Binary eluent analyte retention

The binary eluent adsorption equilibrium is considered to be not disturbed by the injection of a small amount of the analyte (essentially the third component in the system). In an isocratic mode at a fixed eluent composition, the organic adsorbed layer is a stationary phase for the analyte to partition into. The analyte can partition into the adsorbed layer followed by consequent adsorption on the surface of the reversed-phase adsorbent. The overall retention is a superposition of two consecutive processes. Since the eluent component adsorption could be measured independently and adsorbed layer volume could be represented as a function of the mobile phase composition, the analyte retention also could be expressed as a function of the eluent composition. 

Applying this function into the mass-balance equation (2-33) and performing the same conversions [Eqs. (2-34)-(2-39)], the final equation for the analyte retention in binary eluent is obtained. In expression (2-67) the analyte distribution coefficient (Kp) is dependent on the eluent composition. The volume of the acetonitrile adsorbed phase is dependent on the acetonitrile adsorption isotherm, which could be measured separately. The actual volume of the acetonitrile adsorbed layer at any concentration of acetonitrile in the mobile phase could be calculated from equation (2-52) by multiplication of the total adsorbed amount of acetonitrile on its molar volume. Thus, the volume of the adsorbed acetonitrile phase (Vj) can be expressed as a function of the acetonitrile concentration in the mobile phase (V, (Cei)). Substituting these in equation (2-67) and using it as an analyte distribution function for the solution of mass balance equation, we obtain... 

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. 

In a binary eluent system (acetonitrile-water), an adsorbed organic phase with finite thickness and composition different from the bulk mobile phase is preferentially accumulated near the surface of the bonded phase. The organic layer accumulated near the bonded ligands could behave as a liquid stationary phase in reversed-phase HPLC, and it contributes to the overall analyte retention process. 

The reliable range of is less than one and a half decades. When dealing with a series of analytes of diverse retentive properties it is hence necessary to determine Rm values at several compositions of binary eluents and next to extrapolate linearly the relationship between R and the volume percent of one of the eluent components to a fixed value. In the case of reversed-phase TLC extrapolation is usually performed to pure water (buffer) as a hypothetical eluent. Such an extrapolated Rm value is usually denoted by... 

HPLC retention data for QSRR analysis are usually obtained by measuring log at several eluent compositions (isocratic conditions) and then extrapolating the dependence of log on a binary eluent composition to a fixed mobile phase composition, common for all the analytes studied, based on the Soczewinski-Snyder model ... 

The separation of polar analytes can also be optimized by applying ternary eluent mixtures. In this case, the retention is adjusted with binary mixtures with methanol-water for k in the desired range (2polar analytes resolution [57]. 

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