Meng Q C, Chen Y F, Delucas L J, Oparil S
Department of Medicine, University of Alabama, Birmingham 35294.
J Chromatogr. 1988 Jul 8;445(1):29-36. doi: 10.1016/s0021-9673(01)84505-2.
On the basis of studying the retention and band broadening of proteins on the TSK SW column, diffusion coefficients (Ds) 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 Ds 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 lambda dp (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 = CsU + 2 lambda dp + f(alpha M)T (where CsU represents mass transfer resistance caused by solute diffusion in the stationary phase and f(alpha M)T an added term for polydisperse solutes as proposed by Knox and McLennan [Chromatographia, 10 (1977) 75]) reflect impurities in the proteins.
在研究蛋白质在TSK SW柱上的保留和谱带展宽的基础上，获得了溶质在固定相中的扩散系数（Ds），这阐明了通过亲水尺寸排阻色谱法（SEC）对蛋白质进行色谱分离的流体动力学过程。在计算Ds与溶质分子量之间的相关性之后，可以预测色谱分离过程中蛋白质的分子尺寸。蛋白质扩散系数与计算值的偏差反映了测量的分子尺寸与根据SEC柱校准曲线预测的分子体积之间的差异。本研究说明了一种通过SEC估算蛋白质纯度的简便方法。理论塔板高度（H）与流速（U）曲线的截距（由谱带展宽方程H = CsU + 2λdp + f(αM)T得出，其中CsU表示溶质在固定相中扩散引起的传质阻力，f(αM)T是Knox和McLennan [《色谱学》，10 (1977) 75]提出的多分散溶质的附加项）偏离2λdp（其中dp是颗粒直径）反映了蛋白质中的杂质。
