EFFECT OF STATIONARY-PHASE ON PREPARATIVE PROTEIN SEPARATION IN REVERSED-PHASE CHROMATOGRAPHY

A matrix of Zorbax Pro-10 (10 mu m) packing materials with differing alkyl chain lengths (C-3, C-8, C-18) and pore sizes (60 Angstrom, 150 Angstrom, 300 Angstrom) was utilized to determine the influence of chain length and pore size on chromatographic performance (i.e., resolution) and column capacity. Proteins ranging in size from M, 5700 to 68 000 were used as model compounds. For each category of packing material, retention times of the proteins decrease and peak widths increase with increasing sample loading. Among the matrix materials, C-3/300 Angstrom and C-8/300 Angstrom provide the highest resolution of a mixture of proteins with total protein loading from 60 mu g to 6 mg on analytical columns (25 x 0.46 cm). The influence of alkyl chain length and pore size on column capacity was investigated by frontal chromatography, For a fixed chain length, materials with larger pore size provide greater column capacity of the model proteins. In addition, for materials of the same pore size, the longer the alkyl chain, the greater the column capacity. Column capacity for a given alkyl chain length and pore size is indirectly correlated to protein size; the greater column capacity is found for the smaller tested proteins.