Mechanistic Considerations on the Reversed Phase Liquid Chromato-Graphic Separation of Proteins

The effect of column length, stationary phase chemistry, flow rate and gradient variation and direction on the separation of seven proteins was evaluated. In general, resolution increased with shorter columns and higher flow rates. Nontraditional reverse gradients (from acetonitrile to buffer) sometimes produced better protein separations than traditional reversed phase gradients. Bonded phase chemistry has little effect on gradient protein separations, but silica gel properties (e.g., surface area, pore size, etc.) did. Both retention and resolution varied considerably with the nature of the gradient used.