Selection of HPLC Method in Chemical Analysis

Selection of the HPLC Method in Chemical Analysis......Page 1
Preface......Page 2
Copyright......Page 3
The Purpose of Analysis......Page 4
General Information About the Samples......Page 6
The Sample Constituents......Page 7
Instrumentation Availability, Expertise in the Laboratory, and Funding......Page 8
Information About Various Methods of Analysis......Page 9
1.2 Overview of an Analytical Technique......Page 10
Sample Preparation Step......Page 11
The Core Analytical Operation......Page 12
Qualitative and Quantitative Analysis......Page 14
1.3 Statistical Evaluation of Data and Criteria for Method Validation......Page 17
Precision and Accuracy in Quantitative Chemical Analysis......Page 18
Sensitivity and Limit of Detection......Page 20
Linearity of the Instrumental Response and Least Square Regression......Page 25
Validation of an Analytical Method......Page 27
References......Page 31
Types of Instrumental Techniques Not including an Independent Separation (Nonhyphenated Techniques)......Page 33
Light Absorption Spectroscopy......Page 34
Light Emission Spectroscopy......Page 37
2.3 Mass Spectrometry......Page 39
Electron Impact Ionization......Page 40
Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI) in LC-MS......Page 42
Ion Suppression in LC-MS......Page 44
Ion Separation in Mass Spectrometry......Page 45
2.4 Electrochemical Methods......Page 47
Potentiometric Methods......Page 48
Amperometric and Coulometric Methods......Page 49
2.5 Other Analytical Techniques Not Including the Separation of Sample Components......Page 51
Radiochemical Methods......Page 52
2.6 Selection of a Nonhyphenated Method of Analysis versus one Containing a Separation Step......Page 53
References......Page 54
Chromatographic Separations......Page 56
Solid-Phase Extraction Online With Analytical Chromatography......Page 60
Electro Separations......Page 61
Selection of a Separation for the Core Analysis......Page 63
3.2 Gas Chromatography......Page 64
Injectors for Gas Chromatography......Page 65
Gas Chromatography Oven and Columns......Page 67
Detectors for Gas Chromatography......Page 68
3.3 Supercritical Fluid Chromatography......Page 70
Classification of High-Performance Liquid Chromatography Types Based on Separation Mechanism......Page 72
Other Classifications of Analytical High-Performance Liquid Chromatography Techniques......Page 77
Electrophoresis......Page 78
Selection of GC as the Method for Analysis Versus HPLC......Page 80
Selection of SFC as the Method for Analysis Versus HPLC......Page 82
Advantages and Disadvantages in Selecting High-Performance Liquid Chromatography as a Method of Analysis......Page 83
References......Page 85
4.1 Basic Information About Instrumentation in HPLC......Page 87
Solvent Supply System......Page 88
Pumping System and the Mobile Phase......Page 89
Injectors and Autosamplers......Page 94
Tubing and Connectors......Page 95
Chromatographic Columns......Page 96
The Main Characteristics of the Detectors Used in HPLC and UPLC......Page 99
Types of Detectors Used in HPLC and UPLC......Page 102
Selection of a Detector in HPLC......Page 115
Other Devices That Can Be Part of the HPLC System......Page 119
More Complex HPLC Setups......Page 120
Selection of the HPLC System and Transition From HPLC to UPLC......Page 121
4.2 Parameters Describing the Chromatographic Process......Page 122
Retention Time......Page 123
Retention Volume......Page 124
Migration Rate......Page 125
Equilibrium Constant and Phase Ratio in HPLC Separations......Page 126
Retention Factor......Page 127
Characteristics of an Ideal Peak Shape in Chromatography......Page 129
Efficiency of a Chromatographic Column......Page 134
Factors Contributing to Peak Broadening and van Deemter Equation......Page 135
Peak Asymmetry......Page 138
Selectivity (Separation Factor)......Page 139
Resolution......Page 140
Peak Capacity......Page 142
Peak Characteristics for Gradient Separations......Page 143
Quantitation in HPLC......Page 145
Sample Volume and Amount Injected in the Chromatographic Column......Page 148
Partition Equilibrium and Its Thermodynamic Aspects......Page 150
Adsorption Equilibrium......Page 154
The Role of Polarity in Separation Mechanisms......Page 155
Mechanism in Reversed-Phase HPLC......Page 156
Retention Factor in RP-HPLC as Predicted by Solvophobic Theory......Page 160
Mechanisms in Ion-Pair Chromatography (IP)......Page 163
Mechanisms in Normal-Phase HPLC (NP-HPLC or NPC) and in HILIC......Page 165
Equilibria in Ion-Exchange Chromatography......Page 167
Mechanism in Size-Exclusion Chromatography (SEC)......Page 170
Mechanism in Chiral Chromatography......Page 173
The Influence of pH on Retention Equilibria......Page 178
The Influence of Temperature on Retention Equilibria......Page 181
Influence of Additives Not Involved in the Equilibrium......Page 182
References......Page 183
Chemical Composition and Structure......Page 188
Molecular Weight......Page 189
Isoelectric Point......Page 190
Hydrogen Bonding Capability as Part of Molecular Polarity......Page 191
Octanol/Water Partition Constant and Its Use for Polarity Estimation......Page 192
Solubility of Nonelectrolytic Compounds......Page 194
5.2 Physicochemical Properties Related to Detection......Page 198
UV–Vis Absorption......Page 199
Fluorescence......Page 200
Mass Spectra......Page 201
Electrochemical Properties......Page 202
Classification of Samples Based on Their Role in Everyday Life......Page 203
Analysis Selection Based on the Role of the Sample in Everyday Life......Page 208
Role of Chemical Nature of the Analyte......Page 209
Role of Octanol/Water Partition Coefficient......Page 212
The Role of Analyte Concentration in the Selection of HPLC Separation......Page 217
Role of the Chemical Nature of the Matrix......Page 218
Role of the Amount of the Matrix......Page 220
5.6 Review of Sample Properties with the Goal of Selection of a Detector in HPLC......Page 222
Detector Selection for Qualitative or Quantitative Analysis......Page 223
Detector Selection Based on Specific Physicochemical Properties of the Analyte......Page 224
Role of Analyte Concentration in the Selection of Detection in HPLC......Page 226
References......Page 227
External Body of the Column......Page 230
Physical Characteristics of the Solid Supports for the Packed Columns......Page 231
Chemical Characteristics of the Solid Supports for the Packed Columns......Page 234
Silica as Solid Support for the Stationary Phase......Page 235
Silica-Based Monolithic Chromatographic Columns......Page 241
Derivatization of Silica Solid Support......Page 242
Organic Polymers as Stationary Phases......Page 248
Study of Physicochemical Characteristics of a Stationary Phase......Page 249
Dimensions of the Column Body Affecting Separation......Page 250
Physical Properties of Stationary Phase Affecting Separation......Page 254
Chemical Properties of Stationary Phase Affecting Separation......Page 259
Characterization of Stationary Phase Polarity With Octanol/Water Distribution Constant......Page 262
Parameters to Consider for Column Selection......Page 266
Summary of Criteria for Column Selection......Page 270
Column Protection and Storing......Page 271
References......Page 273
Preparation of RP Stationary Phases......Page 277
Basic Physical Properties of Hydrophobic Stationary Phases and Columns......Page 281
Basic Chemical Properties of Hydrophobic Stationary Phases......Page 282
Hydrophobicity......Page 283
End-Capping and Silanol Activity......Page 284
Enhancing the pH and Salt Stability of the Stationary Phase......Page 286
Phase Ratio......Page 287
Equilibrium Constant K(X)......Page 290
Wettability......Page 291
7.3 Parameters Used for the Characterization of Reversed-Phase HPLC Columns......Page 293
Retention Capability of Hydrophobic Columns......Page 294
Formulas for the Prediction of logk′......Page 295
General Selectivity and Methylene Selectivity of Hydrophobic Columns......Page 297
Peak Asymmetry for RP-HPLC Columns......Page 301
Various Other Parameters and Tests for RP-HPLC Column Characterization......Page 302
Hydrophobic Subtraction Model for Selectivity Characterization......Page 304
Common RP Columns......Page 308
Special Types of Hydrophobic Phases......Page 310
7.5 Selection of an RP-HPLC Column......Page 314
Selection of Physical Column Characteristics......Page 316
Columns for the Analysis of Small Molecules With a Hydrophobic Moiety......Page 317
Columns for the Analysis of Peptides and Proteins......Page 320
References......Page 321
Main Types of Stationary Polar Phases......Page 327
Preparation of Polar Stationary Phases......Page 328
Chemical Properties of Polar Stationary Phases......Page 331
Parameters and Tests for HILIC Column Characterization......Page 333
Neutral HILIC Stationary Phases With a Bonded Phase......Page 337
Cation Exchange HILIC Type Stationary Phases......Page 338
Stationary Phases With More Than One Type of Group (Mixed-Mode HILIC Phases)......Page 339
8.4 Selection of a Polar Column......Page 340
Selection of the Nature of Stationary Phase for the Column......Page 342
New Developments......Page 343
References......Page 344
Types of Ion Exchange Phases......Page 346
Stationary Phases with More than One Type of Group (Mixed-Mode Ion Exchange Phases)......Page 347
Summary of Procedures for the Synthesis of Ion Exchange Phases......Page 348
9.2 Characterization of Ion Exchange Phases......Page 350
Ionic Capacity Measurement......Page 351
Phase Affinity for Specific Ions......Page 352
Cation Exchange Phases Based on Silica......Page 353
Organic Polymeric Anion Exchange Phases......Page 354
9.4 Selection of an Ion Exchange Phase......Page 355
References......Page 358
Types of Chiral Phases......Page 360
Brush or “Pirkle” Chiral Phases......Page 361
Cellulose Chiral Phases......Page 363
Cyclodextrin and Cyclofructan Chiral Phases......Page 364
Crown Ether Chiral Phases......Page 365
Ligand Exchange Chiral Phases......Page 366
10.2 Characterization of Chiral Phases......Page 367
The Role of Column Selection in the Development of a Method for Chiral Separations......Page 368
References......Page 371
Silica-Based SEC Stationary Phases and Glass Phases......Page 374
Polymer-Based Phases Used in SEC......Page 375
11.2 Characterization of Size-Exclusion Phases and Columns......Page 377
Inertness and Recovery......Page 378
Selection Factors for SEC Columns......Page 379
References......Page 382
Supports for Stationary Phases in Immunoaffinity Chromatography......Page 384
The Active Phase in Immunoaffinity Chromatography......Page 385
References......Page 388
13.1 Characterization of Liquids as Solvents......Page 390
Miscibility of Solvents......Page 391
Characterization of Solvents with Hildebrand Solubility Parameter......Page 392
Solvent Characterization Using Octanol/Water Partition Constant Kow......Page 396
Solvent Characterization Based on Liquid–Gas Partition......Page 397
Solvatochromic Model and Kamlet–Taft Parameters......Page 400
Solvent Viscosity......Page 401
Superficial Tension......Page 403
Hydrogen Bonding of Solvent Molecules......Page 405
Refractive Index......Page 406
UV Cut-Off......Page 407
Solvent Influence in MS Detection......Page 408
13.4 Buffers and Additives......Page 410
Buffer pH......Page 411
Common Buffers Used in HPLC......Page 413
Buffers in Partially Aqueous Solvent Mixtures......Page 414
The Influence of Temperature on the pH of Buffers......Page 417
Solubility of Buffers in Partially Organic Mobile Phases......Page 418
Additives......Page 419
Influence of the Buffer and Additives on Column Stability and Properties......Page 420
Suitability of the Buffers and Additives for Detection in HPLC......Page 421
Solvent Purity in HPLC......Page 422
Mobile Phases Used in RP-HPLC......Page 423
Solvents, Ion Pairing Agents, and Additives Used in Ion-Pair HPLC......Page 426
Mobile Phases Used in HILIC and NPC......Page 428
Mobile Phases Used in Ion Exchange and Ion-Moderated Chromatography......Page 429
Mobile Phase in Chiral Chromatography......Page 432
Mobile Phase for Size-Exclusion Separations......Page 434
Flow Rate, Temperature, and Degassing of the Mobile Phase......Page 436
The Role of Sample Solvent in the Chromatographic Process......Page 437
Focusing of Sample at the Column Head by Other Procedures......Page 440
References......Page 441
Purposes for the Use of Gradients......Page 448
Practice of Gradient Elution......Page 451
Gradient of pH......Page 452
14.2 Parameters Characterizing the Gradient Separation......Page 453
Retention Factor in Gradient Separations......Page 454
Other Parameters for the Characterization of Chromatograms in Gradient Separations......Page 455
Gradient in RP-HPLC and Nonaqueous Reversed-Phase Chromatography......Page 456
Gradient in HILIC......Page 457
Gradient in Ion Chromatography......Page 458
References......Page 459
Implementation of a Method from the Literature......Page 460
Improvement of a Method from the Literature......Page 462
Development of a New HPLC Method......Page 463
15.2 Special HPLC Techniques......Page 465
Postcolumn Derivatization in HPLC......Page 466
References......Page 467
1: USP Classification of HPLC Columns......Page 468
2: Hydrophobic Stationary Phases......Page 475
3: HILIC and NPC Stationary Phases......Page 518
4: Ion Exchange and Ion-Moderated Stationary Phases......Page 524
5: Chiral Stationary Phases......Page 532
6: Size-Exclusion Stationary Phases......Page 538
7: Properties of Mobile Phase Components......Page 542
A......Page 557
C......Page 559
D......Page 562
E......Page 563
G......Page 565
H......Page 566
I......Page 568
L......Page 569
M......Page 570
N......Page 571
O......Page 572
P......Page 573
Q......Page 575
R......Page 576
S......Page 577
T......Page 580
V......Page 581
Z......Page 582