Counteranion basic analyte retention

The analysis of Dorzolamide HCl at pH 2 with phosphoric acid shows early elution. The addition of hexafluorophosphate to the mobile phase leads to an enhancement of the retention. Figure 4-59 is an overlay of Dorzolamide HCl chromatograms at four increasing PFe concentrations. As the concentration increased, peak tailing decreased, and peak efficiency and analyte retention increased. Figure 4-60 shows the effect of different counteranions on basic analyte retention and peak efficiency. Depending upon the desired selectivity between a neutral component and a charged basic analyte, a particular chaotropic counteranion could be employed. 

R. LoBrutto, A. Jones, and Y. V. Kazakevich, Effect of Counteranion Concentration on HPLC Retention of Protonated Basic Analytes, J. Chromatogr. A 913 (2001), 191-198. 

With the increase of the counteranion concentration, the solvation of the protonated basic analyte decreases. The primary sheath of water molecules around the basic analytes is disrupted, and this decreases the solvation of the basic analyte. The decrease in the analyte solvation increases the analyte hydrophobicity and leads to increased interaction with the hydrophobic stationary phase and increased retention for the basic analytes. 

The chaotropic effect is dependent on the concentration of the free counteranion and not the concentration of the protons in solution at pH < basic analyte Ka. This suggests that change in retention of the protonated basic analyte may be observed with the increase in concentration of the counteranion by the addition of a salt at a constant pH as shown in Figure 4-47 for a pharmaceutical compound containing an aromatic amine with a pKa of 5. 

In the example in Figure 4-47, the retention of pharmaceutical analyte X was first altered by decrease of mobile-phase pH (Figure 4-47A), and in the second case (Figure 4-47B) the pH was maintained constant and the concentration of counteranion was increased via addition of its sodium salt. The resulting effect on the retention of basic analyte is strikingly similar if both dependencies are plotted against the concentration of free counteranions of CIOt, as shown in Figure 4-48. 

Figure 4-47. Variation of the retention of basic analyte >Ka > 5) with mobile-phase pH (A) and counteranion concentration (B). (Reprinted from reference 185, with permission.)...

As was shown above, the chaotropic effect is related to the influence of the counteranion of the acidic modifier on the analyte solvation and is independent on the mobile-phase pH, provided that complete protonation of the basic analyte is achieved. Analyte interaction with a counteranion causes a disruption of the analyte solvation shell, thus affecting its hydrophobicity. Increase of the analyte hydrophobicity results in a corresponding increase of retention. This process shows a saturation limit, when counteranion concentration is high enough to effectively disrupt the solvation of all analyte molecules. A further increase of counteranion concentration does not produce any noticeable effect on the analyte retention. 

Retention of the Counteranions. Three distinct processes could be envisioned in the effect of chaotropic ions on the retention of basic analytes ... 

It is an effect caused primarily by the concentration and type of counteranion. As a result of addition of acid there is an increase in the concentration of the counteranion of the acid and a simultaneous decrease in the pH. pH is a factor affecting the protonation of the analyte. Only when the analyte is protonated it can undergo ionic association with the counteranion of the acid that was used. Hence, when the protonated base interacts with the counteranion this leads to changes in its solvation and increase in its hydrophobicity. At higher concentrations of the acidic counteranion, the protonated basic analyte is desolvated to a greater extent. This ultimately causes an increase in analyte retention. 

Figure 5-21. Change of the retention of basic analytes at low pH with increase of the concentration of counteranion. Concentration region 0.08 mM to 44 mM perchlorate anion. Column 15 x 0.46 cm Zorbax XDB-C18 mobile phase methanohaqueous adjusted with perchloric acid pH 1.4-2.9 (90 10) flow rate ... 

The overall retention factor increase for the basic compound increases within the temperature range from 5 5 C. It is speculated that at the lower temperatures the solvation around the basic analytes is more ordered and that small changes in the environment would lead to significant changes in the retention of the basic compound with increased concentration of the counteranion. 

Each constant in the equation above represents single equilibrium process, which is assumed to be independent on other equilibria in the column. Equation (2-93) describes the retention of basic ionizable analytes in reversed-phase chromatographic system with binary eluents and liophilic counteranions added. Similar expression could be derived for the behavior of anionic analytes in the presence of liophilic countercation. 

Effect of Different Counteranions. The chaotropic theory was shown to be applicable in many cases where small inorganic ions were used for the alteration of the retention of basic pharmaceutical compounds [153-157]. Equation (4-39) essentially attributes the upper retention limit for completely desolvated analyte to the hydrophobic properties of the analyte alone. In other words, there may be a significantly different concentration needed when different counterions are employed in the eluent for complete desolvation of the analyte. Therefore, the resulting analyte hydrophobicity and thus retention characteristics of analyte in completely desolvated form should be essentially independent on the type of counteranion employed. Experimental results, on the other hand, show that the use of different counterions... 

---