Changes in the cis-trans isomer selectivity of a reversed-phase liquid chromatography column during use with acidic mobile phase conditions.

Reversed-phase liquid chromatography (RPLC) is the analytical tool of choice for monitoring process-related organic impurities and degradants in pharmaceutical materials. Its popularity is due to its general ease-of-use, high performance, and reproducibility in most cases, all of which have improved as the technique has matured over the past few decades. Nevertheless, in our work we still occasionally observe situations where RPLC methods are not as robust as we would like them to be in practice due to variations in stationary phase chemistry between manufactured batches (i.e., lot-to-lot variability), and changes in stationary phase chemistry over time. Over the last three decades several models of RPLC selectivity have been developed and used to quantify and rationalize the effects of numerous parameters (e.g., effect of bonded phase density) on separation selectivity. The Hydrophobic Subtraction Model (HSM) of RPLC selectivity has been used extensively for these purposes; currently the publicly available database of column parameters contains data for 750 columns. In this work we explored the possibility that the HSM could be used to better understand the chemical basis of observed differences in stationary phase selectivity when they occur - for example, lot-to-lot variations or changes in selectivity during column use. We focused our attention on differences and changes in the observed selectivity for a pair of cis-trans isomers of a pharmaceutical intermediate. Although this is admittedly a challenging case, we find that the observed changes in selectivity are not strongly correlated with HSM column parameters, suggesting that there is a gap in the information provided by the HSM with respect to cis-trans isomer selectivity specifically. Further work with additional probe molecules showed that larger changes in cis-trans isomer selectivity were observed for pairs of molecules with greater molecular complexity, compared to the selectivity changes observed for simpler molecules. These results do not provide definitive answers to questions about the chemical basis of changes in stationary phase chemistry that lead to observed differences in cis-trans isomer selectivity. However, the results do provide important insights about the critical importance of molecular complexity when choosing probe compounds and indicate opportunities to develop improved selectivity models with increased sensitivity for cis-trans isomer selectivity.

Measuring and using scanning-gradient data for use in method optimization for liquid chromatography.

The use of scanning gradients can significantly reduce method-development time in reversed-phase liquid chromatography. However, there is no consensus on how they can best be used. In the present work we set out to systematically investigate various factors and to formulate guidelines. Scanning gradients are used to establish retention models for individual analytes. Different retention models were compared by computing the Akaike information criterion and the prediction accuracy. The measurement uncertainty was found to influence the optimum choice of model. The use of a third parameter to account for non-linear relationships was consistently found not to be statistically significant. The duration (slope) of the scanning gradients was not found to influence the accuracy of prediction. The prediction error may be reduced by repeating scanning experiments or - preferably - by reducing the measurement uncertainty. It is commonly assumed that the gradient-slope factor, i.e. the ratio between slopes of the fastest and the slowest scanning gradients, should be at least three. However, in the present work we found this factor less important than the proximity of the slope of the predicted gradient to that of the scanning gradients. Also, interpolation to a slope between that of the fastest and the slowest scanning gradient is preferable to extrapolation. For comprehensive two-dimensional liquid chromatography (LC × LC) our results suggest that data obtained from fast second-dimension gradients cannot be used to predict retention in much slower first-dimension gradients.