Retention of basic analytes

FIGURE 14.7 Retention of basic analytes at different mobile phase pH values against pKa for basic molecules. 

Table 1 gives the tailing factor for the basic analyte amitriptyline at neutral pH on several commercially available packings. One can clearly see the difference between the older packings and the newer packings based on high-purity silicas. It is unquestionable today that surface silanols on a packing participate in the retention of basic analytes, and... 

FIGURE 2.12 Influence of pH value on retention of basic analytes. Samples A,A-dimethyl aniline A-ethyl aniline. Column symmetry shield CIS mobile phase acetonitrile-20 mM phosphate buffers-water, 35-35-... 

Changes in mobile-phase components such as pH, ionic strength, and water content have been systematically studied [3,310,316,317]. These studies indicate that retention of basic analytes is mediated primarily by the cation-exchange properties of the silica [2]. Interestingly, it has been suggested from retention data of various pharmaceuticals that the retention mechanisms of silica with aqueous eluents and reversed-phase systems are similar [317,318]. Due to the ion-exchange properties of silica, mobile-phase pH adjustments are useful in changing the retention of ionic compounds. 

LoBrutto, R. et al. Effect of the eluent pH and acidic modifiers in high-performance liquid chromatography retention of basic analytes. J. Chromatogr. A. 2001, 913, 173-187. 

Figure 2-20. Retention of basic analyte as a fnnction of mobile phase pH at three different counterion concentrations.

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.)...

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

Overall, liophilic ions (usually small ions capable for dispersive interactions) provide a useful means for selective alteration of the retention of basic analytes. Influence of these ions on the column properties is fully reversible, and equilibration requires minimal time (usually less than an hour, or about 10 to 20 column volumes). On the other hand, the mechanism of their effect is very complex and is dependent on the type of organic modifier used and on the concentration applied. Theoretical description and mathematical modeling of this process is a subject for further studies. 

Figure 5-1. General dependence of the retention of basic analytes on the pH of the mobile phase. The inflection point of the curve corresponds to the component p f of the component. 

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 ... 

Figure 5-29. Influences of change in concentration on retention of basic analytes.

Retention can also be influenced by other components of the mobile phase present in small concentrations. The user may not be aware of the presence of these components. In such a case, troubleshooting can be quite difiicult. A common example of this problem is water in normal-phase chromatography. Water is always present in all solvents (see Table 9.3) and can shift retention substantially. However, this is not the only example. For instance, a contamination of methanol with amines that influences the retention of basic analytes in reversed-phase chromatography has been observed. To avoid this situation, the use of HPLC-grade solvents is generally recommended for HPLC applications. 

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