Beyond C18—Increase Retention of Hydrophilic Compounds Using Biphenyl Columns

By Amanda Rigdon, Pharmaceutical Innovations Chemist and Rick Lake, Pharmaceutical Market Development Manager

Searching for a better way to retain hydrophilic aromatic drug compounds? Biphenyl phases, such as the Pinnacle™ DB Biphenyl column, provide greater retention than alkyl phases. Use a Biphenyl column to separate difficult-to-retain polar aromatics from unretained matrix contaminants.

Many drug classes include compounds with aromatic ring structures, some of which also contain a sulfone or sulfoxide group. Both sulfur groups have dipole moments, adding a hydrophilic character to compounds containing these functional groups. The analysis of hydrophilic compounds on a traditional alkyl column (e.g., C18) can be problematic, since alkyl columns depend on hydrophobic (dispersive) interactions for retention. Since the sulfone and sulfoxide groups contain ? bonds, the Biphenyl column’s affinity toward compounds containing these bonds makes it a logical choice when increased retention of compounds containing these groups is desired.

To explore the selectivity of the biphenyl phase towards sulfur-containing aromatic compounds, phenyl sulfone, a simple probe, was analyzed on alkyl (C18), phenyl, phenyl hexyl, and Biphenyl columns to determine the relative retention of each phase, as measured by capacity factor (k'). In order to ensure separation of analytes from unretained contaminants, a minimum k' value of 2 is recommended for most analyses, however in cases where there is little to no matrix interference, a k' of 1 may be acceptable. The data in Figure 1 show that phenyl sulfone is retained to a much greater degree on the Pinnacle™ DB Biphenyl column, than on the other phases tested (k' = 2.08). This is due to the unique retention mechanism of the biphenyl stationary phase, which can interact with both the hydrophobic aromatic ring and the hydrophilic sulfone group through ?-? interactions. Although the phenyl stationary phase also allows for the use of ?-? interactions, the biphenyl phase has a larger electron cloud and is significantly more retentive.