246 THE COLUMN Table 5.10 Approximate Plate Number for Well-Packed Columns under Optimized Test Conditions Particle Type a Particle Diameter (μm) Column Length (mm) a Plate Number N Totally porous 5 30 2,500–3,000 Totally porous 5 50 4,500–5,000 Totally porous 5 100 8,000–10,000 Totally porous 5 150 12,000–15,000 Totally porous 5 250 20,000–25,000 Totally porous 3.5 30 3,000–4,000 Totally porous 3.5 50 5,500–7,000 Totally porous 3.5 100 10,500–14,000 Totally porous 3.5 150 17,000–21,000 Fused-core 2.7 30 7,000–8,000 Fused-core 2.7 50 9,000–11,000 Fused-core 2.7 100 18,000–22,000 Fused-core 2.7 150 28,000–34,000 Totally porous 1.8 b 30 6,000–7,000 Totally porous 1.8 50 10,000–12,000 Totally porous 1.8 100 20,000–25,000 Note: Small, neutral test-solute, low viscosity mobile phase, ambient temperature, measured at the plate-height minimum. a Estimated values for 4.6 mm i.d. columns. b An average for commercially available, sub-2-μmparticles. new column that met the manufacturer specifications. If problems are encountered with a routine method that might be caused by the column, values of N and either A s and TF can be compared with prior values for a ‘‘good’’ column. In this way a ‘‘bad’’ column can be confirmed as the cause of the problem. Every column used for a routine method has a finite lifetime (number of injected samples before column failure) that depends on separation conditions—especially mobile-phase pH and temperature. For ‘‘clean’’ samples, 1000 to 2000 analy- ses should be possible for must silica-based columns, particularly reversed phase columns. However, for other samples (with minimal sample preparation), such as extracts of blood, plant or animal tissue, or soil, 200–500 analyses is a more typical column lifetime. 5.8 COLUMN HANDLING The performance and life of the column depend on how it is used and handled. During heavy use with ‘‘dirty’’ samples (especially samples from biological sources), columns can develop severe peak tailing (Fig. 5.26a) or double peaks for each component (Fig. 5.26b)—usually the result of a partly blocked frit, a contaminated 5.8 COLUMN HANDLING 247 (a) (b) peak tailing split peaks Figure 5.26 Examples of peak tailing (a) and split peaks (b). column, or deterioration of the column packing. The following restorative measures are sometimes effective, but the time and effort involved are often not cost-effective. Usually it is more economical to simply replace the column. A blocked frit or contaminated column can sometimes be restored by periodi- cally purging the column with a strong solvent. A 20-column-volume purge (about 30 mL for a 150 × 4.6-mm column) with a mixture of 96% dichloromethane and 0.1% ammonium hydroxide in 4% methanol is often effective for RPC columns. Pure methanol or isopropanol can be used for normal-phase columns. Flushing a RPC column (at least) daily with a strong solvent, such as methanol or acetoni- trile, can enhance column performance and lifetime for isocratic separations. This approach removes strongly retained sample components that can build up at the column inlet. Back-flushing a column with a strong solvent at 0.2 to 0.5 mL/min may be more effective, so as to avoid driving the column contaminants into the column. However, some manufactures recommend against back-flushing because their columns are fitted with an inlet frit that has larger pores that might allow particles to be swept out (consult the column care-and-use instructions for a specific column). In gradient elution, clearing the column of strongly retained components can be accomplished by using a steep or step gradient (Section 9.2.2.5). The use of a 0.5-μm porosity in-line filter (Section 3.4.2.3) can be highly effective in preventing blockage of the column inlet frit, and is highly recommended. To reduce the possible impact of ‘‘dirty’’ samples on column lifetime, sample pretreatment is commonly used (Chapter 16). A guard column canalsobeusedto protect the column, and it is recommended for routine analysis. A guard column is short (e.g., 10–20 mm) and preferably contains a packing that is the same as or similar to that in the main column (Section 5.4.2). The guard column captures strongly retained sample components (and particulates), and prevents these from fouling the analytical column. Guard columns must be replaced at regular intervals, before the column becomes saturated with strongly retained sample components that then pass into the analytical column. However, because of their added expense and inconvenience, some users prefer to avoid guard columns and replace the main column more frequently. The use of guard columns with low-volume, high-efficiency columns (e.g., sub-2-μm columns) requires special care, because of the greater importance of extra-column peak broadening when small columns are used. For columns that are not well packed, a sudden pressure surge (as during sample injection) can cause a void at the column inlet, with a decrease in column performance. Fortunately, this problem is no longer common for columns from 248 THE COLUMN established manufacturers. While pressure-related problems are uncommon for silica-based columns, other types of particles (e.g., organic gels, graphitized carbon) are more fragile and less able to withstand sudden changes in flow, pressure, or temperature. Column performance can be reduced significantly by a loss of the bonded phase during use, leading to a short column lifetime. The recommendations of the manufacturer, especially with regard to mobile-phase pH and temperature, should be observed. A low-pH mobile phase (e.g., pH < 2.5) can cause some hydrolysis of Si–O bonds, with loss of bonded silane (see Fig. 5.16a). Short-chain ligands (e.g., –C 3 , –C 3 –C≡N) are least stable in a low-pH environment, while longer chain alkyl groups (C 18 ,C 8 ) are usually adequately stable for a mobile-phase pH between 2.5 and 7, and a temperature ≤ 40 ◦ C. As previously noted, sterically protected stationary phases (Fig. 5.16b) provide additional stability at low pH. High-pH mobile phases (e.g., pH > 8) can slowly dissolve silica-based packings, again resulting in a degradation of column performance. Columns of hybrid silica-silane particles (Section 5.3.2.2) and those based on zirconia (Section 5.2.5.1) are especially resistant to degradation by high-pH mobile phases; some other special stationary phases (e.g., bonded bidentate silanes, Fig. 5.14b or c) are also more stable for high-pH applications [44]. Stationary-phase loss from silica-based columns at low pH is accelerated at higher temperatures. Therefore higher temperatures as a means of improving col- umn performance or separation selectivity should be used carefully. Many workers have reported good results for conventional C 18 and C 8 columns when operated at 40–60 ◦ C. Sterically protected stationary phases can be used routinely with temper- atures up to about 90 ◦ C, and polymer-coated zirconia can be used satisfactorily at temperatures up to at least 150 ◦ C [34]. Degradation of silica-based columns at higher pH occurs via dissolution of the silica, and its degradation is also accelerated by higher temperatures. Figure 5.27 provides an example where the cumulative loss of silica at 60 ◦ C is almost 20-fold greater than at 40 ◦ C. When using a column at intermediate and high pH with both phosphate and carbonate buffers, temperatures above 40 ◦ C with silica-based 0 5 10 15 500 400 300 200 100 0 Mobile-phase volume (L) Dissolved silica (mg) 60°C 40°C Figure 5.27 Effect of temperature on silica-support dissolution. Column: Zorbax Rx-C18, 15 × 0.46 cm; continuous nonrecycled 20% acetonitrile/80% sodium phosphate buffer, 0.25 M, pH 7.0. Flow rate: 1.0 mL/min. Adapted from [78]. 5.8 COLUMN HANDLING 249 columns should be avoided because of rapid dissolution of the silica support [78]. On the other hand, use of organic buffers (e.g., TRIS, HEPES, citrate) may increase column lifetime over that with phosphate and carbonate buffers when operating at intermediate and higher pH [79]. One speculation is that these basic, partially hydrophobic, organic buffer compounds tend to bind to unreacted silanol groups on the packing so as to create an additional barrier to the dissolution of the silica support [79]. However, the advantage of the organic buffers may be misleading, as the actual pH of the buffer/organic solvent mobile phase may be somewhat lower than that measured for the buffer solution itself (the pH of phosphate and carbonate buffers in organic-containing solvents is somewhat higher that the aqueous buffer [80]); see the further discussion of Section 7.2.3. The performance and lifetime of a column can also be affected by improper storage of the mobile phase. Aqueous buffers (especially with pH ≈7) encourage microbial growth if they are stored for more than a day at room temperature. The resulting particulates can in turn block the column inlet, reducing N and increasing the column pressure. Therefore it is good practice to formulate buffers daily. However, the presence of ≥20% organic solvent in the mobile phase, or an absence of oxygen due to helium sparging, can inhibit bacterial growth and prolong buffer life. When removed from the system, the column is best stored in a nonprotic solvent such as acetonitrile (100% B). For short-term applications (overnight or a few days) it is convenient (and acceptable) to leave the mobile phase in place. However, prolonged storage with buffered solutions, particularly those with high concentrations of water or alcohols, should be avoided. Prior to storage, the column should be flushed with 5 to 10 column volumes of the same aqueous-organic mobile phase but without buffer before an additional 5-column-volume flush with 100% organic phase (this avoids precipitation of the buffer within the column). Flushing columns with pure water for long periods should be avoided because of stationary-phase de-wetting (especially more hydrophobic columns, e.g., C 18 ). To prevent columns from drying out, they should be tightly capped for storage. Special handling is required for columns of <2-μm particles. Such columns are fitted with inlet and outlet frits that have very narrow pores (e.g., 0.2 μm) in order to retain these very small particles. Therefore both the sample and mobile phase should be passed through 0.2-μm filters to ensure that particulates do not block the frits and degrade column performance. Small-particle columns are often used for fast separations (run times of <1 min), in which case resulting peaks have very narrow widths—measured either in time or volume. This requires instrumental conditions that minimize potential extra-column effects that artificially broaden peaks: • short, low-volume tubing that connects the sampling valve, column, and detector • detector microcells of low volume (e.g., 1 μL) • small sample volumes (1–2 μL preferred) • fast detector response (e.g., ≤0.1 sec) • high data-capturing rate (at least 20 points/sec or 20 Hz) For further details, see [81, 82]. 250 THE COLUMN REFERENCES 1. K. K. Unger, Porous Silica, Elsevier, Amsterdam, 1979, p. 294. 2. K. Kalghatgi and C. Horv ´ ath, J. Chromatogr., 443 (1988) 343. 3. K. K. Unger and H. Giesche, Ger. Pat. DE-3543 143.2 (1985). 4. J. J. Kirkland, F. A. Truszkowski, C. H. Dilks, Jr., and G. S. Engel, J. Chromatogr. A, 890 (2000) 3. 5. J. J. Kirkland, T. J. Langlois, and J. J. DeStefano, Amer. Lab., 39 (2007) 18. 6. N. B. Afeyan, N. F. Gordon, I. Mazsaroff, L. Varady, S. P. Fulton, Y. B. Yang, and F. E. Regnier, J. Chromatogr., 519 (1990) 1. 7. D. Whitney, M. McCoy, N. Gordon, and N. Afeyan, J. Chromatogr. A, 807 (1998) 165. 8. L. R. Snyder and M. A. Stadalius, in High-performance Liquid Chromatography: Advances and Perspectives, Vol. 4, C. Horvath, ed., Academic Press, San Diego, 1986. 9. R. K. Iler, The Chemistry of Silica, Wiley, New York, 1979, p. 639. 10. J. Billen, P. Gzil, F. Lynen, P. Sandra, P Van der Meeren, and G. Desmet, Poster, HPLC 2006, San Francisco, June 2006. 11. K. K. Unger and E. Weber, A Guide to Practical HPLC, Git Verlag, Darmstadt, 1999, p. 45. 12. U. D. Neue, HPLC Columns, Wiley-VCH, New York, 1997, p. 82. 13. J. J. DeStefano, T. J. Langlois and J. J. Kirkland, J. Chromatogr. Sci., 46 (2008) 1. 14. J. K ¨ ohler,D.B.Chase,R.D.Farlee,A.J.Vega,andJ.J.Kirkland,J. Chromatogr., 352 (1986) 275. 15. J. K ¨ ohler and J. J. Kirkland, J. Chromatogr., 385 (1987) 125. 16. J. Nawrocki, Chromatographia, 31 (1991) 177. 17. J. Nawrocki, Chromatographia, 31 (1991) 193. 18. H. Engelhardt, H. Low, and W. G ¨ otzinger, J. Chromatogr., 544 (1991) 371. 19. J. J. Kirkland, B. E. Boyes, and J. J. DeStefano, Amer. Lab., 26 (1994) 36. 20. D. W. Sindorf and G. E. Maciel, J. Am. Chem. Soc., 105 (1983) 1487. 21. P. G. Dietrich, K H. Lerche, J. Reusch, and R. Nitzsche, Chromatographia, 44 (1997) 362. 22. A. M ´ endez, E. Bosch, M. Ros ´ es, and U. D. Neue, J. Chromatogr. A, 986 (2003) 33. 23. M. Jacoby, Chemical and Engineering News, Dec. 11, 2006. 24. F. Svec and C. G. Huber, Anal. 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Carr, J. Chromatogr. A, 1028 (2004) 31. 35. J. Nawrocki, C. Dunlap, A. McCormick, and P. W. Carr, J. Chromatogr. A, 1028 (2004) 1. 36. U. Bien-Vogelsang, A. Deege, H. Figge, J. K ¨ ohler, and G. Schomburg, Chromatographia, 19 (1984) 170. 37. J. J. Pesek and M. T. Matyska, J. Chromatogr. A, 952 (2001) 1. 38. P. Ross, LCGC, 18 (2000) 14. 39. J. H. Knox, B. Kaur, and G. R. Millward, J. Chromatogr., 352 (1986) 3. 40. M. J. Wirth and H. O. Fatunmbi, Anal. Chem,. 65 (1993) 822. 41. J. J. Kirkland, J. L. Glajch, and R. D. Farlee, Anal. Chem., 61 (1988) 2. 42. J. L. Glajch and J. J. Kirkland, US Pat. 4,705,725, 1987 43. J. L. Glajch and J. J. Kirkland, US Pat. 4,847,159, 1989. 44. J. J. Kirkland, J. B. Adams, M. A. Van Straten, and H. A. Claessens, Anal. Chem., 70 (1998) 4344. 45. L. C. Sanders and S. A. Wise, Anal. Chem., 56 (1984) 504. 46. N. S. Wilson, J. Gilroy, J. W. Dolan, and L. R. Snyder J. Chromatogr. A, 1026 (2004) 91. 47. L. Brown, B, Ciccone, J. J. Pesek, and M. T. Matyska, Amer. Lab., Dec. (2003) 23. 48. K. Miyabe and N. Orita, Talanta, 36 (1989) 897. 49. M. R. Buchmeiser, J. Chromatogr. A, 918 (2001) 233. 50. K. K. Unger and J. Schick-Kalb, US Pat. 4,017,528, 1977. 51. K. D. Wyndham, J. E. O’Gara, T. H. Walter, K. H. Glose, N. L. Lawrence, B. A. Alden, G. S. Izzo, C. J. Hudalla, and P. C. Iraneta, Anal. Chem., 75 (2003) 6781. 52. R. E. Majors, LCGC, 20 (2002) 516. 53. B. A. Bidlingmeyer and A. D. Broske, J. Chromatogr. Sci., 42 (2004) 100. 54. T. H. Walter, P. Iranetaund, and M. Capparella, J. Chromatogr. A, 1075 (2005) 177. 55. R. Eksteen and J. Thoma, in: Chromatography, the State of the Art,Vol.1,Akademia Kiado, Budapest, 1985. 56. L. Zhang, L. Sun, J. I. Siepmann, and M. R. Shure, J. Chromatogr. A, 1079 (2005) 127. 57. K. Croes, A. Steffens, D. H. Marchand, and L. R. Snyder, J. Chromatogr. A, 1098 (2005) 123. 58. M. R. Euerby, P. Petersson, W. Campbell, and W. Roe, J. Chromatogr. A, 1154 (2007) 138. 59. U. D. Neue, J. Sep. Sci., 30 (2007) 1611. 60. 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. 61. L. R. Snyder, J. W. Dolan, and P. W. Carr, J. Chromatogr. A, 1060 (2004) 77. 62. L. R. Snyder, J. W. Dolan, and P. W. Carr, Anal. Chem., 79 (2007) 3255. 63. D. H. Marchand and L. R. Snyder, J. Chromatogr. A, 1209 (2008) 104. 64. L. R. Snyder and J. W, Dolan, High Performance Gradient Elution , Wiley-Interscience, Hoboken, NJ, 2007, p. 420. 65. L. C. Sander and S. A. Wise, J. Chromatogr. A, 656 (1993) 335. 66. J. J. Kirkland, Amer. Lab., 26 (1994) 28K. 252 THE COLUMN 67. U. D. Neue, E. Serowik, P. Iraneta, B. A. Alden, and T. H. Walter, J. Chromatogr. A, 849 (1999) 87. 68. L. R. Snyder, A. Maule, A. Heebsch, R. Cuellar, S. Paulson, J. Carrano, L. Wrisley, C. C. Chan, N. Pearson, J. W. Dolan, and J. Gilroy, J. Chromatogr. A, 1057 (2004) 49. 69. 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. 70. Y. Mao and P. W. Carr, LCGC, 21 (2003) 150. 71. J. W. Dolan and L. R. Snyder, J. Chromatogr. A, 1216 (2009) 3467. 72. D. V. McCalley, Adv. Chromatogr., 46 (2008) 305. 73. D. M. Marchand, L. R. Snyder, and J. W. Dolan, J. Chromatogr. A, 1191 (2008) 2. 74. H. Colin. P. Hilaireau, and J. De Tournemire, LCGC, 8 (1990) 302. 75. F. Couillard, Chromatography Apparatus, US Patent 4,597,866, 1986-07-01. 76. J. J. Kirkland and J. J. DeStefano, J. Chromatogr. A, 1126 (2006) 50. 77. L. R. Snyder and J. J. Kirkland, Introduction to Modern Liquid Chromatography, 2nd ed., Wiley, New York, 1979, ch. 5. 78. H. A. Claessens, M. A. Van Straten, and J. J. Kirkland, J. Chromatogr. A, 728 (1996) 259. 79. J. J. Kirkland, M. A. Van Straten, and H. A. Claessens, J. Chromatogr. A, 797 (1998) 111. 80. G. W. Tindall and R. L. Perry, J. Chromatogr. A, 988 (2003) 309. 81. J. J. Kirkland, J. Chromatogr. Sci., 38 (2000) 535. 82. F. Gerber, M. Krummen, H. Potgeter, A. Roth, C. Siffrin, and C. Spoendlin, J. Chro- matogr. A, 1036 (2004) 127. CHAPTER SIX REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES 6.1 INTRODUCTION, 254 6.1.1 Abbreviated History of Reversed-Phase Chromatography, 255 6.2 RETENTION, 256 6.2.1 Solvent Strength, 257 6.2.2 Reversed-Phase Retention Process, 259 6.3 SELECTIVITY, 263 6.3.1 Solvent-Strength Selectivity, 263 6.3.2 Solvent-Type Selectivity, 265 6.3.3 Temperature Selectivity, 270 6.3.4 Column Selectivity, 273 6.3.5 Isomer Separations, 276 6.3.6 Other Selectivity Considerations, 278 6.4 METHOD DEVELOPMENT AND STRATEGIES FOR OPTIMIZING SELECTIVITY, 284 6.4.1 Multiple-Variable Optimization, 286 6.4.2 Optimizing Column Conditions, 295 6.5 NONAQUEOUS REVERSED-PHASE CHROMATOGRAPHY (NARP), 295 6.6 SPECIAL PROBLEMS, 297 6.6.1 Poor Retention of Very Polar Samples, 297 6.6.2 Peak Tailing, 298 Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R. Snyder, Joseph J. Kirkland, and John W. Dolan Copyright © 2010 John Wiley & Sons, Inc. 253 254 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES 6.1 INTRODUCTION This chapter describes the separation of neutral samples by means of reversed-phase chromatography (RPC). By a ‘‘neutral’’ sample, we mean one that contains no molecules that carry a positive or negative charge—usually as the result of the ionization of an acid or a base. Although a neutral sample implies an absence of acidic and basic solutes, this is not necessarily the case. Depending on mobile phase pH, any acids or bases in the sample may be present largely (e.g., 90%+) in the neutral (non-ionized) form—in which case their chromatographic behav- ior is similar to that of non-ionizable compounds. The separation of ‘‘ionic’’ samples (which contain one or more ionized compounds) by RPC is covered in Chapter 7. RPC is usually a first choice for the separation of both neutral and ionic samples, using a column packing that contains a less polar bonded phase such as C 8 or C 18 . The mobile phase is in most cases a mixture of water and either acetonitrile (ACN) or methanol (MeOH); other organic solvents (e.g., isopropanol [IPA], tetrahydrofuran [THF]) are used less often. A preferred organic solvent for an RPC mobile phase will be water-miscible, relatively nonviscous, stable under the conditions of use, transparent at the lowest possible wavelength for UV detection, and readily available at moderate cost. Commonly used B-solvents can be ranked in terms of these properties as follows: ACN (preferred) > MeOH > IPA THF (less useful) In a few countries, ACN is considered sufficiently toxic to limit its general use, but this is not true elsewhere. See Appendix I for further information concerning solvent properties and the choice of B-solvent for a given application. Samples that contain acids or bases normally require a buffered mobile phase, in order to maintain a constant pH throughout the separation (Chapter 7). Strongly retained, very hydrophobic samples may require a water-free mobile phase (nonaqueous reversed-phase chromatography [NARP], Section 6.5). Normal-phase chromatog- raphy (Chapter 8) can also provide acceptable separations of very hydrophobic samples, as sample hydrophobicity contributes little to retention for this HPLC mode. Preferred conditions for the isocratic separation of neutral samples by RPC are listed in Table 6.1. Compared to other forms of HPLC (normal-phase, ion-exchange chromatog- raphy, etc.; Table 2.1), separations by RPC are usually more convenient, robust, and versatile. RPC columns also tend to be more efficient and reproducible, and are available in a wider range of choices that include column dimensions, particle size, and stationary-phase type (C 1 –C 30 , phenyl, cyano, etc.; Section 5.3.3). The solvents used for RPC tend to be less flammable or toxic, and are more compatible with UV detection at wavelengths below 230 nm for increased detection sensitivity (Table I.2 of Appendix I). An additional advantage of RPC is generally fast equilibration of the column after a change in the mobile phase—or between runs when using gradient elution (Section 9.3.7). Finally, because RPC has been the dominant form of HPLC since the late 1970s, a better practical understanding of this technique has evolved. This usually means an easier development of better separations. All of the foregoing reasons have contributed to the present popularity of RPC. 6.1 INTRODUCTION 255 Table 6.1 Preferred Conditions for the Separation of Neutral Samples by Reversed-Phase Chromatography Condition Comment Column a Type: C 8 or C 18 (type-B) Dimensions: 100 × 4.6-mm particle size: 3 μm Pore diameter: 8–12 nm Mobile phase Acetonitrile/water Flow rate 2.0 mL/min b Temperature 30 or 35 ◦ C b %B To be determined c Sample Volume ≤ 25 μL Weight ≤ 50 μg k 1 ≤ k ≤ 10 a Alternatively, use a 150 × 4.6-mm column of 5-μm particles; flow rate, column dimensions, and particle size can be varied, depending on the anticipated difficulty of the separation and the maximum allowable column pressure (Section 2.4.1) b Initial values, which may be changed during method development (Section 2.5); a temperature 5–10 ◦ C above ambient is suggested in most cases. Also consult the column manufacturer’s recommendations for a maximum column temperature. c Varies with the sample; start with 80%B and adjust further as described in Section 2.5.1. Many organic compounds have limited solubility in either water or the water-organic mobile phases used for RPC, but this is rarely a practical con- cern. Thus very small weights (nanograms or low micrograms) of individual solutes are usually injected, so the required sample concentration is usually only a few micrograms/mL or less. In those cases where sample solubility in water or water-organic mixtures is exceptionally poor (very hydrophobic samples), the use of normal-phase chromatography with nonaqueous mobile phases may be preferred (Section 8.4.1). Some samples are less well separated by RPC. For example, very polar molecules may be retained weakly in RPC (k 1), even with 100% water as mobile phase; these samples may require a different approach (Section 6.6.1). Similarly enantiomers require separation conditions that exhibit chiral selectivity (Chapter 14). While many achiral isomers can be separated by RPC (Section 6.3.5), these compounds are often better separated by normal-phase chromatography using an unbonded silica column (Section 8.3.4.1). Finally, normal-phase chromatography is often a better choice for preparative HPLC (Chapter 15). 6.1.1 Abbreviated History of Reversed-Phase Chromatography Prior to the invention of RPC in 1950 by A. J. P. Martin [1], the chromatographic separation of neutral samples was carried out with a polar column (or stationary phase) and a less polar mobile phase; such separations are now referred to as . REVERSED-PHASE CHROMATOGRAPHY (NARP), 295 6.6 SPECIAL PROBLEMS, 297 6.6.1 Poor Retention of Very Polar Samples, 297 6.6.2 Peak Tailing, 298 Introduction to Modern Liquid Chromatography, Third Edition, by. 1986-07-01. 76. J. J. Kirkland and J. J. DeStefano, J. Chromatogr. A, 1126 (2006) 50. 77. L. R. Snyder and J. J. Kirkland, Introduction to Modern Liquid Chromatography, 2nd ed., Wiley, New York, 1979, ch. 5. 78 Conditions Particle Type a Particle Diameter (μm) Column Length (mm) a Plate Number N Totally porous 5 30 2,500–3,000 Totally porous 5 50 4,500–5,000 Totally porous 5 100 8,000–10,000 Totally porous