Avoiding Reversed-Phase Chromatography Problems Through Informed Method Development Practices: Choosing the Stationary-Phase Chemistry


Many problems encountered executing high performance liquid chromatography (HPLC) methods are the result of decisions made during early method development. This instalment of "Column Watch" discusses a critical variable in method development: the choice of stationary-phase chemistry. We discuss two stationary phase classes, embedded polar group (EPG) and perfluorophenyl (PFP), that are highly complementary to alkyl phases from a fundamental molecular interaction point of view. An understanding of the contrasting interactions that these different classes of stationary phase chemistries provide then leads to more accurate decisions regarding what phases may be most appropriate for a given set of analytes.

In practice, most chromatographers will initially reach for their favourite C18 column when commencing method development (1). An analyst will possibly evaluate a column using a scouting gradient, adjusting parameters such as organic percentage, pH, and organic modifier type until the desired retention and selectivity are obtained. In cases where the desired results are not obtained, many scientists will then choose their second favourite C18 column and repeat this process. If the practice is unsuccessful, it becomes clear that use of a C18 stationary phase will not provide the necessary separation and the analyst is then faced with a choice: change the stationary phase or force the C18 phase to do something it does not inherently do.

Forcing a C18 phase to provide interactions that are not inherent to a significant degree, such as ion exchange, can be induced by such means as the addition of ion-pairing reagents. Although ion-pair chromatography usually works, it often comes at a significant price. Because of the complexity of the interactions, ion-pairing methods tend to be difficult to reproduce, transfer, and troubleshoot (2). C18 systems can also be "made" to work by moving to highly complex mobile phases or extreme temperatures and pH levels. Again, such methods tend to lack robustness and ruggedness, causing extensive headaches later in the method lifetime.

A preferred approach is to use alternative stationary phases to obtain the desired retention and selectivity. This approach is not often chosen because of a lack of knowledge of interactions provided by the plethora of stationary phases available from many manufacturers. The objective of this work is to compare and contrast interactions provided by two main classes of alternative stationary phases to commonly used alkyl phases; namely, polar-embedded phases and fluorinated aromatic phases. By understanding the interactions that are available on such column chemistries, method development analysts can readily identify phases most appropriate for a given sample. The use of the correct tools facilitates method development and validation. In addition, using the right column generates an increase in method robustness and ruggedness because of the simpler mobile phases that are generally required.