296 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES L = 150 mm 2.0 mL/min R s = 1.1 P = 1050 psi Time (min) 1 12340 2 3 4 5 L = 250 mm 2.0 mL/min R s = 1.5 P = 1750 psi 0246 Time (min) 1 2 3 4 5 L = 100 mm 4.0 mL/min R s = 2.1 P = 1580 psi Time (min) 0123 6 7 8 9 10 L = 250 mm 1.0 mL/min R s = 4.8 P = 990 psi Time (min) 6 7 8 9 10 0 102030 (a) (b) (c) (d) Figure 6.28 Illustrations of a change in column conditions to either improve resolution or decrease run time. Sample components (non-ionized for these conditions; pH-2.6): 1, phthalic acid; 2, 2-nitrobenzoic acid; 3, 2-fluorobenzoic acid; 4, 3-nitrobenzoic acid; 5; 2-chlorobenzic acid; 6, 4-chloroaniline; 7, 3-fluorobenzic acid; 8, 2,6-dimethylbenzoic acid; 9, 2-chloroaniline; 10, 3,4-dichloroaniline. Conditions: 4.6-mm C 18 columns (5-μm) with indicated lengths L; mobile phase is 30% ACN-buffer for (a)and(b); 40% ACN-buffer for (c) and (d); 40 ◦ Cin(a)and(b), 30 ◦ Cin(c)and(d); flow rates indicated in figure. Chromatograms recreated from data of [73]. less polar (B-solvent) organic solvents. Often the A-solvent will be ACN or MeOH, while the B-solvent can be THF, methylene chloride, chloroform, methyl-t-butyl ether (MTBE), or other less polar organic solvents. Sample retention is controlled by varying %B and/or the polarity of the B-solvent, which can be approximated by the value of P in Table I.4 of Appendix I. Figure 6.29 shows an example of NARP for the separation of various carotenes (Fig. 6.29a) in a mixture of standards (Fig. 6.29b) and in an extract from tomato (Fig. 6.29c). Very hydrophobic samples are often insoluble in aqueous solutions, 6.6 SPECIAL PROBLEMS 297 (b) (a) (c) Figure 6.29 Non-aqueous reversed-phase (NARP) separations of carotenes. Conditions: 250 × 4.6-mm C 18 column; 8% chloroform-ACN mobile phase; 2.0 mL/min; ambient tem- perature. Adapted from [75]. which can be another reason to use NARP for such samples. From a practical standpoint, if NARP is chosen for a separation, all water must be washed from the HPLC system and column prior to switching to the nonaqueous mobile phase. Generally a 30-minute flush with ACN or MeOH is sufficient. 6.6 SPECIAL PROBLEMS One reason why RPC is more popular than other HPLC separations is that there are fewer problems in its use. Two possible problems with RPC that require attention are (1) poor retention for very polar samples and (2) peak tailing. 6.6.1 Poor Retention of Very Polar Samples This problem was noted in Section 6.1. Solutes that are very polar may not be retained with k ≥ 1, even when pure water (0% B) is used as mobile phase. This problem is more often encountered in the case of ionized solutes, which are much 298 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES less retained than their non-ionized counterparts (e.g., R–COO − vs. R–COOH). For ionized solutes their RPC retention can usually be increased by a change in mobile-phase pH (so as to decrease solute ionization; Section 7.2), or the addition of an ion-pair reagent to the mobile phase (Section 7.4). When attempting the separation of very polar, non-ionic samples by RPC, some columns exhibit a decrease in retention when mobile phases with < 5%B are used (‘‘stationary-phase de-wetting’’). Some columns are designed to avoid this problem, while the problem can be further minimized by following certain procedures (Section 5.3.2.3). When sample retention must be increased, even with the use of water as a mobile phase, the choice of column can provide some further control over sample retention. For example, columns with a higher surface area (smaller pore diameter) provide generally larger values of k. Graphitized-carbon columns (Section 5.2.5.3) are known to retain some very polar non-ionized solutes preferentially, although the use of these columns is constrained by their high cost and limited stability. When the sample is poorly retained by RPC, the preferred approach is often the use of normal-phase chromatography—because polar solutes are preferentially retained by the more polar stationary phase. Hydrophilic interaction chromatogra- phy (HILIC; Section 8.6), which is a variation of NPC, is especially useful in this connection; it can be used with aqueous mobile phases, and has other advantages when used in combination with mass spectrometric detection (LC-MS). 6.6.2 Peak Tailing Tailing peaks can arise for a number of different reasons (Section 17.4.5.3), often for acids or bases as solutes (Sections 5.4.4.1, 7.3.4.2). Whenever markedly tailing peaks are observed (e.g., with asymmetry factors A s > 2), steps should be taken to correct the problem. When peak tailing is observed during routine analysis, usually a replacement of the column or guard column will solve the problem. If peak tailing is encountered during method development, it is important restore good peak shape by a change in conditions, before carrying out further experiments. For further information on peak tailing, see Sections 7.3.4.2, 7.4.3.3, and 17.4.5.3. REFERENCES 1. G.A.HowardandA.J.P.Martin,Biochem. J., 46 (1950) 532. 2. L. R. Snyder and J. J. Kirkland, Introduction to Modern Liquid Chromatography, Wiley-Interscience, New York, 1974, ch. 8. 3. J. J. Kirkland and J. J. DeStefano, J. Chromatogr. Sci., 8 (1970) 309. 4. J. A. Schmit, R. A. Henry, R. C. Williams, and J. F. Dieckmann, J. Chromatogr. Sci.,9 (1971) 645. 5. W. R. Melander and C. Horv ´ ath, in High-Performance Liquid Chromatography. Advances and Perspectives,Vol.2,C.Horv ´ ath, ed., Academic Press, New York, 1980, pp. 113–319. 6. C. Horv ´ ath, W. Melander, and I. Molnar, J. Chromatogr., 125 (1976) 129. 7. M. T. W. Hearn, in Ion-pair Chromatography,M.T.W.Hearn,ed.,Dekker,New York, 1985. REFERENCES 299 8. N. S. Wilson, M. D. Nelson, J. W. Dolan, L. R. Snyder, R. G. Wolcott, and P. W. Carr, J. Chromatogr. A, 961 (2002) 171. 9. N. S. Wilson, M. D. Nelson, J. W. Dolan, L. R. Snyder, and P. W. Carr, J. Chromatogr. A, 961 (2002) 195. 10. L. R. Snyder and M. A. Quarry, J. Liq. Chromatogr., 10 (1987) 1789. 11. K. Valk ´ o, L. R. Snyder, and J. L. Glajch, J. Chromatogr., 656 (1993) 501. 12. P. W. Carr, D. E. Martire, and L. R. Snyder, eds., Retention in Reversed-Phase HPLC (J. Chromatogr., Vol. 656, 1993). 13. C. F. Poole, The Essence of Chromatography, Elsevier, Amsterdam, 2003. 14. L. C. Tan and P. W. Carr, J. Chromatogr. A, 775 (1997) 1. 15. A. Ailaya and C. Horv ´ ath, J. Chromatogr. A, 829 (1998) 1. 16. P. Nikitas, A. Pappa-Louisi, and P. Agrafiotou, J. Chromatogr. A, 1034 (2004) 41. 17. I. Molnar and Cs. Horv ´ ath, J. Chromatogr., 142 (1977) 623. 18. A. Tchapla, H. Colin, and G. Guiochon, Anal. Chem., 56 (1984) 621. 19. J. Ko and J. C. Ford, J. Chromatogr. A, 913 (2001) 3. 20. B. A. Bidlingmeyer and A. D. Broske, J. Chromatog,. Sci. 42 (2004) 100. 21. T. H. Walter, P. Iraneta, and M. Capparella, J. Chromatogr. A, 1075 (2005) 177. 22. L. C. Sander, K. A. Lippa, and S. A. Wise, Anal. Bioanal. Chem. , 382 (2005) 646. 22a. J. L. Rafferty, J. I. Siepmann, and M. R. Schure, J. Chromatogr. A, 1204 (2008) 11. 23. A. Klimek-Turek, T. H. Dzido, and H. Engelhardt, LCGC Europe, 21 (2008) 33. 24. T. Braumann, G. Weber, and L. H. Grimme, J. Chromatogr., 261 (1983) 329. 25. L. R. Snyder and J. W. Dolan, High-Performance Gradient Elution, Wiley, New York, 2007, pp. 19–21. 26. L. R. Snyder, Today’s Chemist at Work, 5 (1996) 29. 27. P. J. Schoenmakers, H. A. H. Billiet, and L. de Galan, J. Chromatogr., 185 (1979) 179. 28. P. J. Schoenmakers, H. A. H. Billiet, and L. de Galan, J. Chromatogr., 218 (1981) 259. 29. R. G. Wolcott, J. W. Dolan, and L. R. Snyder, J. Chromatogr. A, 869 (2000) 3. 30. J. Chmielowiec and H. Sawatzky, J. Chromatogr. Sci., 17 (9790) 245. 31. J. W. Dolan, L. R. Snyder, N. M. Djordjevic, D. W. Hill, D. L. Saunders, L. Van Heukelem, and T. J. Waeghe, J. Chromatogr. A, 803 (1998) 1. 32. J. W. Dolan, J. Chromatogr. A, 965 (2002) 195. 32a. S. Heinisch, and J. L. Rocca, J. Chromatogr. A, 1216 (2009) 642. 33. S. Heinisch, G. Puy, M P. Barrioulet, and J. L. Rocca, J. Chromatogr. A, 1118 (2006) 234. 34. J. W. Coym and J. G. Dorsey, J. Chromatogr. A, 1035 (2004) 23. 35. J. W. Dolan, L. R. Snyder, N. M. Djordjevic, D. W. Hill, D. L. Saunders, L. Van Heukelem, and T. J. Waeghe, J. Chromatogr. A , 803 (1998) 1. 36. L. R. Snyder, J. Chromatogr., 179 (1979) 167. 37. L. R. Snyder and J. W. Dolan, J. Chromatogr. A, 892 (2000) 107. 38. P. L. Zhu, J. W. Dolan, L. R. Snyder, N. M. Djordjevic, D. W. Hill, J T. Lin, L. C. Sander, and L. Van Heukelem, J. Chromatogr. A, 756 (1996) 63. 39. D. W. Armstrong, W. Demond, A. Alak, W. L. Hinze, T. E. Riehl, and K. H. Bui, Anal. Chem., 57 (1985) 234. 40. F. C. Marziani and W. R. Cisco, J. Chromatogr., 465 (1989) 422. 41. M. Paleologou, S. Li, and W. C. Purdy, J. Chromatogr. Sci., 28 (1990) 311. 300 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES 42. B. Nikolova-Damyanov, in HPLC of Acyl Lipids, J T. Lin and T. A. McKeon, eds., HNB Publishing, New York, 2005, p. 221. 43. B. Voach and G. Schomburg, J. Chromatogr. 149 (1978) 417. 44. L. C. Sander and S. A. Wise, J. Chromatogr. A 656 (1993) 335. 45. L. C. Sander, M. Pursch, and S. A. Wise, Anal. Chem., 71 (1999) 4821. 46. L. C. Sander, K. A. Lippa, and S. A. Wise, Anal. Bioanal. Chem. , 382 (2005). 646. 47. J. W. Dolan and L. R. Snyder, J. Chromatogr. A, 1216 (2009) 3467. 48. J. W. Dolan, A. Maule, L. Wrisley, C. C. Chan, M. Angod, C. Lunte, R. Krisko, J. Winston, B. Homeierand, D. M. McCalley, and L. R. Snyder, J. Chromatogr. A, 1057 (2004) 59. 49. J. Pellett, P. Lukulay, Y. Mao, W. Bowen, R. Reed, M. Ma, R. C. Munger, J. W. Dolan, L. Wrisley, K. Medwid, N. P. Toltl, C. C. Chan, M. Skibic, K. Biswas, K. A. Wells, and L. R. Snyder, J. Chromatogr. A 1101 (2006) 122. 50. J. W. Dolan, L. R. Snyder, T. H. Jupille, and N. S. Wilson, J. Chromatogr. A 960 (2002) 51. 51. J. W. Dolan, L. R. Snyder, and T. Blanc, J. Chromatogr. A, 897 (2000) 51. 52. G. Xue, A. D. Bendick, R. Chen, and S. S. Sekulic, J. Chromatogr. A, 1050 (2004) 159. 53. E. Van Gyseghem, M. Jimidar, R. Sneyers, D. Redlich, E. Verhoeven, D. L. Massart, and Y, Vander Heyden, J. Chromatogr. A, 1074 (2005) 117. 54. D. H. Marchand, L. R. Snyder, and J. W. Dolan, J. Chromatogr. A, 1191 (2008) 2. 55. J. Zhao and P. W. Carr, LCGC, 17 (1999) 346. 56. L. R. Snyder, J. Chromatogr. B, 689 (1997) 105. 57. J. L. Glajch, J. J. Kirkland, K. M. Squire, and J. M. Minor, J. Chromatogr., 199 (1980) 57. 58. A. C. J. H. Drouen, H. A. H. Billiet, P. J. Schoenmakers, and L. de Galan, Chro- matographia, 10 (1982) 48. 59. L. R. Snyder, M. A. Quarry, and J. L. Glajch, Chromatographia, 24 (1987) 33. 60. P. L. Zhu, J. W. Dolan, L. R. Snyder, N. M. Djordjevic, D. W. Hill, J T. Lin, L. C. Sander, and L. Van Heukelem, J. Chromatogr. A, 756 (1996) 63. 61. J. W. Dolan, L. R. Snyder, N. M. Djordjevic, D. W. Hill, D. L. Saunders, L. Van Heukelem, and T. J. Waeghe, J. Chromatogr. A, 803 (1998) 1. 62. J. W. Dolan, L. R. Snyder, N. M. Djordjevic, D. W. Hill, L. Van Heukelem, and T. J. Waeghe, J. Chromatogr. A, 857 (1999) 1. 63. R. G. Wolcott, J. W. Dolan, and L. R. Snyder, J. Chromatogr. A, 869 (2000) 3. 64. A. Gonzalez, K. L. Foster, and G. Hanrahan, J. Chromatogr. A, 1167 (2007) 135. 65. J. W. Dolan, L. R. Snyder, T. Blanc, and L. Van Heukelem, J. Chromatogr. A, 897 (2000) 37. 66. J. W. Dolan, L. R. Snyder, N. M. Djordjevic, D. W. Hill, D. L. Saunders, L. Van Heukelem, and T. J. Waeghe, J. Chromatogr. A, 803 (1998) 1. 67. P. L. Zhu, L. R. Snyder, J. W. Dolan, N. M. Djordjevic, D. W. Hill, L. C. Sander, and T. J. Waeghe, J. Chromatogr. A, 756 (1996) 21. 68. J. J. DeStefano, J. A. Lewis, and L. R. Snyder, LCGC, 10 (1992) 130. 69. L. R. Snyder, J. W. Dolan, and P. W. Carr, J. Chromatogr. A, 1060 (2004) 77. 70. K. Valk ´ o, S. Espinosa, C. M. Du, E. Bosch, M. Ros ´ e s,C.Bevan,andM.H.Abraham, J. Chromatogr. A, 933 (2001) 73. 71. Sz. Nyiredy, A. Szucs, and L. Szepesy, J. Chromatogr. A, 1157 (2007) 122. 72. Y. Mao, and P. W. Carr, Anal. Chem., 73 (2001) 1821. REFERENCES 301 73. P. L. Zhu, J. W. Dolan, and L. R. Snyder, D. W. Hill, L. Van Heukelem, and T. J. Waeghe, J. Chromatogr. A, 756 (1996) 51. 74. N. A. Parris, J. Chromatogr., 157 (1978) 161. 75. M. Zakaria, K. Simpson, P. R. Brown, and A. Krstulovic, J. Chromatogr., 176 (1979) 109. 76. N. E. Craft, S. A. Wise, and J. H. Soares, J. Chromatogr., 589 (1992) 171. 77. H. J. A. Philipsen, J. Chromatogr. A, 1037 (2004) 329. CHAPTER SEVEN IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY 7.1 INTRODUCTION, 304 7.2 ACID–BASE EQUILIBRIA AND REVERSED-PHASE RETENTION, 304 7.2.1 Choice of Buffers, 309 7.2.2 pK a as a Function of Compound Structure, 317 7.2.3 Effects of Organic Solvents and Temperature on Mobile-Phase pH and Sample pK a Values, 317 7.3 SEPARATION OF IONIC SAMPLES BY REVERSED-PHASE CHROMATOGRAPHY (RPC), 319 7.3.1 Controlling Retention, 320 7.3.2 Controlling Selectivity, 320 7.3.3 Method Development, 327 7.3.4 Special Problems, 329 7.4 ION-PAIR CHROMATOGRAPHY (IPC), 331 7.4.1 Basis of Retention, 334 7.4.2 Method Development, 339 7.4.3 Special Problems, 347 7.5 ION-EXCHANGE CHROMATOGRAPHY (IEC), 349 7.5.1 Basis of Retention, 351 7.5.2 Role of the Counter-Ion, 352 7.5.3 Mobile-Phase pH, 354 7.5.4 IEC Columns, 354 7.5.5 Role of Other Conditions, 354 Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R. Snyder, Joseph J. Kirkland, and John W. Dolan Copyright © 2010 John Wiley & Sons, Inc. 303 304 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY 7.5.6 Method Development, 355 7.5.7 Separations of Carbohydrates, 355 7.5.8 Mixed-Mode Separations, 355 7.1 INTRODUCTION Chapter 6 dealt with the separation of neutral (non-ionized) molecules by means of reversed-phase chromatography (RPC). The present chapter extends this treatment to the HPLC separation of ‘‘ionic’’ samples; these are mainly mixtures that contain acids and/or bases (with or without neutral compounds), but they can include compounds that are totally ionized between pH-2 and pH-12 (e.g., tetralkylammonium salts, sulfonic acids). In the early days of HPLC, ionic samples often presented special problems—partly the result of less suitable column packings that were available at that time but also because of a limited understanding of how such separations are best carried out. Although these past limitations have been largely overcome, the separation of ionic samples remains somewhat more demanding when compared with separations of neutral samples. Before 1980, ion-exchange chromatography (IEC, Section 7.5) was commonly selected for the separation of acids and bases, but today RPC (Section 7.3) and—to a lesser extent—ion-pair chromatography (Section 7.4) have become preferred procedures for the separation of ‘‘small,’’ ionizable molecules (<1000 Da). However, IEC is still used heavily for the separation of large biomolecules such as proteins (Chapter 13); for additional details on IEC separation, see Sections 13.4.2, 13.5.1, and 13.6.3. 7.2 ACID–BASE EQUILIBRIA AND REVERSED-PHASE RETENTION The RPC retention of neutral samples decreases for less hydrophobic (more polar) molecules (Sections 2.3.2.1, 6.2). When an acid (HA) or base (B) undergoes ionization (i.e., is converted from an uncharged to a charged species), the compound becomes much more polar or hydrophilic. As a result its retention factor k in RPC can be reduced 10-fold or more: uncharged molecule ionized molecule (acids) HA ⇔ A − + H + (7.1) (bases) B + H + ⇔ BH + (7.1a) hydrophobic (more retained in RPC) hydrophilic (less retained in RPC) 7.2 ACID– BASE EQUILIBRIA AND REVERSED-PHASE RETENTION 305 Acids lose a proton and become ionized when the mobile-phase pH is increased; bases gain a proton and become ionized when mobile-phase pH decreases. The ionization of an acid (HA) or base (B) can be related to its acidity constant K a : (acids) K a = [A − ][H + ] [HA] (7.2) or (bases) K a = [B][H + ] [BH + ] (7.2a) Here [HA] and [A − ] are the concentrations of the free and ionized acidic solute HA; [B] and [BH + ] refer to the concentrations of the free and protonated basic solute B. The pK a value (=−log K a ) of an acid or base is given by the Henderson–Hasselbalch equation: (acids) pK a = pH − log [A − ] [HA] (7.3) or (bases) pK a = pH − log [B] [BH + ] (7.3a) For example, the pK a value in water of a (weakly basic) substituted aniline will fall within a range of about 4 ≤ pK a ≤ 6, while the pK a of a (strongly basic) aliphatic amine will usually lie between 9 and 11. Values of pK a in the literature for different acids or bases usually refer to solutions in buffered-water at near-ambient temperatures. If the mobile phase contains organic solute, or if the temperature is much different from ambient, values of both pH and pK a can change significantly (Section 7.2.3). Retention as a function of pH and sample ionization is illustrated in Figure 7.1 for the separation of a hypothetical sample composed of carboxylic acid HA (solid curve in Fig. 7.1a) and aliphatic-amine B (dashed curve in Fig. 7.1a). In Figure 7.1a, solute ionization (left-hand scale) is plotted against mobile-phase pH for each solute; the dark circles mark the pH where each compound is half ionized (pH ≡ pK a = 5.0 for HA, and 9.0 for B). Values of k (right-hand scale in Fig. 7.1a) decrease with increasing solute ionization and are given as a function of pH and pK a by (acids, bases) k = k 0 (1 − F ± ) + k ± F ± (7.4) Here k 0 is the value of k for the non-ionized molecule (HA or B), k ± is the value of k for the fully ionized molecule (A − or BH + ), and F ± is the fractional ionization of the molecule (0 ≤ F ± ≤ 1). (acids) F ± = 1 1 + [H + ]/K a (7.4a) . CHROMATOGRAPHY (IEC), 349 7.5.1 Basis of Retention, 351 7.5.2 Role of the Counter-Ion, 352 7.5.3 Mobile-Phase pH, 354 7.5.4 IEC Columns, 354 7.5.5 Role of Other Conditions, 354 Introduction to Modern. Snyder and J. J. Kirkland, Introduction to Modern Liquid Chromatography, Wiley-Interscience, New York, 1974, ch. 8. 3. J. J. Kirkland and J. J. DeStefano, J. Chromatogr. Sci., 8 (1970) 309. 4 354 7.5.4 IEC Columns, 354 7.5.5 Role of Other Conditions, 354 Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R. Snyder, Joseph J. Kirkland, and John W. Dolan Copyright © 2010