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Reversed-phase chromatography of proteins on resin-based wide-pore packings

1986; Elsevier BV; Volume: 359; Linguagem: Inglês

10.1016/0021-9673(86)80066-8

1873-3778

Kathleen A. Tweeten, T. N. Tweeten,

Protein purification and stability

PLRP-S, a macroporous poly(styrene—divinylbenzene) HPLC packing material, was evaluated for analysis of proteins. Materials with a particle size of 8 μm and pore sizes of 100 Å, 300 Å, and 1000 Å were tested under similar chromatographic conditions to determine the effect of pore size and surface area on retention, selectivity, and efficiency. This resin-based material has a highly homogeneous hydrophobic surface, based on phenyl moieties contributed by the copolymers of the packing. Thus, unlike silica, PLRP-S has no bonded alkyl groups or residual silanol sites. In addition, the PLRP-S material is stable over a wide range of solvents, pH, and ionic strengths. The elution order of a series of proteins on PLRP-S 300 Å was similar to that observed on a variety of alkyl-bonded silica-based reversed-phase columns. The percentage of acetonitrile in the mobile phase necessary to elute three of these proteins (ribonuclease A, cytochrome c, and ovalbumin) was determined. Protein desorption occurred over a very narrow range of 2–4% increase in concentration of the organic modifier. No apparent change in protein selectivity or peak area occurred with repetitive injections of a mixture of proteins, indicating little relative loss of protein on the column. Pore size appeared to have little effect on the selectivity or retentivity of the test proteins. It appeared that the loss of total surface area due to increased pore size was offset by the increased availability of pore surface area for protein-packing interaction. The resin-based PLRP-S 300 Å was used for analysis of wheat proteins, bovine pancreatic enzymes, high-molecular-weight proteins, and whey proteins. Over the broad range of molecular weights and hydrophobicities in these applications, proteins were eluted as sharp, symmetrical peaks. Thus, PLRP-S offers an effective alternative to reversed-phase silica for the analysis of proteines.