Properties of Reversed-Phase Columns

The following examples will be used to demonstrate that medium-polarity reversed phases have great potential for protein separations. In Fig. 7 the separation of the C apolipoproteins is shown on a phenyl and on a Ci8 column. The Cig column gave almost no separation of the protein mixture, despite the use of a variety of mobile-phase conditions, whereas the phenyl column gave an excellent separation. Lewis et al. (34) found that the columns of intermediate polarity were effective in the separation of tyrosinase, collagen i, cytochrome c, and bovine serum albumin. In this study the diphenyl column was more retentive than the octyl or nitrile 

The chromatography of apolipoprotein A-1 on a Zorbax ODS (A), Zorbax C8 (B), and a lu-Bondapak alkylphenyl (C) column. In each case the mobile phase was 1% triethylammonium phosphate, pH 3.2, and an acetonitrile gradient was used. The flow rate was 1.5 ml/min. Each analysis was performed on 100 /ig of protein, and the concentration of acetonitrile required to elute the sample is indicated in each panel. Adapted from Hancock and Sparrow (1981d). 

Several other parameters, apart from the nature of the reversed phase, are important in the determination of the exact column best suited for a given separation. Such parameters as particle size, shape of particles, range of particle size, pore size, surface area of the silica particles per gram, amount of reversed phase per gram, the presence of polymerized surface coating, the use of a secondary silanization reagent, the stability of the reversed phase to aqueous conditions, and the column efficiency are all important details. In the following discussion, we will attempt to clarify the relevance of these column parameters to a protein separation. 

Pore Size, Flow Rate, and Temperature Effects 

As the molecular weight of the species to be separated increases, the pore size of the reversed-phase support becomes crucial, particularly 

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