316 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY A − or BH + by ion-pairing: ionized solute ion pair (acids) A − + X + ⇔ A − X + (7.5) (bases) BH + + X − ⇔ BH + X − (7.5a) hydrophilic (less retained in RPC) hydrophobic (more retained in RPC) Buffers that ion-pair are usually organic ions, and they tend to be more hydrophobic than inorganic ions. For example, trifluoroacetate is a significant ion-pairing agent (Section 13.4.1.2), whereas phosphate is not. Ion-pairing by inorganic buffers is usually not significant, although some studies suggest that even phosphate may undergo weak ion-pairing with protonated bases [8]. For further information on ion-pairing by the buffer, see Section 7.4.2.1. Buffer Stability and Equipment Compatibility. Inorganic buffers and carboxylic acids are usually stable, but amines are prone to oxidation with a consequent increase in their UV absorbance at wavelengths <250 nm. For example, although pure triethylamine (TEA) should be transparent at wavelengths ≥ 200 nm, one study [9] reported that a 1% aqueous solution of TEA (90 mM) has an absorbance > 2 AU at 220 nm. Citrate buffers have been claimed to attack stainless steel, whereas other reports contradict this claim as applied to HPLC equipment [10]. One laboratory has experienced occasional equipment problems that appeared associated with the use of citrate buffer [11], but this behavior has not been confirmed. While it appears likely that citrate concentrations <10 mM can be used without concern, and its use is convenient for applications where pH is to be varied continuously over the range 2 < pH < 7, caution is nevertheless advised. Overall, the (real or imagined) problems in the use of citrate, its relatively high UV cutoff of 230 nm (Table 7.1), and the availability of favorable alternative buffers (e.g., phosphate plus acetate) make citrate a seldom-used buffer by most workers. 7.2.1.3 Preferred Buffers UV detection in the 200 to 220 nm region is often used for samples with low concentrations of compounds that absorb poorly at higher wavelengths. Phosphate buffers have been preferred for this application, as long as the mobile-phase pH can be accommodated within the ranges specified in Table 7.1 (pH ≤ 3.5, 6.0 ≤ pH ≤ 8.5, or pH ≥ 11.0). Formate and acetate cover the pH range of 2.5 to 6.0, can be used at wavelengths of 210 nm or higher, and are volatile for LC-MS detection; for LC-MS detection at low pH with basic solutes, ammonium acetate or formate buffers provide an increase in ionic strength that allows higher sample concentrations without peak tailing [12]. Phosphate, acetate, and formate are the most commonly used buffers for separations with a mobile-phase pH < 8.0. There is increasing use of higher pH mobile phases (pH > 8) as a result of the development of RPC columns that are stable at high pH (Section 5.2.5). Borate and ammonia have been used to some extent as buffers for high-pH operation, but note the further discussion of Section 5.8 concerning column stability at high pH. See 7.2 ACID –BASE EQUILIBRIA AND REVERSED-PHASE RETENTION 317 also Appendix II for details on the more convenient preparation of some common buffers of required pH. 7.2.2 pK a asaFunctionofCompoundStructure When selecting a range of mobile-phase pH values within which to carry out method development (i.e., optimization of pH), it can be useful to know the approximate pK a values of the various sample components. This information allows the mobile phase to be restricted within an appropriate range of pH values. For example, a mobile-phase pH range that varies from pK a − 1topK a + 1 (for the sample) is useful for controlling retention and selectivity by changes in mobile-phase pH. Similarly a mobile-phase pH outside this range will result in a more robust method that is less sensitive to small (unintended) changes in pH. Values of pK a vary widely for different organic compounds, but a large number of pK a values have been determined experimentally [13], and additional pK a values can be estimated on the basis of solute molecular structure. One very extensive source of experimental plus predicted pK a values (ACD/pK a DB: pK a prediction) is available from Advanced Chemistry Development (Toronto, Canada). Exact pK a values for sample components are not required in RPC method development, and in many cases the chemical structures of sample components may not even be known. Values of pK a are determined by ionizable acid or base groups attached to the solute molecule, for example, –COOH, –NH 2 . This means that estimates of pK a can be obtained from literature pK a values for compounds of similar functionality (e.g., benzoic acid, as a representative of aromatic carboxylic acids). Table 7.2 summarizes pK a values in water for some common acid- or base-substituent groups present in typical sample molecules. It is also possible to infer values of pK a from experimental plots of retention against pH, as for peaks 2and4inFigure7.3a. 7.2.3 Effects of Organic Solvents and Temperature on Mobile-Phase pH and Sample pK a Values This detailed section is less essential in everyday operation, so the reader may wish to skip to Section 7.3. Nevertheless, the conclusions of this section are potentially useful for an accurate interpretation of the relationship between sample retention and mobile-phase pH. Literature values of pK a for different compounds (as in Table 7.2) are usually reported for aqueous solutions at near-ambient temperatures. Quite often, however, RPC separations of ionic samples are carried out at higher temperatures, with mobile phases that contain varying amounts of organic solvent. Both mobile-phase pH and values of pK a for the sample can be affected by added organic (specifically, the value of %B) and by temperature. However, a knowledge of the true (mobile-phase) pH and solute pK a values as a function of %B and temperature has little practical importance, so far as routine RPC assays are concerned; it is only important that %B and temperature are maintained constant for all runs so that solute ionization and retention remain unchanged from run to run. In the case of method development, however, approximate values of pK a can be useful for selecting the pH of the mobile phase (as discussed above). It is useful in this connection to define an ‘‘effective’’ pK a value for the solute, which takes into account the effects of %B 318 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY Table 7.2 Approximate pK a Values for Acidic or Basic Functional Groups (aqueous solutions) pK a Acid Base Group Aliphatic Aromatic Aliphatic Aromatic Sulfonic acid, –SO 3 H11 Amino acid, –C(NH 2 )–COOH a 310 Carboxylic acid, –COOH 5 4 Thiol, –SH 10 7 Purine 3 9 Phenol, –OH 11 Pyrazine 1 Sulfoxide, –SO– 2 Thiazole 2 Amine, –NH 2 ,–NR 2 10 5 Pyridine 5 Imidazole 5 Piperazine 10 Note: Values can vary by 1 to 2 pK a units or more as a result of adjacent groups in the molecule. a See Figure 13.1 for values of individual amino acids. and temperature on the pK a values of both solute and buffer (Tables 7.1 and 7.2). Effective pK a values for the solute can be used with the pH of the buffer (not the buffer-organic mobile phase) to estimate solute ionization and retention as a function of pH. An effective pK a value is equivalent to the value that can be inferred from an experimental plot of retention against mobile-phase pH (i.e., buffer pH, as in Fig. 7.2). 7.2.3.1 Effect of %B on Values of Effective pK a for the Solute The pH of the mobile phase depends on the pK a value for the buffer, while a change in %B will affect pK a values of both the buffer and the solute [14–16]. If the buffer and solute are each acidic (e.g., phosphate buffer and a carboxylic acid solute), changes in pK a with %B will be similar for both solute and buffer—and hence cancel approximately. For the latter case, effective pK a values for the solute can be assumed equal to the values of Table 7.2. The same will be true when both the buffer and solute are basic (i.e., effective solute pK a values that are similar to values measured in water at room temperature). However, when the buffer is acidic and the sample is basic, and vice versa, the apparent change in pK a with %B can be substantial, so that effective pK a values for the solute will no longer be the same as literature values measured in water. Commonly used RPC buffers are more often acidic than basic (e.g., phosphate, acetate, formate). For the case of basic solutes and acidic buffers (with methanol as B-solvent), a decrease in the apparent pK a by about 0.03 units can be expected for 7.3 SEPARATION OF IONIC SAMPLES BY REVERSED-PHASE CHROMATOGRAPHY (RPC) 319 each 1% increase in %B (experimental data of [17, 18]). For example, consider the separation of several substituted anilines, using 25% methanol-phosphate buffer, as reported in [17]. Effective pK a values for these solutes would be expected to be 25 × 0.03 = 0.75 units lower than literature values. For eight different solutes with literature pK a values of 2.7 to 5.3, experimental pK a values (as in Fig. 7.2) for this sample were lower by an average 0.7 ± 0.1 units (1 SD). Similarly, for six substituted benzoic acids separated with 40% methanol and acetate buffer [17], effective pK a values were the same as literature values (±0.1 unit, 1 SD). The effect of added acetonitrile on effective pK a values is similar to that for the addition of methanol. The effect on relative retention of changes in effective pK a values with %B is equivalent to a change in mobile-phase pH (the ‘‘effective’’ pH of the mobile phase), which suggests that a change in %B can have a larger effect on relative retention and selectivity for ionic samples than for neutral samples. This has been observed for gradient elution as a function of gradient steepness [19, 20], which is equivalent to a change in %B for isocratic elution (Section 9.1.3). In the discussion above, the mobile-phase pH is equated to that of the measured pH of the aqueous buffer, a procedure that we recommend for reasons given in Section 7.2.1. The pH of the final, water-organic mobile phase could be measured instead (less conveniently, and less reproducibly), as suggested by some workers [14–16]. However, possible changes in solute pK a values with %B would still require correction; that is, the use of pH values measured in the mobile phase does not solve the problem of solute pK a values that vary with %B. Again, we strongly recommend measuring the pH of the buffer, not the final mobile phase. 7.2.3.2 Effect of Temperature on Values of pK a Values of pK a for both the buffer and sample can vary with temperature [21–25]. For protonated basic solutes, a lowering of effective pK a values with increasing temperature [24] can lead to a decrease in solute ionization and an atypical increase in retention at higher temperatures. Because of such changes in pK a with temperature, significant changes in relative retention with temperature can also result for ionic samples—more so than for neutral samples [19, 20]. That is, temperature selectivity will generally be greater for ionic samples. While the correction of pK a values for a change in temperature should be possible (as above for a change in %B), at present there are no simple guidelines for this purpose. As temperature is seldom varied over a very wide range (e.g., 30–50 ◦ C is typical), the effect of temperature on solute pK a values will usually be small and can therefore be ignored when using estimated values of pK a for method development. 7.3 SEPARATION OF IONIC SAMPLES BY REVERSED-PHASE CHROMATOGRAPHY (RPC) Reversed-phase chromatography (RPC) should be a first choice for the separation of mixtures of ionizable organic compounds. Method development for the RPC separation of ionic samples (Section 7.3.3) proceeds in similar fashion as for neutral samples, with some important differences that are developed in the remainder of this 320 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY section. The following information on RPC separation (Sections 7.3.1, 7.3.2) should be useful for both method development and in troubleshooting routine separations. 7.3.1 Controlling Retention Following an initial experiment, mobile-phase strength (%B) can be varied to obtain a desirable retention range (e.g., 1 ≤ k ≤ 10), the same way as for neutral samples (Sections 2.5.1, 6.2.1). Alternatively (and generally preferable), initial experiments can be carried out using gradient elution, as discussed in Section 9.3.1. Once a value of %B has been selected for the reasonable retention of the sample, the next step is the adjustment of separation selectivity for optimal relative retention and maximum resolution. 7.3.2 Controlling Selectivity For the separation of neutral samples, selectivity can be varied by changing solvent strength (%B), temperature, the B-solvent, or the column. These variables also affect the separation of ionic samples, usually to a greater extent than for neutral samples. In addition separation selectivity for ionic samples is strongly affected by mobile-phase pH, and to a lesser extent by the kind and concentration of the buffer. In the past, mobile-phase additives for the suppression of silanol activity (alkylamines, quaternary ammonium compounds) have been added to the mobile phase, and these additives can further change selectivity. Today, however, the widespread preference for type-B columns (Section 5.2.2.2) has virtually eliminated the need for silanol suppression by such additives. Depending on the nature of the sample, all of these separation conditions may exert a significant effect on relative retention and resolution, as discussed below. One study [25] ranked the relative importance of different conditions in affecting the selectivity of RPC separations of ionic samples as follows: pH (most important) > solvent type ≈ column type > %B > temperature buffer concentration and type (least important). 7.3.2.1 Mobile-Phase pH The ability of a change in mobile-phase pH to affect the relative retention of ionizable samples is apparent from the example of Figure 7.3. We have noted that solute retention changes with pH only when the pH of the mobile phase is within ≈±1.5 units of the pK a value of the solute (Fig. 7.2). Consequently, if mobile-phase pH is to have an effect on separation selectivity, the pH must be similar to pK a values of the sample constituents. Carboxylic acids and amines are the most commonly encountered examples of ionic solutes; reference to Table 7.2 suggests for a mobile-phase pH between 2 and 3 that bases (pK a ≈ 5–10) will be fully ionized, and acids (pK a ≈ 5) will be in the neutral form. This is only approximately true, since it overlooks the effects of %B and temperature on values of pK a ,aswellas changes in pK a that can result from the presence of different substituents in the solute molecule. Consequently, while pH selectivity is usually reduced at low pH, it can still be significant—depending on the sample and the value of %B. 7.3 SEPARATION OF IONIC SAMPLES BY REVERSED-PHASE CHROMATOGRAPHY (RPC) 321 Another example of a change in relative retention with pH is shown in Figure 7.6 for a mixture of substituted benzoic acids (peaks 1–4) and anilines (peaks 5–7). As mobile-phase pH is increased from 3.2 to 4.3 (Fig. 7.6a–c), the retention of acids 1 to 4 decreases, while the retention of bases 5 to 7 (shaded peaks) increases. For a mobile-phase pH of 4.3 or higher, the acidic compounds 1 to 4 are mainly in the ionized form and therefore retained weakly; similarly, at higher pH the basic compounds 5 to 7 are largely non-ionized and more strongly retained. As a result for a mobile-phase pH > 4 there is a separation of these acids and bases into two groups of peaks. An optimum mobile-phase pH = 3.4(Fig.7.6d)provides acceptable resolution of the sample. However, even at this relatively low pH, the separation of Figure 7.6d is seen to be somewhat sensitive to small changes in pH; 0 2 4 6 8 10 12 14 5 1 2 6 3 4 7 pH-3.2 5 ≤ k ≤ 15 R s = 0.6 pH-3.7 2 ≤ k ≤ 15 R s = 0.9 pH-4.3 0.5 ≤ k ≤ 15 R s = 0.2 pH-3.4 4 ≤ k ≤ 15 R s = 2.7 (a) (b) (c) (d) 024681012 Time (min) Time (min) Time (min) Time (min) 5 1 2 6 3 4 7 02468101214 2 1 3 5 4 6 7 02468101214 2 1 3 4 5 6 + 7 1-4 acids 5-7 bases Figure 7.6 Effect of mobile-phase pH on the RPC separation of a mixture of acids and bases. Sample: 1, 2-fluorobenzoic acid; 2, 3-chlorobenzoic acid; 3, 3-nitrobenzoic acid; 4, 3-fluorobenzoic acid; 5, 3,5-dimethylaniline; 6, 4-chloroaniline; 7, 3-chloroaniline. Con- ditions: 150 × 4.6-mm C 18 column (5-μm particles); mobile phase, 13% acetonitrile-buffer (buffer is citrate plus phosphate); 2.0 mL/min; 35 ◦ C. Peaks for basic compounds 5 to 7 are shaded. Chromatograms based on data of [19]. 322 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY the buffer pH should therefore be maintained within ±0.1 pH units of the specified value for reproducible separation. The presence of partly ionized acids and bases in the same sample for 3 < pH < 4 is the reason for both changes in relative retention with pH and the marginally robust nature of the separation of Figure 7.6d. 7.3.2.2 Solvent Strength (%B) and Temperature In Figure 7.7 the effects of a change in %B and temperature on relative retention are illustrated for the sample of Figure 7.6, in each case for the same mobile-phase pH = 3.2. It is apparent that significant changes in relative retention occur as either temperature or %B is changed. These changes in relative retention can be attributed to the same factors that are operative for the separation of neutral compounds (e.g., Fig. 6.26), plus more important changes in the ‘‘effective’’ mobile-phase pH as a result of change in either %B or temperature (Section 7.2.3). Consequently a change in %B or temperature usually has a larger effect on the relative retention (and resolution) of ionic samples than neutral samples, as noted above. In the examples of Figure 7.7 we see an increase in the relative retentionofbases5to7(shadedpeaks) compared with acids 1–4 as either %B or temperature increase (while the absolute retention of all compounds decreases). This implies that the pK a values of the sample bases have decreased as a result of an increase in either %B or temperature, which is equivalent to an increase in mobile-phase pH for these basic compounds Time (min) 13% B, 35 ° C 5 ≤ k ≤ 16 R s = 0.6 28% B, 35 ° C 1 ≤ k ≤ 5 R s = 0.3 19% B, 49 ° C 3 ≤ k ≤ 9 R s = 3.3 28% B, 60 ° C 1 ≤ k ≤ 4 R s = 1.0 13% B, 60 ° C 5 ≤ k ≤ 11 R s = 0.7 0246810 12 14 0 2 4 6 8 10 Time (min) Time (min)Time (min) 5 1 2 6 3 4 7 1 5 2 3 6 4 7 (a) (c) (b) (d) 024 024 5 1 2 3 4 + 6 7 5 2 3 4 6 7 02468 Time ( min ) 5 1 2 3 6 4 7 (e) Figure 7.7 Effect of mobile-phase strength (%B) and temperature on the separation of a mixture of acids and bases. Sample: same as in Figure 7.6; conditions also the same, except pH = 3.2, and values of %B and temperature are noted in the figure. Peaks for basic com- pounds 5 to 7 are shaded. Chromatograms based on data of [19]. 7.3 SEPARATION OF IONIC SAMPLES BY REVERSED-PHASE CHROMATOGRAPHY (RPC) 323 (see the discussion of Section 7.2.3 for the case of an acidic buffer and basic solutes). A maximum resolution of R s = 3.3 is observed in Figure 7.7e for 19% B and 49 ◦ C. The latter optimized separation can be found by trial-and-error changes in %B or temperature, but a more efficient procedure is the use of computer simulation (Section 10.2.2). The remainder of this section represents an alternative way of interpreting the changes in retention of Figure 7.7. Since it is not essential to an understanding of the effects of %B and temperature on retention, the reader may prefer to skip to the following Section 7.3.2.3. The effects of changes in %B, temperature, or other conditions on relative retention can be further interpreted in terms of (acids, bases) k = k 0 (1 − F ± ) + k ± F ± (7.4) Because k 0 k ± , this relationship can be simplified to k ≈ k 0 (1 − F ± ), (7.6) where k 0 is the value of k for the neutral (non-ionized) molecule, and F ± is the fractional ionization of the solute for a given mobile-phase pH. An increase in either temperature or %B will lead to a decrease in values of k 0 for the solute, regardless of whether it is ionic or neutral. Additionally a change in conditions that also changes the ‘‘effective’’ mobile-phase pH (and therefore values of F ± ) can have a further effect on the separation of an ionic sample. Thus in Figure 7.7 an increase in either %B or temperature appears to increase mobile-phase pH slightly (equivalent to a decrease in pK a values for these solutes)—with a preferential retention of basic solutes 5 to 7. 7.3.2.3 Solvent Type A change in solvent type (e.g., methanol replacing acetonitrile) is expected to have a comparable effect on the relative retention of both ionic and neutral samples. In addition any change in ‘‘effective’’ pK a values as a result of this change in B-solvent can further affect the relative retention of ionic samples—similar to the case of a change in %B or temperature. The latter effect should lead to larger changes in relative retention for ionic as opposed to neutral samples when the B-solvent is changed. This was observed to be the case in one study [26], where the average change in values of α for 45 neutral solutes was ±0.04 units for a change in B-solvent from 50% ACN to 45% ACN + 5% MeOH. The corresponding change in α for 22 ionic compounds was ±0.09 units (twice as large as for neutral solutes). See Sections 6.3.2 and 6.4.1 for a related discussion of the effect of solvent type on the separation of non-ionic samples. 7.3.2.4 Column Type Separations of a neutral sample with four columns of different type (different ligands) were illustrated in Figure 6.14. Similar separations of an ionic sample are shown in Figure 7.8 for three of the same columns (note that an ‘‘ionic’’ sample may also contain neutral solutes, as in this example). A comparison of results for these two 324 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY Time (min) 0246 Time (min) Symmetry C18 2 ≤ k ≤ 9 , R s = 0.3 Altima HP C18 amide 2 ≤ k ≤ 7, R s = 0.5 F s = 35 Luna phenyl-hexyl 2 ≤ k ≤ 8, R s = 0.4 F s = 33 Time (min) 1 5 2 3 4 + 6 7 1 2 + 5 3 4 7 6 1 2 + 3 4 5 6 7 (a) (b) (c) 02 4 6 8 6246 Figure 7.8 Separation of an ionic sample as a function of column type. Sample: 1, 5-phenylpentanol; 2,4-n-hexylaniline; 3, toluene; 4, ethylbenzene; 5,4-n-butylbenzoic acid; 6, trans-chalcone; 7, mefenamic acid. Conditions: 150 × 4.6-mm columns (5-μm particles); 50% acetonitrile-buffer (buffer is pH-2.8 phosphate buffer); 35 ◦ C, 2.0 mL/min. F s values ver- sus Symmetry C18. Recreated separations based on data of [27, 28]. samples (Fig. 6.14 vs. Fig. 7.8) shows somewhat greater changes in relative retention for the ionic sample of Figure 7.8; this is expected, as discussed below. We also see marginal resolution (R s < 1 for the least-resolved peak-pair) for each column in Figure 7.8, similar to the result of Figure 6.14 for the separation of a neutral sample with these same columns. That is, a change in just the column is not likely to provide a significant improvement in overall (‘‘critical’’) resolution. However, a change in column combined with optimized values of other conditions (e.g., %B and temperature) is much more promising, as shown in Figure 6.27 for the separation of a neutral sample on these same columns. Differences in selectivity among these three columns can be described by the column-comparison function F s (Section 5.4.2): Symmetry/Altima, F s = 35; Symmetry/Luna, F s = 33. In each case these F s values suggest significant differences in column selectivity, although much larger differences can be achieved with other pairs of columns. Another example of the effect of the column on selectivity is shown in Figure 7.9 for the separation of a mixture of five, fully protonated strong bases (1–5), five partly ionized weak acids (6–10), and a neutral reference 7.3 SEPARATION OF IONIC SAMPLES BY REVERSED-PHASE CHROMATOGRAPHY (RPC) 325 compound (11; shaded peak). In Figure 7.9a,mixturesofeither the strong bases or weak acids plus neutral compound 11 are separated on each of these three columns. In Figure 7.9b corresponding separations of samples containing all 11 compounds are shown. The relative retention of the fully protonated strong bases (1–5) of Figure 7.9 is most affected by values of the ion-exchange capacity C for the column (Section 5.4.1); larger values of C mean a greater retention of protonated bases. Values of C at pH-2.8 for these columns are, respectively, −0.47 (Inertsil), −0.30 (Symmetry), and 0.18 (Discovery). As expected, the relative retention of basic solutes 1 to 5 increases in proceeding from the Inertsil to the Symmetry to the Discovery column (note the retention ranges for peaks 1–5 in Fig. 7.9b, indicated at the top of each chromatogram by arrows). The relative retention of the weak acids 6 to 10 and neutral compound 11 are quite similar on the three columns because their retention is not affected by values of C. 02 Time (min) 2460 Time (min) 02 Time (min) 024 Time (min) 02 Time (min) 0 2 Time (min) 1 2 3 11 4 5 1 2 11 4 5 11 2 1 3 4 5 6 + 7 11 8 9 10 6 7 11 8 9 10 6 7 11 8 9 10 Strong bases Acids Inertsil ODS-3 C = −0.47 Inertsil ODS-3 Symmetry C18 C = −0.30 Symmetry C1 8 Discovery C18 C = 0.18 Discovery C18 3 (a) Figure 7.9 RPC separation of an ionic sample as a function of column type. Sample: (bases) 1, propranolol; 2, prolintane; 3, diphenhydramine; 4, nortriptyline; 5, amitriptyline; (acids) 6, 2-fluorobenzoic acid; 7, 3-cyanobenzoic acid; 8, 2-nitrobenzoic acid; 9, 3-nitrobenzoic acid; 10, 2,6-dimethylbenzoic acid; (neutral compound, shaded) 11, benzylalcohol. Conditions: 150 × 4.6-mm columns (5-μm particles); 30% acetonitrile-buffer (buffer is pH-2.8 phosphate buffer); 35 ◦ C; 2.0 mL/min. (a)Separationofacids6to10orbases1to5,ineachcasewith neutral-compound 11 added; (b) separation of all 11 compounds. Recreated separations based on data of [26, 29]. . solutes 1 to 5 increases in proceeding from the Inertsil to the Symmetry to the Discovery column (note the retention ranges for peaks 1–5 in Fig. 7.9b, indicated at the top of each chromatogram. C 18 column (5-μm particles); mobile phase, 13% acetonitrile-buffer (buffer is citrate plus phosphate); 2.0 mL/min; 35 ◦ C. Peaks for basic compounds 5 to 7 are shaded. Chromatograms based on. 3.2 to 4.3 (Fig. 7.6a–c), the retention of acids 1 to 4 decreases, while the retention of bases 5 to 7 (shaded peaks) increases. For a mobile-phase pH of 4.3 or higher, the acidic compounds 1 to