DryLab® software, 481–489; see alsoComputer-simulation software gradient optimization, 483–485 isocratic predictions from gradient data, 485–486 method development examples, 492–496 segm
Trang 1Column-comparison function
Fs, 236
Fs (-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 pKa, 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
Trang 2perpendicular 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
Trang 3DryLab® 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
Trang 4best 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
Trang 5HPLC (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
Trang 6problems, 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
Trang 7Method 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
Trang 8Multi-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
Trang 9Particle; 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
pKa 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
Trang 10Plate 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