Critical Evaluation of HPLC Methods: Working with Ionizable Analytes

In chromatography, “like dissolves like”; that is, nonpolar analytes interact well with nonpolar stationary phases and vice versa. Increased and improved interactions with the stationary phase lead to higher distribution constant (k_d) values and generally improved separations. Neutral or ion-suppressed analytes are less polar than ionized analytes and will therefore have improved retention on nonpolar reversed-phase type stationary phases, while in their ionized forms they will exhibit decreased retention. The mobile-phase pH can have a dramatic effect on the ionization state of ionizable analytes, so it must be fixed by buffers to maintain the analyte in the desired state.

Knowledge of the functional group chemistry and the associated pK_a of an analyte will allow for tuning of the mobile-phase pH. The pK_a gives an indication of the strength of the acid or the base; however, it cannot be decided from the pK_a alone if the molecule is an acid or a base-the functional groups contained within the molecule must also be known. For example, the pK_a of aspirin and diazepam are very similar (3.5 and 3.3, respectively). Aspirin is a weak acid because it contains carboxylic acid groups, and diazepam is a weak base and contains basic nitrogen functional groups. The pH will affect the extent of analyte ionization, which will in turn influence elution and retention properties. For an acidic analyte, in a buffered solution, the addition of an acid will lower the pH and the analyte will become less ionized. The change in degree of ionization happens over a limited pH range because of pH and pK_a being logarithmic: 1 pH unit away from the pK_a, the extent of ionization is ~90%; at 2 pH units away from the pK_a, the extent of ionization is ~99%; and at 3 pH units it is 99.9% (Figure 1). At a pH equal to the pK_a an ionizable molecule will be 50% ionized and 50% non-ionized, which can lead to poor peak shape.

Figure 1: 2 pH unit rule for determining the extent of ionization of acidic and basic analytes.

Unlike ionized acids, which are eluted rapidly from the column when charged, protonated bases may have long retention times and poor peak shape because of interaction with silanol species on the silica surface. Separations of basic compounds are not usually carried out under ion-suppression conditions because the increase in pH to produce the neutral species would damage traditional silica columns (although hybrid columns can be used at extremes of pH). Traditionally, the analysis of weak bases is carried out at low pH so that surface silanol species are non-ionized (pK_a 3.5–4.5), which results in improved peak shape.

Mobile-phase pH is controlled using a buffer consisting of a weak acid or base in cosolution with its conjugate acid or base. Buffers are only reliable within 1 pH unit either side of their pK_a, and their concentration must be adequate but not excessive: below 10 mM buffers will have very little buffering capacity; above 50 mM, there is a high risk of precipitation of the salt in the presence of high organic concentration, hence, concentrations of 25–50 mM are typical. For liquid chromatography–mass spectrometry (LC–MS) applications the buffer must be volatile. It is good practice to prepare buffers daily because pH can change on standing due to ingress of carbon dioxide.

Sacrificial bases or ion-pair reagents can be used to improve peak shape or retention of basic analytes. Sacrificial bases are sterically small, highly surface active species (for example, triethylamine) that preferentially interact with surface silanol groups. They are added to the mobile phase in sufficient concentration (10–100 mM) to ensure full surface coverage. Modern, high purity, low-silanol packing materials negate the need for sacrificial bases.

Ion-pair reagents can be used when other methods such as ion suppression have not been successful. Samples containing both anionic and cationic components have one type “masked” by the ion-pair reagent and the other suppressed by pH. This technique is useful if analyte pK_a values are not similar. For example, tetrabutylammonium phosphate at pH 7.5 forms a strong ion pair with acids and pH suppresses weak base ions. Ion-pair reagents do have disadvantages, including long equilibration times (>100 column volumes); difficult removal from columns (it is recommended that a guard column or dedicated column is used); irreversible modification of the stationary phase, which drastically reduces lifetime; neutral analytes precluded from the stationary phase at very high ion pair concentrations, which results in decreased retention; and interference from ultraviolet (UV) activity of some ion-pair reagents at the analytical wavelengths being used (such as trifluoroacetic acid, 210 nm). Finally, ion-pair reagents are not suitable for LC–MS work because they suppress ion formation and reduce sensitivity. Alternative approaches, such as hydrophilic-interaction chromatography (HILIC) and mixed-mode chromatography, can be used for LC–MS. Another alternative is the use of modified column chemistries that have counterions built in as part of the stationary phase; these embedded functional groups can be activated by changing the mobile-phase pH, without the need for additional chemical modifiers.

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