Characterizing Reversed - Phase Column Selectivity C

olumn selectivity varies among different reversed-phase columns, and, sometimes, different batches of the same column. For a reversed-phase liquid chromatography (LC) assay that is to be used for several months or years, replacement columns with the same selectivity must be available for the duration of the method. For example, consider the separation in Figure 1a, which uses an Ace C8 column (Hichrom Ltd., Theale, United Kingdom). If for some reason that column became unavailable (or if batch-to-batch selectivity changed), we would require a replacement column of similar selectivity. As Figure 1b shows, a Precision C8 column (Mac-Mod Analytical, Inc., Chadds Ford, Pennsylvania) would serve this purpose. For method development, a column of very different selectivity could be necessary to achieve acceptable sample resolution. For example, an Inertsil C8 column (GL Sciences, Inc., Tokyo, Japan) provides the separation of Figure 1c, in which peak pairs 1–2 and 8–9 overlap. Changing to a column with very different selectivity can lead to an improved separation, as would be the case for this sample by changing from the Inertsil C8 to either the Precision C8 or the Ace C8 columns.