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Controlling the Extent of Ionization

The pH of a solution will influence the charge state of an acidic or basic analyte.  For example, addition of an acid to an aqueous solution of a basic analyte will increase the concentration of charged analyte in solution as the hydrogen ion concentration increases.

Conversely, raising the pH by addition of a base will increase the concentration of the neutral form of the basic analyte.  This principle was first described by Le Chetalier and the converse applies to acidic analyte species.  Take the example of homovanillic acid shown (Figure 1) – equilibrium between the ionized and non-ionized forms of the analyte will be established according to the solution pH.

 

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Figure 1: The effect of mobile phase pH on extent of ionization for an ionizable analyte in Reversed Phase HPLC.

Key Learning Points

  • At pH 3.74 the analyte is in its 50% ionized form – this is equal to the pKa value of the analyte molecule
  • At approximately pH 5.74 the analyte is 99% ionized – raising the pH further will not significantly affect the degree of ionization (and hence the retention behavior)
  • At around pH 1.74 the analyte is almost fully non-ionized (ion-suppressed) – again lowering the pH further will not significantly affect the degree of ionization (and hence the retention behavior)

Adding an acid to a buffered solution of homovanillic acid (i.e. lowering the mobile phase pH), will cause the equilibrium to shift to the left and the analyte will become less ionized (ion suppressed) as the analyte recombines to reduce the effect of the added hydrogen ions (protons) from the acidic species. 

Adding a base to a buffered solution of homovanillic acid (i.e. increasing the mobile phase pH), will cause the analyte to become more ionized as the solution attempts to regain equilibrium by producing more hydrogen ions to neutralize the added base.  The slider bar can be used to investigate the degree of ionization of homovanillic acid in solution at varying pH values (Figure 2).

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Figure 2: The effect of mobile phase pH on extent of ionization for an ionizable analyte in reversed phase HPLC.

Homovanillic acid has multiple ionizable groups, each of which has its own pKa, the speciation (concentration) of each of the ionized and non-ionized forms of homovanillic acid are shown over the full pH range (Figure 3). 

 
Figure 3: pKa values and ionized and non-ionized forms (top), and speciation plot (bottom) for homovanillic acid. 7

The extent of ionization of basic molecules is also affected by the addition of acidic or basic species to the mobile phase. 
Use the slider to investigate the extent of ionization for the basic beta-blocker molecule Atenolol (Figure 4).

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Figure 4: The effect of mobile phase pH on extent of ionization for a basic analyte in reversed phase HPLC.

Key Learning Points

  • At pH 9.67 the analyte is 50% ionized – this is equal to the pKa value of the analyte molecule
  • At approximately pH 7.67 the analyte is fully ionized (i.e. is fully protonated) – lowering the pH will not significantly affect the degree of ionization (and hence the retention behavior)
  • At around pH 11.67 the analyte is fully non-ionized – again raising the pH will not significantly affect the degree of ionization (and hence the retention behavior)
  • It should be noted that the extent of ionization behavior is exactly the opposite to that encountered with acidic species

Atenolol has two ionizable groups each with its own associated pKa; the speciation (concentration) of each of the ionized and
non-ionized forms of homovanillic acid are shown over the full pH range (Figure 4).

Figure 5: pKa values and ionized and non-ionized forms (top), and speciation plot (bottom) for atenolol. 7
 
 
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