Organic solvent modifier and temperature effects in non-aqueous size-exclusion chromatography on reversed-phase columns.

Common reversed-phase columns (C18, C4, phenyl, and cyano) offer inert surfaces suitable for the analysis of polymers by size-exclusion chromatography (SEC). The effect of tetrahydrofuran (THF) solvent and the mixtures of THF with a variety of common solvents used in high performance liquid chromatography (acetonitrile, methanol, dimethylformamide, 2-propanol, ethanol, acetone and chloroform) on reversed-phase stationary phase characteristics relevant to size exclusion were studied. The effect of solvent on the elution of polystyrene (PS) and poly(methyl methacrylate) (PMMA) and the effect of column temperature (within a relatively narrow range corresponding to typical chromatographic conditions, i.e., 10°C-60°C) on the SEC partition coefficients KSEC of PS and PMMA polymers, were also investigated. The bonded phases show remarkable differences in size separations when binary mixtures of THF with other solvents are used as the mobile phase. The solvent impact can be two-fold: (i) change of the polymeric coil size, and possible shape, and (ii) change of the stationary phase pore volume. If the effect of this impact is properly moderated, then the greatest benefit of optimized solute resolution can be achieved. Additionally, this work provides an insight on solvent-stationary phase interactions and their effects on column pore volume. The only effect of temperature observed in our studies was a decreased elution volume of the polymers with increasing temperature. SEC partition coefficients were temperature-independent in the range of 10°C-60°C and therefore, over this temperature range elution of PS and PMMA polymers is by near-ideal SEC on reversed-phase columns. Non-ideal SEC appears to occur for high molar mass PMMA polymers on a cyano column when alcohols are used as mobile phase modifiers.