge to charge interactions
Ion to dipole interactions
Dipole to dipole interactions
Ion to polarizable molecule interactions
Dipole to molecule interactions
Nonpolar molecule to molecule interactions
Total interaction between two molecules in the absence of ions
Lennard-Jones potential
Formation of hydrogen bonds
Halogen bonding (X-bonding)
Other types of interaction
Solvation energy of an ion
The effect of a solvent on molecular interactions
Solvophobic effects
Chaotropic and kosmotropic interactions
Key points
2.3 A classification of analytical HPLC types
A classification of HPLC types based on the nature of stationary and mobile phase
A classification of HPLC types based on the utilized detection
Key points
2.4 Influence of pH, ionic strength, and temperature on separation equilibria
The role of pH in separation equilibria
The role of ionic strength and of additives on separation equilibria
The role of temperature on separation equilibria
Key points
3. Instrumentation in high-performance liquid chromatography
3.1 Overall description of an HPLC instrument
Standard HPLC instrumentation
More complex HPLC instrumentation
Miniaturization in HPLC instrumentation
Key points
3.2 The separatory section of an HPLC instrument
Mobile phase supply system
Pumping systems
Tubing and connectors (fittings)
Injectors and autosamplers
Column holders
Chromatographic columns
Key points
3.3 Detectors in HPLC other than mass spectrometers
UV-Vis spectrometric detectors
Fluorescence detectors
Chemiluminescence detectors
Refractive index detectors
Electrochemical detectors
Evaporative light-scattering detectors
Other types of detectors
The use of multiple detectors
Key points
3.4 Mass spectrometers used as HPLC detectors
Ion source for mass spectrometers hyphenated with HPLC
Mass analyzers
Collision-induced dissociation cell and ion guide
Ion detection in MS
Types of mass spectrometric instruments
Ion mobility hyphenated with mass spectrometry
Utilization of mass spectrometric detection in HPLC
Key points
4. Performance and utilization of high-performance liquid chromatography separation and detection
4.1 Parameters related to the characterization of the chromatographic peak
Flow rate of the mobile phase
Retention time
Run time
Retention volume
Retention factor
Characteristics of an ideal peak shape
Efficiency of a chromatographic column
Peak asymmetry
Key points
4.2 Parameters related to peak separation in HPLC
Selectivity (separation factor)
Resolution
Peak capacity
Key points
4.3 Parameters for characterization of gradient separation in HPLC
Retention factor in RP-HPLC gradient separation
Peak compression in gradient elution
Key points
4.4 Characterization of detector performance in HPLC
Capability of a detector to provide quantitative and qualitative information
Selectivity
Sensitivity and the stability of sensitivity
Limit of detection and limit of quantitation
Dynamic range and linearity
Precision and reproducibility
Baseline noise, noise, and drift
Suitability to a given mobile phase, use of gradient, and flow rate of the mobile phase
Volume of measuring cell
Low contribution to peak broadening
Dependence of response to changes in the flow rate of mobile phase
Frequency of data collection
Backpressure accepted and the one generated by the detector
The capability to not alter the analyte
The case of more than one detector to select
4.5 Parameters related to quantitation in HPLC
Peak characteristics related to quantitation
Limitations for the sample volume and amount injected in the chromatographic column
Key points
4.6 Utilizations of HPLC in chemical analysis
Application of HPLC in qualitative analysis
Application of HPLC in quantitative analysis
Selection of the internal standards in quantitation using HPLC
Matrix effects on HPLC quantitation
Utilization of size exclusion HPLC for the evaluation of molecular weight of polymers
Key points
4.7 Utilization of HPLC in nontargeted type analysis
Application of nontargeted analysis
Challenges in NTA analysis
Key points
5. Samples in high-performance liquid chromatography analysis
5.1 Compounds characteristics relevant to HPLC separation
Organic or inorganic character
Molecular mass
Acidic, basic, or amphoteric character
Some geometric molecular properties
Isoelectric point
Polarity, dipole moment, and polarizability
Octanol/water partition coefficient
Capability of formation of hydrogen bonds
Diffusion coefficient
Other physical characteristics to consider for compounds in HPLC separations
General chemical structure of the organic compound and related properties
Key points
5.2 Characteristics of compounds relevant for detection in HPLC
UV spectra of the analyte
Fluorescence of the analyte
Chemiluminescence of molecules
Characteristics of molecules relevant in LC-MS detection
The role of compound structure in other detection types
Key points
5.3 Complexity of the injected sample
Sample complexity regarding the chemical nature of its constituents
Sample complexity regarding the number of compounds
Analytes level in the injected sample
Complexity of the matrix
Key points
5.4 Reference materials, commercial reagents, solvents, and additives
Key points
6. Sample preparation for high-performance liquid chromatography analysis
6.1 The role of sample preparation for HPLC
Initial sample processing and sample dissolution
Sample cleanup and sample fractionation
Concentration of analytes in the processed sample
Sample derivatization
Key points
6.2 Mechanical processing in sample preparation
Grinding and sieving
Weighing and volume measuring
Filtration
Centrifugation
Key points
6.3 Phase transfer techniques used in sample preparation
Vaporization, distillation, and drying
Physical dissolution
Crystallization and precipitation
Key points
6.4 Liquid–liquid extraction in sample preparation
Equilibrium in a simple liquid–liquid extraction
Equilibrium in liquid–liquid extraction involving multiple solute species
Properties of solvents used in liquid–liquid extraction
Types of liquid–liquid extraction
Separations using liquid–liquid extraction
Key points
6.5 Liquid–solid extraction in sample preparation
Extraction efficiency in liquid–solid extraction
Solvents used in liquid–solid extraction
Types of liquid–solid extraction
Key points
6.6 Solid-phase extraction in sample preparation for HPLC
Practice of solid-phase extraction
Materials used as SPE sorbent
Special types of SPE techniques
Key points
6.7 Sample derivatization
The role of derivatization in sample preparation for HPLC
Procedures for performing derivatization for liquid chromatography
Types of chemical reactions used in derivatization
Key points
6.8 Automated sample preparation and on-line coupling with the HPLC
Key points
7. Chromatographic columns in high-performance liquid chromatography
7.1 Construction of an HPLC column
Column physical construction
Physical characteristics of the solid support of stationary phase
Chemical characteristics of the solid support of stationary phase
The active component of the stationary phase
Summary of the main characteristics of columns used in HPLC
Key points
7.2 Columns with hydrophobic character
Stationary phases used in RP-HPLC and related techniques
Characterization of columns with hydrophobic stationary phase
Other properties and parameters characterizing hydrophobic stationary phases
Key points
7.3 Columns with polar character
Stationary phases used in HILIC and NPC
Characterization of columns with polar stationary phase
Key points
7.4 Chiral chromatographic columns
Chiral stationary phases
Characterization of chiral columns
Key points
7.5 Columns used in ion exchange chromatography and related techniques
Stationary phases used in ion exchange chromatography and related techniques
Characterization of columns with an ion exchange phase
Key points
7.6 Chromatographic columns used in size exclusion
Stationary phases and columns for SEC
Characterization of columns and stationary phases used in SEC
Key points
7.7 Other types of chromatographic columns
Key points
7.8 Care for the chromatographic column
Column protection
Column cleaning and storage
Key points
8. Mobile phase and the role of solvents in high-performance liquid chromatography
8.1 General aspects regarding mobile phase
Solvents used in HPLC
The use of isocratic or of gradient elution
Flow rate and temperature of the mobile phase
Buffers and additives used in HPLC
Key points
8.2 Properties of solvents relevant for the HPLC separation
General properties of solvents
Miscibility of solvents and solubility one in another
Parameters used for solvent characterization as eluents in HPLC
Key points
8.3 Properties of mobile phase relevant for detection in HPLC
Effects on refractive index detection
Effect on UV absorption detection
Effect on the detection using fluorescence
Solvent influence in MS detection
Solvent properties related to other detection techniques
Key points
8.4 Other uses of solvents in HPLC
The role of sample solvent in the chromatographic process
Solvents for the needle wash and other utilization of solvents
Key points
8.5 Buffers and additives used in HPLC
Buffers use in HPLC
The pH of buffers in partially aqueous media
Additives
Key points
8.6 Gradient use in HPLC
Gradient in solvent composition
Gradient of pH or of additive concentration
Gradient in flow rate or in temperature
Purpose of using gradient separations
Restrictions in using gradient separations
Key points
8.7 Efforts toward “green chemistry” in HPLC laboratory
Green solvents
Greenness evaluation for analytical procedures
Key points
9. Development of a high-performance liquid chromatography analytical method
9.1 General plan for adopting or developing an analytical method
Decision to use an HPLC method from the literature
Decision to develop a new HPLC method
Objectives in developing an HPLC method
Key points
9.2 Stages in developing a new HPLC analytical method
Definition of the analytical problem
Decision on HPLC type based on the nature of the sample
Decision on the type of detection to use with the method
Decision on using HPLC or UPLC and on the type of pumping system
Other decisions regarding the selection of an HPLC method
Key points
9.3 Development of an RP-HPLC analytical method
Selection of a chromatographic column in RP-HPLC
Solvents and general composition of the mobile phase in RP-HPLC
Selection of an internal standard
Isocratic separation in RP-HPLC
Gradient separation in RP-HPLC
The sample solvent in RP-HPLC method
The detection for an RP-HPLC method
Calibration for an RP-HPLC method
Application of RP-HPLC to the real samples
Key points
9.4 Development of an ion-pair HPLC analytical method
Factors affecting the retention process in ion-pair HPLC
Selection of stationary phase and column in ion-pair HPLC
Selection of IPA and mobile phase composition in ion-pair HPLC
Detection in ion-pair chromatography
Other chromatography types similar to ion-pair
Key points
9.5 Development of an analytical method on polar columns
Selection of a chromatographic column in HILIC and NPC
Selection of mobile phase in HILIC and NPC
Detection in HILIC and in NPC
Key points
9.6 Development of an HPLC analytical method for the separation of chiral compounds
Selection of a chromatographic column for a chiral separation
Selection of the mobile phase in chiral separations
Selection of detection in chiral separations
Separation on achiral stationary phases
Key points
9.7 Development of an analytical method for ion chromatography
Separation process in ion exchange chromatography
Selection of a column in ion exchange chromatography
Mobile phase in ion exchange chromatography
Detection in ion exchange chromatography
Method development in ion exclusion chromatography
Method development in ion-moderated and ligand exchange chromatography
Ion chelation chromatography
Key points
9.8 Development of an analytical method for size exclusion HPLC
Selection of a chromatographic column in SEC
Selection of mobile phase in SEC
Selection of detection in SEC
Interaction polymer chromatography
Key points
9.9 Development of a method for immunoaffinity HPLC
Stationary phase in immunoaffinity chromatography
Mobile phase and elution in immunoaffinity chromatography
Detection in immunoaffinity chromatography
Other techniques related to immunoaffinity HPLC
Key points
9.10 Development of a method for mixed-mode HPLC
Selection of a chromatographic column in MMC
Selection of the mobile phase in mixed-mode separations
Key points
9.11 Development of a bidimensional HPLC analytical method
Key points
9.12 Additional issues to consider in developing an HPLC analytical method
Key points
10. Selection of the best options in developing a high-performance liquid chromatography analytical method
10.1 Improvement and optimization processes in an HPLC method
Improvement process
Optimization process
Challenges and solutions in the improvement and optimization process
Key points
10.2 Trial-and-error in method development
Key points
10.3 Theoretical approaches used for prediction of various parameters in HPLC
Prediction of retention factor in RP-HPLC and in other types of HPLC
Prediction of other parameters in HPLC
Key points
10.4 Methodologies in HPLC optimization
Mathematical techniques used in optimization problems
Development and optimization using quality-by-design methodology
Key points
10.5 Computer-assisted method development in HPLC
Key points
10.6 Selection between different HPLC methods
Key points
11. Method and data validation in high-performance liquid chromatography analysis
11.1 An overview of HPLC method validation
Key points
11.2 Validation of an analytical method
Sample stability
Selectivity and specificity
Precision and accuracy of an analytical method
Range of calibration and calibration linearity of an analytical method
Limit of detection, limit of quantitation, and sensitivity
Robustness and ruggedness
Key points
11.3 Validation of the routine data
Formal data validation
Data evaluation requirements
Data presentation
Comparison of data generated by different methods
Special studies on sample stability
Validation of predicted data
Key points
11.4 Other aspects regarding validation
Simplicity
Analysis time
Number of analytes that can be analyzed together
Cost per analysis
Environmental impact
Safety in the laboratory
Key point
Appendix 1
Index

Life Sciences

Physical Sciences & Engineering

Social Sciences & Humanities

Health

Method Development in Analytical HPLC

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Serban C. Moldoveanu

Victor David

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