886 PROPERTIES OF HPLC SOLVENTS are rarely ingested; rather, their primary effect is by contact or inhalation. Rubbing alcohol (i-propanol) with an LD 50 of 400 is clearly not a problem in terms of either contact or inhalation. Material Safety Data Sheets (MSDS) should be consulted before handling any solvent or reagent. REFERENCES 1. J. B. Li, LCGC, 10 (1992) 856. 2. High-Purity Solvent Guide, Burdick & Jackson Laboratories, Muskegon, MI, 1980. 3. J. A. Riddick and W. B. Bunger, Organic Solvents, Wiley-Interscience, New York, 1970. 4. L. R. Snyder, in High-performance Liquid Chromatography: Advances and Perspectives, Vol. 3, C. Horv ¨ ath, ed., Academic Press, New York, 1983, p. 157. 5. K. Valko, L. R. Snyder and J. L. Glajch, J. Chromatogr., 656 (1993) 501. 6. L. R. Snyder, J. Chromatogr. Sci., 16 (1978) 223. 7. H. Colin, J. C. Diez-Masa, G. Guiochon, T. Czajkowska, and I. Miedziak, J. Chromatogr., 167 (1978) 41. 8. M. A. Quarry, R. L. Grob, and L. R. Snyder, J. Chromatogr., 285 (1984) 1. 9. J. Billen, K. Broeckhoven, A Liekens, K. Choikhet, G. Rozing, and G. Desmet, J. Chromatogr. A, 1210 (2008) 30. APPENDIX II PREPARING BUFFERED MOBILE PHASES II.1 SEQUENCE OF OPERATIONS Buffered mobile phases can be prepared by the following sequence of operations: 1. combine the buffer ingredients with water to obtain the aqueous buffer (solution A) 2. confirm or adjust the pH of solution A with a pH meter 3. combine a given volume (e.g., 200 mL) of organic buffer (solution B) with a given volume (e.g., 800 mL) of solution A from step 2 to obtain the final mobile phase (20% organic buffer in this example) 4. check the pH of the final mobile phase (optional) Because a pH measurement for a mobile phase that contains organic buffer is unreliable due to drift of the pH meter, step 4 is only useful for detecting major errors in the formulation or comparing two solutions with the same organic content. Most laboratories elect to skip step 4. The usual approach in step 1 is to formulate aqueous buffers of differing pH (A1 and A2), and then combine these two solutions in the correct proportions to obtain final solution A with the desired pH. If the pH is adjusted in step 2, the same two starting solutions can be used to titrate the final buffer to the desired pH as measured by the pH meter. The precision of a pH measurement (step 2) in most laboratories is usually no better than ±0.05 to 0.10 pH unit, which can cause significant changes in resolution for some samples (Section 7.3.4.1). When an HPLC method is pH sensitive, step 2 should be used only for an approximate confirmation of pH. By combining accurate weights of the buffer ingredients with accurate volumes of distilled and degassed water (without further adjusting pH), the pH of the buffer solution can be controlled within narrow limits (±0.02 unit). Buffer concentrations whose pH is known quite accurately are also commercially available. Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R. Snyder, Joseph J. Kirkland, and John W. Dolan Copyright © 2010 John Wiley & Sons, Inc. 887 888 PREPARING BUFFERED MOBILE PHASES Table II.1 Preparation of Low-pH Phosphate Buffers of Defined pH Required pH Volume (mL) of A1 a Volume (mL) of A b 2.0 565 435 2.2 455 545 2.4 345 655 2.6 250 750 2.8 175 825 3.0 110 890 3.2 55 945 a Solution of 0.1 M phosphoric acid; the phosphoric acid used to prepare this stock solution must be titrated to confirm the amount of phosphoric acid present. b Solution of 0.1 M sodium monophosphate; combine 13.8 g of NaH 2 PO 4 monohydrate with water in a 1-L flask. It is common practice to adjust the buffer pH with a concentrated acid. For example, solution A2 of Table II.1 might be prepared and titrated to the desired pH with concentrated phosphoric acid. This still produces a buffer at the desired pH, but the ionic strength of the buffer will be higher than if equimolar solutions of A1 and A2 are blended. While it is unlikely to make much difference in the chromatographic results obtained by the two techniques (titrating with concentrated acid vs. equimolar blending) for RPC, some separations can be sensitive to differences in ionic strength (especially ion exchange). It is best to describe in the method documentation exactly how a buffer is to be prepared, and to follow these directions—consistency in mobile-phase preparation is generally important and will give more reliable results. Acidic or basic additives are sometimes added to the mobile phase for various purposes. When such additives are not used as the primary buffer, they should be added to the desired quantity (concentration) of the buffer first; the mixture should then be adjusted to the desired pH by titrating with acid or base. II.2 RECIPES FOR SOME COMMONLY USED BUFFERS The pH of a buffered solution remains approximately constant as the buffer is diluted or concentrated, or when one ionized cation (e.g., Na + ,K + ) or anion (e.g., Cl − ,Br − ) is replaced by another. Tables II.1 to II.3 describe the preparation of some buffers that are commonly used in RPC (adapted from [1])—using the mixing of two solutions A1 and A2, each of which have an equal concentration of the buffering species. The specified volumes of solutions A1 and A2 are combined and mixed to give the final buffer solution of a required pH. The formulations of Tables II.1 to II.3 are based on a final buffer concentration of 0.1M and sodium as cation. Formulations for other buffer concentrations and/or the use of different cations (K + is usually preferred) can be inferred from these data. The pH of buffers that are more dilute or more concentrated, or that contain different cations, may differ slightly II.2 RECIPES FOR SOME COMMONLY USED BUFFERS 889 Table II.2 Preparation of Acetate Buffers of Defined pH Required pH Volume (mL) of A1 a Volume (mL) of A2 b 3.6 926 74 3.8 880 120 4.0 820 180 4.2 736 264 4.4 610 390 4.6 510 490 4.8 400 600 5.0 296 704 5.2 210 790 5.4 176 824 5.6 96 904 a Solution of 0.1 M acetic acid; combine 6.0 g (5.8 mL) of glacial acetic acid with water in a 1-L flask. b Solution of 0.1 M sodium acetate; combine 8.2 g of sodium acetate (or 13.6 g sodium acetate trihydrate) with water in a 1-L flask. Table II.3 Preparation of Intermediate-pH Phosphate Buffers of Defined pH Required pH Volume (mL) of A1 a Volume (mL) of A2 b 5.6 948 52 5.8 920 80 6.0 877 123 6.2 815 185 6.4 735 265 6.6 685 315 6.8 510 490 7.0 390 610 7.2 280 720 7.4 190 810 7.6 130 870 7.8 85 915 8.0 53 947 a solution of 0.1 M monobasic sodium monophosphate; combine 13.8 g of monobasic sodium monophos- phate monohydrate with water in a 1-L flask. b solution of 0.1 M dibasic sodium phosphate; combine 26.8 g of Na 2 HPO 4 .7H 2 O with water in a 1-L flask. 890 PREPARING BUFFERED MOBILE PHASES from these values. The exact pH value of the mobile phase is usually unimportant in method development. What is important is that the final pH of the mobile phase can be reproduced (preferably within ±0.02 unit) each time a new batch of mobile phase is prepared. Note that solutions only buffer effectively ±1 pH unit from the pK a value of the ionizable constituent (Section 7.2.1). Although the mobile phase may be used at a temperature other than ambient, the pH at ambient is assumed for the buffers of Tables II.1 to II.3 and should be used to describe the final mobile phase. As an alternative to Tables II.1 to II.3 as guides for buffer preparation, many on-line buffer calculators are available (search for ‘‘HPLC buffer calculator’’) that provide for the use of several additional buffers. For example, one such calculator (‘‘The Buffer Wizard,’’ Zirchrom, Anoka, MN, www.zirchrom.com) provides buffer preparation instructions. Input the acid, base, desired buffer concentration, and pH, and the calculator provides instructions for preparation, along with warnings about buffer capacity, column stability, and so forth. REFERENCE 1. G. Gomori, in Meth. Enzymology, S. P. Colowicxk and N. O. Kaplan, eds., Academic Press, New York, 1955, p. 145. INDEX Accuracy, 508, 535; see also specific method type Acidic glycoprotein (AGP), chiral stationary phase, 693–694 Active pharmaceutical ingredient (API), 535 Adsorption chromatography; see Normal-phase chromatography Albuterol sample, 784–786 Alkyl groups, separation by RPC vs. NPC, 365 Alkyl sulfonates for ion-pairing, 340–342 Alkylsilica columns, 226–227; see also Column Alumina column packing, 215, 217 Amide column, HILIC, 397 Amine modifiers, RPC, 327 Amino acids, pK a values, 571–572, 598 Amperometric detectors; see Detectors, electrochemical Amphoteric solute, 309, 311 Analytical method or procedure; see Test method Analytical method transfer (AMT), 554–561 Acceptable Analytical Practice, 554 acceptance criteria, 557 best practice, 558 documentation, 558 essentials 556 gradient elution, 450 options, 555 pitfalls, 558 protocol, 557 report, 558 Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R. Snyder, Joseph J. Kirkland, and John W. Dolan Copyright © 2010 John Wiley & Sons, Inc. summary, 559–560 waiver, 556 Anion-exchange chromatography; see also Ion-exchange chromatography carbohydrates, 628–629 nucleic acids, 619–620 viruses, 630–631 Antichaotropic salt, 610–611 Artifact peaks; see also Ghost peaks gradient elution, 442, 470 ion-pair chromatography, 347 Assay procedure; see Test method Asymmetry factor, 51 At-column dilution, 744–745 Autosamplers, 113–122; see also Injectors accuracy and precision, 116 carryover, 116 design, 116–119 load-ahead, 117 needle-seal, 118, 119 periodic maintenance, 140 problems; see Troubleshooting, symptoms reproducibility, 138 Axial-compression column, 239 Back-flushing, column, 247 Band, 24; see also Peak migration, 23 migration in gradient elution, 411–412 width, 24 Band-broadening processes, 39–41 Baseline drift; see Drift 891 892 INDEX Baseline noise problems; see Noise; Troubleshooting, baseline noise Batch tests for columns, 244, 245 Beer’s law, 160 Best practice(s), analytical method transfer, 558 best column for method development, 327 biochemical separation, 585–588, 599–603, 607, 609–614, 633–638 blank gradient, 449 buffer choice, 316 buffer solubility, 314 carryover, 818–819 check valve cleaning, 815 chiral columns, 688, 700 column conditions in gradient elution, 418 dedicated columns, 142 degassing, 141 divide-and-conquer, 819–820 equilibration, 142 glassware cleaning, 816–817 ignore first injection, 142 injection, 521 integration, 506 ion-pair chromatography, when to use, 332 leak detection, 816 linear gradients, 407 liquid-liquid extraction, 768–769 matrix-based standards, 520 method adjustment, 562 mobile phase, 312 module substitution, 820 parts replacement, 814 PEEK fittings, 816 percent-error plots, 527 performance tests, 856 pre-mixing mobile phase, 815 preventive maintenance, 138–142 priming injections, 142 problem isolation, 819–821 pump maintenance, 140 put back good parts, 821 reagent quality, 141 removing air from the pump, 814 reservoirs, 138–139 RI detectors, 179 robustness, 542 rules of thumb for problem isolation, 819–821 siphon test, 91, 814 standards and calibrators, 142 system cleanliness, 141 system suitability, 142 temperature control, 345 TFA, 817 troubleshooting tables, 865–888 troubleshooting tips and techniques, 814–819 validation protocol and report, 546 water purity, 817–818 BET procedure, 201 Bidentate silanes, 248 Bioanalytical methods,186, 548–553; see also Biochromatography accuracy and precision, 550 calibration curve, 551 documentation, 553 guidelines, 548 internal standards, 551 QC samples, 552 reference standards, 549 routine use, 552 stability, 551–552 validation, 549 Biochromatography, 570–648 columns, 579–583 sample recovery, 583 Biomacromolecule; see Biomolecule Biomolecule, structure and conformation, 571–579 Boiling point, solvent, 880 Bonded phase ligand, effect of chain length, 222 Bonded stationary phases, ligand concentration, 221–222 Books, HPLC, 13–14 Boxcar chromatography, 79–80 Brunauer-Emmett-Teller procedure, 201 Buffer, 309–317 absorbance, 315 capacity, 311–314 concentration, effect on selectivity, 327 inadequate, 312–314, 848 ion pairing, 315–316 pK a , 311–314 precipitation, 314 preferred, 316–317 preparation, 309–311, 885–888 properties, 313–317 selectivity, 326–327 solubility, 314–315 INDEX 893 stability, 316 volatile, 315 Cahn-Ingold-Prelog priorities, 669 Calibration, 510–529 area normalization, 525 curves, 520–523, 527–529 errors, 510–511 external standardization, 520–523 extrapolation, 515 internal standardization, 523–525 limits (LOD, LOQ, LLOQ, ULOQ), 512–516 limits samples outside limits, 515 and signal-to-noise (S/N), 512 linearity, 510 matrix-based standards, 520 multi-point, 511 peak area vs. peak height, 529 percent peak area, 525 percent-error plot, 527 plot, 521 problems forced-zero, 527 r 2 used improperly, 529 quality control (QC) samples, 521 single-point, 511 standard addition, 526 standard curve; see Calibration, curve standards, 520 trace analysis, 529 two-point, 511 Capacity factor; see Retention factor Capillary electrochromatography, 12 Capillary electrophoresis, 11 Capillary LC, 170 Carbohydrates, 576–578, 625–629 anion-exchange chromatography, 628–629 HILIC, 625–626 ion-exchange chromatography, 350, 355–356 ion-moderated partition chromatography, 626–628 pK a values, 628 Carboxylic acids, ion-exchange chromatography, 350 Carotenes, 297 Carryover, 818–819 Cartridge columns, 238 Cation exchange, 228 Cause-and-effect, troubleshooting tables, 865–888 CCC; (Countercurrent chromatography), 11 CE; see Capillary electrophoresis CEC (Capillary electrochromatography), 12 Cellubiohydrolase I (CBH I), chiral stationary phase, 694 Ceramic hydroxyapatite, 604 Certificate of analysis, 549 Chaotropes, 343 Charge transfer interactions, 33, 228 Charged-aerosol detectors (CAD), 184 Check standards; see Quality control, QC samples, Check valves; see also Troubleshooting, check valves active, 109 ball-type, 106 cleaning (best practice), 815 Chelating solutes, 229 Chemiluminescent nitrogen detector; see Detectors, chemiluminescent Chip, see HPLC, on a chip Chiral columns; see Chiral stationary phases Chiral definitions, 667 classification of isomers, 667 complementarity of size and shape, 680 constitutional isomers, 667 diastereomers, 667, 669 distribution constant, 675 dynamic fit, 680 epimers, 669 Fischer designation, 669 functional fit, 680 helical chirality, 668 homomers, 667 isomerism, 667 levorotatory, 669 Pfeiffer’s rule, 680 Chiral derivatization reagents, 670–675 o-phthaldialdehyde, 672 Chiral detectors, 175–177, 678 Chiral recognition, 667, 669; see also Chiral separation bi-Langmuir adsorption model, 718 enantioselective site, 717 enthalpically controlled, 716 entropically controlled, 716 non-enantioselective sites, 717 site-selective thermodynamics, 717–718 unusual temperature-induced behaviors, 716 894 INDEX Chiral selectors, 666, 675, 677, 682; see also Chiral stationary phases fit with analyte, 679–680 surface attachment, 677, 679 Chiral separation, 665–718 achiral environment, 668–669 cyclodextrin derivatives, 679 direct HPLC enantioemer separation, 666 direct method, 669, 675–681 human serum albumin, 694 hydrophobic fit, 680 indirect method, 669, 670–675 induced fit, 680 mobile-phase-additive mode, 675–677 molecular interactions, 679–680 molecular rigidity, 679 non-racemic mixtures, 668 non-superimposible mirror images, 668 normal phase, 670 peak dispersion and tailing, 681 pi-pi interactions, 679 planar chirality, 668 preparative isolation of enantiomers, 678 principles, 679 quinidine, 711 quinine, 712 racemate, 668 reciprocity principle of chiral recognition, 707 reversed phase, 670 rotation of polarized light, 669 solute-selector association, 715 solvation of interaction sites, 681 specificity of molecular recognition, 666 stereochemical descriptors, 668 stereogenic centers, 667 stereoisomers, 667 stereoselectivity of drugs, 680 steric fit, 680 thermodynamic considerations, 715–718 three-point interaction model, 678, 679,680 topological chirality, 668 transient diastereomeric complexes, 675 Chiral stationary phases, 666, 677, 681–715 acidic glycoprotein (AGP), 693–694 adsorption-desorption kinetics, 681 advantages and disadvantages, 678 amylose, 682 aromatic substitution and chiral recognition, 684–685 association constant, 675 automated screening procedures, 666 best practices, 688, 700 Cahn-Ingold-Prelog priorities, 669 Cellubiohydrolase I (CBH I), 694 Cellulose, 683 ChiraDex, 679 Chiralbiotic, 699 Chiralcel, 683 ChiralDexGamma, 679 ChiralHyun-CR-1, 706 Chiralpak, 683, 690, 715 ChiralSil, 706 ChiraSpher, 690 ChirBase, 666 Chirobiotic, 699 cinchonan carbamates, 711–713 cross-linked polymetharylamide, 690 crown-ether stationary phases, 706, 707 Crownpak CR, 706 Cyclobond, 679 cyclodextrin-based CSPs, 697–699 dinitrobenzoyl (DNB), 707–708 donor-acceptor type, 707–711 dynamically coated, 675 immobilized polysaccharides, 688 ion-exchangers, 711–713 ChirKromasil, 691 ligand-exchange stationary phases, 713–715 macrocyclic antibiotics, 699–705 microcrystalline cellulose triacetate, 682 mobile phase effects, 680–681 network-type, 691 ovomucoid (OVM), 693 Pirkle-type, 707–711 polyacrylamide-based, grafting-from approach, 691 polysaccharide-based, 682–689 normal phase mode, 685–686 polar organic mode, 686 reversed phase mode, 686 protein-based, 691–696 Ristocetin A, 699 screening for method development, 685 synthetic-polymers, 689 teicoplanin, 699, 704, 705 ULMO, 708, 710 vancomycin, 699, 702 WHELK-O1, 708 Chiralpak, chiral stationary phase, 683, 690, 715 ChiralSil, chiral stationary phase, 706 INDEX 895 ChiraSpher, chiral stationary phase, 690 ChirBase, chiral stationary phase, 666 Chirobiotic, chiral stationary phase, 699 Chromatofocusing, polypeptides, 603–604 Chromatogram, 3, 23 Chromatographic mode, selection, 66 Chromatography countercurrent; see Countercurrent chromatography gel permeation; see Gel permeation chromatography hydrophilic interaction; see Hydrophilic interaction chromatography hydrophobic interaction; see Hydrophobic interaction chromatography ion exchange; see Ion-exchange chromatography ligand exchange; see Ligand exchange chromatography normal-phase; see Normal-phase chromatography reversed-phase; see Reversed-phase chromatography Circular dichroism detectors; see Detectors, chiral Cleaning validation, methods, 547 Column, 199–249 axial-compression, 239 back-flushing, 247, 852–854 biochromatography, 579–583 blanks, 238–239 capillary, 595 cartridge, 238 conditions; see Column conditions configuration, 239–240 connectors, 238 contaminated, 247 dead-time, 24, 27–28 dead-volume, 26, 28 dedicated (best practices), 142 degradation, 238 efficiency, 35–54, 205–08; see also Plate number end fittings, 238 equilibration, 74–75 gradient elution, 446–449 equivalent, 235–236, 279–282 fittings, 238–239 flushing, 247 frits and screens, 238–239 glass lined, 239 guard, 247 handling, 246–249 hardware, 238–240 HIC, 609–610 high pressure, 238 ion-exchange capacity, 231 ion-exchange chromatography, 354 irreproducible, 68 isomer selectivity, 277–278 lifetime, 248–249 monolithic; see Monoliths orthogonal, 236–237 ovens, 125–127 periodic maintenance, 140 temperature-control requirements, 125 overload, 69, 726–727; see also Preparative separation severe, 748–751 packing methods, 240–244 packing; see Equipment, column packing; Particle; Stationary phase particle size, 205–207 performance standards, 244 plate height, 37–45 plate number, 245–246; see also Plate number manufacturer’s specifications, 245 polymeric, 582–583 for polypeptides (IEC), 599–601 pore size, 579–581 pressure-surge, 247–248 purging, 247 radial-compression, 239 reproducibility, 235–236 reversal, 852–854 saturation capacity, 737, 740–742 selectivity; see Column selectivity small diameter, 240 specifications, 244–246 stability, 248–249, 331, 582–583; see also Stationary phase, stability storage, 249 sub-2 μm particles, 249 supports, 200–217 temperature-control requirements, 125 warranties, 245 weak and strong (IEC), 600–601 . 558 essentials 556 gradient elution, 450 options, 555 pitfalls, 558 protocol, 557 report, 558 Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R. Snyder, Joseph J. Kirkland, and John. concentrations whose pH is known quite accurately are also commercially available. Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R. Snyder, Joseph J. Kirkland, and John W. Dolan Copyright. chromatography, 628–629 HILIC, 625–626 ion-exchange chromatography, 350, 355–356 ion-moderated partition chromatography, 626–628 pK a values, 628 Carboxylic acids, ion-exchange chromatography, 350 Carotenes, 297 Carryover,