Reversed-phase eluent composition

In most situations the eluent composition is chosen to minimize the effects of hydrophobic interaction, but these secondary effects can be used to advantage. By careful selection of a salt and its concentration, specific selectivities for analytes can be achieved without the use of organic solvents. Therefore, many separations usually run by solvent gradient reversed-phase methods can be completed with a purely aqueous isocratic eluent (13,14). 

Gilpin, R. K., Jaroniec, M., and Lin, S., Dependence of the methylene selectivity on the composition of hydro-organic eluents for reversed-phase liquid chromatographic systems with alkyl bonded phases, Chromatographia, 30,393, 1990. 

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]. 

Analysis of the Copolymerizabilities of Monomers The composition of the copolymers formed was determined by measuring the relative amounts of each monomer, NIPAAM and AAM, that remained in solution after a copolymerization. Copolymerizations were terminated by addition of 1 ml of reaction mix to 9 ml of 0.1% phosphoric acid at 50 C, followed by centrifugation of a 0.4 ml aliquot at 6,500 x g for 5 minutes in an Eppendorf microfuge. After 100 fold dilution of an aliquot of the supernate, 200 pi of this was injected onto an IBM reversed phase Cig HPLC column pre-equilibrated with 2% acetonitrile in 0.1% aqueous phosphoric acid and the eluent monitored at 214 nm. The monomers were eluted using a 0.1% aqueous phosphoric acid (solvent A) acetonitrile (solvent B) gradient as follows for 5 minutes the solvent was 98% solvent A and 2% solvent B, followed by a linear gradient to 80% A and 20% B over 10 minutes. After 5 more minutes at 80% A and 20% B, the solvent was returned to 98% A and 2% B. 

Compensation temperatures have been found to be identical for the retention of a variety of eluites on three different types of reversed phase columns over a wide range of eluent composition (/ 77). The authors noted that if compensation behavior occurs in chromatography, the retention... 

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-... 

TABLE 2-2. Selectivity Between Alkylbenzenes at Different Eluent Compositions on Reversed-Phase Column"... 

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. 

Several different theories have been proposed for the description of the influence of the eluent composition on the analyte retention in reversed-phase... 

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. 

General dependence of the analyte retention on the eluent composition in reversed-phase FIPLC shows an exponential decay with the increase of the organic modifier concentration. This is usually described in the following form ... 

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