IPC versus RP-HPLC

The retention of ionogenic analytes in RP-HPLC is obviously dependent on the eluent pH. The pH value is an important optimization parameter because it controls the extent of ionization of the solute and hence the magnitude of electrostatic interactions. Varying the acidity of the mobile phase can lead to extreme changes in selectivity. Many interdependent parameters can be modulated in IPC and their optimization requires a theory-driven procedure. 

Ion pairing interactions need charged analytes to operate. Let us focus on the dependence of basic analyte retention as a function of pH. The hydrophobic retention of ionogenic bases at pH values two units above the basic analyte pK is the highest on reversed phase materials because the analyte is predominantly neutral. Separations at these pH levels may not be feasible because the pH stability thresholds of common silica-based stationary phases are exceeded, as discussed in Chapter 5. 

Eluent pH is limited to a maximum of 7 to 8 due to the reduced chemical stability of a chromatographic bed in an alkaline medium. The nucleophilic attack of Si-0 bonds by hydroxide ions leads to the erosion of the silica surface as shown by back pressure increases caused by the formation of Si(OH)4. With polystyrene-divinyl-benzene-based stationary phases, pH stability is not an issue and a very wide mobile phase pH range can be used, thereby providing additional selectivity [1]. Several silica-based and polymeric columns claimed to be stable in pH ranges from 1 to 13 are commercially available, however, they are not commonly used. 

In case of a weak monoprotic acid-base analyte, two different species are retained the protonated acid form (HA, uncharged or cationic) and the deprotonated conjugate basic form (A, uncharged or anionic). In other words, if HA is uncharged, A is anionic if HA is charged, A is uncharged. The ionization of the analyte is described by the following equilibrium  

With decreasing eluent pH, [A] decreases and [HA increases. Since the retention factor of the protonated and deprotonated species, ha and k, are respectively 

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