Potschka M
Anal Biochem. 1987 Apr;162(1):47-64. doi: 10.1016/0003-2697(87)90009-1.
Gel permeation chromatography (GPC) has become a routine technique for both biology and polymer chemistry. By comparison our theoretical perception of the separation principle of GPC is still immature and conflicting and so is the assessment of the analytical informational content of this method. In order to discriminate between the various parameters that might influence GPC and thus to decide among the numerous propositions of calibration, several odd biopolymers (tropomyosin, spectrin, DNA, tobacco mosaic virus, alpha-actinin, ovomucoid) were selected. They were characterized by analytical ultracentrifugation as well as quasielastic light scattering, and they were compared to globular proteins including icosahedral viruses (tomato bushy stunt virus, turnip yellow mosaic virus, Q beta, MS2) on several different HPLC column matrices. The results demonstrate that the universal calibration principle of GPC is the viscosity radius, i.e., the molecular volume times a shape function which is defined by the intrinsic viscosity. Alternate propositions such as molecular weight, second virial coefficient, diffusion coefficient (Stokes radius), radius of gyration, mean linear projected length, contour length, and related measures seem to be excluded on the basis of the evidence presented. These results help to focus the physical picture which seems to govern GPC. Finally it is demonstrated that GPC is a versatile and unique tool with which to characterize molecular shape and dynamics in solution.
凝胶渗透色谱法（GPC）已成为生物学和高分子化学领域的常规技术。相比之下，我们对GPC分离原理的理论认识仍不成熟且存在争议，对该方法分析信息含量的评估也是如此。为了区分可能影响GPC的各种参数，从而在众多校准方法中做出选择，我们挑选了几种奇特的生物聚合物（原肌球蛋白、血影蛋白、DNA、烟草花叶病毒、α -辅肌动蛋白、卵类粘蛋白）。通过分析超速离心和准弹性光散射对它们进行了表征，并在几种不同的高效液相色谱柱基质上与包括二十面体病毒（番茄丛生矮化病毒、芜菁黄花叶病毒、Qβ、MS2）在内的球状蛋白质进行了比较。结果表明，GPC的通用校准原理是粘度半径，即分子体积乘以由特性粘度定义的形状函数。基于所提供的证据，诸如分子量、第二维里系数、扩散系数（斯托克斯半径）、回转半径、平均线性投影长度、轮廓长度及相关度量等其他方法似乎被排除。这些结果有助于聚焦似乎支配GPC的物理图景。最后证明，GPC是一种用于表征溶液中分子形状和动力学的通用且独特的工具。
