SALT MEDIATED ELUTION BEHAVIOR OF PROTEINS ON A SILICA-BASED STATIONARY-PHASE FOR SIZE-EXCLUSION HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Salt mediated elution behavior of several proteins on the commercial silica-based size exclusion TSK G 3000 SW column was examined. Depending on the nature and salt concentration, this column exhibits three domains where either sieving effect or electrostatic or hydrophobic interactions are predominant. At sufficiently low salt concentration positively charged proteins were retained by electrostatic interactions, whereas negatively charged proteins were subjected to Donnan exclusion. On the other hand, by using salt with a large molal surface tension increment, at sufficiently high salt concentration proteins were retained by hydrophobic interactions. Under these conditions the hydrophobic parameters, defined as the slope of the linear plot of log K' against salt molality, were evaluated and compared to those obtained on two columns for hydrophobic interaction chromatography , prepared by coupling butyl and phenyl glycidil ethers to the TSK G 3000 SW packing material. Proteins in a narrow molecular weight range were separated on the TSK G 3000 SW column according to their isoelectric point by the use of increasing salt gradient.