Effect of steric exclusion on the separation of proteins by hydrophilic size-exclusion chromatography

✍ Scribed by Qing Cheng Meng; Yiu-Fai Chen; Lawrence J. Delucas; Suzanne Oparil

• Publisher: Elsevier Science
• Year: 1988
• Tongue: English
• Weight: 534 KB
• Volume: 445
• Category: Article
• ISSN: 1873-3778
• DOI: 10.1016/s0021-9673(01)84505-2

✦ Synopsis

On the basis of studying the retention and band broadening of proteins on the TSK SW column, diffusion coefficients (OS) of solute in stationary phase were obtained which elucidate the hydrodynamic process of chromatographic resolution of proteins by hydrophilic size-exclusion chromatography (SEC). After calculating the correlation between D, and the molecular weight of the solute, the molecular dimensions of proteins in the process of chromatographic separation can be predicted. Deviations in diffusion coefficient of a protein from the calculated value reflect differences of measured molecular dimensions from molecular volumes predicted from the calibration curve of the SEC column. This study illustrates a convenient method for estimating the purity of proteins by SEC. Deviations from 2;1 dr (where dp is the particle diameter) in the intercept of the theoretical plate height (H> versus flow-rate (U) curve from the band broadening equation H = C,U + 21 d,, + f(a& {where C,U represents mass transfer resistance caused by solute diffusion in the stationary phase and f(a& an added term for polydisperse solutes as proposed by Knox and McLennan [Chromatographia, 10 (1977) 751) reflect impurities in the proteins.