Essentials in Modern HPLC Separations

Part 1. Basic information about HPLC1. Introductory information regarding HPLC 1.1. Preliminary discussion about HPLCGeneral commentsWhat is chromatography and what is HPLC? Types of equilibria in HPLCRole of polarity in HPLCApplications of HPLC in chemical analysisNon-analytical applications of analytical HPLC1.2. Main types of HPLCCriteria for the classification of HPLC proceduresA classification of HPLC types based on the nature of stationary and mobile phase Relation between the type of HPLC, equilibrium type, and molecular interactions1.3. Flow of a typical HPLC analysisGeneral aspectsSelection of the type of HPLC for a particular applicationSample collection and sample preparation for HPLCReferences

2. Overview of HPLC instrumentation and its use

2.1. Description of main components of HPLC instrumentationGeneral commentsDescription of a typical HPLC instrumentSolvent supply systemPumping systemsTubing and connectorsInjectors and autosamplersColumn holdersChromatographic columnsGeneral comments regarding detectorsUV-Vis spectrometric detectorsFluorescence and chemiluminescence detectorsRefractive index detectorsElectrochemical detectorsMass spectrometric detectorsEvaporative light-scattering (ELS) detectorsOther types of detectorsSelection of a detector in HPLCOther devices that can be part of the HPLC systemMore complex or special HPLC setupsInstrument control and data processing unitSelection of the HPLC system and transition from HPLC to UPLCReferences

3. Parameters for the characterization of HPLC separation

3.1. Parameters describing the chromatographic peakGeneral aspectsFlow rate of the mobile phaseRetention timeRun timeRetention volumeMigration rateEquilibrium constant and phase ratio in HPLC separationsRetention factorGeneral equation of solute retentionCharacteristics of an ideal peak shape in chromatographyEfficiency of a chromatographic columnFactors contributing to peak broadening and van Deemter equationApplication of van Deemter equationPeak asymmetryStatistical moments for the description of peak characteristicsPeak characterization using exponentially modified Gaussian shape3.2. Parameters describing the separationGeneral aspectsSelectivity (separation factor)ResolutionPeak capacity3.3. Summary of chromatographic peak and separation characteristicsGeneral comments3.4. Parameters related to quantitation in HPLCGeneral commentsQuantitation parameters from the peak shapeSample volume and amount injected in the chromatographic columnLimit of detection in HPLCLimit of quantitation3.5. Parameters characterizing the gradient separationGeneral commentsRetention factor in gradient separationsOther parameters for the characterization of chromatograms in gradient separationsReferences

4. Equilibrium types in HPLC4.1 Partition equilibriumGeneral commentsLiquid-liquid partitionDependence of retention factor on mobile phase composition in partition chromatographyDistribution coefficientPeak shape in partition chromatographyEvaluation of retention factor from liquid-liquid distribution constants4.2 Adsorption equilibriumLiquid-solid equilibriumDependence of retention factor on mobile phase composition in adsorption equilibriumPeak shape in adsorption chromatography4.3 Equilibrium involving ionsGeneral aspectsRetention equilibrium involving ionsEquilibrium of ions in the presence of a complexing reagent4.4 Equilibrium in size exclusion processesGeneral aspectsEquilibrium between interstitial mobile phase and pore mobile phase4.5 The influence of pH and of additives on retention equilibriaPreliminary information about pHDependence of compound structure on pHThe influence of pH on partition equilibriumDependence on pH of octanol/water distribution coefficient D_owInfluence on separation of additives not involved in the equilibriumChaotropic salts influence on equilibria 4.6 The influence of temperature on retention equilibriumGeneral aspectsEvaluation of thermodynamic parameters of a separation from van’t Hoff plotsNon-linear dependence of the retention factor on 1/TEvaluation of enthalpy-entropy compensation from van’t Hoff plotsHigh temperature HPLC4.7 Reactions occurring in HPLC columnGeneral commentsEnantiomerization and diastereomerisationTautomerismReferences

5. Intermolecular forces

5.1 Forces between molecules General commentsCharge to charge interactionsEnergy of an ion in a continuous mediumPolar moleculesIon to dipole interactionsDipole to dipole interactionsPolarizability of moleculesIon to molecule interactionsDipole to molecule interactionsNon-polar molecule to molecule interactionsUnified view of interactions in absence of ionsLenard-Jones potentialHydrogen bond interactionsCharge transfer or donor acceptor interactionsStacking and inclusion in supermolecular systemsOther types of bondsThe effect of a solvent on molecular interactionsSolvophobic effectsChaotropic and kosmotropic interactions5.2 Forces between molecules and a charged surfaceGeneral commentsCharge to charged-surface interactionsNeutral molecule to charged-surface interactionsReferences

6. Characterization of analytes and matrices

6.1. Properties of solutes important for HPLC separationSolutes classification based on their chemical structureClassification based on the role of the analyte in everyday lifeComments on physico-chemical properties of analytes and matricesMolecular weightAcidic or basic character of analytesVan der Waals molecular volume and areaMolecular shapeMolar volumeMolecular polarityPartial charge distributionIsoelectric pointOctanol/water partition constant and its use for polarity estimationThermodynamic parameters related to solubility of non-electrolyte compoundsActivity coefficient from enthalpy of mixingSolubility in water from octanol/water partition constantCorrelation between van der Waals molecular surface and octanol/water partition constantSolvatochromic parameters for solute characterizationOther parameters for solute characterization6.2. Physico-chemical properties related to detection General commentsGas-phase acidity and basicity in MS process of ion formationThe role of analyte polarity in MS detection6.3. Properties of matrix related to HPLC separation and detection General commentsMatrix effects on the separation in HPLCMatrix effects on HPLC detectionReferences

7. Mobile phases and their properties7.1. Characterization of liquids as eluents in HPLC

General commentsCharacterization of solvents with Hildebrand solubility parameterMiscibility of solvents and solubility one in anotherSolvent characterization using octanol/water partition constant K_owSolvent characterization based on liquid-gas partitionSolvatochromic model and Kamlet-Taft parametersElutropic strengthSolvent characterization based on other parametersSolvent properties in liquid mixtures7.2 Additional physical properties of liquids affecting separationGeneral commentsSolvent density, viscosity, and diffusion coefficientSurface tensionDielectric constant, dipole moment, and polarizabilityHydrogen bonding of solvent moleculesSolvent boiling point7.3 Properties of liquids affecting HPLC detectionGeneral commentsRefractive indexUV cut-offFluorescenceSolvent influence in MS detectionSolvent properties related to other detection techniques7.4. Buffers and additivesGeneral commentsBuffer pHBuffer capacityCommon buffers used in HPLCBuffers in partially aqueous solvent mixturesThe influence of temperature on the pH of buffersSolubility of buffers in partially organic mobile phasesAdditivesInfluence of buffers and additives on column stability and propertiesSuitability of buffers and additives for the detection in HPLC7.5. General use of solvents as mobile phaseGeneral commentsSolvent purity in HPLCFlow rate, temperature, and degassing of mobile phase The use of "green" solvents as mobile phase in HPLC7.6. Solvents for sample injection and for needle washGeneral commentsThe role of sample solvent in the chromatographic processEffect of sample solvent on detectionSolvents for the needle wash7.7. Gradient elutionGeneral commentsGradient of solvent compositionGradient of pH or of additive concentrationGradient in flow rateGradient in separation temperatureUsefulness of gradient versus isocratic elutionReferences

8. Analytical HPLC columns and their characteristics

8.1. Construction of an HPLC columnGeneral commentsExternal body of the columnPacking of particles in the chromatographic columnPhysical characteristics of the solid supports for the packed columnsChemical characteristics of the solid supports for the packed columnsSilica and ethyl-bridged silica as solid support for the stationary phaseSilica-based monolithic chromatographic columnsCore-shell particles in packed columnsHydride-based silicaOther inorganic support materialsPorous graphitic carbon and other carbon based materialsOrganic polymers used as support for stationary phasesDerivatization of silica solid supportDirect synthesis of silica materials with an active bonded phase surfaceDerivatization of silica hydride supportsDerivatization of pre-synthesized organic polymersSynthesis of organic polymers with active groupsSynthesis of organic polymeric monoliths with active functionalitiesCoated or immobilized polymeric stationary phases on silicaMetal-organic frameworks (MOF) used as stationary phase for HPLC8.2. Column properties affecting separationGeneral commentsDimensions of the column body affecting separationPhysical properties of stationary phase affecting separationChemical characteristics of stationary phase affecting separationOctanol/water distribution constant used to describe polarity of the active groups in the stationary phase8.3. Selection of a column for an HPLC separationGeneral commentsThe use of guard columns and cartridgesColumn protection, cleaning, regeneration, and storingSelection of columns for orthogonal separationsReferences

Part 2. Main types of HPLC separations 9. Reversed-phase HPLC9.1. Retention and elution process in RP-HPLCGeneral commentsRetention/elution in RP-HPLCEquilibrium type in RP-HPLC separationMolecular interactions in RP-HPLCRetention results based on molecular interactions evaluationOther interactions affecting RP-HPLC separations9.2. Stationary phases and columns for RP-HPLCGeneral commentsSpecific procedures for the synthesis of stationary phases in RP-HPLCBasic physical properties of hydrophobic stationary phases and columnsBasic chemical characteristics of RP-type stationary phasesAdvances in the construction of common RP-columnsAvailability of special types of hydrophobic columns9.3. Parameters used for the characterization of RP-HPLC phases and columnsGeneral commentsEfficiency of columns (theoretical plate number)Retention capability of columns used in RP-HPLCMethylene selectivity and general selectivity for hydrophobic columnsPeak asymmetry for RP-HPLC columnsHydrophobic subtraction model for selectivity characterizationVarious other parameters and tests for RP-HPLC column characterizationTests for the evaluation of aging of the chromatographic column9.4. Selection of the column in RP-HPLCGeneral commentsSample nature in RP-HPLC and its influence on column selectionColumn choice from several possibilities9.5. Mobile phases in reversed-phase chromatographyGeneral commentsWater and mobile phases with high water contentAlcohols used in mobile phaseAcetonitrileOther solvents used in RP-HPLC9.6. Selection of mobile phase in RP-HPLCGeneral comments9.7. Prediction of parameters describing the separation in RP-HPLCGeneral commentsEstimation of retention factor k’ for similar systems at different mobile phase compositionsEvaluation of retention factor k’ from octanol/water partition constant K_ow or D_owCalculation of retention factor from van der Waals molecular surface of the analytePrediction of log k’ based on solute, mobile phase, and stationary phase characteristicsEvaluation of the energies of interaction in the separation systemOther approachesReferences

10. Other HPLC separations performed on hydrophobic stationary phases10.1. Non-aqueous RP-HPLC

General commentsMobile phase composition in NARPUtility of NARP compared to RP-HPLC10.2. Ion pair chromatographyGeneral commentsIon pairing mechanismsPartition model in IPElectrostatic model in IPStationary phases in ion pair chromatographyMobile phase in ion pair chromatographyChaotropes in ion pairing10.3. Hydrophobic interaction chromatography (HIC)General commentsRetention mechanism in HIC10.4. Micellar, microemulsion, and other types of HPLC performed on hydrophobic phasesMicellar liquid chromatography (MLC)Microemulsion liquid chromatography (MELC)Other liquid chromatography types on hydrophobic phasesReferences

11. Hydrophilic interaction chromatography (HILIC)

11.1 Retention and elution process in HILICGeneral commentsEquilibrium type for retention/elution in HILICDescription of molecular interactions in HILICRetention results based on molecular interactions in HILIC11.2. Polar stationary phases and columnsGeneral commentsSpecific procedures for the synthesis of polar phasesPhysical properties of polar stationary phases and columnsChemical characteristics of polar stationary phasesBare silica stationary phasesHILIC stationary phases with a bonded surfaceSilica hydride-based phasesAdvances in the construction of HILIC columns11.3. Retention and separation properties of polar stationary phasesGeneral commentsParameters and tests for HILIC column characterization11.4. Selection of the column in HILICGeneral commentsSelection of the nature of stationary phase for the columnSelection of physical column characteristics in HILICOther parameters important in HILIC column selection11.5. Mobile phase in HILICGeneral commentsDouble role of the mobile phase in HILICThe gradient elution in HILIC separationsIon-pairing additives for HILIC separationsInfluence of mobile phase on detection in HILIC11.6. Prediction of parameters describing the separation in HILICGeneral commentsEstimation of retention factor k’ for similar systems at different mobile phase compositionsOther estimation procedures for HILIC parametersReferences

12. Other HPLC separations performed on polar stationary phases12.1. Normal phase liquid chromatography (NPC)

General commentsNPC compared to HILICMobile phase in NPC12.2. Other chromatographic techniques based on polar interactionsAqueous normal phase liquid chromatography (ANPC)Electrostatic repulsion hydrophilic interaction chromatography (eHILIC or ERLIC)References

13. Ion-exchange, ion-moderated, and ligand-exchange liquid chromatography13.1. Retention and elution in ion-exchange liquid chromatography

General commentsRetention/elution in ion exchange chromatographySeparation in ion chromatographyRetention of neutral molecules on ion exchange phasesRetention in ion-moderated chromatographyRetention in ligand exchange and immobilized metal affinity chromatographyIon exclusion13.2. Stationary phases and columns for ion exchange and related techniquesTypes of ion exchange phasesIon-moderated and ligand exchange phasesSummary of procedures for the synthesis of ion exchange phasesLatex-agglomerated ion exchangersCation exchange phases based on silicaOrganic polymeric cation exchange phasesAnion exchange phases based on silicaOrganic polymeric anion exchange phasesZwitterionic stationary phasesCapillary IC columnsOther IC stationary phases13.3. Characterization of ion exchange phases General commentsIonic loading capacity measurementSolvent compatibility of ionic phasesPhase affinity for specific ionsHydrophobicity of IC columns13.4. Selection of an ion exchange phaseGeneral commentsSeparation of small ions by ICSeparation of ionic organic moleculesSeparation of neutral organic moleculesSeparation of proteins and nucleic acids13.5. Mobile phase in ion exchange and ion-moderated chromatographyGeneral commentsMobile phase in cation exchange chromatographyMobile phase in anion exchange chromatographyGradient elution in ion chromatographyChromatofocusingMobile phase in ion-moderated chromatographyReferences

14. Chiral HPLC Separations

14.1. Separation process in chiral liquid chromatographyGeneral commentsChiral recognitionOther mechanisms for chiral separations14.2. Stationary phases and columns for chiral separationsTypes of chiral phasesBrush or "Pirkle" chiral phasesCellulose chiral phasesAmylose chiral phasesCyclodextrins and cyclofructans chiral phasesCrown ether chiral phasesMacrocyclic antibiotics and glycopeptidesProtein chiral phasesLigand exchange chiral phasesChiral synthetic polymers14.3. Characterization of chiral phasesGeneral commentsRetention behavior of enantiomers on chiral stationary phases14.4. Selection of a chiral phasesGeneral commentsThe role of column selection in the development of a method for chiral separations14.5. Mobile phase in chiral chromatographyMobile phase for chiral phasesMobile phase for chiral separations on achiral stationary phaseIon pairing mechanism for enantioseparationReferences

15. Size exclusion HPLC15.1. Separation process in size exclusion chromatography

General comments15.2. Stationary phases and columns for size exclusion chromatographyGeneral commentsSilica-based SEC stationary phases and glass phasesPolymer-based phases used in SECNew developments15.3. Characterization of size exclusion phases and columnsGeneral commentsPorosity and particle sizeInertness and recovery15.4. Selection of a stationary phase in size exclusion HPLCSelection factors for SEC columns15.5. Mobile phase in size exclusion separationsGeneral commentsTypical solvents for gel filtration (GFC)Typical solvents for gel permeation (GPC)15.6. Interaction polymer chromatography (IPC)General commentsGradient temperature in IPCReferences

16. Affinity, immunoaffinity, and aptamer type HPLC16.1. Separation process in immunoaffinity HPLC

General comments16.2. Types of phases and their preparation in affinity and immunoaffinity chromatographyGeneral commentsSupports for stationary phases in immunoaffinity chromatographyThe active phase in immunoaffinity chromatographyOther types of affinity chromatography16.3. Biomimetic liquid chromatographyGeneral commentsStationary phases used for biomimetic LCRetention in immobilized artificial membrane LCReferences

17. Mixed mode HPLC17.1. Stationary phases with more than one type of active groups

General commentsStationary phases with mixed mode including RP and HILIC capabilityStationary phases with mixed mode including ion exchange capability17.2. Mobile phase in mixed mode HPLC General commentsReferences

Part 3. Practice of HPLC analysis 18. Utilization of HPLC in chemical analysis18.1 Steps in development and implementation of an HPLC separationGeneral comments Information for starting the development of an HPLC methodSelection to be made for an HPLC analysisComments on the implementation of a method from the literatureImprovement of a method from the literatureDevelopment of a new HPLC methodMethod optimizationMethod validation18.2. Application of HPLC for quantitative analysisGeneral commentsCalibration proceduresSelection of the internal standards in HPLCReferences

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