Analyte retention factors affecting

HPLC retention expressed either in absolute values (T ) or in relative terms (retention factor) is a complex function of a multitude of parameters. Type of bonded phase is only one of them. Surface area of base material, pore size, bonding density, end-capping, and even the column history all can significantly affect analyte retention. 

The analyte nature and its appearance (e.g., ionization state) in the mobile phase are also factors that affect the retention mechanism. Eluent pH influences the analyte ionization equilibrium. Eluent type, composition, and presence of counterions affect the analyte solvation. These equilibria are also secondary processes that influence the analyte retention and selectivity and are of primary concern in the development of the separation methods for most pharmaceutical compounds. 

The composition of the sample has a dramatic influence on extraction. Solid samples can differ significantly in their physico-chemical properties, the type of compounds they contain and their degree of granulation (particle size). These factors affect sorption and retention of 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. 

The following is a graph showing the retention factors as a function of concentration of perchlorate anion obtained in Figs. 5-17 to 5-19 under the three different experimental conditions. It can be seen that, regardless of pH, the counteranion concentration is the determining factor that affects the solvation and ultimately the retention of the analyte. 

Figure 3.5 illustrates the efficiency loss for 1.0,2.1,3.0, and 4.6 mm i.d. columns using four analytes with different retention factors. The loss in efficiency is affected significantly by the column internal diameter and the retention factor of analytes, as predicted by Eq. (3.8). The columns of 2.1-4.6 mm i.d. provide more than 80%... 

The temperature gradient also affects the analyte diffusivity and retention on the column. Thus, both diffusivity and retention factors are a function of pressure and temperature. Frictional heating can cause a nonuniform increase or radial gradient in temperature inside a column, which can have a detrimental effect on the separation, resulting in band-broadening and poor peak shape (40,41). 

Internal standardization circumvents the effects of time-variant instrument response, but does not compensate for different ionization efficiencies of analyte and standard. For internal standardization, a compound exhibiting close similarity in terms of ionization efficiency and retention time is added to the sample at a known level of concentration, e.g., an isomer eluting closely to the analyte or a homologue may serve for that purpose. It is important to add the standard before any clean-up procedure in order not to alter the concentration of the analyte without affecting that of the standard. For reliable results, the relative concentration of analyte and standard should not differ by more than a factor of about ten. 

First, we will explore the three fundamental factors in HPLC retention, selectivity, and efficiency. These three factors ultimately control the separation (resolution) of the analyte(s). We will then discuss the van Deemter equation and demonstrate how the particle diameter of the packing material and flow rate affect column efficiencies. 

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