266 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES mobile phases will often provide a better separation. This proved to be the case for the present sample. A 1:1 blend of the mobile phases of Figure 6.8a (46% ACN) and Figure 6.9a (61% MeOH) was prepared, and used to obtain the separation of Figure 6.9b. The new mobile phase (containing 23% ACN + 30% MeOH) provides baseline resolution (R s = 1.8). (a) (b) S “regular” r 2 = 1.00 6 4 2 0 0246 log k w 2 1 0 log k %B (100f) 40 60 80 Figure 6.7 Variation of log k with %B for regular and irregular samples. (a )Regularsample(a mixture of herbicides [24]), separated on a C 18 column with methanol-water as mobile phase (see Fig. 2.6 for other conditions); (b)plotofvaluesofS versus log k w for data of (a); (c) irreg- ular sample of Figure 6.6; conditions as in Figure 6.6; (d) plot of values of S versus log k w for data of (c). (a, b) Adapted from [25]. 6.3 SELECTIVITY 267 (d) 1 1.5 2.0 2.5 3.0 4.0 3.5 3.0 2.5 2.0 S log k w “irregular” r 2 = 0.87 (c) %B (100f) 0 10203040506070 2.0 1.5 1.0 0.5 0.0 1 2 3 4 5 6 7 8 log k Figure 6.7 (Continued) While the separation of Figure 6.9b is much improved, further variation in the proportions of mobile phase from Figure 6.8a (46% ACN) and Figure 6.9a (61% MeOH) may provide some more resolution. Peaks 1 to 4 in Figure 6.9b are less resolved than remaining peaks 5 to 10 (i.e., peaks 1–4 are ‘‘critical’’), so we will limit our discussion to these peaks. Figures 6.10a–c replicate earlier chromatograms for peaks 1 to 4 from Figures 6.8 and 6.9, for changes in the relative proportions of ACN and MeOH. As MeOH replaces ACN in going from Figure 6.10a–c,it is seen that peaks 2 and 3 (shaded) move toward the front of the chromatogram. In Figure 6.10b (1:1 blend of mobile phases from Fig. 6.10a,c), the critical (least resolved) peak-pair is 2/3, and we can improve its resolution by a further movement 268 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES 0 2 4 6 8 10 12 14 Time (min) 1 2 3 + 4 5 7 8 9 10 6 (a) 46% ACN 1.1 ≤ k ≤ 4.6 R s = 0.0 0 20 40 Time (min) 1 2 + 3 + 4 5 6 7 8 9 10 (b) 34% ACN 2 ≤ k ≤ 14 R s = 0.3 Figure 6.8 Separation of a mixture of substituted benzenes as a function of solvent strength (%B). Sample: 1, p-cresol; 2, benzonitrile; 3, 2-chloroaniline; 4, 2-ethylaniline; 5, 3,4-dichloroaniline; 6, 2-nitrotoluene; 7, 3-nitrotoluene; 8, toluene; 9,3-nitro-o-xylene; 10, 4-nitro-m-xylene. Conditions: 250 × 4.6-mm (5-μm) C 8 column; acetonitrile–pH-6.5 buffer mobile phase; 35 ◦ C; 1.0 mL/min. Chromatograms recreated from data of [26]. Note that all compounds are non-ionized under these condition, so the sample is effectively neutral. 0 2 4 6 8 10 12 14 Time ( min ) 1 2 3 4 5 6 7 8 9 10 (b) 30% MeOH + 23% ACN 1 ≤ k ≤ 4 R s = 1.8 Time (min) 0246810 1 2 3 4 5 6 7 8 9 10 (a) 61% MeOH 0.8 ≤ k ≤ 3.0 R s = 0.6 Figure 6.9 Solvent-type selectivity. Separation of a mixture of substituted benzenes with methanol or mixtures of methanol-acetonitrile as mobile phase. Same sample and conditions as in Figure 6.8, except as noted in figure. Chromatograms recreated from data of [26]. of peak 2 away from peak 3 and toward peak 1. This can be achieved by increasing the proportion of MeOH in the final mobile phase (with respect to the mobile phase of Fig. 6.10b). As seen in Figure 6.10d, a mixture of 57% Figure 6.10c (61% MeOH) and 43% Figure 6.10a (46% ACN) positions peak 2 midway between peaks 1 and 3 for maximum resolution (R s = 2.0vs.R s = 1.8 in Fig. 6.10b). Achieving an optimum final separation in this example involves simple interpolation between 6.3 SELECTIVITY 269 4 6 Time (min) 1 2 4 3 (c) 61% MeOH 0.8 ≤ k ≤ 3.0 R s = 0.6 6 Time (min) 1 2 3 4 (d) 57/43 mixture of (c) and (a) 1 ≤ k ≤ 4 R s = 2.0 4 6 (a) 46% ACN 1.1 ≤ k ≤ 4.6 R s = 0.0 Time (min) 1 2 3 + 4 6 Time (min) (b) 50/50 mixture of (a) and (c) (30% MeOH + 23% ACN) 1 ≤ k ≤ 4 R s = 1.8 1 2 3 4 Figure 6.10 Solvent-type selectivity: fine-tuning the B-solvent. Same sample and conditions as in Figures 6.8 and 6.9 (peaks 1–4 only), plus added figure (d); (b) is 30% MeOH + 23% ACN, and (d) is 35% MeOH + 20% ACN. Chromatograms recreated from data of [26]. the two preceding experiments (Fig. 6.10b,c), much like the example of Figure 6.6 where %B was optimized. Similar optimizations of selectivity can be carried out more conveniently by the use of computer simulation (Chapter 10). Although improvements in resolution may be explored by changing the B-solvent (solvent-type selectivity), the new mobile phase must have a similar solvent strength in order to maintain comparable values of k and run time. In Figure 6.9a, a higher %-MeOH was used (61% vs. 46% ACN) because methanol is a weaker (more retentive) B-solvent than acetonitrile. When changing solvent type, we can estimate the necessary change in %B for the new B-solvent by means of the solvent nomograph of Figure 6.11. Here similar %B values for different B-solvents fall on vertical lines. Recall that in the previous example we needed to replace 46% ACN with a similar strength MeOH-water mobile phase. From the diagram of Figure 6.11, we see that 46% ACN should be about equivalent in strength to 57% MeOH (each of these two mobile phases is marked by • in Fig. 6.11); this is close to the mobile phase actually selected in Figure 6.9a (61% MeOH). Note that the run time in Figure 6.9a (11 min) is somewhat shorter than that in Figure 6.8a (15 min), 270 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES 100% 100% 100% 0 102030405060708090 0102030405060708090 0 20 30 40 50 60 70 80 90 THF/H 2 O MeOH/H 2 O ACN/H 2 O Figure 6.11 Solvent-strength nomograph for reversed-phase HPLC (adapted from [28]). Two mobile phases of equal strength (46% ACN and 57% MeOH) marked by • ,asanexample. as could be expected from Figure 6.11; that is, 61% MeOH is stronger than the 57% MeOH recommended by Figure 6.11. To summarize, when using a change in solvent type to change selectivity and increase resolution, proceed as follows: If ACN was used in the initial separation, and MeOH is to be substituted, Figure 6.11 shows how to estimate the best %-MeOH for use in the second separation. Next examine the ACN and MeOH chromatograms to determine if a mixture of ACN and MeOH is likely to give a better separation than either B-solvent alone (as in Fig. 6.9b). Conditions can be improved as illustrated by Figure 6.10 and discussed further in Section 6.4.1. If MeOH was used initially as the B-solvent, use Figure 6.11 to estimate the equivalent % ACN for a change in B-solvent without affecting the solvent strength. Solvent-type selectivity arises from interactions among solute, B-solvent, and column, as described in Sections 2.3.2.1 and 5.4.1. The preferential retention of the B-solvent in the stationary phase means that a stronger interaction of the B-solvent and solute will normally lead to increased retention (this may at first seem counterintuitive, in terms of the discussion of Fig. 2.7a,b). However, even qualitative predictions of the effect of a given B-solvent on the relative retention of different solute classes (e.g., phenols, ethers) are difficult at best. For further discussion, see [23]. 6.3.3 Temperature Selectivity Retention as a function of temperature can usually be described by log k = A + B T K (6.2) where A and B are constants for a given compound when only the absolute temperature T K (K) is varied. Plots of log k versus 1/T K should therefore yield straight lines, as in Figure 6.12a for a number of polycyclic aromatic hydrocarbons (PAHs). An increase in column temperature by 1 ◦ C usually results in a decrease in values of k by 1–2% for each peak in the chromatogram [29]. The effect of a change in temperature on selectivity is usually minor for the RPC separation of neutral samples. However, exceptions have been noted, as in the case of PAHs [30] and plant pigments [31]. Figure 6.12b is an extension of Figure 6.12a,withdatafor three additional PAHs (dashed curves A–C); compounds 1 to 6 are flat, fused-ring 6.3 SELECTIVITY 271 1.0 0.5 3.2 3.4 3.6 1000/T K 45°C35°C25°C15°C 6 5 4 3 2 1 A B C log k 22°C 1.0 0.5 3.2 3.4 3.6 1000/T K 45°C35°C25°C15°C 6 5 4 3 2 1 log k (a) (b) Figure 6.12 Retention of polycyclic aromatic hydrocarbons as a function of separation tem- perature. Conditions: 250 × 4.6-mm (5-μm) Chromegabond-C 18 column; 80% ACN-water; 1.0 mL/min. (a) Sample (fused-ring aromatic hydrocarbons): 1, anthracene; 2, fluoranthene; 3, triphenylene; 4, chrysene; 5, 3,4-benzofluoranthene; 6, 1,2,5,6-dibenzoanthracene. (b) Sam- ple same as (a), plus added poly-aryls: A,1,1  ,-dinaphthyl; B, 1,3,5-triphenylbenzene; C, 9,10-diphenylanthracene. Adapted from [30]. PAHs such as anthracene (peak 1), while compounds A to C are three-dimensional (nonflat) poly-aryls such as 1,1  ,-dinaphthyl (peak A). It is seen that retention reversals (marked by • ) occur in Figure 6.12b for five adjacent peak-pairs as temperature is varied between 15 and 45 ◦ C. (Note the ◦ C scale at top of figure): B/5 at 42 ◦ C, A/3 at 36 ◦ C, C/6 at 31 ◦ C, B/4 at 16 ◦ C, and C/5 at 6 ◦ C. In this example there are major changes in selectivity as temperature is varied. As a result several 272 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES temperatures exist between 10 and 70 ◦ C where baseline resolution (R s ≥ 1.5) is possible, for example, a temperature of 22 ◦ C, as shown in Figure 6.12b by a vertical dotted line. Changes in resolution with temperature for this sample are better seen in the resolution map of Figure 6.13a: a plot of critical resolution as a function of temper- ature. The resolution map of Figure 6.13a is enhanced in Figure 6.13b,toprovide additional insight into this very useful tool. Thus potentially critical peak-pairs (e.g., B/4, C/6, A/3) that correspond to different line segments in Figure 6.13a are identified in Figure 6.13b. Similarly arrows in Figure 6.13b indicate four temperatures where baseline resolution (R s ≥ 1.5) is possible. Resulting separations for the latter four preferred temperatures are shown in Figures 6.13c–f (note the changed retention order in each of these examples). The three poly-aryls A–C (shaded peaks) are seen to be retained more strongly at higher temperatures relative to the fused-ring PAHs 1–6 (see the further discussion of following Section 6.3.3.1). The best separation (R s = 2.1) is provided by a temperature of 22 ◦ C. While changes in temperature sometimes can be effective in altering separation selectivity for neutral samples, changes in selectivity with temperature are much more likely for partly ionized acids or bases (Section 7.3.2.2). For a further discussion of temperature selectivity in RPC, see [32, 32a]. 6.3.3.1 Further Observations The following treatment provides a more complete picture of temperature selectivity for neutral samples. However, the reader may prefer to skip this discussion and continue with Section 6.3.4. While Equation (6.2) is generally an adequate representation of RPC retention as a function of temperature, more complex changes in k with temperature are sometimes observed [7, 33–35]. Sometimes kincreaseswhen the temperature is increased, or plots of log k versus 1/T may be curved instead of linear. These exceptions to Equation (6.2) are often associated with acidic or basic solutes and a mobile phase pH that is close to the pK a value of the solute (see Section 7.2). In such cases a change in temperature may result in a change in the relative ionization of an acid or base (Section 7.2.3), with a large resulting change in solute retention—in addition to (and sometimes opposing) the normal effect of temperature that is described by Equation (6.2). The dependence of log k on temperature is determined by the value of B in Equation (6.2), which is proportional to the enthalpy of retention, H.Values of B usually increase for larger values of k; as a result plots of log k versus 1/T for different solutes (other conditions the same) often resemble the fan-shaped plots of Figure 6.12a for several fused-ring PAHs. The latter behavior can be compared with the similar example of Figure 6.7a forregularsamplesas%Bis varied; in both cases (change in %B or T) a change in condition has the largest effect on k for the most retained solute (with the largest values of k). For most neutral samples, changes in retention order with temperature are not expected (asobservedinFig.6.12a), but maximum resolution may still be observed at an intermediate temperature. However, a change in temperature is generally less useful for improving the resolution of neutral samples (similar to the case of ‘‘regular’’ samples and solvent-strength selectivity). 6.3 SELECTIVITY 273 As noted in Figure 6.12b, values of B tend to be relatively smaller for more com- pact molecules such as phenylnaphthalene (a poly-aryl), compared to less compact molecules such as anthracene (a fused-ring PAH). Similar temperature-selectivity effects have been observed for gas chromatography [36], where values of B in Equation (6.2) decrease in the order n-alkynes > n-alkanes > branched alkanes > cycloalkanes. That is, values of B in GC also decrease for less extended, more compact molecule—possibly for similar reasons. 6.3.4 Column Selectivity During the early days of HPLC, a change of column was often used as a means of varying selectivity and improving resolution. Indeed column selectivity represents a powerful means for altering relative retention and improving the separation of neutral samples. However, the use of column selectivity alone for the purpose of systematically improving separation has a serious limitation, compared to changes in %B, solvent type, or temperature. When any of the latter conditions are varied, 2.0 1.0 0.0 R s 10 20 30 40 50 60 70 T (°C) R s = 1.5 B/4 C/5 A/3 C/6 A/3 B/5 A/4 B/6 2.0 1.0 0.0 R s 10 20 30 40 50 60 70 T (°C) (a) (b) 3/4 A/4 Figure 6.13 Separation of polycyclic aromatic hydrocarbons as a function of temperature. Sample and conditions of Figure 6.12. (a, b) resolution map; (c–f ) chromatograms for differ- ent optimum temperatures. Adapted from [30]. 274 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES 024681012 02 0 02 0 40 024681012 Time (min) Time (min) Time (min) Time (min) T = 11°C R s = 1.9 T = 22°C R s = 2.1 T = 46°C R s = 1.7 T = 61°C R s = 1.7 (c) (d) (e) (f) 1 2 A 3 B 4 5 C 6 1 2 A 3 4 B 5 C 6 1 2 3 A 5 B 6 C 1 2 3 4 A 5 6 B C 4 10 30 14 16 Figure 6.13 (Continued) continuous changes in selectivity are possible, and there will often be an intermediate condition that corresponds to maximum resolution. In the examples of Figure 6.13, this greatly increased the likelihood of an acceptable separation, compared to the use of only one or two arbitrary temperatures. When the column is changed, the selection of an intermediate separation (between that provided by either column) is inconvenient. As a result a change in just the column is less likely to result in an improved separation (but see the further discussion of Section 6.4.1.4). Changes in the column are also less convenient than changes in continuous variables. Examples of a change in selectivity when only the column is changed are shown in Figure 6.14 for a 10-component neutral sample and four columns of varying selectivity. Despite significant changes in selectivity for this sample when the column is changed, no single column provides a resolution of R s > 0.8, as long as other conditions remain fixed (as in this example). If two different columns are connected together, additional separation possibilities are created, but this procedure 6.3 SELECTIVITY 275 Time (min) Symmetry C18 1.6 ≤ k ≤ 19, R s = 0.4 1 2 3 4 5 + 6 7 8 9 (a) 10 0 2 4 6 8 10121416 1 ≤ k ≤ 7, R s = 0.4 Alltima HP C18 amide Time (min) 1 3 2 + 4 5 7 6 8 9 + 10 (b) 0246 1 ≤ k ≤ 11, R s = 0.8 Luna phenyl-hexyl Time (min) 1 2 3 4 5 6 7 8 10 9 (c) 0246810 1 ≤ k ≤ 14, R s = 0.0 Spherisorb ODS-2 Time ( min ) 2 4 5 6 7 1 8+ 9 10 3 (d) 024681012 Figure 6.14 Separation of a mixture of 10 organic compounds of diverse structure on four different columns. Sample: 1, 4-nitrophenol; 2, 5,5-diphenylhydantoin; 3, acetophenone; 4, benzonitrile; 5, 5-phenylpentanol; 6,anisole;7, toluene; 8, cis-chalcone; 9, ethylbenzene; 10, trans-chalcone. Conditions: 150 × 4.6-mm (5-μm) columns; 45% acetonitrile-water; 35 ◦ C; 2.0 mL/min. Chromatograms recreated from data of [8, 9]. is inconvenient and only marginally better than the use of a single column (in the example of Fig. 6.14, no combination of these four columns can improve the separation of Fig 6.14c). The real advantage of a change in column selectivity is only achieved when a change in column is accompanied by a simultaneous change in one or more other conditions, for example, %B, solvent type, and/or temperature for the case of neutral samples (Section 6.4.1.4). . 5, 3,4-dichloroaniline; 6, 2-nitrotoluene; 7, 3-nitrotoluene; 8, toluene; 9,3-nitro-o-xylene; 10, 4-nitro-m-xylene. Conditions: 250 × 4.6-mm (5-μm) C 8 column; acetonitrile–pH-6.5 buffer mobile. Figure 6.11 shows how to estimate the best %-MeOH for use in the second separation. Next examine the ACN and MeOH chromatograms to determine if a mixture of ACN and MeOH is likely to give a better. 6.13a is enhanced in Figure 6.13b,toprovide additional insight into this very useful tool. Thus potentially critical peak-pairs (e.g., B/4, C/6, A/3) that correspond to different line segments in