Universal calibration of gel permeation chromatography and determination of molecular shape in solution

Abstract

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, α-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, Qβ, 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.