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 a lkyl chain lengths (C-3, C-8, C-18) and pore sizes (60 Angstrom, 150 A ngstrom, 300 Angstrom) was utilized to determine the influence of chai n length and pore size on chromatographic performance (i.e., resolutio n) and column capacity. Proteins ranging in size from M, 5700 to 68 00 0 were used as model compounds. For each category of packing material, retention times of the proteins decrease and peak widths increase wit h increasing sample loading. Among the matrix materials, C-3/300 Angst rom and C-8/300 Angstrom provide the highest resolution of a mixture o f proteins with total protein loading from 60 mu g to 6 mg on analytic al columns (25 x 0.46 cm). The influence of alkyl chain length and por e size on column capacity was investigated by frontal chromatography, For a fixed chain length, materials with larger pore size provide grea ter column capacity of the model proteins. In addition, for materials of the same pore size, the longer the alkyl chain, the greater the col umn capacity. Column capacity for a given alkyl chain length and pore size is indirectly correlated to protein size; the greater column capa city is found for the smaller tested proteins.