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Essentials in Modern HPLC Separations

By: Victor David, Serban Moldoveanu

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Hardcover | 21 September 2012 | Edition Number 1

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Hardcover
550 Pages

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24.13 x 19.05 x 3.18

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Essentials in Modern HPLC Separations discusses the role of separation in high performance liquid chromatography (HPLC). This up-to-date reference systematically covers new developments in types and characteristics of stationary phases, mobile phases, and other factors of this technique that influence separation of compounds being analyzed. The volume also considers the selection process for stationary and mobile phases in relation to the molecules being separated and examined, as well as their matrices.

The book includes a section on the contemporary applications of HPLC, particularly the analysis of pharmaceutical and biological samples, food and beverages, environmental samples, and more.

Discusses key parameters in HPLC separation
Describes interrelation between various HPLC features (solvent pressure, separation, detection)
Includes a large number of references

Industry Reviews

"This book is clearly written, easy to understand, and comprehensive. Because of the extensive descriptions, it is a helpful resource for scientists conducting research in separation science, workers engaged in separation-related industries, and students studying analytical chemistry." --Analytical and Bioanalytical Chemistry online, June 23, 2013

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You Can Find This Book In

Non-Fiction Science Science in General Engineering & Technology Biochemical Engineering Industrial Chemistry & Manufacturing Technologies Industrial Chemistry Chemical Engineering Chemistry Analytical Chemistry

Preface	p. xv
Basic Information about HPLC	p. 1
Introduction to HPLC	p. 2
What is Chromatography?	p. 2
Types of Equilibria in HPLC	p. 3
Criteria for the Classification of HPLC Procedures	p. 6
Role of Polarity in HPLC	p. 7
Qualitative Analysis and HPLC Main Use as a Quantitative Analytical Technique	p. 8
Main Types of HPLC	p. 9
A Classification of HPLC Types	p. 9
Relation between the Type of HPLC and Equilibrium Mechanism	p. 14
Practice of HPLC	p. 14
General Aspects	p. 14
Selection of the Type of HPLC for a Particular Application	p. 15
Sample Collection and Sample Preparation for HPLC	p. 15
Injection	p. 16
Column Selection in HPLC	p. 17
Mobile Phase Selection	p. 17
Detection in HPLC and Quantitation Procedures	p. 18
Overview of HPLC Instrumentation	p. 20
General Comments	p. 20
Schematic Description of an HPLC Instrument	p. 20
Solvent Supply Systems	p. 20
Pumping Systems	p. 22
Injectors	p. 27
Tubing and Connectors	p. 28
Chromatographic Columns	p. 29
Setups for Multidimensional Separations	p. 31
Other Devices that are Part of the HPLC System	p. 31
General Comments on Detectors	p. 33
Spectrophotometric Detectors	p. 34
Fluorescence and Chemiluminescence Detectors	p. 36
Refractive Index Detectors	p. 38
Electrochemical Detectors	p. 39
Mass Spectrometric Detectors	p. 42
Other Types of Detectors	p. 46
Selection of a Detector for the HPLC Separation	p. 47
Fraction Collectors	p. 50
Controlling and Data Processing Units	p. 50
References	p. 50
Parameters that Characterize HPLC Analysis	p. 53
Parameters Related to HPLC Separation	p. 53
General Comments	p. 53
Flow Rate	p. 54
Retention Time	p. 54
Retention Volume	p. 55
Migration Rate	p. 56
Capacity Factor (Retention Factor)	p. 57
Equilibrium Constant and Phase Ratio	p. 58
General Equation of Solute Retention	p. 59
Characteristics of Ideal Peak Shape and Definition of Efficiency	p. 60
Selectivity	p. 64
Resolution	p. 64
Peak Capacity	p. 66
Statistical Moments for the Description of Peak Characteristics	p. 67
Description of Peak Characteristics for Gradient Separations	p. 68
Quantitation in HPLC	p. 69
Experimental Peak Characteristics in HPLC	p. 71
Van Deemter Equation	p. 71
Kinetic Plots	p. 75
Peak Asymmetry	p. 76
Application of van Deemter Equation	p. 78
Peak Characterization Using Exponentially Modified Gaussian Shape	p. 79
Summary of Chromatographic Peak Characteristics	p. 81
References	p. 83
Equilibrium Types in HPLC	p. 85
Partition Equilibrium	p. 86
General Comments	p. 86
Liquid-Liquid Partition	p. 86
Distribution Coefficient	p. 89
Peak Shape in Partition Chromatography	p. 89
Evaluation of Capacity Factor k from Liquid-Liquid Distribution Constants	p. 90
Adsorption Equilibrium	p. 91
Liquid-Solid Equilibrium	p. 91
Peak Shape in Adsorption Chromatography	p. 94
Equilibria Involving Ions	p. 95
General Aspects	p. 95
Retention Equilibrium in Ion Chromatography	p. 95
Equilibrium in the Presence of a Complexing Reagent	p. 97
Gibbs-Donnan Effect	p. 98
Equilibrium in Size-Exclusion Processes	p. 99
General Aspects	p. 99
Partition Equilibrium between Interstitial Mobile Phase and Pore Mobile Phase	p. 100
The Influence of pH on Retention Equilibria	p. 102
Preliminary Information	p. 102
Dependence of Compound Structure on pH	p. 103
The Influence of pH on Partition	p. 103
Octanol/Water Partition Constant K_ow and Distribution Coefficient Dow	p. 107
The Influence of Temperature on Retention Equilibria	p. 109
General Aspects	p. 109
Evaluation of Thermodynamic Parameters of a Separation from van't Hoff Plots	p. 109
Nonlinear Dependence of the Capacity Factor on 1/T	p. 110
Evaluation of Enthalpy-Entropy Compensation from van't Hoff Plots	p. 112
References	p. 112
Intermolecular Interactions	p. 115
Forces Between Ions and Molecules	p. 115
General Comments	p. 115
Charge to Charge Interaction	p. 117
Energy of an Ion in a Continuous Medium	p. 118
Polar Molecules	p. 119
Ion-to-Dipole Interaction	p. 121
Dipole-to-Dipole Interaction	p. 122
Polarizability of Molecules	p. 123
Ion-to-Molecule Interaction	p. 124
Dipole-to-Molecule Interaction	p. 125
Nonpolar Molecule-to-Molecule Interaction	p. 125
Unified View of Interactions in the Absence of Ions	p. 127
Lennard-Jones Potential	p. 127
Hydrogen-Bond Interactions	p. 129
Charge Transfer or Donor-Acceptor Interactions	p. 131
Stacking and Inclusion in Supermolecular Systems	p. 131
The Effect of a Solvent on Molecular Interaction	p. 133
Solvophobic Interaction	p. 135
Chaotropic Interactions	p. 140
Forces Between Molecules and a Surface	p. 140
General Comments	p. 140
Charge to Surface-Charged Interaction	p. 140
Neutral Molecule-to-Surface Interaction	p. 141
References	p. 143
Retention Mechanisms in Different HPLC Types	p. 145
Retention in Reversed-Phase Chromatography	p. 146
General Comments	p. 146
Evaluation of Equilibrium Constant Based on Solvophobic Theory	p. 147
Qualitative Remarks Regarding Retention in RP-HPLC	p. 151
Elution Process in RP-HPLC	p. 153
Separation in RP-HPLC Based on Solvophobic Theory	p. 155
Nonaqueous Reversed-Phase Chromatography (NARP)	p. 156
Other Theories Applied for Explaining RP Separation	p. 156
Retention and Separation Process in Ion-Pair Chromatograpy	p. 156
General Aspects	p. 156
Ion-pairing Mechanisms	p. 158
Partition Model	p. 158
Electrostatic Model	p. 161
Chaotropes in Ion Pairing	p. 168
Retention and Separation on Polar Stationary Phases	p. 168
General Comments	p. 168
Retention Process in NPC and HILIC	p. 169
Separation in NPC and HILIC	p. 171
Retention Process in Ion-Exchange Chromatography	p. 172
General Comments	p. 172
The Mechanism of Ion-Exchange Retention and Elution	p. 172
Separation in Ion Chromatography	p. 175
Retention of Neutral Molecules on Ion-Exchange Phases	p. 176
Separation Process in Chiral Chromatography	p. 176
General Comments	p. 176
Types of Mechanisms in Chiral Separations	p. 178
Retention Process in Size-Exclusion Chromatography	p. 181
General Comments	p. 181
Dependence of Retention Volume on the Molecular Weight in SEC	p. 182
Retention Process in Other Chromatography Types	p. 183
General Comments	p. 183
Hydrophobic Interaction Chromatography (HIC)	p. 184
Electrostatic Repulsion Hydrophilic Interaction Chromatography	p. 184
Aqueous-Normal-Phase Chromatography	p. 185
Ion Exclusion	p. 186
Ligand-Exchange and Immobilized Metal Affinity Chromatography	p. 186
Ion-Moderated Chromatography	p. 187
Displacement Chromatography	p. 187
Affinity Chromatography	p. 187
Multimode HPLC	p. 188
References	p. 188
Stationary Phases and Their Performance	p. 191
Solid Supports for Stationary Phases	p. 192
General Comments	p. 192
Silica Support for Stationary Phases	p. 193
Organic/Inorganic Silica Supports	p. 201
Coated or Immobilized Polymeric Stationary Phases on Silica	p. 203
Hydride-based Silica	p. 203
Other Inorganic Support Materials	p. 204
Inorganic Monolithic Supports (Silica-based Monoliths)	p. 206
Organic Polymers Used as Support for Stationary Phases	p. 207
Reactions Used for Obtaining Active Groups of Stationary Phases	p. 211
General Comments	p. 211
Derivatizations to Generate Bonded Silica-based Stationary Phases	p. 211
Direct Synthesis of Silica Materials with an Active Bonded Phase Surface	p. 220
Derivatization of Silica Hydride Supports	p. 221
Derivatization of Presynthesized Organic Polymers	p. 222
Synthesis of Organic Polymers with Active Groups	p. 223
Synthesis of Organic Polymeric Monoliths with Active Functionalities	p. 224
Silica Covered with a Bonded Active Polymer	p. 225
Properties of Stationary Phases and Columns	p. 226
General Comments	p. 226
Packing of the Stationary Phase in the Column and Flow Direction	p. 226
Physical Properties of the Stationary Phase	p. 227
Physical Dimensions of the Chromatographic Column	p. 227
Types of Stationary Phases and USP Classification	p. 228
Characterization of the Polarity of a Column Using K_ow for Models of Stationary Phase	p. 235
Performance of Stationary Phases and Columns	p. 237
Selection of a Column for an HPLC Separation	p. 242
The Use of Guard Columns and Cartridges	p. 244
Column Protection, Cleaning, Regeneration, and Storing	p. 245
Selection of Columns for Orthogonal Separations	p. 246
Physicochemical Characterization of Stationary Phases	p. 247
Hydrophobic Stationary Phases and Columns	p. 249
Types of Hydrophobic Stationary Phases	p. 249
End-capping of Free Silanols	p. 250
Preparation of Polar-Embedded Hydrophobic Phases	p. 253
Physical Characteristics of Hydrophobic Stationary Phases	p. 254
Retention and Separation Properties of Hydrophobic Stationary Phases	p. 255
Methylene Selectivity	p. 262
Hydrophobic Subtraction Model	p. 264
Other Tests for Comparing Hydrophobic Columns	p. 279
Other Properties of Reversed-Phase Columns Critical for Separation	p. 281
Common Hydrophobic Stationary Phases	p. 285
Hydrophobic Stationary Phases with Some Polarity	p. 289
Other Types of Hydrophobic Phases	p. 293
Organic Polymer-based Hydrophobic Stationary Phases	p. 296
Special Hydrophobic Stationary Phases	p. 296
Selection of a Column for RP-HPLC Separations	p. 299
Polar Stationary Phases and Columns	p. 300
General Aspects	p. 300
Specific Procedures for Polar-Phase Synthesis	p. 301
Retention and Separation Properties of Polar Stationary Phases	p. 304
Bare Silica Stationary Phase	p. 311
HILIC Stationary Phases with a Bonded Surface	p. 312
Silica Hydride-based Phases	p. 316
Stationary Phases and Columns for Ion-Exchange, Ion-Moderated, and Ligand-Exchange Chromatography	p. 318
General Aspects	p. 318
Silica-based Ion Exchangers	p. 319
Synthesis of Organic Polymeric Ion Exchangers	p. 320
Latex-Agglomerated Ion Exchangers	p. 325
Commercial Polymeric Stationary Phases Used in Ion-Exchange HPLC	p. 326
Ion-Moderated Phases	p. 326
Ligand-Exchange and Immobilized Metal Affinity Phases	p. 330
Stationary Phases and Columns for Chiral Chromatography	p. 331
General Comments	p. 331
Procedures for the Synthesis of Phases with Chiral Properties	p. 331
Main Types of Stationary Phases Used in Chiral HPLC	p. 336
Stationary Phases and Columns for Size-Exclusion Chromatography	p. 344
General Comments	p. 344
Silica-based SEC Phases and Glass Phases	p. 346
Polymeric-based SEC Phases	p. 346
Stationary Phases and Columns in Immunoaffinity Chromatography	p. 350
General Aspects	p. 350
Synthesis of Stationary Phases Used in Affinity and Immunoaffinity Chromatography	p. 350
References	p. 353
Mobile Phases and Their Properties	p. 363
Characterization of Liquids as Solvents	p. 364
General Comments	p. 364
Solubility Based on Thermodynamic Concepts	p. 365
Miscibility of Solvents	p. 367
Estimation of Partition Constant and Selectivity in HPLC from the Solubility Parameter	p. 367
Elution "Strength" of a Solvent	p. 373
Solvent Characterization Using Octanol/Water Partition Constant K_ow	p. 373
Solvent Characterization Based on Liquid-Gas Partition	p. 374
Solvatochromic Model	p. 380
Eluotropic Strength	p. 385
Estimation of Selectivity Based on Eluotropic Strength	p. 388
Solvent Properties of Liquid Mixtures	p. 389
Additional Properties of Liquids Affecting Separation	p. 394
General Comments	p. 394
Solvent Viscosity	p. 395
Dielectric Constant	p. 397
Dipole Moment	p. 398
Polarizability	p. 398
Superficial Tension	p. 398
Properties of the Mobile Phase of Importance in HPLC, not Related to Separation	p. 400
General Comments	p. 400
Refractive Index and UV Cutoff	p. 400
Fluorescence	p. 402
Solvent Selection for MS Detection	p. 403
Solvent Purity in HPLC	p. 404
Buffers and other Additives in HPLC	p. 404
General Comments	p. 404
Buffer pH	p. 405
Buffer Capacity	p. 406
Buffers in Partially Aqueous Solvent Mixtures	p. 408
Solubility of Buffers in Partially Organic Mobile Phases	p. 410
Influence of the Buffer on Column Stability and Properties	p. 411
Suitability of the Buffers for Detection in HPLC	p. 412
Other Additives in the Mobile Phase	p. 414
Gradient Elution	p. 414
General Comments	p. 414
Parameters Characterizing the Gradient Separation	p. 416
Usefulness of Gradient versus Isocratic Elution	p. 419
Mobile Phase in Reversed-Phase Chromatography	p. 422
General Aspects	p. 422
Water and Mobile Phases with High Water Content	p. 423
Alcohols	p. 423
Acetonitrile	p. 423
Other Solvents Used in RP-HPLC and NARP	p. 424
Mobile Phase Composition and Detection in RP-HPLC	p. 425
Mobile Phase in Ion-Pair Liquid Chromatography	p. 425
General Aspects	p. 425
Ion-Pairing Agents (Hetaerons)	p. 426
Organic Modifiers of the Mobile Phase in IP	p. 426
The pH of the Mobile Phase, Chaotropes and Other Additives in IP	p. 428
Accordance of Mobile Phase in IPC with the Detection Mode	p. 428
Mobile Phase in HILIC and NPC	p. 429
General Comments	p. 429
Double Role of the Mobile Phase in HILIC and NPC	p. 430
The Gradient Elution in HILIC Separations	p. 431
Influence of Mobile Phase on Detection in HILIC and NPC	p. 432
Mobile Phase in Ion-Exchange and Ion-Moderated Chromatography	p. 433
General Comments	p. 433
Mobile Phase in Cation-Exchange Chromatography	p. 433
Mobile Phase in Anion-Exchange Chromatography	p. 434
Gradient Elution in Ion Chromatography	p. 435
Chromatofocusing	p. 436
Mobile Phase in Ion-Moderated Chromatography	p. 437
Mobile Phase in Chiral Chromatography	p. 437
General Comments	p. 437
Mobile Phase for Chiral Separations on Chiral Stationary Phases	p. 438
Mobile Phase for Chiral Separations on Achiral Stationary Phase	p. 439
Ion-Pairing Mechanism for Enantioseparation	p. 440
Mobile Phase for Size-Exclusion Separations	p. 441
General Comments	p. 441
Typical Solvents for Gel Filtration (GFC)	p. 442
Typical Solvents for Gel Permeation (GPC)	p. 443
References	p. 443
Solutes in HPLC	p. 449
Nature of the Solute	p. 449
General Aspects	p. 449
Solutes Classification Based on Their Chemical Structure	p. 450
Classification Based on the Role of the Solute in Everyday Life	p. 450
Parameters for Solute Characterization in the Separation Process	p. 451
General Aspects	p. 451
Molecular Weight	p. 451
Acidic or Basic Character of Solutes	p. 452
Isoelectric Point	p. 452
Molecular Polarity and Octanol/Water Partition Constant	p. 452
Solubility in Water from Octanol/Water Partition Constant	p. 454
Van der Waals Molecular Volume and Surface	p. 454
Correlation between van der Waals Molecular Surface and Octanol/Water Partition Constant	p. 456
Partial Charge Distribution	p. 459
Parameters for Solute Hydrophobicity, Steric Effects, Hydrogen Bonding, and Ion-Exchange Character	p. 459
Molar Volume, Hildebrand Solubility Parameter	p. 459
Evaluation of Solubility of Nonpolar Compounds from Hildebrand Solubility Parameter	p. 460
Solvatochromic Parameters for the Solute	p. 461
Other Solute Properties Affecting Separation	p. 461
Other Parameters for Solute Characterization	p. 462
General Aspects	p. 462
Physical Properties of the Analyte Determining Detection	p. 463
References	p. 463
HPLC Analysis	p. 465
Chemical Nature of the Analytes and the Choice of HPLC Type	p. 466
General Aspects	p. 466
Use of Polarity of the Analyte in the Choice of HPLC Type	p. 468
The Quantity of Sample Injected for HPLC Analysis	p. 469
General Aspects	p. 469
Sample Volume and Amount	p. 469
Nature of Sample Solvent and Its Importance in Separation	p. 471
Estimation of Parameters Describing the Separation	p. 475
General Comments	p. 475
Estimation of Capacity Factor k for Similar Systems at Different Mobile Phase Compositions	p. 476
Evaluation of Capacity Factor k from Octanol/Water Partition Constant K_ow	p. 478
Calculation of Capacity Factor from van der Waals Molecular Surface of the Analyte	p. 483
Prediction of log k Based on Solute, Mobile Phase, and Stationary Phase Characteristics	p. 484
Evaluation of the Energies of Interaction in the Separation System	p. 487
Steps in Development and Implementation of an HPLC Separation	p. 487
General Comments	p. 487
Information for Starting the Development of an HPLC Method	p. 487
Choice of HPLC Type	p. 489
Choice of the Chromatographic Column	p. 489
Choice of Mobile Phase and Achieving Separation of Standards	p. 490
Application of the Method to Real Samples	p. 491
Method Validation	p. 492
Method Transfer	p. 493
Separations by RP-HPLC	p. 493
General Comments	p. 493
Application of RP-HPLC to Analysis of Small Polar Molecules	p. 493
Analysis of Small Molecules with Average or Strong Hydrophobic Character	p. 494
Analysis of Small Sugar Molecules, Oligo, and Polysaccharides	p. 495
Analysis of Amino Acids, Peptides, and Proteins	p. 496
Analysis of Nucleobases, Nucleosides, Deoxynucleosides, Nucleotides, and Nucleic Acids	p. 501
Separations by Ion-Pair Chromatography	p. 502
General Comments	p. 502
Separations by HILIC and NPC	p. 505
General Comments	p. 505
Analysis of Small Polar Molecules Using HILIC	p. 505
Analysis of Small Sugar Molecules, Oligo, and Polysaccharides	p. 506
Analysis of Amino Acids, Peptides, and Proteins	p. 507
Separations Using the eHILIC (ERLIC) Technique	p. 507
The Use of NPC Technique	p. 507
Separations by Ion-Exchange Chromatography	p. 507
General Comments	p. 507
Separation of Small Ions by IC	p. 508
Separation of Ionic Organic Molecules	p. 508
Separation of Neutral Organic Molecules	p. 509
Separation of Proteins and Nucleic Acids	p. 509
Chiral Separations	p. 510
General Comments	p. 510
Separations by Size-Exclusion Chromatography	p. 512
General Comments	p. 512
Examples of Applications of SEC	p. 514
References	p. 515
Symbols	p. 521
Index	p. 523
Table of Contents provided by Ingram. All Rights Reserved.

Essentials in Modern HPLC Separations

At a Glance

New Edition

Hardcover

Industry Reviews

Shipping

How to return your order

Defective items

You Can Find This Book In

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