326 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY Inertsil ODS-3 Symmetry C18 Discovery C18 0246 Time (min) Time (min) 1 2 3 6 + 7 11 4 8 9 10 5 bases 1–5 024 1 2 3 + 6 7 11 8 4 5 + 9 10 bases 1–5 02 Time (min) 7 6 11 8 2 1 + 9 3 4 5 + 10 bases 1–5 (b) Figure 7.9 (Continued) 7.3.2.5 Other Conditions That Can Affect Selectivity Conditions that are less used today for the control of RPC selectivity include: • buffer type (e.g., phosphate, acetate, ammonium) • buffer concentration • amine modifiers Buffer type is not commonly considered as a means of controlling selectivity for the separation of ionic samples. As discussed in Section 7.2.3, however, buffer type can affect the ‘‘effective’’ pK a of a solute, which is equivalent to a change in pH. The largest changes in relative retention will occur when a basic buffer (e.g. ammonium, triethylamine, etc.) replaces an acidic buffer such as phosphate or acetate, and vice versa. Another way in which buffer type can contribute to selectivity is by ion pairing (Section 7.2.1.2). More hydrophobic buffers such as trifluoroacetate TFA or (especially) heptafluorobutyrate HFBA can ion-pair with protonated bases BH + and selectively increase their retention [30]. The increase in retention for protonated bases increases with the positive charge on the solute molecule, as in the case of peptides which contain multiple, basic amino-acid residues (Fig. 13.8; Section 13.4.1.2). The use of TFA and HFBA as buffers is not subject to problems that are common for other ion-pair separations (Section 7.4.3). 7.3 SEPARATION OF IONIC SAMPLES BY REVERSED-PHASE CHROMATOGRAPHY (RPC) 327 Buffer concentration usually has only a minor effect on relative retention for separations at low pH on modern (type-B) alkylsilica columns [26]. However, for separations at pH > 6 and/or older, type-A columns (Section 5.2.2.2), protonated bases can be retained by ion exchange as a result of interaction with ionized silanols of silica-based column packings. Ion-exchange retention decreases as mobile-phase ionic strength increases (Section 7.5.1), with the result that an increase in buffer concentration will tend to decrease the retention of protonated bases [31]. Amine modifiers, such as triethylamine or tetrabutylammonium salts, have been added to the mobile phase in the past, primarily as a means of suppressing unwanted silanol interactions (Section 7.3.4.2). By interacting with stationary-phase silanol groups, amine modifiers can suppress ion exchange by the sample, thereby resulting in decreased retention for protonated bases. These modifiers are little used today because (1) modern RPC columns (type-B) are largely free of unwanted silanol interactions and (2) the use of amine modifiers can be inconvenient, requiring long column-equilibration times in some cases. 7.3.3 Method Development Method development is similar for the RPC separation of either ionic or neutral samples, as summarized in Figure 6.21a. Seven method-development steps are defined there, of which only one (step 3: choosing separation conditions) differs significantly for the separation of ionic samples. The choice of separation conditions for either ionic or neutral samples is summarized in Figure 6.21b and includes the following steps: 1. choose starting conditions 2. select %B for 1 ≤ k ≤ 10 3. adjust conditions for improved selectivity and resolution 4. vary column conditions for a best compromise between resolution and run time Method development for ionic samples differs from that for neutral samples mainly with respect to steps 1 and 3 above. Method development should always start with a new (unused) column, as exposure of a column to previous samples and conditions can change its selectivity so as to make it impossible to replicate the column at a later time. 7.3.3.1 Starting Conditions (Step 1) Table 7.3 suggests conditions for the initial separation of a mixture of acids and/or bases, conditions that are similar to those recommended for the initial separation of neutral samples (Table 6.1). The main difference for ionizable samples is the need for a buffered mobile phase. Because ionic samples are usually less strongly retained in RPC, the value of %B for the initial mobile phase is likely to be a bit lower than for neutral samples. However, it is best to start development at 80% B, so as to reduce the risk of missing a late-eluted solute with a mobile phase that is too weak. Alternatively (and preferably), an initial gradient-elution run can be used to determine the best value of %B for isocratic separation (Section 9.3.1). 328 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY Table 7.3 Representative Conditions for the Separation of Ionic Samples by Means of 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 80 % acetonitrile-buffer; buffer is 10 mM potassium phosphate, adjusted to pH−2.5 b Flow rate 2.0 mL/min b Temperature 30 or 35 ◦ C b %B Determined by trial and error c Sample Volume ≤ 50 μL weight ≤ 10 μg k 1 ≤ k ≤ 10 a Alternatively, use a 150 × 4.6-mm column of 5-μmparticles;note that a new (unused) column should always be selected at the start of method development. b Initial values will be varied during method development (Section 2.5); the starting pH of the mobile phase can also be varied. c Start with 80%B and adjust further as described in Section 2.5.1. The choice of starting mobile-phase pH and buffer depends on (1) the sep- aration goals and (2) what the chromatographer knows about the sample. We recommend carrying out initial separations with a mobile-phase pH of 2.5 to 3.0, using phosphate for UV detection, or ammonium formate for LC-MS. Problems aris- ing from peak tailing (Section 7.3.4.2), column instability (Section 5.3.1), or a lack of method robustness (Section 12.2.6) are somewhat less likely for a mobile-phase pH of 2.5 to 3.0. For samples that contain strong bases, there is increasing use of high-pH mobile phases (pH > 8), in order to minimize the ionization of basic solutes during separation. Decreased sample ionization results in stronger retention, and can favor symmetrical peaks and more robust RPC methods. However, when the mobile phase pH is > 8, special columns are required to avoid the dissolution of the silica particles with resulting failure of the column (Sections 5.2.5, 5.3). The main advantage of a mobile-phase pH > 8 for strongly basic samples is that a larger sample weight can be injected (Section 15.3.2.1), with a resulting increase in detection sensitivity for an assay procedure, or increase in yield for preparative separations. Whatever mobile-phase pH is used, care should be taken to ensure adequate buffering capacity (Section 7.2.1.1). 7.3.3.2 Optimizing Selectivity (Step 3) Any of the separation conditions described in Section 7.3.2 can be varied in order to improve relative retention and maximize resolution. The simultaneous variation of two different separation conditions will generally prove more effective; the same 7.3 SEPARATION OF IONIC SAMPLES BY REVERSED-PHASE CHROMATOGRAPHY (RPC) 329 two-variable procedures described for neutral samples in Section 6.4.1 can be applied for ionic samples. We recommend that temperature and %B should be varied first over a range that results in solute k-values within the range 0.5 ≤ k ≤ 20; see the example of Figure 7.7 and the discussions of Section 6.4.1.3 and [32]. If further changes in selectivity are needed, simultaneous changes in pH and solvent strength can be used for ionizable samples [33]. When varying two conditions simultaneously, simulation software (Section 10.2) is especially helpful for determining conditions that correspond to maximum resolution. More than two conditions can be simultaneously optimized for the control of selectivity; for example, varying %B and temperature for different columns (Section 6.4.1.4) is a popular and effective strategy. A few other examples have been reported of the simultaneous optimization of three different variables [34, 35], each of which can be varied continuously (i.e., excluding column type as a variable). However, this approach can require a formidable number of experiments, for example, 32 experiments for the simultaneous optimization of %B, temperature, and pH in one example [34]. 7.3.4 Special Problems RPC separations of ionic samples are subject to two problems that do not occur for the separation of neutral samples. 7.3.4.1 pH Sensitivity As noted in Section 7.2 for the RPC separation of ionizable samples, relative retention can be quite sensitive to small (unintended) variations in mobile-phase pH. The ability of most laboratories to replicate the pH of the buffer by means of a pH meter is typically no better than ±0.05 to 0.10 units; variations in mobile-phase pH of this magnitude may be unacceptable for some separations. For this reason the robustness of the final method in terms of pH should be a major concern during method development for ionic samples. There are several ways in which the problem of pH sensitivity can be minimized. First, determine the pH sensitivity of the method. If the mobile-phase pH must be held within narrow limits (±0.1 unit or less), precise pH control can be achieved by accurately measuring the buffer ingredients (either by weight or volume), rather than by using a pH meter to adjust the buffer to a desired pH (see Appendix II for some examples). Second, as an alternative to the precise adjustment of pH in this way, carry out separations with mobile phases that are, respectively, 0.2 pH units higher and lower than the required pH. The inclusion of these chromatograms in the method procedure can be used by an operator to guide the correction of mobile-phase pH when needed (Section 12.8). Finally, the best approach for a method that proves to be too pH sensitive is to re-optimize conditions so as to obtain a method that is more robust. This will sometimes require a change in pH to a value that differs by more than ±1 pH unit from the pK a values of critical solutes (those whose resolution can be compromised by small changes in pH). Minor changes in other conditions can also result in a more robust separation. See the further discussion of Section 12.2.2.6 and [36]. 330 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY 7.3.4.2 Silanol Effects Protonated basic solutes BH + can interact with stationary-phase silanols by ion exchange (a buffer in the potassium form is assumed): BH + + SiO − K + ⇔ BH + SiO − + K + (7.7) This interaction can lead to increased retention, peak tailing, and column-to-column irreproducibility. Problems of this kind are most pronounced when older, type-A columns (Section 5.2.2.2) are used, because type-A silica is contaminated by Al 3+ , Fe 2+ , and other heavy metals. Metal contamination increases silanol acidity, results in a higher concentration of SiO − groups for all mobile-phase pH values, and likely contributes to poor column reproducibility. Newer columns made from purer, type-B silica are largely free of metal contamination, and fewer associated problems are encountered in their use. Even when newer, type-B columns are used, the separation of basic compounds can lead to peak tailing [37]. The origin of peak tailing with type-B columns appears to differ for separations with mobile phases of high or low pH. For a mobile-phase pH < 5, tailing peaks usually resemble rounded right triangles, as in the example of Figure 2.15e.ForapH≥ 6, exponential peak tailing as in Figure 2.15a is more often seen. Low-pH tailing is now believed due to charge repulsion between retained ionized molecules (Section 15.3.2.1; [38]). As a result the column overloads more quickly for basic samples than for neutral samples, and peak tailing can become noticeable for injections of more than 0.5 μg of a basic compound (assumes a column diameter of 4–5 mm). Low-pH peak tailing can be reduced somewhat by an increase in mobile-phase ionic strength. For example, the use of buffers at a mobile-phase pH that favors buffer ionization results in a higher ionic strength, even when buffer molarity is unchanged; this approach for reduced peak tailing of bases has been recommended when volatile formate buffers are used for LC-MS [39]. The tailing of basic samples on type-B columns at pH-7 and above is less well understood, but may be the result of slow sorption-desorption of molecules of BH + [37]. The extent of tailing is affected by the nature of the B-solvent [40, 41], with acetonitrile (worst) > methanol ≈ tetrahydrofuran (best) Peak tailing is generally decreased by the use of higher column temperatures or higher %B, conditions that also favor lower values of k. The use of columns with smaller values of C (cation-exchange capacity values; Section 5.4.1) is likely to minimize peak tailing for a mobile-phase pH < 5. ‘‘Hybrid’’ particles (Section 5.3.2.2) do not exhibit silanol ionization below pH-8, and the peak shape of protonated bases is good for pH < 8 [16]. For a good review of peak tailing for basic solutes, see [16]. Small weights of undissociated carboxylic acids can exhibit tailing peaks for some columns (Section 5.4.4.1), but peak shape improves for larger samples. The origin of such peak tailing is as yet unknown. It is possible nevertheless to identify columns that are less likely to exhibit this problem (Section 5.4.4.1; [42]). In the case of type-A columns, various means have been employed in the past to reduce ion exchange (Eq. 7.7) and associated deleterious effects [43, 44]: 7.4 ION-PAIR CHROMATOGRAPHY (IPC) 331 • suppress silanol ionization (use low-pH mobile phases) • suppress the ionization of basic solutes B (use high-pH mobile phases) • suppress ion exchange (use high–ionic-strength mobile phases) • block ionized silanols (add amine modifiers to the mobile phase) • use end-capped columns Silica-based RPC columns can degrade more rapidly when the mobile phase pH is <2.5 or > 8.0, which limits the use of extreme pH to control silanol or solute ionization. Some type-B columns are now available for operation outside these pH limits (see the discussion of Section 5.3). Ion-exchange and related adverse silanol effects can also be minimized by the use of higher buffer concentrations; the buffer cation competes with the solute in the equilibrium of Equation (7.7). Buffer cations such as K + ,Li + and NH + 4 aremoreeffectivethanNa + in suppressing silanols and minimizing peak tailing. The addition to the mobile phase of amine modifiers such as triethylamine and dimethyloctylamine was popular at one time for improving the separation of basic samples, but today the predominant use of type-B columns has rendered these (inconvenient) additives unnecessary. End-capping the column (Section 5.3.1) tends to shield silanols from the solute and typically improves peak shape. 7.3.4.3 Poor Retention of the Sample Very polar samples are poorly retained in RPC, as noted for neutral samples in Section 6.6.1. The same problem is even more common for ionic samples—because of the greater polarity of ionized molecules. However, there are additional means for increasing sample retention in this case. Poor retention of an ionic solute is usually due to its ionization, which can result in more than a 10-fold decrease in values of k. The simplest approach for solutes that are acidic or basic is a change in mobile-phase pH that results in decreased solute ionization. Alternatively, ion-pair chromatography (IPC, Section 7.4) can be used to similar effect, especially for per- manently ionized solutes such as quaternary-ammonium compounds. Hydrophilic interaction chromatography (HILIC) is also effective for very polar samples (Section 8.6) and is usually a better choice than IPC for this purpose. 7.3.4.4 Temperature Sensitivity The relative retention of ionized solutes tends to be more dependent on temperature than is the case for neutral samples. Therefore the need for accurate column thermostatting (Section 3.7) can be more important for ionic samples. The use of (unthermostatted) separations at ambient temperature is not recommended for any sample, and it is especially problematic for the separation of ionic samples. 7.4 ION-PAIR CHROMATOGRAPHY (IPC) Ion-pair chromatography (IPC) can be regarded as a modification of RPC for the separation of ionic samples. The only difference in conditions for IPC is the addition 332 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY of an ion-pairing reagent R + or R − to the mobile phase, which can then interact with ionized acids A − or bases BH + in an equilibrium process: ionized solute ion pair (acids) A − + R + ⇔ A − R + (7.8) (bases) BH + + R − ⇔ BH + R − (7.8a) hydrophilic solute hydrophobic ion-pair (less retained in RPC) (more retained in RPC) The use of IPC can thus create similar changes in sample retention as by a change in mobile-phase pH (Section 7.2), but with greater control over the retention of either acidic or basic solutes, and without the need for extreme values of mobile-phase pH (e.g., pH < 2.5 or > 8). Typical ion-pairing reagents include alkylsulfonates R–SO − 3 (R − ) and tetraalkylammonium salts R 4 N + (R + ), as well as strong (normally ionized) carboxylic acids (trifluoroacetic acid, TFA; heptafluorobutyric acid, HFBA [R − ]), and so-called chaotropes (BF − 4 ,ClO − 4 ,PF − 6 ). When first introduced in the 1970s, high-performance IPC was found to reduce peak tailing for basic solutes. This and its ability to increase the retention of weakly retained ionized acids and bases for acceptable values of k were primary reasons for its use at that time. Additionally IPC provides further options for the control of selectivity in the separation of ionic samples. Today the predominant use of type-B columns has reduced the importance of peak tailing, and we now have a better understanding of how best to control RPC selectivity. The poor RPC retention of very hydrophilic acids and bases (especially strong bases that remain ionized for pH < 8) can also be addressed in other ways, for example, (1) by the use of high- or low-pH mobile phases in order to minimize solute ionization and increase retention (combined with the use of columns that are stable at pH extremes) and (2) by the use of hydrophilic interaction chromatography (HILIC, Sect. 8.6). Consequently there is much less need for IPC today because of its greater complexity and other problems (Section 7.4.3). When developing an HPLC separation, we recommend starting with RPC, followed by the addition of an ion-pairing reagent only when necessary. When, or for what applications, might IPC be recommended? IPC separation involves two additional variables (type and concentration of the IPC reagent) that can be used for further control of selectivity. As will be seen below, the effects of an added IPC reagent on solute retention are reasonably predictable, when we know whether a particular peak corresponds to an acid, base, or neutral. Consequently the retention of both acidic and basic solutes can be varied continuously so as to optimize their separation, when other changes in RPC conditions fail to achieve acceptable resolution. IPC can also be used to narrow the retention range of a sample, so samples that might otherwise require gradient elution can be separated isocratically. An example is shown in Figure 7.10, for a proprietary sample that includes a drug-product X plus several preservatives and degradants. In Figure 7.10a, RPC separation is shown with a mobile phase of 30% methanol-buffer (pH-3.5). The neutral preservative, propylparaben PP, is strongly retained, while the basic drug X and its degradants 7.4 ION-PAIR CHROMATOGRAPHY (IPC) 333 02040 Time (min) no ion-pairing (0 < k < 30) ion-pairing (0.6 < k < 9) (a) (b) X X 1 X 3 + HB B MP PP 0246810 Time (min) HB B MP X PP X 2 X 2 X 3 X 1 Figure 7.10 RPC separation of a proprietary mixture of acids, strong bases and neutrals. Sam- ple: X, strongly basic (proprietary) drug substance; X1, X2, X3, strongly basic degradants of X; MP and PP, methyl and propyl paraben preservatives; B, benzoic acid; HB, hydroxyben- zoic acid (degradant of MP and PP). Conditions: (a) 150 × 4.6-mm column (5-μm particles); 30% methanol-buffer mobile phases (buffer is pH-3.5 acetate); 30 ◦ C; 2.0 mL/min. (b) Same as (a), except mobile phase is 45% methanol-buffer plus 65-mM octane sulfonate; 1.5 mL/min. Adapted from [46]. X 1 –X 3 are weakly retained (because they are in the protonated form as BH + ). Two other sample compounds, benzoic acid B (another preservative) and hydroxybenzoic acid HB (a paraben degradant), are acidic, while methyl paraben MP is also a neutral preservative. The separation of Figure 7.10a exhibits an excessive retention range (0 ≤ k ≤ 30), which would normally suggest gradient elution as an alternative (Section 9.1). Because compounds X–X 3 are strongly basic, an increase in their isocratic retention (relative to the rest of the sample) by an increase in pH was deemed impractical. Thus a mobile-phase pH > 8 would be necessary (requiring a column that is stable at high pH), but this would lead to k 1 for acidic compounds B and HB, while having no effect on the retention of neutral compounds MP and PP. Thus no practical change in pH is able to narrow the retention range of this sample so as to provide k-values for all compounds in an acceptable range of values (e.g., 0.5 ≤ k ≤ 20). Isocratic elution was preferred for the sample of Figure 7.10, so the use of IPC was investigated as an alternative to gradient elution. The addition of a sulfonate IPC reagent would be predicted to lead to strongly increased retention for the sample cations (X–X 3 ), accompanied by a modest decrease in the retention of both sample acids (B and HB) and neutral compounds (MP and PP); see Section 7.4.1.2 below. The separation of Figure 7.10b was therefore carried out with octane sulfonate as IPC reagent (for a preferential increase in the retention of X–X 3 ), plus a stronger mobile phase (45% B vs. 30% B in Fig. 7.10a) for a reduction in k for the neutral solute PP. The resulting decrease in retention range (0.6 ≤ k ≤ 9) now allows the baseline separation of this sample within a reasonable time (11 min). 334 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND ION-EXCHANGE CHROMATOGRAPHY The remainder of this section provides a short description of the basis of ion-pair separation, followed by a discussion of how separation depends on various conditions. For further details, see [45] and Chapter 7 of [46]. 7.4.1 Basis of Retention Two possible retention processes or ‘‘mechanisms’’ exist for separation by IPC. As an example, we will use the ion-pairing of an ionized acidic solute A − by a tetraalkylammonium IPC reagent R + . The ion-pairing of a protonated basic solute B + by an alkylsulfonate IPC reagent R − can be described similarly. One hypothesis for IPC retention assumes that an ion-pair forms in solution, as described by Equation (7.8a). The resulting ion-pair A − R + is retained by the column; that is, the solute retention equilibrium as described by Equation (2.2) in Section 2.2 is replaced by A − R + (mobile phase) ⇔ A − R + (stationary phase) (7.9) According to this hypothesis, retention is governed by (1) the fraction of solute molecules A in the mobile phase that are ionized (determined by mobile-phase pH and the solute pK a value), (2) the concentration of the IPC reagent and its tendency to form an ion pair (the equilibrium constant for Eqs. 7.8 or 7.8a), and (3) the value of k for the ion-pair complex A − R + (which will be greater for more hydrophobic IPC reagents). An alternative picture of IPC retention assumes that the IPC reagent is retained by the stationary phase, with retention then occurring by an ion-exchange process (Section 7.5.1), for example, for an ionized acid A − and IPC reagent R + X − : A − (mobile phase) + R + X − (stationary phase) ⇔ A − R + (stationary phase) + X − (mobile phase) (7.9a) That is, the ion-pair reagent R + X − first attaches to the stationary phase, and then the sample ion A − replaces the counter-ion X − in the stationary phase. Either of these two IPC retention processes (Eqs. 7.9 or 7.9a) might predominate for a given separation, but which mechanism plays the more important role is neither easy to determine nor important in practice. It has been shown that these two retention mechanisms are virtually equivalent [47], and both provide similar predictions of retention as a function of experimental conditions. Consequently either process can be assumed in practice. We will use the ion-exchange process of Equation (7.9a) in the following (simplified) discussion, because this retention mechanism appears to us to be easier to understand and to apply in practice. 7.4.1.1 pH and Ion Pairing Further insight into IPC retention as a function of mobile-phase pH is provided by Figure 7.11 for the case of an acidic sample (a carboxylic acid RCOOH) and a positively charged IPC reagent (tetrabutylammonium, TBA + ). In Figure 7.11a,no IPC reagent is added to the mobile phase (i.e., RPC separation), so the non-ionized acid RCOOH is preferentially retained by the C 8 stationary phase (shown as a 7.4 ION-PAIR CHROMATOGRAPHY (IPC) 335 RCOOH k pK a C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 C 8 k pK a RCOO − + H + RCOO − + H + RCOOH (a) (b) TBA + TBA + TBA + TBA + TBA + TBA + TBA + TBA + TBA + TBA + TBA + TBA + TBA + TBA + TBA + TBA + TBA + TBA + pH 2468 pH 246 8 Figure 7.11 Representation of retention for an acidic solute RCOOH as a function of mobile-phase pH; (a)RPCand(b) IPC with tetrabutylammonium ion (TBA + ) as IPC reagent in high concentration. surface with attached C 8 groups). Also shown on the right side of Figure 7.11a is aplotofretentionk as a function of mobile-phase pH; retention decreases with increased pH, due to the greater ionization of RCOOH (i.e., characteristic RPC retention for an acidic solute as pH is varied). In Figure 7.11b, the retention of the same compound RCOOH is shown, except that the IPC reagent TBA + has been added to the mobile phase in sufficient concentration to cover the entire stationary phase surface—hence blocking RPC interaction of the sample (non-ionized RCOOH) with the column C 8 groups. Now the ionized acid RCOO − is preferentially retained by ion exchange with TBA + in the stationary phase (Eq. 7.9a). The dependence of k on mobile-phase pH is seen to be the reverse of that in Figure 7.11a for RPC (no ion-pairing); retention in Figure 7.11b increases with increasing mobile-phase pH and the consequent increasing ionization of the solute, due to ion-pairing of the ionized solute. If fully protonated bases BH + are present in the sample (e.g., strong bases), their retention will decrease for increasing concentrations of the IPC reagent (TBA +) , due to the repulsion of the positively charged BH + ions by the positively charged TBA + ions in the stationary phase, as well as by the decreased availability of C 8 groups—which are covered by . chromatograms in the method procedure can be used by an operator to guide the correction of mobile-phase pH when needed (Section 12.8). Finally, the best approach for a method that proves to be too. discussion, because this retention mechanism appears to us to be easier to understand and to apply in practice. 7.4.1.1 pH and Ion Pairing Further insight into IPC retention as a function of mobile-phase. likely to be a bit lower than for neutral samples. However, it is best to start development at 80% B, so as to reduce the risk of missing a late-eluted solute with a mobile phase that is too weak.