Basic Analytes effect

The pH value also affects the ionization of acidic and basic analytes and their electromigration. Since this migration can be opposite to that of the electroos-motic flow, it may both improve and impair the separation. This effect is particularly important in the separation of peptides and proteins that bear a number of ionizable functionalities. Hjerten and Ericson used monolithic columns with two different levels of sulfonic acid functionalities to control the proportion of EOF and electromigration. Under each specific set of conditions, the injection and detection points had to be adjusted to achieve and monitor the separation [117]. Another option consists of total suppression of the ionization. For example, an excellent separation of acidic drugs has been achieved in the ion-suppressed mode at a pH value of 1.5 [150]. 

For analytical properties to be consistent with the quality expected from an analytical process and the results derived from it, they should be considered in a hierarchical way [4,50]. Thus, there are three primary types of analytical properties, namely (a) capital properties (accuracy and representativeness), which are directly related to quality of the results (b) basic properties (sensitivity, selectivity and precision), which support accuracy and are related to the analytical process and (c) accessory properties (expeditiousness, cost-effectiveness and personnel safety/comfort), which are also related to the properties are related to one another in an additive or contradictory way. The best way of envisaging the ensuing relationships is by means of two analytical tetrahedra sharing a common apex (see Fig. 1.16.A). The apices of the tetrahedron on the left hold the basic analytical properties that define the accuracy triangle, whereas those of the tetrahedron on the right accommodate the accessory analytical properties, which delimit the analytical... 

With some stationary phases at low pH values (<4) benzyl amine as benzyl ammonium ion can be excluded by a Donnan potential from the pores, when positive charges are present at the surface. These could have stemmed from the manufacturing process or could have been introduced on purpose to shield amines from interacting with silanols. With an increasing pH, the Donnan exclusion decreases and at pH >5 benzyl amine is retarded increasingly. An example of this effect with a modern RP with low silanophilic properties is demonstrated in Figure 2.22, where the elution peaks of benzyl amine are presented as a function of pH. With these stationary phases, basic analytes cannot be separated at low pH values. 

LoBrutto, R., Jones, A., and Kazakevich, Y.V. Effect of counter-anion concentration on retention in high-performance liquid chromatography of protonated basic analytes. J. Chmmatogr. A. 2001, 913,189-196. 

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

Corresponding dependencies in van t Hoff coordinates, although almost linear, show different directions in their slope, which is dependent on the particular pH chosen for this particular model (Figure 2-f8). Note that in this model the effect of temperature on the change of the dissociation constants of the buffer species and model basic analyte species were taken into account on the basis of standard relationship of the equilibrium constant with the temperature (Ki = exp[AG/i r]). It has been shown that the dissociation constants of particular acidic species and basic species show some specific variations of their ionization constants with temperature in methanoFwater and acetoni-trUe/water mobile phases [40,41],... 

The pH dependencies of the basic analyte retention in Figure 2-20 are not classic sigmoidal, which is supposed to plateau at low pH, and no change in retention should occur for ionized bases with a further decrease in mobile-phase pH. However, as shown in Figure 2-20, a slight increase of the retention is observed with decrease of mobile-phase pH. This effect has been observed... 

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. 

The second reason in the introduction of polar groups in the bonded ligands is that these groups interact with residual silanols, which make the silanols effectively inactive for the interaction with polar or basic analytes. Sometimes these phases are also end-capped with polar end-capping groups. These phases also show a significant difference in selectivity compared to conventional CIS-type phases. In some cases they show improvement in the peak shape for basic components. 

The third overloading effect is usually associated with the presence of accessible residual sUanols or other strong adsorption sites on the surface of stationary phase. If the surface concentration of these sites is very low, then a small portion of components sensitive to these sites (usually protonated basic analytes) will get adsorbed on them, thus increasing the formation of the peak tail. Neutral and nonionizible analytes are usually not sensitive for these effects. 

A.3 Effects in Region A. Basic analytes show relatively low retention (analyte in its ionic form) and may even elute in the void. The employment of chaotropic additives may be needed to enhance the retention of the protonated basic analytes (see Section 4.10) However, acidic analytes show longer retention times because the acidic analyte would be analyzed in its neutral form. 

Liquid chromatography has also been widely used for the determination of dissociation constants [88-92] since it only requires small quantity of compounds, compounds do not need to be pure, and solubility is not a serious concern. However, the effect of an organic eluent modifier on the analyte ionization needs to also be considered. It has been shown that increase of the organic content in hydro-organic mixture leads to suppression of the basic analyte pKa and leads to an increase in the acidic analyte pK compared to their potentiometric pKa values determined in pure water [74]. 

Effect of Organic Modifier on Basic Analyte pA Shift... 

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. 

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