Evaluation of an embedded polar C4 phase for hydrophobic protein analysis by reversed-phase liquid chromatography

A C4 column (InertsilWP300 C4) was developed for a protein analysis in reversed-phase chromatography. Polar groups were embedded on this phase. And this column was evaluated with low concentration of trifluoroacetic acid (TFA) and formic acid. Test C4 phases that controlled carbon loading and commercially available columns were compared with this embedded C4 column. The test column with higher carbon loading showed good separations of proteins at low concentrations of TFA, although hydrophobic proteins were broadened. On the other hand, embedded polar C4 packings showed ideal retention behavior for proteins, which is independent of ion-pairing effects of additives in mobile phase. The reason was concluded to be the shielding effects of polar groups on the sorbents, which reduce silanophilic interactions with polar groups of proteins.