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,

, 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

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

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

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

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

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

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

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-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

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

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

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

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

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

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

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

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

Primary sequence, polypeptide, 571–572
Priming valve, 108
Problems, 297–298 ; see also specific
problems; Troubleshooting
gradient elution, 470
HILIC, 401
ionic samples, 329–331
NPC, 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, 654
Pump(s), 104–113
active check valves, 109
check valves, 106
periodic maintenance (best practice), 140
priming valve, 108
purge valve, 108
reciprocating-piston, 104–109
seal replacement, 823–824

Regnier rules, 583–584
Regular sample, 60–61, 265–267
gradient elution, 414
Relative retention; see Selectivity
Repairs,
143
personnel, 143, 144
record keeping, 143, 144
system configuration record, 143
Reproducibility
column, 68
gradient elution, 449–451
gradient method development, 449–450
NPC, 392–394
routine gradient assays, 450–451
Reservoir, 89–91
frit, 90
periodic maintenance, 138
Resolution map, 271–273
Resolution, 54–57
INDEX 907
critical, 55
equations, 54, 55
gradient elution, 434
map, 271–273
vs. retention, 57–59
selectivity, see Selectivity
size-exclusion chromatography, 636
Restricted diffusion, 203
Retention, 24–35, 256–263

assumed)
advantages, 254
history, 255–256
nucleic acids, 620–624
polypeptides, 584–597
preferred conditions (isocratic), 255
preferred solvents, 254
retention process, 259–263
Reviewer guidance, 534, 543
Reviews, HPLC, 13
RI detectors; see Detectors, refractive index
RI values, solvent, 879–880
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, minimizing295
Safety, 819, 883
Sample(s); see also Solute
irregular; see Irregular sample
non-ionic, 254–298
regular; see Regular sample
Sample degradation, 855–856
Sample enrichment, 122
Sample injection, preparative separation,

solvent selection, 767
special approaches, 770–771
theory, 766
membrane techniques, 790
908 INDEX
Sample preparation (contd.)
overview, 758–759
particle-size reduction, 760, 762
precipitation, 802
preliminary processing, 760–764
restricted access media (RAM), 802
solid samples, 791–796; see also
Samples, solid
solid-phase extraction; see Solid-phase
extraction
turbulent-flow, 802
types of samples, 759–760
ultrafiltration (UF), 800
Sample pretreatment; see Sample
preparation
Sample size, 69–74; see also Preparative
separations
problems, 73–74
size effects, 119–121
Saturation capacity, column, 740–742
Scale-up, 728
Secondary equilibria, 78–79
Secondary structure, polypeptide, 573
Segmented gradients, 422–428,
445

conditions, choosing, 67
large molecules, 570–658
modes (HPLC separation modes), 22
orthogonal, 68, 282–284
process, 20–24
Sequence failures, nucleic acids, 619
Service contracts, 143
SFC; see Supercritical fluid chromatography
Shape selectivity, 232–235, 277–278
Shell particles, 202–203, 211
Short courses, HPLC, 13
Signal measurement, 500–516
Signal-to-noise, 155–156
error contribution, 512
improving, 156
and precision, 156
Silanol, 208–209
acidity, 209–210, 231
effect on separation, 330–331
endcapping, 222
ionization, 210
Silica
acidity, 210
advantages (NPC), 362
column, HILIC. 397–398
column, use with organic buffers, 249
comparison with other metal oxides, 215
contaminating metals, 209
dissolution, 248–249
measurement of metal contaminants, 209

pressurized solvent extraction (PSE), 795
shake-flask method, 793
solid samples, supercritical fluid
extraction (SFE), 794–795
sonication, 793
Soxhlet extraction, 791–794
Solid-phase extraction (SPE), 771–790
advantages and disadvantages, 771
apparatus, 777–778
automation, 778
cartridges, 774
class-specific cartridges, 789
coated fibers (SPME), 776
conditioning, 779–780
derivatization in-situ, 774
desalting, 774
devices, 774–776
disks, 775
dispersive SPE, 789–790
elution, 781
enrichment, 773
example, 784–786
vs. HPLC, 772
immunoaffinity extraction, 788–789
interference removal, 772–773
loading, 780
method development, 778–785
micropipette tip, 776
mixed-mode, 781
molecular-imprinted polymers (MIPs),

nomograph (NPC), 371–373
nomograph (RPC), 269–270
non-basic (NPC), 372, 379
non-localizing (NPC), 372, 378–380
polarity values (P

), 33–34, 880–881
preferred, 254, 287–288
properties, 877–883
RI values, 879–880
safety, 883
selectivity triangle, 33–34
selectivity values, 880–881
strength, 28–29, 257–259
ion-pair chromatography, 344
NPC, 370–373
parameter ε, 370–373
prediction of, 258
strong, 29
UV absorbance, 877–879
viscosity values, 880
water-saturated, 392–393
weak, 28
Solvent-selectivity triangle, 288
Solvent-strength selectivity; see Selectivity,
solvent-strength
SPE; see Solid-phase extraction (SPE)
Specifications, column, 244–246
Specificity, 539
comparison to known materials, 539

steric protected, 220–221
synthesis; see Stationary phase,
preparation of
Step-test; see Performance tests, gradient,
step-test
Steric exclusion, 227, 228, 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
chemical composition vs. molecular
weight, 656–657
critical conditions, 655
interactive liquid chromatography,
653–655
molecular structure vs. chromatography,
650–651
structures, 648–649
two-dimensional chromatography,

stability-indicating, 535
synonyms, 532
Tetraalkyl-o-silicate, 211
TFA (trifluoroacetic acid)
concentration effects, 848
ion pairing with polypeptides, 587
polypeptides (RPC), 586
troubleshooting
, 817
Thermally-tuned tandem-column, 294
Thin-layer chromatography, 2, 373–376
R
F
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 911
Trace analysis, 73–74, 529
Triazine herbicide sample, 788
Tricarboxymethylethylenediamine (TED),
607
Trifluoroacetic acid; see TFA
Triple-quadrupole detector, see Detectors,

module substitution, 820
new-column test, 819–820
performance tests, 811–812, 856–865
flow-rate problems, 864
GPV test failure, 858–859
gradient linearity, 857–861
gradient, 812
gradient test failure, 857–864
IQ, OQ, PQ, 812
other, 812
pressure bleed-down problems, 864
retention-time reproducibility,
864–865
step-test failure, 857–859
prioritizing tests, 820
problem isolation, 819–821
problem prevention, 811–819
problem symptoms; see Troubleshooting,
symptoms
quick fix, 812–813
records, 813–814
retention-time, changes, 836–838
siphon test, 90–91, 814
stationary-phase problems, 835
symptoms; see Troubleshooting
symptoms
system suitability, 813
tables, 865–888
TFA, 817
tips and techniques, 814–819

peak-area problems, 838–841
peak-area ratios changing, 854–855
peak fronting, 849–851
peak shape, 847–856
peak splitting, 850, 852–854
peak tailing, 847–849
pressure problems 830–834
pump seal problems, 823–824
random noise, 844
912 INDEX
Troubleshooting symptoms (contd.)
retention problems, 833–838, 854–855
spikes in baseline, 845
temperature changes, 844
temperature problems, 836
Tswett, Mikhail, 7
Tubing, 96–99
diameters, 98
high-pressure, 97–99
length, 99
low-pressure, 96–97
PEEK, 97
periodic maintenance, 140
stainless steel, 97
21 CFR Part 11,506–507
Two-dimensional HPLC, 76–77, 457–464,
657–658; see also Multidimensional
liquid chromatography
equipment, 461–462
examples, 460

Viscosity
acetonitrile-water mixtures vs.
temperature, 882
effect on diffusion, 44
effect on pressure, 38–39
methanol-water mixtures vs.
temperature, 882
solvent values, 880
Vitamins, water soluble, 342–343
Waste container, periodic maintenance, 141
Waste diversion, 124
Water sample, 193, 773, 775
Water-saturated solvents, 392–393
Weak solvent, 28
WHELK-O1 chiral stationary phase, 708
Wilke-Chang equation, 44
Zirconia particles, 215–217
Zwitterion; see Solute, amphoteric