4 Column characterization and selection

Abstract

Reversed-phase high performance liquid chromatography (RP-HPLC) methods are used extensively during quality control of pharmaceutical products. During HPLC method development one of the biggest challenges is to select a stationary phase that has the desired selectivity, suitable, reproducibility and stability. Column selection presents difficulties since more than 600 RP-LC column brands are available on the market. Selectivity of the column and peak shapes of injected compounds is highly dependent on the characteristics of the column. Good understanding of the most significant features of the stationary phases facilitates fast solution of chromatographic issues that occur during method development (e.g. bad peak shape or poor resolution). Properties such as column efficiency, hydrophobicity, silanol activity, ion-exchange capacity, steric selectivity and the amount of metal impurities can be characterized by rather simple chromatographic tests. Calculating representative parameters of the tests allows one to classify stationary phases with similar or dissimilar characteristics. Consideration of classes of chromatographic phases can be beneficial if a column has to be replaced with a similar selectivity column or with an orthogonal stationary phase. Such chromatographic test procedures can also be used to control the performance of a column at any time of its life cycle. This chapter focuses on reviewing the most important characteristics of RP-HPLC columns that play important roles in real separations. These characteristics can be determined with different chromatographic test methods, which are described in the literature and also summarized here. The results of the test methods provide excellent basis for classification of RP-HPLC columns according to their performance. This type of classification and the existing databases offer great practical assistance in selection of appropriate RP-HPLC columns. One of the databases is freely accessible through the Internet.