Binary eluent composition

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 ... 

Another important parameter is the eluent composition. Binary mixtures (and obviously pure solvents) should be preferred to complex mixtures, since new systems perform an on-line analysis of the composition of binary eluents. These eluent systems allow the automatic eluent recycling, with a reduced number of controls. 

Assuming that the adsorbent surface is occupied by an adsorbed solute molecule (Ag) and a molecule of a stronger solvent (n ) which are equal to one another, the elution strength of a binary eluent, shows the following dependence on its quantitative composition ... 

Coupling Equation 2.16 and Equation 2.17, we can obtain the following relationship, which describes the dependence of the solute s retention parameter, R, on the quantitative composition of a given binary eluent ... 

By contrast, it is often not possible to standardize cleanup steps based on adsorption chromatography. Altered volumes of elution solvent, small deviations in the water content of the adsorbent and minor changes in the composition of binary eluents are often necessary and should be regarded as minor changes. 

FIGURE 13.2 Plots of viscosity versus eluent composition for binary mixtures of water and methanol (top curve) and water and acetonitrile (bottom curve). 

Fig. 5. Transition from the exclusion to the adsorption separation mode through critical conditions for polystyrene standards at a varying composition of the binary eluent (CCI4—CHC13). (Column Si-300, flow rate u = 0.5 rnl/min, volume of.santple 10 pi, UV detector, >, = 275 nm, t = 27 °C)...

Fig. 18a-c. Transition through the critical conditions for hydroxyl-containing polyibutylene tereph-thalate) at a varying composition of heptane-tetrahydrofuran binary eluent. (Column Si-60, u = 1 mi/ min, volume of the sample 10 pi, UV detector, X = 254 nm, t = 24 °C)... 

In binary eluents, k depends on the composition of the mobile phase. This dependence is approximately given through ... 

Equation (2-46) is applicable for the description of adsorption behavior of binary eluent in the column. In the isocratic mode, the column is equilibrated at given composition of the eluent. Any small volume of the mixture of eluent... 

Effect of the eluent composition could be discussed on the basis of equation (2-55). In the simple case of binary eluent (organic/water mixture), we can consider in the first approximation that Gibbs free energy of the eluent interaction with the packing material surface is a linear function of the eluent composition... 

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... 

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... 

The Rf of a secondary front depends on the eluent composition. It was found that a plot of versus the logarithm of the mole fraction of polar constituent used in the mixture did not show a linear relationship, unlike the compounds migration in the P zone. Rf increases with increasing concentration of polar modifier as well as with decreasing specific surface areas of the sorbent (42). Similar results were found by Wawrzynowicz and Soczewinski (43) in the case of a sandwich chamber and binary eluents. 

Mobile phase composition The same experiments are performed on the same plates using the same chamber system and environmental conditions changing the mobile phase composition. Analytical performance parameters Rf, / 5, precision, lowest detectable quantity Sample Model A and B or C. %-nonpolar 5% (rel) (normal-phase) or polar [preverse-phase] organic solvent in binary eluent system... 

With just a few exceptions, there is a dearth of published information providing systematic studies of retention volumes as a function of composition of the eluent over the whole composition range of binary solvents. To rectify this situation, a general equation for HPLC binary solvent retention behavior has been proposed [59] that should help generate a chromatographic retention model to fit Eq. (15.20) ... 

The retention indices, measured on the alkyl aryl ketone scale, of a set of column test compounds (toluene, nitrobenzene, p-cresol, 2-phenyl ethanol, and IV-methylaniline) were used to determine the changes in selectivity of a series of ternary eluents prepared from methanol/0.02M phosphate buffer pH 7 (60 40), acetonitrile/0.02 M phosphate buffer pH 7 (50 50) and tetrahydrofuran/0.02 M phosphate buffer pH 7 (25 65). The analyses were carried out on a Spherisorb ODS reversed-phase column. The selectivity changes were often nonlinear between the binary composition [83]. 

The first results of optimization in chromatography were published in 1975 Since then a growing number of optimization experiments in HPLC using the Simplex procedure has been reported (table 9). The examples are mainly reversed-phase separations, in which the composition of the ternary or binary mobile phase composition is optimized. The factors optimized are usually a selection from flow rate, column temperature and length, the eluents constitution (e.g. organic modifier content, buffer concentration and pH), the gradient shape. Seven years after the first applications of Simplex optimization had appeared, the first fully automated optimization of HPLC separations was published by Berridge in 1982. This development coincid-... 

The relationships between eluent strength and composition for binary (one to two) and ternary (one to three) solvents have been derived ... 

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