Investigation of Retention Anomalies With Ternary Mobile Phases in Reverse Phase Liquid Chromatography
Chappell, Mark D.
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Retention and selectivity for a set of analytes in reversed-phase HPLC can be adjusted by changing the mobile phase composition. In one approach to finding an optimum ternary mobile phase, two different binary mobile phases (such as methanol/water and acetonitrile/water) are identified which give the same (suitable) retention time for one key analyte of interest. Then these isoeluotropic mobile phases are mixed in varying proportions, selectivity differences are observed, and the mobile phase providing the best separation is selected. Assiuning that the isoeluotropic mobile phases have identical Hildebrand solubility parameters, this theory would predict that the retention times would remain approximately constant in this experiment. However, the retention times are normally greater than predicted, showing a maximum difference near the 50:50 mixture of the isoeluotropic solvents. This anomalous retention time increase has been noted occasionally in the literature and possible explanations have been hypothesized, but it does not appear to be well understood. In this work, retention was measured as a function of mobile phase composition for 5 sets of analytes (19 total compounds) using a ternary solvent system consisting of acetonitrile, methanol and buffer. The anomalous increase in retention was noted for each system, although the extent of the anomaly varied considerably. The magnitude of the retention anomaly was quantitated by calculating the maximum difference between experimental retention and the expected constant retention, using a quadratic regression model to fit the experimental retention data. Correlations were sought between the direction and magnitude of the retention anomaly and solvent and solute properties. The positive direction of the retention anomaly is accounted for by nonlinear changes in solvent polarity. Solvent polarity values were measured for the solvent systems of interest using absorption spectral shifts for the solvatochromic Et(30) dye. These polarity values showed a maximum in polarity at intermediate solvent compositions, corresponding to increased retention in reversed phase HPLC. However, the solvent Et(30) polarity values are not correlated with the magnitude of the anomaly for the compounds investigated. Solute molecular weight was not well correlated with the extent of the anomaly, even for sets of similar compounds, suggesting that hydrophobic interactions are not a controlling factor. The magnitude of the retention anomaly is correlated with the sensitivity of solute retention to the change in organic composition of either of the binary mobile phases.
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