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Reversed Phase HPLC of Ionizable Samples

So far in this section the focus has been on the analysis of neutral samples by reversed phase HPLC.  Ionizable analytes present their own particular separation challenges and opportunities.

Before we examine how ionizable samples are typically analyzed, a few fundamental principles should be reviewed.

The concept of pH is used to indicate the relative acidity or basicity (of a mobile phase in HPLC), and is defined as the negative logarithm of the hydrogen ion concentration in an aqueous solution. Adding an acid or base to a mobile phase will change the solution pH (Figure 1-2).

Figure 1: The link between pH and hydrogen ion concentration in solution.

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Figure 2: Effect of addition of acidic and basic modifiers to mobile phase pH.

Addition of acids, which are proton donors, will increase the hydrogen ion concentration, thus lowering the pH.  Addition of bases, which are proton acceptors, will reduce the hydrogen ion concentration, thus raising the pH.

The mobile phase pH can be used to influence the charge state of ionizable species in solution.  The extent of analyte ionization can be used to affect retention and selectivity.

Acid Base Chemistry

When working with ionizable analytes, it is important to consider the pH of the mobile phase to optimize separation; therefore, knowledge of the analyte is of utmost importance.  Knowledge of the functional group chemistry and the associated pKa of an analyte will allow for tuning of the pH of the mobile phase.

All acids and bases have a unique “ionization constant” (Ka), which specifies the degree to which the species ionizes in aqueous solution.  The greater the ionization, the greater the influence of the species on the hydrogen ion concentration, and thus, the “stronger” the acid or base.

For example, a strong acid will completely ionize, creating a high hydrogen ion concentration, and a very low pH in the solution.  Conversely, a strong base will completely ionize by removing hydrogen ions from the solution, thus, lowering the hydrogen ion concentration and raising the pH of the solution to a very high value.  Weaker acids and bases will ionize to a lesser degree and, therefore, have less effect on changing the pH.

Brønsted was the first to demonstrate the advantage of expressing the ionization of both acids and bases using the same scale.  He made an important distinction between strong and weak acids and bases:

  • Strong acids and bases are completely ionized over the pH range 0-14
  • Weak acids and bases are incompletely ionized within the pH range 0-14

When an acid or base is dissolved in water the equilibria shown are established (Equation 1 and 4).  
The equilibrium constants (dissociation constants,Ka andKb) are given by Equation 2 and 5.  The partial acid (or base) dissociation (ionization) constant is defined as the negative logarithm of the equilibrium coefficient of the neutral and charged forms of a compound (Equation 3 and 6).  This allows the proportion of neutral and charged species at any pH to be calculated, as well as the basic or acidic properties of the compound to be defined.  It is very important to note the extent of ionization at different pH values around the analyte pKa

A strong base will have a large Kb and exists in equilibrium with its conjugate acid which is a weak acid with a small Ka.  The converse is true for acidic species.  All acid-base reactions in aqueous solutions can be viewed from the standpoint of the conjugate acid form losing a proton to form the conjugate base. When this is done pKa can always be used in calculations and Kb or pKb is not required, making comparisons of acidic and basic molecules much more facile. 

For Acids



For Bases



The stronger the acid the smaller the pKa
The stronger the base the larger the pKa

The pH corresponding to the point at which the two forms of the analyte (ionized and non-ionized) are present in equal concentrations (i.e. the analyte is 50% ionized) is the called the pKa value.  Each ionizable functional group on the analyte molecule will have its own pKa value.  Since the pKa represents an equilibrium value, when the pH is equal to the pKa the analyte will be rapidly converting between the ionized and non-ionized form, which can result in poor peak shape in HPLC.

As can be seen from Figure 3 the pKa gives an indication of the strength of the acid or the base (relative to its conjugate acid or base), however, it cannot be decided from the pKa if the molecule is an acid or a base, the functional groups contained within the molecule must be known.  For example, the pKa of aspirin and diazepam are very similar (3.5 and 3.3 respectively).  Aspirin is a weak acid as it contains acidic carboxylic acid groups, while diazepam is a weak base as it contains basic nitrogen functional groups.

Ibuprofen, pKa4.9   Aspirin, pKa 3.5   Diazepam, pKa 3.3   Amphetamine, pKa 9.8
Figure 3:  Ibuprofen, aspirin, diazepam, and amphetamine.  pKa values are shown for each molecule.  Acidic groups are shown in red and basic groups are shown in blue.

Table 1 details the pKa range of several common functional groups. 

Functional Group

pKa range (H2O)

Carboxylic acids


















Table 1: pKa ranges for selected functional groups* = Values in DMSO.6

Each molecule will have a specific pKa and there are several resources which detail physicochemical data of known compounds.



  • CRC Handbook of Chemistry and Physics 4
  • Lange’s Handbook of Chemistry 3

It is important to realize that the two forms of ionizable analyte molecules give different retention characteristics.  The ionized form is much more polar, and its retention in reversed phase HPLC is much lower (shorter retention time (tR), smaller retention factor (k)).  This behavior is expected, as the more polar analyte has a higher affinity for the mobile phase and moves more quickly through the column.  The converse is true of the non-ionized form as it is much more hydrophobic, relative to the ionized form.

Further information on functional group chemistry can be found in our Sample Prep Channel >
Sample Prep - Solid Phase Extraction Molecular Properties

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