Anomalies of 2,2-bipyridyl in reverse-phase high-performance liquid chromatography
Abstract
Using the example of the retention features of 2,2¢-bipyridyl in reverse-phase HPLC, it was established for the first time that the observed anomalies at the maximum water content in the eluent depended on the flow rate of the mobile phase. To identify these anomalies, we used a recurrent approximation of the dependence of retention times on the methanol content in the eluent. The formation of the hydrate is typical for 2,2¢-bipyridyl, therefore the points corresponding to the highest water content in the eluent deviated downwards from the regression line on the graphs of the recurrent approximation of its retention times when using aqueous acetonitrile eluents. This feature is typical for all analytes forming hydrates. A similar anomaly was observed when using water-methanol eluents with a minimum eluent flow rate (0.7 cm3/min). However, with an increase in the eluent flow rate (up to 1.0 ml/min), the corresponding points on the graphs of the recurrent approximation of retention times were located above the regression line, which has never been observed before. We proposed a possible explanation of the effect.
If the deviations of the points on the graphs of the recurrent approximation down from the regression line were caused by the properties of analytes (formation of hydrates), the dependence of the nature of the observed anomalies on the eluent flow cannot be the only explanation for the deviations. It was also necessary to take into account the greater influence of eluent flow rate on the values of the asymmetry factor of chromatographic peaks of 2,2¢-bipyridyl when using aqueous methanol eluents as compared to aqueous acetonitrile. The combination of two identified anomalies, a change in the sign of deviations of points on the graphs of recurrent approximation and an increase in the asymmetry factor with an increase in eluent flow rate, could be interpreted as a consequence of the nonequilibrium nature of chromatographic separation. With a decrease in eluent consumption, the state of the system became more consistent with equilibrium.
Such a detailed study of the features of chromatographic retention of 2,2¢-bipyridyl did not seem to be a particular problem, as similar anomalies could be observed for compounds of a different chemical nature. As a result, 2,2¢-bipyridyl could not be recommended as a test component for monitoring the inertness of chromatographic systems in reverse-phase HPLC.
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References
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