RETENTION BEHAVIOR OF PROTEINS UNDER CONDITIONS OF COLUMN OVERLOAD INHYDROPHOBIC INTERACTION CHROMATOGRAPHY

The sample solvent has a significant effect on band profiles in prepar ative liquid chromatography. This effect has been described by Guiocho n and Jandera in non-aqueous reversed-phase high-performance liquid ch romatography. Similar behaviour was observed in hydrophobic interactio n chromatography of cytochrome c and lysozyme under conditions of colu mn overloading. If the sample solvent contains a lower concentration o f salt than the starting mobile phase in gradient hydrophobic interact ion chromatography, the protein peak will be broken to two peaks. One peak is longer-retarded, another is shorter-retarded by hydrophobic in teraction. The latter changes its retention time with the different in jection volumes. This results in a loss in the recovery of protein in preparative chromatography. The possible mechanism of the band-splitti ng was investigated.