Joscha Breibeck
University of Vienna, Austria
Title: The value of size exclusion chromatography in hydrodynamic characterization of molecules
Biography
Biography: Joscha Breibeck
Abstract
Statement of the Problem: In size exclusion chromatography (SEC), macromolecules are sorted according to their size distribution. Therefore, SEC is also a measure for the hydrodynamic volume and yields valuable analytical information. Column calibration with well-defined polymer standards allows for estimation of apparent molecular weights. However, SEC analysis is usually considered an isolated methodology, overlooking interconnections with related methods. The present study aimed in the detailed hydrodynamic characterization of a novel recombinant biopolymer in comparison with poly(ethylene glycol) by elaborating the correlation between various techniques, including SEC, in order to obtain an integrated overall picture of hydrodynamic molecular properties. Methodology & Theoretical Orientation: In polymer sciences, a universal SEC calibration procedure is used, keeping in mind the dependence of the hydrodynamic volume on both the molecular weight and the intrinsic viscosity. Though essentially defining the solution viscosity of a macromolecule, the latter parameter is often neglected in biochemical sciences. It can be obtained from viscosity data and yields an expectation value for the hydrodynamic radius rh, assuming an ideal spherical molecule shape. Dynamic light scattering (DLS) as a shape-sensitive technique leads to diverging rh values for non-ideal, elongated molecular shapes.
Findings: The discrepancy between the rh data obtained by DLS and viscometric methods was evaluated for molecular shape estimation. SEC for itself is less sensitive to molecular shape and conformation than DLS, but provided useful indications for non-ideal molecule shapes in combination with DLS. In addition, SEC data confirmed independently obtained results from microviscometry.
Conclusion & Significance: In measuring the hydrodynamic volume, SEC data holds relevant conformational information exceeding the mere molecular weight information. To access shape information, it is advisable to use complementary hydrodynamic techniques. Especially the biosciences can only benefit from changing their view on SEC from an isolated standard technique to an integrated hydrodynamic characterization tool.