Introduction to Modern Liquid Chromatography, Third Edition part 42 pot

366 NORMAL-PHASE CHROMATOGRAPHY hydrocarbons—according to the number of double bonds in the molecule, but with little effect of differences in alkyl substitution or solute molecular weight. Similarly lipid samples can be resolved into mono-, di-, and tri-glycerides (as well as other compound classes). Isomeric solutes are usually much better separated by NPC than by RPC, as seen in Figure 8.2b (C 1 or methyl-substituted anilines), Figure 8.2c (C 2 or dimethyl- plus ethyl-substituted anilines), and Figure 8.2d (C 3 or trimethyl-, methylethyl-, and n-propyl-substituted anilines). The RPC retention of isomers (e.g., C 1 anilines) is generally similar (with marginal separation), while the reverse is true for NPC (more varied values of k for different isomers, and better separation). The greater isomer-selectivity of NPC versus RPC is also shown by the range in values of k for each set of isomers; that is, the ratio of k-values for the most retained and least retained isomers (‘‘range in k,’’ Fig. 8.2e). The spread in k values for a group of isomers (C 1 ,C 2 ,orC 3 ) ranges from 1.1- to 1.2-fold for RPC, versus 2.0- to 3.4-fold for NPC, which is many times larger for NPC. When two isomeric compounds prove difficult to separate by RPC (Section 6.3.5), NPC will usually prove more effective. Thus, for preparative separations (Chapter 15), where the largest possible values of α are desirable, NPC with a silica column is strongly recommended for the separation of achiral isomers (for chiral isomers, see Chapter 14). For a review of the RPC separation of isomers, see Section 6.3.5; for a further discussion of isomer separation by NPC, see Section 8.3.5. If in doubt as to whether a separation is based on NPC or RPC retention, a simple test is to vary the polarity of the mobile phase. If retention increases with increased mobile-phase polarity, RPC is involved; if retention decreases with increased mobile-phase polarity, NPC can be assumed. This test holds whether the stationary phase is unbonded or bonded silica, and whether the mobile phase is aqueous or nonaqueous (note that water is the most polar of all solvents). 8.2.1 Theory Retention in NPC is best described by a displacement process, based on the fact that the silica surface is covered by a monolayer of solvent molecules that are adsorbed from the mobile phase [1, 5, 6]. Consequently, for a solute molecule to be retained in NPC, one or more previously adsorbed solvent molecules must be displaced from (leave) the silica surface in order to make room for the adsorbing solute. Displacement in NPC is illustrated in Figure 8.3a, b for a relatively nonpolar solute (chlorobenzene) and a weaker, less-polar mobile-phase solvent methylene chloride. When a molecule of chlorobenzene moves from the mobile phase in Figure 8.3a to the stationary phase in Figure 8.3b, one or more pre-adsorbed solvent molecules CH 2 Cl 2 must be displaced from the stationary phase and return to the mobile phase. In this example the adsorbed solute molecule is assumed to lie flat on the surface of the silica, and to occupy an area that is indicated in Figure 8.3b by the dotted rectangle that surrounds the molecule of retained chlorobenzene. By reference to Figure 8.3a, it is seen that this same area was originally occupied by (approximately) two retained molecules of CH 2 Cl 2 . Consequently the resulting retention equilibrium canbewrittenas Y (m) + nM (s) ⇔ Y (s) + nM (m) (8.1) 8.2 RETENTION 367 where n is the number of solvent molecules M displaced by a retained solute molecule Y (n = 2 in the example of Fig. 8.3a,b). Y (m) and Y (s) refer to a molecule of solute Y in the mobile and stationary phases, respectively, while M (m) and M (s) refer to a molecule of solvent M in the mobile and stationary phases, respectively. The quantity n in Equation (8.1) is thus the ratio of molecular areas for the solute with relation to the mobile phase. Retention differs for a more-polar mobile-phase solvent such as tetrahydrofu- ran (THF) and a more polar solute such as phenol (Fig.8.3c,d). Here the interaction of solvent and solute molecules with surface silanols will be stronger, as indicated by the arrows that connect the two interacting species—in contrast to the weaker (a)(b) HO H O Mobile phase Stationary phase Mobile phase Stationary phase Y (m) + 2 M (s) Y s + 2 M(m) H O H O H O Y (m) + M (s) Y (s) + M (m) (c)(d) H O C l H O H O H O H O H O Cl CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 CH 2 Cl 2 HO Figure 8.3 Hypothetical examples of solute retention on silica for chlorobenzene (a,b non- localized) and phenol (c,d localized). Mobile phase in (a,b) is a less-polar solvent (CH 2 Cl 2 ); mobile phase in (c,d) is a more-polar solvent (tetrahydrofuran, THF). 368 NORMAL-PHASE CHROMATOGRAPHY and less specific interactions shown in Figure 8.3a,b. As a result there is a ratio 1 : 1 interaction of a surface silanol with a polar group in a molecule of either solute or mobile phase—called localized adsorption. Under these conditions adsorbed molecules can assume a vertical rather than flat configuration, as illustrated by the retention of phenol in Figure 8.3d. In the example of Figure 8.3a,b, the entire solute molecule (chlorobenzene) is attracted more strongly to the silica surface than are molecules of the less-polar solvent CH 2 Cl 2 . In Figure 8.3c,d, the very polar –OH group of the phenol solute interacts strongly with a silanol (–OH) group on the silica surface, while the less-polar phenyl group is attracted less strongly than are molecules of the strong (very polar) solvent THF. As a result the phenyl group cannot compete with molecules of THF for a place on the silica surface; the phenyl group therefore dangles out into the mobile phase, tethered to the surface by the interaction of a silanol with solute-hydroxyl groups. For solutes with some number n of more strongly retained (very polar) substituent groups (e.g., –OH, –NH 2 ), Equation (8.1) will still apply, with n referring now to the number of polar groups in the solute molecule that can simultaneously interact with silica silanols. However, for less-polar molecules of the mobile phase as in Figure 8.3a,b, n refers to the ratio of areas required by molecules of the solute and mobile-phase, respectively. The competition of solute and solvent molecules for a place on the silica surface will be affected by the interactions of each molecule with the mobile and stationary phases. Because polar interactions predominate in NPC, polar molecules of solute or solvent will interact much more strongly with the more-polar silica surface than with the less-polar mobile phase. As a first approximation we can ignore interactions between solute and solvent molecules and consider only interactions of the solute and solvent with the stationary phase. This allows the derivation of a simple equation for k as a function of the concentration of the B-solvent in a binary mobile phase A–B [1, 6]: log k = log k A − A s ε (8.2) where k A is the value of k for a nonpolar A-solvent (for which ε is zero), A s refers to the molecular area of the solute molecule, and the mobile-phase solvent strength ε can be calculated as a function of (1) the solvent strengths ε A and ε B of the pure A- and B-solvents, respectively, (2) the molecular area of the B-solvent, and (3) the concentration (%B) of the B-solvent in the mobile phase (see Eq. 8.5 in Section 8.2 following). The remainder of this section provides a quantitative, somewhat detailed, discussion of retention as a function of the solute and mobile phase. The reader may prefer to skip to Section 8.2.2 (which provides a practical summary of the discussion below) and return to this section as appropriate. Equation (8.2) applies approximately for all solvents and solutes. For more polar solutes and B-solvents, as in Figure 8.3c,d, polar solute groups can attach to individual silanols. So Equation (8.1) now applies with n equal to the number of polar groups in the solute molecule (rather than the area of the solute molecule). If %B is large enough, and the B-solvent is strong enough, the silica surface will be covered almost entirely by the B-solvent (to the exclusion of the A-solvent); under these conditions the concentration of adsorbed B-solvent will not vary much when 8.2 RETENTION 369 %B is changed. For this case the Soczewinski equation can be derived from Equation (8.1) [7, 8]. Thus the equilibrium for Equation (8.1) can be written as K eq = X Y,s X M,m n X Y,m X M,s n (8.3) where X Y,s and X Y,m refer to the concentrations (mole-fractions) of solute Y in the stationary (s) and mobile (m) phases, respectively. Similarly X M,s and X M,m are the mole-fractions of the B-solvent M in the stationary (s) and mobile (m) phases. The retention factor k can be written as k = ψ  X Y,s X Y,m  (8.3a) where ψ is the phase ratio: the volume-ratio of stationary phase to mobile phase within the column. If the silica surface is covered almost entirely by the B-solvent, X M,s ≈ 1, Equations (8.3) and (8.3a) then yield log k = log k B − n log X B (Soczewinski equation) (8.4) where n is the number of B-solvent molecules displaced by the solute (approximately equal to the number of polar substituent groups in the solute molecule); k B refers to the value of k for pure B-solvent as the mobile phase (100% B), and X B is the mole-fraction of the B-solvent in the mobile phase. Equation (8.4) can be regarded as a special case of Equation (8.2), whenever X M,s ≈ 1 (i.e., for larger concentrations of the B-solvent). Equation (8.4) is more conveniently (and approximately) expressed as log k ≈ log k B − n log φ (8.4a) where φ is the volume-fraction of the (polar) B-solvent in the mobile phase (= 0.01× %B). An example of the application of Equation (8.4a) is shown in Figure 8.4 for two different (polar) solutes, and a mobile phase composed of hexane (A) plus tetrahydrofuran (B), with %B varying between 8 and 27%. In each case an approximately linear plot of log k against φ is observed. Values of n from Equation (8.4a) for the two plots of Figure 8.4 are each equal to about 1.4, which is somewhat greater than the expected value of n = 1.0 (as there is just one polar –OH group in each solute molecule); this is a consequence of the approximate nature of Equation (8.4a). Equations (8.4) and (8.4a) are reliable for more-polar B-solvents (ε 0 B > 0.4) and higher concentrations of the B-solvent ( > 10% B)—conditions that assure that the surface of the silica will be covered almost entirely by molecules of the B-solvent (instead of the A-solvent). Typically values of n in Equation (8.4a) fall between one and two for representative small-molecule solutes (molecular weights <500 Da), so that on average a change in %B by a factor of two (e.g., a change in mobile phase from 100% B to 50% B) will change values of k by a factor of 2 to 4. Similar plots of log k against log %B as in Figure 8.4 are shown in Figure 8.5a for two less-polar solute molecules, using the weaker B-solvent chloroform (ε 0 B = 0.26 with lower values of %B. Because these conditions fail to meet the above 370 NORMAL-PHASE CHROMATOGRAPHY 1.4 1.2 1.0 0.8 0.6 0.4 0.2 log k benzyl alcohol 3-phenyl-1-propanol y = x –1.0 log φ 10 15 20 25 30 %-THF –0.8 –0.6 –0.4 Figure 8.4 Dependence of log k on log %B for solutions of a polar (localizing) B-solvent. Sample: benzyl alcohol and 3-phenyl-1-propanol. Conditions: silica column; tetrahydrofuran (THF)-hexane mobile phases; 25 ◦ C. Data from [11]. requirements for the application of Equation (8.4a), the resulting plots are curved rather than straight (as required by the theory upon which Eqn. 8.4a is based), and the best-fit values of n are 1. That is, a change in %B by a factor of 2 for this example produces a change in k by much less than 2- to 4-fold. However, the use of the more accurate Equation (8.2) for these data (Fig. 8.5b) yields a near-linear fit of values of log k versus ε 0 as expected. 8.2.2 Solvent Strength as a Function of the B-Solvent and %B Solvent strength in NPC depends on the polarity of the solvent; more polar solvents are stronger, resulting in smaller values of k for a sample. The strength of a pure solvent for unbonded silica as column packing can be expressed by the solvent-strength parameter ε of Equation (8.2); for pure solvents, ε will be referred to as ε 0 . As the value of ε increases, the solvent becomes stronger, and solute k-values decrease. Values of ε 0 for some commonly used NPC solvents are listed in Table 8.1; for additional values of ε 0 for other pure B-solvents, see Appendix I. The value of ε for a mixture of A- and B-solvents is given by [1, 6] ε = ε O A + log[N B 10 n B (ε 0 B −ε 0 A ) + 1 − N B ] n B (8.5) Here, ε 0 A and ε 0 B refer to the ε 0 values of pure solvents A and B, N B is the mole-fraction of solvent B in the mobile phase, and n B represents the relative size (area) of solvent B (relative to a value of n B = 6 for benzene as B-solvent). Equation (8.5) assumes a fully active (non–water-deactivated) adsorbent such as silica. When a weaker A-solvent (e.g., hexane) is mixed with a stronger B-solvent (e.g., CH 2 Cl 2 ), the resulting mobile phase will have an intermediate strength and 8.2 RETENTION 371 value of ε that is given by Equation (8.5). Figure 8.6 is a solvent nomograph for NPC with a silica column (similar to that for RPC in Fig. 6.11), which compares the strengths of different mobile-phase mixtures in terms of their values of ε (see values at top of Fig. 8.6, calculated from Eq. 8.5). A change in ε by 0.05 units will change values of k by roughly a factor of 2. For example, a mobile phase of 50% methylene chloride-hexane has ε = 0.24 (dotted vertical line of Fig. 8.6). If a change nitrobenzene phenanthrene (a) ( b ) 2.5 2.0 1.5 1.0 0.5 0.0 log k 0.00 0.02 0.04 0.06 0.08 ε 0 %B = 0.5% 1% 2% 3% 4% 5% –2.5 –2.0 –1.5 log %B 2.0 1.5 1.0 0.5 log k y = x nitrobenzene phenanthrene %B = 0.5% 1% 2% 5% Figure 8.5 Dependence of log k on log %B for dilute solutions of a lesspolar (nonlocalizing) B-solvent. Sample: nitrobenzene and phenanthrene. Conditions: silica column; CHCl 3 -hexane mobile phases; 25 ◦ C. (a) unsatisfactory correlations of log k with log φ (Eq. 8.4); (b)improved correlations of log k with ε (Eq. 8.2). Data from [11]. 372 NORMAL-PHASE CHROMATOGRAPHY Table 8.1 Solvent Strength (ε 0 ), Molecular Area (n B ), and UV Absorbance as a Function of Wavelength for Normal-Phase Solvents Solvent ε 0g n B Absorbance (AU) at Indicated Wavelength (nm) 200 210 220 230 240 250 260 Acetonitrile a,b 0.52 3.1 0.03 0.02 0.00 0.00 0.00 0.00 0.00 Ethoxynonafluorobutane 0.01 h Presumed similar to ethyl ether Chloroform c 0.26 5.0 > 1.0 > 1.0 > 1.0 > 1.0 > 1.0 0.25 0.08 Ethyl acetate b 0.48 5.2 > 1.0 > 1.0 > 1.0 > 1.0 > 1.0 > 1.00.10 Ethyl ether d,e 0.43 4.4 > 1.0 > 1.0 0.46 0.27 0.18 0.10 0.05 Hexane, heptane 0.00 h 0.35 0.20 0.07 0.03 0.02 0.01 0.00 Methanol a 0.70 3.7 > 1.0 0.53 0.23 0.10 0.04 0.02 0.01 Methylene chloride c 0.30 4.1 > 1.0 > 1.0 > 1.0 > 1.0 0.09 0.00 0.00 Methyl-t-butyl ether d 0.48 4.1 > 1.0 0.70 0.54 0.45 0.28 0.10 0.05 n-Propanol f 0.60 4.4 > 1.0 0.65 0.35 0.15 0.07 0.03 0.01 i- Propanol f 0.60 4.4 > 1.0 0.44 0.20 0.11 0.05 0.03 0.02 Tetrahydrofuran d,e 0.53 5.0 > 1.0 > 1.0 0.60 0.40 0.21 0.18 0.09 Sources: Data from [6, 13]. a immiscible with hexane. b Nonbasic localizing. c Nonlocalizing. d Basic localizing. e Easily oxidized and therefore less useful in practice. f Very strong (localizing), proton-donor solvent; classification as ‘‘basic’’ or ‘‘nonbasic’’ may not be relevant. g Values from [6], derived as described in [1]. h Values of n B for A-solvents are not required in Equation (8.5). in values of k by 2-fold is needed, a mobile phase of 30% methylene chloride-hexane (ε = 0.19) will increase k by about 2-fold, while 95% methylene chloride-hexane (ε = 0.29) will decrease k by about 2-fold. Mobile phases with the same values of ε in Figure 8.6 should provide similar values of k for a given sample; for example, suppose that 50% CH 2 Cl 2 -hexane (ε = 0.24) has been found to provide 1 ≤ k ≤ 10 for given sample and a silica column. From Figure 8.6 we can predict that 3.5% MTBE-hexane, 6% THF-hexane, or 4% ethyl acetate-hexane will each have a similar solvent strength (ε = 0.24); each of these four mobile phases should therefore provide a retention range of about 1 ≤ k ≤ 10 for the same sample. Figure 8.6 is thus useful for selecting a different B-solvent in order to change selectivity (Section 8.3.2), while maintaining the same solvent strength and similar values of k. Because of potentially large changes in solvent-type selectivity for NPC (Section 8.3.2), the NPC solvent-nomograph of Figure 8.6 is somewhat more approximate than the corresponding nomograph of Figure 6.11 for RPC. An example of NPC separation as a function of %B is shown in Figure 8.7 for an arbitrary mixture of organic compounds, using a silica column with mixtures of 8.2 RETENTION 373 2 4 6 10 20 50 100 1 2 4 6 10 50 100 1 2 4 6 10 50 100 1 2 4 6 10 50 100 0.5 1 2 3 5 10 20 50 100 0 0.1 0.2 0.3 0.4 0.5 0.6 e %v CH 2 Cl 2 -hexane %v MTBE-hexane %v THF-hexane %v Ethyl acetate-hexane %v propanol- hexane 10 20 50 100 10 10 20 %v MTBE-CH 2 Cl 2 %v THF-CH 2 Cl 2 %v Ethyl acetate-CH 2 Cl 2 %v propanol- CH 2 Cl 2 50 100 10 50 100 10050 Figure 8.6 Solvent nomograph for normal-phase chromatography and silica columns. Adapted from [12]. ethyl acetate (B) and cyclohexane (A) as mobile phase. An increase in %B by a factor of two (40% B in Fig. 8.7b vs. 20% B in Fig. 8.7a) leads to a decrease in values of k by a factor of 2 to 3 in this example. Changes in relative retention with %B are also seen in Figure 8.7, as discussed in following Section 8.3.1 (solvent-strength selectivity). Because of these changes in relative retention with %B, an intermediate mobile-phase composition (Fig. 8.7c) provides the best resolution for this sample. 8.2.3 Use of TLC Data for Predicting NPC Retention Thin-layer chromatography (TLC) can be a useful complement to the use of HPLC with a silica column. Corresponding separations by TLC and column chromatogra- phy (involving the same sample, mobile phase, temperature, and especially the same silica as stationary phase) should yield similar values of k for each compound in the sample. As illustrated in Figure 8.8, the positions of separated bands (spots) on a TLCplatecanbeusedtoestimatevaluesofk for a corresponding column separa- tion. The R F value of a solute in TLC is defined as its fractional migration from the original sample spot (point at which the sample is applied) toward the solvent front (end of solvent migration during TLC). Thus a sample band that migrates half as far as the solvent front (e.g., spot D in Fig. 8.8) would have R F = 0.5; the R F values of solutes A through D in Figure 8.8 are 0.15, 0.25, 0.38, and 0.50, respectively. 374 NORMAL-PHASE CHROMATOGRAPHY 0 Time (min) 024 Time (min) 024 Time ( min ) 20% B, e = 0.31 3 ≤ k ≤ 5, R s = 0.1 40% B, e = 0.38 1 ≤ k ≤ 3, R s = 1.3 25% B, e = 0.33 2 ≤ k ≤ 4, R s = 1.6 1 2 3 + 4 5 6 1 4 2 3 5 6 1 4 2 3 5 6 (a) (b) (c) 123 Figure 8.7 Solvent-strength selectivity in normal-phase chromatography. Sample: 1, 2-aminonaphthalene; 2, 2,6-dimethylquinoline; 3, 2,4-dimethylquinoline; 4, 4-nitrophenol; 5, quinoline; 6, isoquinoline. Conditions: 150 × 4.6-mm silica column (5-μm particles); ethy- lacetate (B)-cyclohexane (A) mixtures as mobile phase; ambient temperature; 2.0 mL/min. Peaks 1 and 4 are shaded to emphasize their change in relative retention as %B is varied. Chro- matograms recreated from data of [14]. Corresponding values of k can be obtained from the relationship k = 1 − R F R F (8.6) In the example of Figure 8.8, the R F values of the spots vary from 0.15 to 0.50, corresponding to 1 ≤ k ≤ 5.7. Similar k-values are expected for a corresponding HPLC separation (use of the same mobile phase with a silica column), hence providing an acceptable retention range (1 ≤ k ≤ 10) for this sample. TLC is sometimes used prior to NPC separation with a column, as a convenient way of anticipating possible problems and/or exploring different mobile phases. Sample components (bands) that do not move (R F ≈ 0.0) will be visible in TLC, whereas any strongly retained solutes in column NPC will remain in the column and hence be undetected and perhaps missed. A related advantage of a preliminary TLC separation is that samples that might otherwise contaminate a column—and therefore require pretreatment for column chromatography (Chapter 16)—can usually be applied directly in TLC because a TLC plate is used only once and then discarded. By trial-and-error changes in %B (e.g., for methylene chloride-hexane mixtures) a value of %B that can provide values of 1 ≤ k ≤ 10 for HPLC separation 8.2 RETENTION 375 solvent front original sample spo t R F k 1.0 0.0 0.9 0.1 0.8 0.2 0.7 0.3 0.6 0.7 0.5 1.0 0.4 1.5 0.3 2.3 0.2 4.0 0.1 9.0 0.0 ∝ D C B A Figure 8.8 Use of thin-layer chromatography (TLC) for selecting a mobile phase to be used for HPLC with a silica column (hypothetical sample). can be obtained in a short time by means of TLC—provided that isocratic elution is possible. If 1 ≤ k ≤ 10 is the goal for all peaks in isocratic column chromatography, R F values in a corresponding TLC separation should fall between 0.1 and 0.5. TLC experiments may show that the sample cannot be separated isocratically by column NPC (no value of %B results in sample bands with 0.1 ≤ R F ≤ 0.5). Changes in relative retention as a result of solvent-strength selectivity (Section 8.3.1) or solvent-type selectivity (Section 8.3.2) can also be explored by TLC—often more conveniently than by HPLC (see the example of Section 8.4.3). While the use of TLC as described above is reasonably reliable, it should be noted that solvent demixing (Section 8.5.2) can lead to misleading results in some cases. When dilute solutions (<10% B) of strong B-solvents (ε 0 > 0.4) are used as the mobile phase in TLC, the B-solvent can be strongly retained by the silica, leading to its removal from the mobile phase during separation. As a result the concentration of B-solvent in the mobile phase will be lowered, the mobile-phase strength will be reduced, and observed values of R F will be too small; thus values of k that are estimated from R F values may be too large when solvent demixing occurs. This problem can be minimized by first equilibrating the TLC plate with the mobile phase. The plate should be placed in the developing chamber for 15 minutes—but without allowing the mobile phase to touch the plate and initiate sample migration [9]. This way the vapor above the solvent will equilibrate the plate and minimize any solvent demixing, after which the plate is lowered to contact the mobile phase and begin the separation. TLC can be especially useful for monitoring preparative separations. Thus several fractions from a separation by column chromatography can be conveniently analyzed at one time, by spotting a single (suitably large) plate with multiple samples—each in a different lane of the plate. Fractions that are seen to contain the desired product, uncontaminated by adjacent impurity peaks, can be combined for further processing. . so that on average a change in %B by a factor of two (e.g., a change in mobile phase from 100% B to 50% B) will change values of k by a factor of 2 to 4. Similar plots of log k against log %B. assumed to lie flat on the surface of the silica, and to occupy an area that is indicated in Figure 8.3b by the dotted rectangle that surrounds the molecule of retained chlorobenzene. By reference to Figure. solute and mobile phase. The reader may prefer to skip to Section 8.2.2 (which provides a practical summary of the discussion below) and return to this section as appropriate. Equation (8.2)