Essentials in Modern HPLC Separations

Essentials in Modern HPLC Separations

2nd Edition - June 1, 2022

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  • Authors: Serban Moldoveanu, Victor David
  • Paperback ISBN: 9780323911771

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Essentials in Modern HPLC Separations, Second Edition discusses the role of separation in high performance liquid chromatography (HPLC). This new and updated edition systematically presents basic concepts as well as new developments in HPLC. Starting with a description of basic concepts, it provides important guidance for the practical utilization of various HPLC procedures, such as the selection of the HPLC type, proper choice of the chromatographic column, selection of mobile phase and selection of the method of detection, all of which are in correlation with the physico-chemical characteristics of the compounds separated. Every chapter has been carefully reviewed, with several new sections added to bring the book completely up-to-date. Hence, it is a valuable reference for students and professors in chemistry.

Key Features

  • Provides a thoroughly updated resource, with an entirely new section on Computer-aided Method Development in HPLC and new subsections on miniaturization and automation in HPLC, chemometric aspects of HPLC, green solvent use in HPLC, and more
  • Includes insights into the chromatographic process to find the optimum solution for analyzing complex samples
  • Presents a basis for understanding the utilization of modern HPLC for applications, particularly for the analysis of pharmaceutical, biological, food, beverage and environmental samples


Chemists of a wide range of levels of specialization, from industry, research and academic sectors; students and professors in PhD and Masters programs in chemistry

Table of Contents

  • Part 1. Basic information about HPLC
    1. Introductory information regarding HPLC
    1.1. Preliminary discussion about HPLC
    General comments
    What is chromatography and what is HPLC?
    Types of equilibria in HPLC
    Role of polarity in HPLC
    Applications of HPLC in chemical analysis
    Non-analytical applications of analytical HPLC
    1.2. Main types of HPLC
    Criteria for the classification of HPLC procedures
    A classification of HPLC types based on the nature of stationary and mobile phase
    Relation between the type of HPLC, equilibrium type, and molecular interactions
    1.3. Flow of a typical HPLC analysis
    General aspects
    Selection of the type of HPLC for a particular application
    Sample collection and sample preparation for HPLC

    2. Overview of HPLC instrumentation and its use
    2.1. Description of main components of HPLC instrumentation
    General comments
    Description of a typical HPLC instrument
    Solvent supply system
    Pumping systems
    Tubing and connectors
    Injectors and autosamplers
    Column holders
    Chromatographic columns
    General comments regarding detectors
    UV-Vis spectrometric detectors
    Fluorescence and chemiluminescence detectors
    Refractive index detectors
    Electrochemical detectors
    Mass spectrometric detectors
    Evaporative light-scattering (ELS) detectors
    Other types of detectors
    Selection of a detector in HPLC
    Other devices that can be part of the HPLC system
    More complex or special HPLC setups
    Instrument control and data processing unit
    Selection of the HPLC system and transition from HPLC to UPLC

    3. Parameters for the characterization of HPLC separation
    3.1. Parameters describing the chromatographic peak
    General aspects
    Flow rate of the mobile phase
    Retention time
    Run time
    Retention volume
    Migration rate
    Equilibrium constant and phase ratio in HPLC separations
    Retention factor
    General equation of solute retention
    Characteristics of an ideal peak shape in chromatography
    Efficiency of a chromatographic column
    Factors contributing to peak broadening and van Deemter equation
    Application of van Deemter equation
    Peak asymmetry
    Statistical moments for the description of peak characteristics
    Peak characterization using exponentially modified Gaussian shape
    3.2. Parameters describing the separation
    General aspects
    Selectivity (separation factor)
    Peak capacity
    3.3. Summary of chromatographic peak and separation characteristics
    General comments
    3.4. Parameters related to quantitation in HPLC
    General comments
    Quantitation parameters from the peak shape
    Sample volume and amount injected in the chromatographic column
    Limit of detection in HPLC
    Limit of quantitation
    3.5. Parameters characterizing the gradient separation
    General comments
    Retention factor in gradient separations
    Other parameters for the characterization of chromatograms in gradient separations

    4. Equilibrium types in HPLC
    4.1 Partition equilibrium
    General comments
    Liquid-liquid partition
    Dependence of retention factor on mobile phase composition in partition chromatography
    Distribution coefficient
    Peak shape in partition chromatography
    Evaluation of retention factor from liquid-liquid distribution constants
    4.2 Adsorption equilibrium
    Liquid-solid equilibrium
    Dependence of retention factor on mobile phase composition in adsorption equilibrium
    Peak shape in adsorption chromatography
    4.3 Equilibrium involving ions
    General aspects
    Retention equilibrium involving ions
    Equilibrium of ions in the presence of a complexing reagent
    4.4 Equilibrium in size exclusion processes
    General aspects
    Equilibrium between interstitial mobile phase and pore mobile phase
    4.5 The influence of pH and of additives on retention equilibria
    Preliminary information about pH
    Dependence of compound structure on pH
    The influence of pH on partition equilibrium
    Dependence on pH of octanol/water distribution coefficient Dow
    Influence on separation of additives not involved in the equilibrium
    Chaotropic salts influence on equilibria
    4.6 The influence of temperature on retention equilibrium
    General aspects
    Evaluation of thermodynamic parameters of a separation from van’t Hoff plots
    Non-linear dependence of the retention factor on 1/T
    Evaluation of enthalpy-entropy compensation from van’t Hoff plots
    High temperature HPLC
    4.7 Reactions occurring in HPLC column
    General comments
    Enantiomerization and diastereomerisation

    5. Intermolecular forces
    5.1 Forces between molecules
    General comments
    Charge to charge interactions
    Energy of an ion in a continuous medium
    Polar molecules
    Ion to dipole interactions
    Dipole to dipole interactions
    Polarizability of molecules
    Ion to molecule interactions
    Dipole to molecule interactions
    Non-polar molecule to molecule interactions
    Unified view of interactions in absence of ions
    Lenard-Jones potential
    Hydrogen bond interactions
    Charge transfer or donor acceptor interactions
    Stacking and inclusion in supermolecular systems
    Other types of bonds
    The effect of a solvent on molecular interactions
    Solvophobic effects
    Chaotropic and kosmotropic interactions
    5.2 Forces between molecules and a charged surface
    General comments
    Charge to charged-surface interactions
    Neutral molecule to charged-surface interactions

    6. Characterization of analytes and matrices
    6.1. Properties of solutes important for HPLC separation
    Solutes classification based on their chemical structure
    Classification based on the role of the analyte in everyday life
    Comments on physico-chemical properties of analytes and matrices
    Molecular weight
    Acidic or basic character of analytes
    Van der Waals molecular volume and area
    Molecular shape
    Molar volume
    Molecular polarity
    Partial charge distribution
    Isoelectric point
    Octanol/water partition constant and its use for polarity estimation
    Thermodynamic parameters related to solubility of non-electrolyte compounds
    Activity coefficient from enthalpy of mixing
    Solubility in water from octanol/water partition constant
    Correlation between van der Waals molecular surface and octanol/water partition constant
    Solvatochromic parameters for solute characterization
    Other parameters for solute characterization
    6.2. Physico-chemical properties related to detection
    General comments
    Gas-phase acidity and basicity in MS process of ion formation
    The role of analyte polarity in MS detection
    6.3. Properties of matrix related to HPLC separation and detection
    General comments
    Matrix effects on the separation in HPLC
    Matrix effects on HPLC detection

    7. Mobile phases and their properties
    7.1. Characterization of liquids as eluents in HPLC
    General comments
    Characterization of solvents with Hildebrand solubility parameter
    Miscibility of solvents and solubility one in another
    Solvent characterization using octanol/water partition constant Kow
    Solvent characterization based on liquid-gas partition
    Solvatochromic model and Kamlet-Taft parameters
    Elutropic strength
    Solvent characterization based on other parameters
    Solvent properties in liquid mixtures
    7.2 Additional physical properties of liquids affecting separation
    General comments
    Solvent density, viscosity, and diffusion coefficient
    Surface tension
    Dielectric constant, dipole moment, and polarizability
    Hydrogen bonding of solvent molecules
    Solvent boiling point
    7.3 Properties of liquids affecting HPLC detection
    General comments
    Refractive index
    UV cut-off
    Solvent influence in MS detection
    Solvent properties related to other detection techniques
    7.4. Buffers and additives
    General comments
    Buffer pH
    Buffer capacity
    Common buffers used in HPLC
    Buffers in partially aqueous solvent mixtures
    The influence of temperature on the pH of buffers
    Solubility of buffers in partially organic mobile phases
    Influence of buffers and additives on column stability and properties
    Suitability of buffers and additives for the detection in HPLC
    7.5. General use of solvents as mobile phase
    General comments
    Solvent purity in HPLC
    Flow rate, temperature, and degassing of mobile phase
    The use of "green" solvents as mobile phase in HPLC
    7.6. Solvents for sample injection and for needle wash
    General comments
    The role of sample solvent in the chromatographic process
    Effect of sample solvent on detection
    Solvents for the needle wash
    7.7. Gradient elution
    General comments
    Gradient of solvent composition
    Gradient of pH or of additive concentration
    Gradient in flow rate
    Gradient in separation temperature
    Usefulness of gradient versus isocratic elution

    8. Analytical HPLC columns and their characteristics
    8.1. Construction of an HPLC column
    General comments
    External body of the column
    Packing of particles in the chromatographic column
    Physical characteristics of the solid supports for the packed columns
    Chemical characteristics of the solid supports for the packed columns
    Silica and ethyl-bridged silica as solid support for the stationary phase
    Silica-based monolithic chromatographic columns
    Core-shell particles in packed columns
    Hydride-based silica
    Other inorganic support materials
    Porous graphitic carbon and other carbon based materials
    Organic polymers used as support for stationary phases
    Derivatization of silica solid support
    Direct synthesis of silica materials with an active bonded phase surface
    Derivatization of silica hydride supports
    Derivatization of pre-synthesized organic polymers
    Synthesis of organic polymers with active groups
    Synthesis of organic polymeric monoliths with active functionalities
    Coated or immobilized polymeric stationary phases on silica
    Metal-organic frameworks (MOF) used as stationary phase for HPLC
    8.2. Column properties affecting separation
    General comments
    Dimensions of the column body affecting separation
    Physical properties of stationary phase affecting separation
    Chemical characteristics of stationary phase affecting separation
    Octanol/water distribution constant used to describe polarity of the active groups in the stationary phase
    8.3. Selection of a column for an HPLC separation
    General comments
    The use of guard columns and cartridges
    Column protection, cleaning, regeneration, and storing
    Selection of columns for orthogonal separations

    Part 2. Main types of HPLC separations
    9. Reversed-phase HPLC
    9.1. Retention and elution process in RP-HPLC
    General comments
    Retention/elution in RP-HPLC
    Equilibrium type in RP-HPLC separation
    Molecular interactions in RP-HPLC
    Retention results based on molecular interactions evaluation
    Other interactions affecting RP-HPLC separations
    9.2. Stationary phases and columns for RP-HPLC
    General comments
    Specific procedures for the synthesis of stationary phases in RP-HPLC
    Basic physical properties of hydrophobic stationary phases and columns
    Basic chemical characteristics of RP-type stationary phases
    Advances in the construction of common RP-columns
    Availability of special types of hydrophobic columns
    9.3. Parameters used for the characterization of RP-HPLC phases and columns
    General comments
    Efficiency of columns (theoretical plate number)
    Retention capability of columns used in RP-HPLC
    Methylene selectivity and general selectivity for hydrophobic columns
    Peak asymmetry for RP-HPLC columns
    Hydrophobic subtraction model for selectivity characterization
    Various other parameters and tests for RP-HPLC column characterization
    Tests for the evaluation of aging of the chromatographic column
    9.4. Selection of the column in RP-HPLC
    General comments
    Sample nature in RP-HPLC and its influence on column selection
    Column choice from several possibilities
    9.5. Mobile phases in reversed-phase chromatography
    General comments
    Water and mobile phases with high water content
    Alcohols used in mobile phase
    Other solvents used in RP-HPLC
    9.6. Selection of mobile phase in RP-HPLC
    General comments
    9.7. Prediction of parameters describing the separation in RP-HPLC
    General comments
    Estimation of retention factor k’ for similar systems at different mobile phase compositions
    Evaluation of retention factor k’ from octanol/water partition constant Kow or Dow
    Calculation of retention factor from van der Waals molecular surface of the analyte
    Prediction of log k’ based on solute, mobile phase, and stationary phase characteristics
    Evaluation of the energies of interaction in the separation system
    Other approaches

    10. Other HPLC separations performed on hydrophobic stationary phases
    10.1. Non-aqueous RP-HPLC
    General comments
    Mobile phase composition in NARP
    Utility of NARP compared to RP-HPLC
    10.2. Ion pair chromatography
    General comments
    Ion pairing mechanisms
    Partition model in IP
    Electrostatic model in IP
    Stationary phases in ion pair chromatography
    Mobile phase in ion pair chromatography
    Chaotropes in ion pairing
    10.3. Hydrophobic interaction chromatography (HIC)
    General comments
    Retention mechanism in HIC
    10.4. Micellar, microemulsion, and other types of HPLC performed on hydrophobic phases
    Micellar liquid chromatography (MLC)
    Microemulsion liquid chromatography (MELC)
    Other liquid chromatography types on hydrophobic phases

    11. Hydrophilic interaction chromatography (HILIC)
    11.1 Retention and elution process in HILIC
    General comments
    Equilibrium type for retention/elution in HILIC
    Description of molecular interactions in HILIC
    Retention results based on molecular interactions in HILIC
    11.2. Polar stationary phases and columns
    General comments
    Specific procedures for the synthesis of polar phases
    Physical properties of polar stationary phases and columns
    Chemical characteristics of polar stationary phases
    Bare silica stationary phases
    HILIC stationary phases with a bonded surface
    Silica hydride-based phases
    Advances in the construction of HILIC columns
    11.3. Retention and separation properties of polar stationary phases
    General comments
    Parameters and tests for HILIC column characterization
    11.4. Selection of the column in HILIC
    General comments
    Selection of the nature of stationary phase for the column
    Selection of physical column characteristics in HILIC
    Other parameters important in HILIC column selection
    11.5. Mobile phase in HILIC
    General comments
    Double role of the mobile phase in HILIC
    The gradient elution in HILIC separations
    Ion-pairing additives for HILIC separations
    Influence of mobile phase on detection in HILIC
    11.6. Prediction of parameters describing the separation in HILIC
    General comments
    Estimation of retention factor k’ for similar systems at different mobile phase compositions
    Other estimation procedures for HILIC parameters

    12. Other HPLC separations performed on polar stationary phases
    12.1. Normal phase liquid chromatography (NPC)
    General comments
    NPC compared to HILIC
    Mobile phase in NPC
    12.2. Other chromatographic techniques based on polar interactions
    Aqueous normal phase liquid chromatography (ANPC)
    Electrostatic repulsion hydrophilic interaction chromatography (eHILIC or ERLIC)

    13. Ion-exchange, ion-moderated, and ligand-exchange liquid chromatography
    13.1. Retention and elution in ion-exchange liquid chromatography
    General comments
    Retention/elution in ion exchange chromatography
    Separation in ion chromatography
    Retention of neutral molecules on ion exchange phases
    Retention in ion-moderated chromatography
    Retention in ligand exchange and immobilized metal affinity chromatography
    Ion exclusion
    13.2. Stationary phases and columns for ion exchange and related techniques
    Types of ion exchange phases
    Ion-moderated and ligand exchange phases
    Summary of procedures for the synthesis of ion exchange phases
    Latex-agglomerated ion exchangers
    Cation exchange phases based on silica
    Organic polymeric cation exchange phases
    Anion exchange phases based on silica
    Organic polymeric anion exchange phases
    Zwitterionic stationary phases
    Capillary IC columns
    Other IC stationary phases
    13.3. Characterization of ion exchange phases
    General comments
    Ionic loading capacity measurement
    Solvent compatibility of ionic phases
    Phase affinity for specific ions
    Hydrophobicity of IC columns
    13.4. Selection of an ion exchange phase
    General comments
    Separation of small ions by IC
    Separation of ionic organic molecules
    Separation of neutral organic molecules
    Separation of proteins and nucleic acids
    13.5. Mobile phase in ion exchange and ion-moderated chromatography
    General comments
    Mobile phase in cation exchange chromatography
    Mobile phase in anion exchange chromatography
    Gradient elution in ion chromatography
    Mobile phase in ion-moderated chromatography

    14. Chiral HPLC Separations
    14.1. Separation process in chiral liquid chromatography
    General comments
    Chiral recognition
    Other mechanisms for chiral separations
    14.2. Stationary phases and columns for chiral separations
    Types of chiral phases
    Brush or "Pirkle" chiral phases
    Cellulose chiral phases
    Amylose chiral phases
    Cyclodextrins and cyclofructans chiral phases
    Crown ether chiral phases
    Macrocyclic antibiotics and glycopeptides
    Protein chiral phases
    Ligand exchange chiral phases
    Chiral synthetic polymers
    14.3. Characterization of chiral phases
    General comments
    Retention behavior of enantiomers on chiral stationary phases
    14.4. Selection of a chiral phases
    General comments
    The role of column selection in the development of a method for chiral separations
    14.5. Mobile phase in chiral chromatography
    Mobile phase for chiral phases
    Mobile phase for chiral separations on achiral stationary phase
    Ion pairing mechanism for enantioseparation

    15. Size exclusion HPLC
    15.1. Separation process in size exclusion chromatography
    General comments
    15.2. Stationary phases and columns for size exclusion chromatography
    General comments
    Silica-based SEC stationary phases and glass phases
    Polymer-based phases used in SEC
    New developments
    15.3. Characterization of size exclusion phases and columns
    General comments
    Porosity and particle size
    Inertness and recovery
    15.4. Selection of a stationary phase in size exclusion HPLC
    Selection factors for SEC columns
    15.5. Mobile phase in size exclusion separations
    General comments
    Typical solvents for gel filtration (GFC)
    Typical solvents for gel permeation (GPC)
    15.6. Interaction polymer chromatography (IPC)
    General comments
    Gradient temperature in IPC

    16. Affinity, immunoaffinity, and aptamer type HPLC
    16.1. Separation process in immunoaffinity HPLC
    General comments
    16.2. Types of phases and their preparation in affinity and immunoaffinity chromatography
    General comments
    Supports for stationary phases in immunoaffinity chromatography
    The active phase in immunoaffinity chromatography
    Other types of affinity chromatography
    16.3. Biomimetic liquid chromatography
    General comments
    Stationary phases used for biomimetic LC
    Retention in immobilized artificial membrane LC

    17. Mixed mode HPLC
    17.1. Stationary phases with more than one type of active groups
    General comments
    Stationary phases with mixed mode including RP and HILIC capability
    Stationary phases with mixed mode including ion exchange capability
    17.2. Mobile phase in mixed mode HPLC
    General comments

    Part 3. Practice of HPLC analysis
    18. Utilization of HPLC in chemical analysis
    18.1 Steps in development and implementation of an HPLC separation
    General comments
    Information for starting the development of an HPLC method
    Selection to be made for an HPLC analysis
    Comments on the implementation of a method from the literature
    Improvement of a method from the literature
    Development of a new HPLC method
    Method optimization
    Method validation
    18.2. Application of HPLC for quantitative analysis
    General comments
    Calibration procedures
    Selection of the internal standards in HPLC

    Part 4. Appendices
    Appendix to Chapter 6
    Appendix to Chapter 7
    Appendix to Chapter 8
    Appendix to Chapter 9
    Appendix to Chapter 11
    Appendix to Chapter 13
    Appendix to Chapter 14
    Appendix to Chapter 15

Product details

  • Language: English
  • Copyright: © Elsevier 2022
  • Published: June 1, 2022
  • Imprint: Elsevier
  • Paperback ISBN: 9780323911771

About the Authors

Serban Moldoveanu

Serban Moldoveanu
Dr. Serban C. Moldoveanu is Senior Principal Scientist at R. J. Reynolds Tobacco Company. His research activity is focused on various aspects of chromatography including method development for the analysis by GC/MS, HPLC, and LC/MS/MS of natural products and cigarette smoke. He has also performed research on pyrolysis of a variety of polymers and small molecules. He has over 100 publications in peer reviewed journals, eleven books, and several chapter contributions. He is a member of the editorial board of the Journal of Analytical Methods in Chemistry.

Affiliations and Expertise

Senior Principal Scientist, RJ Reynolds Tobacco Co., Winston-Salem, NC, USA

Victor David

Victor David
Dr. Victor David is a Professor and Head of the Department of Analytical Chemistry, University of Bucharest, Romania. He is the author of more than 120 publications, including 92 scientific papers in ISI international journals and 10 books and chapters in various Encyclopedias. He is also a reviewer at several international journals.

Affiliations and Expertise

Professor and Head of the Department of Analytical Chemistry, University of Bucharest, Romania

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