896 INDEX Column-comparison function F s , 236 F s (-C), 236, 279 Column conditions, 46–50, 61–63, 295 gradient elution, 415–418, 445–446 Column packing; see Particle; Stationary phase Column selectivity, 227–238, 345–346; see also Selectivity comparison of, 236 differences, 235 interactions, 227–228 for ionic samples, 323–326 for isomers, 277–278 neutral sample, 273–276 NPC, 381–382 parameters, 233 Column switching, 79–80, 122–123; see also Multidimensional HPLC; Two-dimensional HPLC boxcar chromatography, 79–80 column regeneration, 122 column selection, 123 fraction collection, 123 mobile phase recycling, 125 multidimensional liquid chromatography, 618 parallel columns, 123 sample enrichment, 122 sample preparation by, 796–797 waste diversion, 124 Compendial methods, 533, 561 Complex sample, 442; see also Multidimensional liquid chromatography Compound class separation, 365–366 Comprehensive two-dimensional HPLC; see Two-dimensional HPLC Computer-simulation software, 476–490; see also DryLab advantages and disadvantages, 479–481 application, 478–481 based on molecular structure, 491 column selectivity, 492 commercial sources, 489–490 DryLab, 481–489 examples 492–496 experimental design, 479, 481 expert systems, 492 history, 478 method development, 492–497 method robustness, 480 options, 486 peak tracking, 489 predictions of pK a , 491–492 resolution map, 476–477 robustness, 496–497 Condensation nucleation light-scattering detectors; see Detectors, light-scattering Conditional peak capacity; see Equivalent peak capacity Conductivity detectors; see Detectors, conductivity Conformation, polypeptides (RPC), 593–595 Controlled surface porosity particle, 202–203, 211 Copolymer, 648 graft, 649 random, 649 Corona-discharge detectors; see Detectors, charged-aerosol Corresponding separations isocratic vs. gradient, 409, 413–418 TLC vs. NPC, 373 Corrosion, by citrate, 316 Coulombic interactions, 32 Counter ion, ion-exchange chromatography, 351–354 Countercurrent chromatography, 11 Critical peak-pair, 55 Critical resolution, 55 Crossing isotherms, 750–751 Crownpak CR, chiral stationary phase, 706 Cyano column, 233 HILIC, 397 Cyclobond, chiral stationary phase, 679 Data processor, 127–130; see also Data systems Data systems data bunching, 501 data collection, 129, 501 data processing, 130 data rate, 129 data slices, 501 integration, 500–508 errors, 505–506; see also Error, sources peak area vs. area, 508, 529 peak recognition, 503 peak size, 508 peak skimming, 504 INDEX 897 perpendicular drop, 505 retention measurement, 507 LIMS, 128 method-development software, 128 Part 11 compliant, 130 report generation, 130 sampling rate, 129, 154–155, 501–503 signal measurement, 500–516 system control, 129 21 CFR Part 11, 130 Daughter ion, 189 Dead time, 24, 27–28 Dead volume, 26, 28 Deamidation products, polypeptides (RPC), 587 Degassing, 92–96 benefits, 814 Degradation; see Column, degradation; Sample degradation Denaturing HPLC, 621–623 Derivatization, 194; see also Sample preparation, derivatization Desalting, gel filtration, 641 Detector(s) back-pressure regulator, 150 bulk property, 151 characteristics, 149–159 charged-aerosol (CAD), 184 chemiluminescent, 174–175 chiral, 175–177, 678 condensation nucleation light-scattering, (CNLSD), 182–183 conductivity, 174 detection limits, 157–159; see also Calibration, limits drift, 153–55 electrochemical, 170–172 error sources, 509 flow cell, 150, 161 fluorescence, 167–170 quenching, 170 FTIR, 191–192 heat exchangers, 150 hyphenated, 152, 185, 191 light-scattering, 180–184 evaporative (ELSD), 181–182 laser (LLSD), 183–184 limits, 157–158; see also Calibration, limits linearity (calibration plot); see Calibration, linearity linearity(detector), 158–159 mobile-phase modification detectors, 152 MS (mass spectral), 185–191 flow rate considerations, 188 interfaces, 186–188 nitrogen; see Detector(s), chemiluminescent NMR, 192–193 noise, 153–55 overload, 727 peak identification; see Peak identification peak purity, 539 periodic maintenance, 141 preparative separation, 733–734 problems; see Troubleshooting, symptoms radioactivity, 172–173 reaction 194–196 refractive index (RI), 177–180 sample-specific, 152 selection, 66 sensitivity, 157 time constant, 153 UV, 160–167 characteristics, 166 history, 148 maintenance, 167 selectivity, 161, 165 wavelength selection, 161 visible; see Detectors, UV De-wetting; see Stationary phase, dewetting Diastereomers, 667, 669 Dielectric constant, solvent values, 881 Differential migration, 24 Diffusion coefficient, 44 Diffusive pores, 203 Dinitrobenzoyl (DNB), chiral stationary phases, 707–708 Diol column, HILIC, 397 Dipole interactions, 32, 228–229 Direct method, chiral separation, 669, 675–681 Dispersion interactions, 30 Displacement, in NPC, 366–368 Distribution coefficient, SEC, 632 DNA, 620–623, Documentation; see Validation, documentation Dorsey-Foley equation, 53 Drift baseline, 155 gradient elution, 470, 847–850 Drug product vs. drug substance, 535 898 INDEX DryLab ® software, 481–489; see also Computer-simulation software gradient optimization, 483–485 isocratic predictions from gradient data, 485–486 method development examples, 492–496 segmented gradients, 485, 488 two-run procedure, 488–489 Dwell time, 424; see also Dwell volume Dwell volume, 69, 112, 424–425; see also Gradient, dwell volume effect on reproducibility, 450–451 measurement, 134 Eddy diffusion, 40 Effective buffer capacity, 312–314 Electrochemical detectors; see Detectors, electrochemical Electrostatic interaction, 231–232 Electrostatic repulsion, 227, 228 hydrophilic-interaction chromatography (ERLIC), 614–616 Eluent, see Mobile phase Embedded-polar-group (EPG) column, 226–227, 233 Enantiomer separation; see Chiral separation Enantiomeric detectors; see Detectors, chiral End-capping, 222 Epimers, 669 Equilibration; see also Column equilibration gradient elution, 446–449 ion-pair chromatography, 347–349 NPC, 394 Equilibrium, acid-base, 304–309 Equipment; see also specific modules (pumps, detectors, etc.) column packing, 241 variation of, 69 Equivalent column, 235–236, 279–282 Equivalent peak capacity, 452 Equivalent separation; see Equivalent column; Method adjustment Error sources, 508–512 calibration; see Calibration, errors Evaporative light-scattering detector; see Detectors, light-scattering, evaporative Excipient peak, 27 Excipients, 535 Experimental design, 286–290, 479, 481 Expert systems, 492 Extra-column effects, 39–40, 42, 131 symptoms, 848, 851 Fast HPLC (fast LC), 63–65 Fast separation, gradient elution, 456–457 Fatty acids, isomer separations, 277 Filters, in-line, 247 Filtration; see specific type, Sample, Mobile phase, etc. Final %B, gradient elution, 420–422 Fischer designation, 669 Fittings, 99–104; see also Column, fittings in-line filter, 103 low-volume mixer, 103 periodic maintenance, 140 Flow rate optimum, 37 and pressure, 37 verification; see Performance tests, flow rate Fluorescence detectors; see Detectors, fluorescence Fraction collection, 123, 734–745, 747 Frit, inlet-line, 90 FTIR detector; see Detectors, FTIR Fused-core ™ particle; see Shell particle Gas chromatography (GC), 8 Gaussian peak shape, 36, 50, 503 Gel filtration, 631–641; see also Size-exclusion chromatography applications, 639–641 columns, 633–636 mobile phases, 636–637 non-ideal behavior, 636, 638 operational considerations, 637–638 Gel permeation chromatography (GPC), 651, 653; see also Size-exclusion chromatography history, 7 General elution problem, 75 Generic separation, 406 Ghost peaks; see also Artifact peaks gradient elution, 470 Global optimum, 57 Glycoproteins, 577–578 GPC; see Gel permeation chromatography Gradient elution, 75–76, 404–470 applications, 404–407, 423 artifact peaks, 442, 470 band migration, 411–412 baseline drift, 470, 847–850 biochemical separation (RPC), 585–588 INDEX 899 best practices, 418, 449 column conditions, 415–418 complex sample, 442 corresponding separations, 413–418 curved gradients, 407–408 dwell volume, 112, 424–425; see also Dwell volume problems, 450–451, 861–864 early elution, 440–441 effect of experimental conditions, 412–434 equations, 430–434 equilibration, 446–449 fast separation, 456–457 final %B, 420–422 generic separation, 406 ghost peaks, 470; see also Troubleshooting, ghost peaks gradient conditions, 418–430 gradient delay, 407–408, 422–424 gradient distortion, 860–862 gradient shape, 407–409 gradient volume and gradient performance, 860–862 high-molecular-weight samples, 406 HILIC, 467–469 IEC, 470 initial %B, 419–420 initial separation, 437–442 irregular samples, 414–415, 428–430 vs. isocratic elution, 404–405, 409–413, 437–440 large molecules, 464–465 late elution, 421, 441 linear gradients (best practices), 407–408 method development, 406, 434–463 method development outline, 435–436 mixer, 112 NPC, 466–467 optimization, 442–446 with DryLab, 483–485 peak capacity, 451–456 peak tailing, 407, 440 polypeptides (RPC), 589–593 pre-mixing mobile phase (best practice), 815 problems, 440–442, 470; see also Troubleshooting steep gradients, 860–861 program, 409 range, 407–408, 410, 444–445 regular samples, 414 reproducibility, 449–451 resolution, 434 retention factor k ∗ , 411–412 as sample preparation replacement, 406–407 segmented gradients, 407–408, 425–428, 445 selectivity, 426–428 solvent demixing, 470 starting %B, 419–420 steepness b, 410 step gradient, 407–408 temperature, 443–444 tests; see Performance tests, gradient theory, 430–434 transfer problems, 424–425, 861–864 when to use, 437–440 Graphitized carbon, 217 particle preparation, 212 Guard column, 247 Guidelines, regulatory, 534, 548 Harmonization, 532 Helium sparging, 170; see also Degassing Henderson-Hasselbalch equation, 305 Herbicides, fast separation of, 208 Hidden peak; see Peak, missing High-flow HPLC, 112 High-pressure HPLC, 112; see also U-HPLC High-pressure liquid chromatography, 8 High-priced liquid chromatography; see U-HPLC High-speed liquid chromatography, 8 HILIC, see Hydrophilic interaction chromatography Homologs, retention, 82, 260–261 Homopolymer, 648 Horv ´ ath, Csaba, 7 HPLC, 2–7 books, 13–14 characteristics, 1 growth of, 4 history, 6–8 journals, 13 layout, 88–89 method; see Test method on a chip, 12 precursors, 7 publication growth, 4 sales of, 4 separation modes, 22 900 INDEX HPLC (contd.) short courses, 13 vs. SPE, 772 Huber, Josef, 7 Human growth hormone, 426, 590 Human serum albumin (HSA), 694 Humidity, effect on NPC, 392–394 Hummel-Dreyer method, 640 Hybrid particles, 211, 223–224 Hydrodynamic chromatography, 654 Hydrodynamic diameter, 580 in SEC, 633, 634 Hydrofluoric acid cleavage, for ligand characterization, 222 Hydrogen bonding interactions, 32, 231 Hydrogen-bond acidity, solvent values, 881 Hydrogen-bond basicity, solvent values, 881 Hydrophilic interaction chromatography (HILIC), 395–401, 613–614 adsorption vs. partition, 396–397 advantages, 395 carbohydrates, 625–626 columns, 397–398 gradient elution, 467–469 method development, 398–400 problems, 401 retention, 396–397 solvent-type selectivity, 399–400 Hydrophobic interaction chromatography (HIC), 608–612 antichaotropic salt, 610–611 columns, 609–610 nucleic acids, 624 pH, 611 surfactants, 611–612 temperature, 612 Hydrophobic interaction, 31, 230–231 Hydrophobic interferences, 387 Hydrophobic-subtraction model, 229–232 Hydroxyapatite chromatography, polypeptides, 604–605 Hydroxytestosterone isomers, 276 Hyphenated detectors; see Detectors, hyphenated IEC; see Ion-exchange chromatography Immobilized-metal affinity chromatography, 605–608 Indirect method, chiral separation, 669, 670–675 Infrared detector; see Detectors, FTIR Initial conditions; see Starting conditions Injection solvent, 121–122 Injection valves; see Injectors Injection volume, 120 problems, 70–71, 852 Injectors, 112–118; see also Autosamplers accuracy, 115 filled-loop, 114, 117, 118 laminar flow, 115 operation, 114 partial-loop, 115, 118 Inlet-line frit, 90 In-line filter, 103 Inorganic particles, 214–217 Installation qualification; see Performance tests Insulin, production-scale separation, 642–648 Integrators; see Data systems Interactions charge transfer, 33 column, 227–228 dipole, 32 dispersion, 30 hydrogen bonding, 32 hydrophobic, 31 ionic, 32 molecular, 30–35 pi-pi, 33 Internet resources, 13 Interstitial volume, 201, 632 Ion chromatography, 12, 349–350 detection, 174 Ion pairing, buffer, 315–316 Ion suppression, LCMS, 801 Ion trap detectors; see Detectors, MS Ion-exchange chromatography (IEC), 349–357, 597–607; see also Nucleic acids; Carbohydrates Ion-exchange chromatography applications, 349–351 columns, 354, 600–601 counter-ion, 351–354 gradient elution, 470 method development, 355 mixed-mode, 355–357 pH effect, 354 retention, 351–352 Ionic interactions, 32 Ionic samples IEC, 349–356 IPC, 331–349 method development, 327–331 INDEX 901 problems, 329–331 RPC, 319–327 Ionization effect of %B, 318–319 effect of temperature, 319 sample, 304–309 Ion-moderated partition chromatography, 626–628 Ion-pair chromatography (IPC), 331–349 advantages, 332–334 artifact peaks, 347 buffer effect, 345–346 chaotropes, 343 column type, 345–346 ion-pair reagent, 334–339 method development, 339–347 peak tailing, 349 pH effect, 334–339 problems, 347–349 retention, 334–339 selectivity, 343–346 slow equilibration, 347–349 solvent strength, 344 solvent type, 344 temperature, 345 when to use (best practice), 332 Ion-pair reagent, 332 concentration effect, 336–337 effect on separation, 334–339 type effect, 336–337 Irregular sample, 60–61, 265–267, 428–430 gradient elution, 414–415 Isocratic elution, 20 prediction from gradient run, 437–440 Isomer separations NPC, 365–366, 382–385 RPC, 276–278 temperature dependence, 276 Isomerism, chiral, 667 Isomers, constitutional, 667 Isotherms crossing, 750–751 sorption, 737–738 Journals, HPLC, 13 Junk peak, (excipient peak), 27 Kinetic plot, 47–48 Knox equation, 43 Laminar flow, 115 Large-molecule separations, 570–658 Laser light-scattering detectors; see Detectors, light-scattering Late elution, gradient elution, 421 LC x LC; see Two-dimensional HPLC LC-MS detectors, 185–191 ion suppression, 801 Ligand; see also Stationary phase alkyl, 226–227 characterization after cleavage with hydrofluoric acid, 222 cyano, 226 embedded-polar-group column, 226–227 phenyl, 226 type, 225–227 Light-scattering detectors; see Detectors, light-scattering Limit of detection, see LOD Limit of quantification, see LLOQ Limits; see Calibration, limits LIMS; see Data systems, LIMS Linearity, 540 Linearity, detector, 158 Linear-solvent-strength (LSS) model, 409–411 LLOQ, 540; see also Detectors, limits Local optimum, 57 Localized adsorption, 367–368, 378–380 LOD, 539; see also Detectors, limits Longitudinal diffusion, 40 LOQ; see LLOQ Lower limit of quantification; see LLOQ Low-flow HPLC, 112 Macrocyclic antibiotics, chiral stationary phases, 699–705 Maintenance, 131–138; see also Repairs periodic, 812; see also Preventive maintenance Map, resolution; see Resolution, map Martin equation, 82 Martin, Archer (A. J. P.), 7 Mass overload, 71–73, 726, 746 Mass spectral detectors; see Detectors, MS Mass transfer, mobile phase, 40 Mechanically held polymers, 223 Mercury intrusion, 201 Method (routine); see Test method Method adjustment, 279–282, 534, 561–564; see also Method modification 902 INDEX Method adjustment (contd.) best practices, 562 buffer, 563 column size, 564 detector wavelength, 564 mobile phase, 563 non-equivalent columns, 282 particle size, 564 pH, 563 temperature, 564 Method change; see Method modification; Method adjustment Method development, 65–69, 284–295 column conditions, 61–62 column conditions (gradient), 445–446 computer assisted, 475–497 gradient elution, 434–463 gradient range, 444–445 gradient steepness and k∗, 442 HILIC, 398–400 IEC, 355 initial gradient separation, 437–442 ionic samples, 327–331 IPC, 339–347 neutral samples, 284–295 NPC, 385–392 polypeptides (IEC), 597–603 polypeptides (RPC), 595–597 preparative separation, 745–747 segmented gradients, 445 selectivity (NPC), 389–390 selectivity in gradient elution, 442–444 selectivity, 328–329 starting conditions, 327–328 strategy, 284–286 using gradient elution, 406 Method modification, 534, 561–564; see also Method adjustment Method performance precision; see Precision robustness; see Robustness specificity; see Specificity Method robustness, see Robustness Method transfer; see also Analytical method transfer gradient elution, 450 Methods bioanalytical, see Bioanalytical methods category 1, 546 category 2, 547 category 3, 547 category 4, 548 cleaning validation, 547 content uniformity, 546 degradants, 547 dissolution, 547 identification tests, 548 impurities, 547 limit tests, 547 potency, 546 product performance characteristics, 547 quantitative tests, 547 validation, see Validation Micellar liquid chromatography, 12 Micro HPLC, 112, 170 Micro-column chromatography, 12 Mixer, low-volume, 103 Mixing; see also Mobile phase, mixing gradient, 112 high-pressure, 109 hybrid systems, 111 low-pressure, 111 on-line, 109–111 Mobile phase, 20; see also Solvent best practices, 312, 141 degassing, 92 filtration, 89, 91–92 optimum pH, 307–308 recycling, 125 reduced velocity, 44 reservoirs, 90 selectivity; see Selectivity; Solvent selectivity solvents for RPC, 254 pre-mixing, 109, 815 velocity, 26 Mobile-phase-additive mode, chiral separation, 675–677 Modes (HPLC separation modes), 22 Molecular structure, retention predictions from, 80–83, 491 Molecular weight, biopolymers, 633, 637 Molecular weight, synthetic polymers average, 653–654 determination, 632–633 distribution, 651–654 Monolayer, adsorbed, 366, 737 Monolith(s), 200, 212–214 advantages and disadvantages, 214 silica based, 213 Monomeric column; see Stationary phase, monomeric MS detectors; see Detectors, MS INDEX 903 Multi-angle light-scattering detectors (MALS); see Detectors, light-scattering Multidimensional liquid chromatography, 616–618; see also Two-dimensional chromatography column switching, 618 directly coupled, 617–618 discontinuous, 617 polypeptides, 616–618 Multi-variable optimization, neutral samples, 286–295 Myoglobin tryptic digest, 592 Nano HPLC, 112 Nanospray ionization sources, polypeptides (RPC), 595 Naphthalenes, substituted, 289 NARP; see Non-aqueous RPC Nernst distribution law, 766 New-column test; see Performance tests, new column test Nitrogen detector; see Detectors, chemiluminescent Nitro-substituted benzenes, 258, 264 NMR detector; see Detectors, NMR Noise, 153–155; see also Troubleshooting, symptoms, Nomograph; see Solvent nomograph Non-aqueous reversed-phase (NARP), 182, 295–297 Non-localizing solvents, 378–380 Normal-phase chromatography (NPC), 362–401 applications, 362 column selectivity, 381–382 example (Paclitaxel), 390–392 gradient elution, 466–467 isomers, 365–366, 382–385 method development, 385–392 problems, 392–395 reproducibility, 392–394 retention, 363–370 vs. RPC, 363–366 selectivity, 376–385 solvent demixing, 394–395 solvent nomograph, 371–373 solvent strength, 370–373 solvent-strength selectivity, 376 solvent-type selectivity, 376–380 starting conditions, 387–389 tailing peaks, 394–395 temperature, 380–381 Nucleic acids, 574–576, 618–624 anion exchange, 619–620 double stranded, 575–576 hydrophobic interaction chromatography, 624 RPC, 620–624 sequence failures, 619 single stranded, 574–575 transfer (tRNAs), 619, 620 Oligomers (synthetic), 648 Oligonucleotides, 621; see also Nucleic acids Oligosaccharides; see Carbohydrates Operational qualification; see Performance tests o-Phthaldialdehyde, 672 Optical isomers separation; see Chiral separation Optical rotary dispersion detectors, 175–177 Optimization, 57; see also Method development; Retention; Selectivity; Plate Number; Column conditions; Multi-variable optimization Orthogonal separation, 68, 236–237, 282–284, 386–387 Ovens; see Column, ovens Overload, 70–73 column; see Column, overload Ovomucoid (OVM), chiral stationary phase, 693 Oxygen, problems with dissolved, 93 Packing methods, column, 240–244 PAH; see Polycyclic aromatic hydrocarbon Paper chromatography, 2, 7 Parent ion, 189 Particle; see also Column characterization, 201 configurations; see Particle, type diameter, 201, 203 electron micrographs, 206, 208 for highly aqueous mobile phases, 224 hybrid, 211 pellicular, 65, 201–202 pore diameter, 201 preparation, 211–212 shell, 65, 202–203 silica sol aggregation, 211 size distribution, 201, 203, 205–207 sol-gel preparation, 211 904 INDEX Particle; see also Column (contd.) spherical vs. irregular, 205 spray drying, 211 superficially porous; see Particle, shell surface area, 201 totally porous, 201 type, 201–203 Peak, 24; see also Band area measurement, 500–516 area reproducibility; see Performance tests, peak-area reproducibility hidden, 68 missing, 236, 282–283 overlooked, 68 shape problems; see Troubleshooting, symptoms; Peak tailing size, 508 split, 247 tailing; see Peak tailing Peak capacity, 76–77, 451–456 optimized, 453–456 polypeptides, 616 two-dimensional HPLC, 461 Peak fronting, 52 Peak height, vs. retention, 57 Peak identification, 516–519; see also specific detector types by co-injection, 517 method of standard addition, 517 off-line analysis, 519 by on-line qualitative analysis, 517 by retention time, 516 UV spectra, 518 Peak matching, 77–78, 489 Peak shape, 50–54 Gaussian, 36 Peak tailing, 51, 246–247, 298, 330–331, 854–856 anticipation of, 237 buffer inadequate, 312–314 causes, 52, 237, 854–856 exponential, 52 ion-pair chromatography, 349 overload, 52, 69–73, 739–740 Peak tracking, 77–78, 489 Peak width, 35–54 detector contribution, 158 preparative separation, 738–739 vs. sample weight, 738–739 Pellicular particle, 65, 201–202 Peptides, see Polypeptides Performance qualification; see Performance tests Performance tests, 131–138 flow rate, 135 gradient, 132–135 linearity, 132 proportioning valve test (GPV), 135 step-test, 134 installation qualification (IQ), 132 new column test, 138 operational qualification (OQ), 132 peak area reproducibility, 138 performance qualification (PQ), 132 pressure bleed-down, 136 problems, 856–865; see also Troubleshooting, performance tests retention reproducibility, 137 Perfusion particle, 203 Periodic maintenance, 138 Pesticide sample, 773, 775 Pfeiffer’s rule, 680 pH; see also Ionization, sample; Equilibrium, acid-base; Mobile phase pH ion-exchange chromatography, 354 ion-pair chromatography, 334–339 meter, use of, 309–310 sensitivity, 329 Pharmaceutical mixture, 492–496 Phase ratio, 26 and surface area, 201 Phase-optimized liquid chromatography, 294 Phenyl columns, 226, 233 Phosphate buffer, 312–317 Phosphopeptides, immobilized-metal affinity chromatography, 606 Phosphoproteins, immobilized-metal affinity chromatography, 606 Physicochemical data for particles, 244 pi - pi (π - π) interactions, 33, 228,229 Pirkle-type chiral stationary phase, 707–711 pK a amino acids, 571–572 buffer, 311–314 carbohydrates, 628 estimation of, 308–309 predictions from solute structure, 491–492 solute, 307–309, 317–318 Plate height, 37–45 reduced, 44 INDEX 905 Plate number, 35–54; see also Column efficiency; Peak width approximate, 246 chiral columns, 671, 681 and experimental conditions, 40 optimization, 61–65 well-packed column, 246 Polysaccharide-based chiral stationary phases, 682–689 Polar endcapped columns, 233, 237 Polar-bonded-phase columns, 362–363 Polarimeters; see Detectors, chiral Polarity, solvent (P  ), 33–34, 880–881 Polycyclic aromatic hydrocarbons separation of, 234 shape selectivity, 278 temperature dependence, 273 Polymer (synthetic); see Synthetic polymers Polymerase chain reaction (PCR), RPC, 621 Polymeric particles, 212 Polymeric stationary phase, 218–219, 221–222, 232–233 Polypeptide-bonded column, HILIC, 397 Polypeptides, 571–574 capillary columns, 595 chromatofocusing, 603–604 columns (IEC), 599–601 columns (RPC). 585 conformation effect, 593–595 ERLIC, 614–616 gradient elution (RPC), 589–593 HILIC applications, 614 hydrophilic interaction chromatography (HILIC), 613–614 hydrophobic interaction chromatography (HIC), 608–612 hydroxyapatite chromatography, 604–605 immobilized-metal affinity chromatography, 605–608 method development (IEC), 597–603 method development (RPC), 595–597 mixed-mode retention (RPC), 593 mobile phases (IEC), 601–603 mobile phases (RPC), 585–588 molecular weight effect, 589–591 multidimensional liquid chromatography, 616–618 post-translational modification, 574 preparative separation, 641–648 primary sequence, 571–572 quaternary structure, 574 retention vs. pH (IEC), 598–599 RPC, 584–597 secondary structure, 573 separation, 584–618 starting conditions (RPC), 596 surfactants (RPC), 588 temperature effects, 588–589, 593–595 tertiary structure, 574 Polyphosphates, ion-exchange chromatography of, 351–352 Poor retention gradient elution, 440–441 RPC, 331 Poppe plot, 47–48 Pore diameter, 201 Pore size columns for biochromatography, 579–581 SEC columns, 635 Pore volume, 632 Porous polymers, 212 Post-translational modification polypeptide, 574 polypeptide variants (RPC), 587 Precision, 508, 536–538 bioanalytical methods, 550 intermediate, 537 intra-assay, 536 repeatability, 536 reproducibility, 537 ruggedness, 538 and signal-to-noise, 156 Precursor ion, 189 Preparative separation, 112, 726–755; see also Column, overload applications, 726 column diameter, 728 column saturation capacity, 737 columns, 730–731 crossing isotherms, 750–751 detectors, 733–734 equipment, 730–736 fraction collection, 734–745, 747 gradient elution, 751–754 initial conditions, 745 isocratic elution, 736–747 isocratic vs. gradient elution, 752 mass overload, 726, 746 method development, 745–747 mobile phase, 743 optimized conditions, 728 peak width, 738–739 [...]... 392– 395 troubleshooting tables, 865–888 Product ion, 189 Production-scale separation, rh-insulin, 642–648 Protein; see also Polypeptides aggregation, gel filtration, 640 contact area, 583 folding, gel filtration, 639–640 Regnier rules, 583–584 structure vs chromatography, 583–584 Proteomics, multidimensional liquid chromatography, 616–618 Protocol; see Validation, protocol Pseudocritical chromatography,. .. 655 interactive liquid chromatography, 653–655 molecular structure vs chromatography, 650–651 structures, 648–649 two-dimensional chromatography, 657–658 System controller; see Data systems System maintenance; see Maintenance System suitability, 69, 542–543, 813 Tacticity, 649 Tailing factor, 51 Tailing peaks; see also Peak tailing gradient elution, 440 NPC, 394– 395 Tandem MS; see Detectors, MS Temperature... troubleshooting , 817 Thermally-tuned tandem-column, 294 Thin-layer chromatography, 2, 373–376 RF values, 373–375 Three-point interaction model, chiral separation, 678, 679,680 Through-pores, 203 Time-of-flight detector, see Detector(s), MS Titania particles, 217 TOF detector; see Detectors, MS, time-of-flight Touching-peak separation, 727, 739–748 Toxicology standards, 405 INDEX Trace analysis, 73–74, 529 Triazine... guidance, 534, 543 Reviews, HPLC, 13 RI detectors; see Detectors, refractive index RI values, solvent, 879–880 907 Ribonuclease, conformational effects (RPC), 593– 595 Ristocetin A, chiral stationary phase, 699 Robustness, 540–542 computer-simulation, 480 lack of, 68 RPC-5 chromatography, 623–624 Rule of one, 820 Rule of2.5, 58–59, 121 Run time, minimizing 295 Safety, 819, 883 Sample(s); see also Solute... techniques, 800 biochromatography, 797–800 column switching, 796–797 derivatization, 194, 802–804 dialysis, 800 dilute and shoot, 760 drying, 762–763 electrodialysis, 800 filtration, 763–764 ion-exchange, 802 LC-MS samples, 800–802 liquid samples, 764 liquid- liquid extraction (LLE), 764–771 counter-current distribution, 768 emulsions, 769 extraction efficiency, 766 flowchart, 765 immobilized liquid extraction... UV detectors; see Detectors, UV UV-visible detectors; see Detectors, UV Validation, 69 bioanalytical methods, 548–554 cleaning, 547 documentation, 543–546 overview, 532–534 pharmaceutical methods, 546–548 regulatory guidelines, 533 terms and definitions, 534–542 Valves, switching, 122 van Deemter equation, 43 Vancomycin chiral stationary phases, 699, 702 Viruses, 578–579, 630–631 Viscosity acetonitrile-water... Segmented gradients, 422–428, 445 Selectivity, 55, 263–284 amine modifiers, 327 buffer concentration, 327 buffer type, 326–327 column, 293– 295, 323–325, 345–346 gradient elution, 442–444 ion exchange chromatography, 352–355 ionic samples, 320–327 ion-pair chromatography, 343–346 isomer, 276–278 NPC, 376–385 optimization, 59–61 pH, 320–322 RPC, 263–284, 320–327 shape, 232–235 solvent-strength, 322–323... 231 Steric interaction, 232–235 Sterically protected stationary phase, 220–221 Storage, column, 249 Strong solvent, 29 Substituted anilines, 364–366 Substituted naphthalenes, 289 Supercritical fluid chromatography, 10 Superficially porous particle, 202–203, 211 Support, 200 Surface area, 201 Surrogate biopolymer, 583 Symptoms, troubleshooting tables, 865–888 Synthetic polymers, 648–658 analysis, 651–653... Data systems, integration Retention factor, 25–28 Retention factor, desired range, 57 gradient; see Gradient, retention factor k∗ optimizing, 57 small values, 297–298 Retention range, narrowing of, 332–333 Retinol isomers, 383–385 Reversed-phase chromatography (RPC), 20, 253–355; see also specific items (e.g., Columns; RPC normally assumed) advantages, 254 history, 255–256 nucleic acids, 620–624 polypeptides,... identification; Detectors Quaternary ammonium salts for ion-pairing, 340–342 Quaternary structure, polypeptide, 574 Quick fix, 809–810, 865–888 Radial-compression column, 239 Radioactivity detectors, 172–173 Range gradient, 407–408, 444–445 method, 540 Reaction detectors, 194–196 Reciprocity principle of chiral recognition, 707 Reduced parameters, 44 Refractive index detectors; see Detectors, refractive . U-HPLC High-pressure liquid chromatography, 8 High-priced liquid chromatography; see U-HPLC High-speed liquid chromatography, 8 HILIC, see Hydrophilic interaction chromatography Homologs, retention,. rules, 583–584 structure vs. chromatography, 583–584 Proteomics, multidimensional liquid chromatography, 616–618 Protocol; see Validation, protocol Pseudocritical chromatography, 654 Pump(s), 104–113 active. 229–232 Hydroxyapatite chromatography, polypeptides, 604–605 Hydroxytestosterone isomers, 276 Hyphenated detectors; see Detectors, hyphenated IEC; see Ion-exchange chromatography Immobilized-metal affinity chromatography, 605–608 Indirect